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A mutant EGF-receptor defective in ubiquitylation and endocytosis unveils a role for Grb2 in negative signaling

Hadassa Waterman,Patent Attorney, Ehrlich & Fenster 2001

Ligand-induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c-Cbl, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c-Cbl, here we report that defective Tyr1045 of EGFR, an inducible c-Cbl docking site, enhances the mitogenic response to EGF. Signaling potentiation is due to accelerated recycling of the mutant receptor and a concomitant defect in ligand-induced ubiquitylation and endocytosis of EGFR. Kinetic as well as morphological analyses of the internalization-defective mutant receptor imply that c-Cbl-mediated ubiquitylation sorts EGFR to endocytosis and to subsequent degradation in lysosomes. Unexpectedly, however, the mutant receptor displayed significant residual ligand-induced ubiquitylation, especially in the presence of an overexpressed c-Cbl. The underlying mechanism seems to involve recruitment of a Grb2 c-Cbl complex to Grb2-specific docking sites of EGFR, and concurrent acceleration of receptor ubiquitylation and desensitization. Thus, in addition to its well-characterized role in mediating positive signals, Grb2 can terminate signal transduction by accelerating c-Cbl-dependent sorting of active tyrosine kinases to destruction.

Keywords: growth factor/SH2 domain/signal transduction/tyrosine kinase/ubiquitin ligase

Introduction

Polypeptide growth factors mediate cell-to-cell interactions by initiating an ordered cascade of membranal and cytoplasmic events culminating in altered gene expression (van der Geer et al., 1994). While events involved in signal generation and maintenance are extensively characterized, our understanding of processes that terminate signaling is relatively limited. The role for such negative signaling pathways extends beyond the ability to terminate intracellular signals. For example, analyses of one of the major signaling pathways, the mitogen-activated protein kinase (MAPK) cascade, led to the realization that the amplitude and duration of MAPK activation critically determine not only the kinetics but also the identity of cellular responses to hormonal signals (Marshall, 1995). The ErbB family of growth factor receptors exemplifies the importance of negatively acting regulatory pathways and their significance to human diseases (reviewed in Yarden and Sliwkowski, 2001). The four ErbB proteins bind a large group of growth factors all sharing an epidermal growth factor (EGF) domain. Interestingly, the four receptors differ in their signaling potency in accordance with distinct mechanisms that negatively regulate the receptor’s fate. For example, only ErbB-1 (also called EGFR) is strongly coupled to the c-Cbl adaptor protein, and this receptor, unlike other ErbB members, is effectively targeted to lysosomal degradation (Levkowitz et al., 1998). Similarly, the ortholog of ErbB proteins in Caenorhabditis elegans, LET-23, is negatively regulated by SLI-1, the ortholog of mammalian Cbl proteins (Jongeward et al., 1995). Recent studies that made use of an in vitro ubiquitylation system uncovered the role of c-Cbl as an E3 ubiquitin ligase that recruits ubiquitin-loaded E2 enzymes to ligand-activated receptors (Joazeiro et al., 1999; Levkowitz et al., 1999; Waterman et al., 1999; Yokouchi et al., 1999). Apparently, Cbl proteins bind ligand-activated receptor tyrosine kinases through their N-terminally located phosphotyrosine-binding domain, whereas the flanking RING finger enables close apposition of an E2 enzyme, permitting transfer of ubiquitin to target proteins.

Exactly how c-Cbl-induced poly-ubiquitylation of EGFR regulates delivery to the lysosome remains an open question. Internalization of yeast membrane proteins is initiated by protein mono-ubiquitylation (reviewed by Hicke, 2001). In line with the possibility that a similar mechanism operates in mammalian cells, internalization of the macrophage growth factor receptor is retarded in c-Cbl-defective cells (Lee et al., 1999). However, although overexpression of c-Cbl enhanced ubiquitylation of EGFR, no concurrent increase in receptor internalization rate could be demonstrated (Levkowitz et al., 1998; Thien et al., 2001). Likewise, it is unclear whether c-Cbl is recruited to EGFR at the plasma membrane or only when the receptors reach the early endosome (Levkowitz et al., 1998; Lee et al., 1999; Stang et al., 2000). To address the function of c-Cbl in negative signaling, we made use of a mutant EGFR, the c-Cbl docking site of which—Tyr1045—was defective (Levkowitz et al., 1999). Here we provide evidence that this site restricts the mitogenic action of EGFR by enabling recruitment of c-Cbl to the plasma membrane-localized receptors, thereby accelerating their internalization and delivery to lysosomes. By employing the defective receptor, we uncovered a secondary pathway that allows indirect coupling of c-Cbl to activated receptors. This surrogate pathway involves the adaptor function of Grb2. Thus, our studies help resolve the role of c-Cbl in receptor desensitization and reveal a previously uncharacterized negative function of Grb2.

Results

An EGFR mutant defective at Tyr1045 elicits stronger mitogenic signals

Replacement of Tyr1045 of EGFR with a phenylalanine reduced ligand-induced down-regulation in living cells and significantly reduced receptor ubiquitylation (Levkowitz et al., 1999). To resolve the relationship between receptor ubiquitylation and signaling capacity, we stably expressed the mutant receptor (Y1045F) in interleukin-3 (IL-3)-dependent 32D myeloid cells. These cells are originally devoid of any ErbB protein, but ectopic expression of ErbB receptors confers mitogenic responsiveness to the respective ligands. Cell lines expressing comparable numbers of wild-type and mutant receptors were established by drug selection and their growth examined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. When cultured for 24 h in the absence of IL-3, but in the presence of increasing concentrations of EGF, cells expressing a wild-type (wt) EGFR generated a moderate mitogenic signal (Figure 1A). However, the mutant receptor defective in c-Cbl binding elicited significantly higher proliferative signals. The relatively potent mitogenic potential of the mutant receptor was also reflected in a long-term cell growth assay. Whereas EGF only slightly extended survival of cells expressing wt-EGFR, ligand stimulation of 32D cells expressing Y1045F-EGFR exerted marked cell proliferation, albeit lower than the effect observed with IL-3 (Figure 1B). To examine the possibility that Y1045F-EGFR is endowed with relatively potent mitogenic signaling because it escapes inhibition by c-Cbl, we tested the effect of an ectopic c-Cbl on signaling downstream of EGFR (Figure 1C and D). The biochemical assays employed were MAPK activation and transcription of a reporter gene controlled by the serum response element (SRE). The assays were performed with Chinese hamster ovary (CHO) cells, because these cells express no endogenous EGFR. As expected, stimulation of wt-EGFR with EGF resulted in marked activation of MAPK, but introduction of an exogenous c-Cbl significantly reduced the effect of EGF. Likewise, transcription from the SRE was markedly elevated by EGF and almost completely suppressed when c-Cbl was overexpressed. The Y1045F mutant mediated comparable MAPK activation, as well as marked transcription from the SRE, but both activities were unaffected by an ectopically overexpressed c-Cbl. Taken together with the results of the cell growth assays, these observations indicate that c-Cbl acts as a suppressor of EGFR signaling.

Fig. 1. A mutant EGFR defective at Tyr1045 elicits potent signals and is refractory to c-Cbl. (A) Sublines of 32D cells that express either a wt-EGFR (squares), or a Tyr1045 mutant (Y1045F; triangles) were deprived of IL-3 and plated at a density of 5 × 10⁵ cells/ml in media containing serial dilutions of EGF. The MTT assay was performed 24 h later. The results obtained are presented as fold induction relative to a control culture maintained in the absence of added growth factor. (B) The indicated sublines of 32D cells (5 × 10⁵ cells/ml) were incubated for various time intervals with EGF at 100 ng/ml (circles). Control cultures were incubated in the absence (squares) or presence of IL-3 (triangles). Cell growth was determined daily by using the colorimetric MTT assay, and compared with the signal observed at time zero. The data presented are the mean ± SD of four determinations. (C) CHO cells were transiently transfected with plasmids encoding either a wt-EGFR (WT) or a Tyr1045 mutant (Y1045F). Alongside, we used a vector encoding c-Cbl or a control empty vector. Cell monolayers were stimulated with EGF (100 ng/ml) for the indicated time intervals at 37°C. Whole-cell lysates were analyzed with an antibody specific to the active form of MAPK. (D) CHO cells were co-transfected in triplicates as in (C), along with a reporter plasmid (SRE-luc). Thirty-six hours later cells were untreated or treated with EGF (20 ng/ml). Following an additional 12 h, cells were harvested for a luciferase assay. Signals obtained were normalized to protein concentrations and are presented as average ± SD.

Abrogation of c-Cbl’s interaction with EGFR retards receptor endocytosis and accelerates recycling

To address the relationships between c-Cbl recruitment and receptor endocytosis, we made use of a previously described fusion protein comprised of a full-length EGFR fused to green fluorescence protein (GFP) (Brock et al., 1999). A mutation was introduced in the codon corresponding to Tyr1045 and the plasmid used to co-transfect CHO cells. Consistent with previous reports, the plasma membrane-associated wt-EGFR translocated into large endocytic vesicles upon short exposure of cells to EGF (Figure 2). Prior to stimulation with a ligand, c-Cbl was localized primarily to the cytoplasm (Wang et al., 1999). However, shortly after stimulation with EGF, a large fraction of c-Cbl translocated into cytoplasmic vesicles, many of which contained the GFP–EGFR. These results are consistent with observations made with a GFP–Cbl fusion protein (Levkowitz et al., 1998). The behavior of GFP–Y1045F revealed remarkable differences: upon stimulation with EGF, this mutant underwent only limited translocation into endosomal vesicles. Furthermore, in line with a major defect in internalization, the mutant receptor was unable to induce translocation of c-Cbl.

Fig. 2. The c-Cbl docking site of EGFR is necessary for ligand-induced receptor endocytosis and for translocation of c-Cbl to endocytic vesicles. CHO cells transiently expressing HA-tagged c-Cbl along with a wild-type GFP–EGFR (upper panel), or a similar fusion protein containing a mutation at Tyr1045 (Y1045F; lower panel), were grown on cover slips. Cells were incubated for 15 min at 37°C without or with EGF (100 ng/ml). To visualize c-Cbl, cells were fixed, permeabilized and incubated with an anti-HA antibody, followed by incubation with a Cy3-conjugated secondary antibody (red, middle column). The GFP–EGFR fluorescence is represented in the left column (green). The right column presents the overlay of GFP and Cy3 fluorescence, generating a yellow color in areas of co-localization.

To elucidate the defect in internalization of the Y1045F mutant receptor, we performed several kinetic assays. For a reference mutant whose ligand-induced internalization is defective we used an EGFR devoid of tyrosine kinase activity due to a mutation in the ATP-binding site (K721A) (Chen et al., 1989; Felder et al., 1990). A short-term ligand internalization assay confirmed the relatively slow internalization rate of EGF by K721A (Figure 3A). In the same assay Y1045F displayed an intermediate rate of ligand endocytosis. Likewise, whereas the wt-EGFR mediated rapid ligand degradation, which was completely inhibited by an inhibitor of lysosomal hydrolases, both mutant receptors mediated significantly slower rates of EGF degradation (Figure 3B). In concordance with the internalization assay, Y1045F was slightly more effective than the kinase-dead mutant, but even the latter mediated ligand destruction, probably because it can enter the basal route of endocytosis (Wiley et al., 1991). In agreement with previous reports that documented a defect in translocation of a kinase-defective EGFR to late endosomal compartments (Felder et al., 1990; Hopkins et al., 1990), this mutant recycled EGF more efficiently (Figure 3C). Interestingly, Y1045F was as effective as the kinase-dead receptor in recycling an intact ligand. In conclusion, Tyr1045-mediated interaction of EGFR with c-Cbl seems essential for rapid endocytosis of ligand-receptor complexes and their translocation to lysosomal sites of EGF degradation.

Fig. 3. The c-Cbl docking site of EGFR is essential for rapid ligand endocytosis and degradation, and it enables sequestration from the recycling pathway. (A) CHO cells transiently expressing a wt-EGFR (squares), a kinase-defective mutant receptor (circles) and a Tyr1045 mutant (Y1045F; triangles) were incubated at 37°C with a radiolabeled EGF (2 ng/ml). At the indicated times, cell monolayers were acid-washed to remove surface-bound EGF. Radioactivity present in the acidic fraction was quantified in triplicates and designated surface-associated ligand. The remaining cell-associated radioactivity (internalized) was similarly quantified following cell solubilization. The ratio obtained at each time point is presented (average ± SD). (B) Monolayers of CHO cells transfected as in (A) were incubated for 1 h at 20°C with a radiolabeled EGF. Sister monolayers were pre-incubated for 30 min with chloroquine (0.2 mM; open symbols). Thereafter, cells were washed and maintained at 37°C for the indicated time intervals. Media were then collected, and the cells were solubilized. The trichloroacetic acid-soluble radioactivity was determined and the fraction of degraded ligand presented. (C) CHO cells transiently expressing wt-EGFR (squares), the Y1045F mutant receptor (circles) or a kinase-defective mutant (triangles) were allowed to internalize a radiolabeled EGF (1 ng/ml) for the indicated time periods. The remaining surface bound ligand was removed by mild acid-washing. The EGF-loaded cells were then incubated with an unlabeled EGF at 4°C, followed by 1 h at 37°C. Intact radioactive ligand was harvested from the medium following a 1 h chase period. Similarly, the fraction of surface-bound radioactivity was determined and the combined fractions designated recycled ligand. The fraction of recycled ligand is presented as average ± SD (bars) of triplicate determinations.

Grb2 enhances a surrogate mechanism of c-Cbl-dependent receptor ubiquitylation

Several results we obtained with the Y1045F mutant receptor raised the possibility that ligand-induced endocytosis of the mutant receptor is only partially impaired. Assuming that c-Cbl and receptor ubiquitylation are essential for the residual ligand-dependent endocytosis of Y1045F, we examined the status of ubiquitylation of this mutant receptor upon high-level ectopic expression of c-Cbl. The results depicted in Figure 4A confirmed the ability of an overexpressed c-Cbl to enhance ligand-induced ubiquitylation and degradation of wt-EGFR. Likewise, faint but reproducible ubiquitylation of Y1045F was detectable upon short stimulation with EGF, and this modification was further enhanced when c-Cbl was overexpressed. In line with weak ligand-induced ubiquitylation, Y1045F underwent undetectable degradation following stimulation with EGF (Figure 4A). These results suggest the existence of a secondary mechanism of c-Cbl-induced receptor ubiquitylation. Because the surrogate pathway is sensitive to EGF but independent of Tyr1045, it seemed likely that another tyrosine phosphorylation site of EGFR is engaged.

Fig. 4. An alternative receptor ubiquitylation pathway independent of Tyr1045 may involve Grb2. (A) CHO cells were transfected with plasmids encoding a wt-EGFR (WT) or the Y1045F mutant, along with vectors encoding c-Cbl and an HA-tagged ubiquitin. Following 48 h, cell monolayers were treated for 10 min at 37°C without or with EGF (100 ng/ml) and cell lysates subjected to immunoprecipitation (I.P.) and immunoblotting (I.B.) with the indicated antibodies. Whole-cell lysates were also analyzed (lower panel). (B) wt-EGFR or the Y1045F mutant receptor were immunopurified from transfected HEK-293 cells. Receptor immunoprecipitates were subjected to an in vitro ubiquitylation assay with a radiolabeled ubiquitin. c-Cbl, Grb2 or a combination of the two proteins was added to the reaction mixtures in the form of GST fusion proteins. Receptor immunoprecipitates were resolved by electrophoresis and proteins transferred to filters, which were first autoradiographed (upper panel, ¹²⁵I-Ub) and then immunoblotted with anti-EGFR antibodies (lower panel). (C) Immuno precipitates of Y1045F were subjected to an in vitro ubiquitylation assay in the presence of one or two of the indicated GST fusion proteins. As a control, GST was added alone (Cont.) or it was omitted from the reaction (unlabeled lane).

The above results led us to suspect that a c-Cbl-associated protein, which is capable of binding a phosphotyrosine distinct from Tyr1045, is involved in recruiting c-Cbl to Y1045F. Because two autophosphorylation sites of EGFR can bind Grb2 (Batzer et al., 1994; Okutani et al., 1994), and this adaptor is constitutively and stoichiometrically bound to c-Cbl (Meisner and Czech, 1995; Donovan et al., 1996), we reasoned that Grb2 may be involved in the surrogate pathway of EGFR ubiquitylation. To test this prediction, we utilized a previously described in vitro ubiquitylation assay (Levkowitz et al., 1999; Waterman et al., 1999). Figure 4B shows that incubation of an immuno-affinity purified wt-EGFR with reticulocyte lysate in the presence of a radiolabeled ubiquitin resulted in faint receptor ubiquitylation. However, addition of c-Cbl strongly promoted receptor ubiquitylation, as has been reported previously (Joazeiro et al., 1999; Levkowitz et al., 1999; Waterman et al., 1999; Yokouchi et al., 1999). In contrast, a recombinant Grb2 protein was ineffective, but its combination with c-Cbl moderately enhanced receptor ubiquitylation. This synergistic effect of Grb2 and c-Cbl was more conspicuous when the Y1045F mutant receptor was used in vitro as a substrate (Figure 4B). To test which domains of Grb2 are involved in Y1045F ubiquitylation, we used proteins carrying partially inactivating point mutations at each of the three domains of Grb2. Of the three mutants we tested, a protein mutated at the single SH2 domain (mutant denoted R86K-Grb2) was severely impaired in its ability to ubiquitylate Y1045F (Figure 4C), in line with binding to a phosphotyrosine of EGFR. On the other hand, a Grb2 protein mutated at the C-terminal SH3 domain (G203R-Grb2) was almost as active as wild-type Grb2, but a mutation within the N-terminal SH3 domain (mutant denoted P49L-Grb2) partly inactivated Grb2. Taken together, these results support recruitment of c-Cbl to Y1045F by simultaneous binding of Grb2 to c-Cbl (primarily via the N-terminal SH3 domain) and EGFR (via the SH2 domain).

The synergistic in vitro effect of Grb2 and c-Cbl prompted us to examine their combined action on EGFR in living cells. Overexpression of c-Cbl exerted only a moderate effect on ubiquitylation of the wt-EGFR (Figure 5A). However, co-expression of Grb2 and c-Cbl enhanced receptor ubiquitylation and significantly increased its degradation. Moreover, when singly overexpressed, neither c-Cbl nor Grb2 could strongly enhance EGF-dependent ubiquitylation and degradation of Y1045F, but their combination effectively enhanced both activities (Figure 5A). Because the effect of Grb2 was especially strong when cells were stimulated with EGF, we assumed that at least one of the two Grb2 association sites of EGFR [tyrosines 1068 and 1086 (Batzer et al., 1994; Okutani et al., 1994)] is involved in recruiting a complex of Grb2 and c-Cbl. This possibility was indirectly supported by the inability of a combination of Grb2 and c-Cbl to reconstitute ligand-induced ubiquitylation of a receptor lacking the whole C-terminus (ΔCT, residues 1–972), including all Grb2 and Shc association sites (Figure 5A). In line with loss of ligand-induced ubiquitylation, we observed no co-immunoprecipitation of the tail-less mutant receptor with Grb2 (Figure 5B), but both Y1045F and the wt-EGFR displayed physical association with the adaptor. In other experiments we analyzed EGFR mutants whose seven (mutant denoted F7) or eight (F8) potential phosphorylation sites, including Tyr1045, were replaced by phenylalanines. Whereas F8 lost ligand-induced ubiquitylation, F7 displayed some EGF-induced modification (data not shown), probably because its Tyr1114, a known Grb2 and Shc docking site, was intact.

Fig. 5. Grb2 enhances c-Cbl-dependent ubiquitylation and degradation of EGFR in living cells. (A) Monolayers of CHO cells were transiently transfected with expression vectors encoding the indicated forms of EGFR. Alongside, plasmids encoding c-Cbl, Grb2, HA-ubiquitin and control empty vectors were used as indicated. Cell monolayers were treated for 10 min at 37°C without or with EGF (100 ng/ml). Subsequently, cell lysates were directly subjected to immunoblotting (I.B.; panels labeled NONE). Alternatively, EGFR was first isolated, and the immunoprecipitates analyzed with the indicated antibodies. (B) CHO cells were transfected with plasmids encoding the indicated forms of EGFR, along with plasmids encoding Grb2 and c-Cbl. A control culture was treated with an empty expression vector. Following stimulation with EGF (100 ng/ml; 10 min at 37°C), EGFR was isolated from cell lysates and the immuno-complexes analyzed by using antibodies to either Grb2 or EGFR.

Grb2 and c-Cbl co-operatively accelerate down-regulation and endocytosis of a mutant EGFR

To substantiate the combined effect of Grb2 and c-Cbl, we examined receptor down-regulation and endocytosis in CHO cells ectopically expressing the combination. As is evident from Figure 6A, overexpression of Grb2 alone exerted no significant effect on the extent of ligand-induced down-regulation of the wt-EGFR. This contrasts with c-Cbl, whose overexpression enhanced receptor down-regulation, in line with previous reports (Levkowitz et al., 1998; Yokouchi et al., 1999; Lill et al., 2000). However, co-transfection of Grb2 and c-Cbl slightly enhanced receptor down-regulation (Figure 6A). The synergistic effect was more significant in the case of Y1045F, as this mutant underwent almost no ligand-induced down-regulation upon overexpression of either c-Cbl or Grb2 (Figure 6A). A complementary short-term ligand internalization assay reflected the lower rate of Y1045F internalization relative to wt-EGFR (Figures 3A and and6B).6B). As noted previously (Levkowitz et al., 1998; Thien et al., 2001), we observed no significant effect of an overexpressed c-Cbl on the rate of internalization of the wt-EGFR. Likewise, Grb2 exerted no effect on internalization of either receptor form. However, the combination of Grb2 and c-Cbl reproducibly accelerated the rate of Y1045F internalization (Figure 6B). Taken together, these results extend the combined effect of c-Cbl and Grb2 to receptor endocytosis, implying causative relationships between receptor ubiquitylation and endocytosis.

Fig. 6. Grb2 accelerates internalization and down-regulation of the Y1045F mutant EGFR. (A) CHO cells were transfected with expression vectors encoding wt-EGFR (WT) or the Y1045F mutant. Alongside we used plasmids encoding c-Cbl (closed circles), Grb-2 (open squares), a combination of Grb2 and c-Cbl (closed squares) or an empty control vector (open circles). Forty-eight hours post-transfection, cultures were incubated at 37°C with EGF (25 ng/ml) for the indicated periods of time. Cell-bound ligand was removed, and the level of surface receptors was determined by binding of a radiolabeled EGF at 4°C. The average ± SD (bars) of triplicate determinations is shown for each time point. (B) Monolayers of CHO cells were transfected as in (A) with vectors driving expression of the indicated version of EGFR. After 48 h, cells were incubated at 37°C with a radiolabeled EGF (2 ng/ml). At the indicated time points, monolayers were acid-washed to remove surface-bound ligand. Radioactivity present in the acidic fraction was designated surface-associated ligand. The remaining cell-associated radioactivity was determined in triplicates following cell solubilization and designated internalized ligand. Symbols are as in (A). (C) CHO cells transiently overexpressing HA-tagged c-Cbl and histidine-tagged Grb2, along with a GFP–EGFR, or a fusion protein containing a mutation at Tyr1045 (Y1045F), were grown on cover slips for 48 h after transfection. Thereafter, cells were incubated for 15 min at 37°C without or with EGF (100 ng/ml). To visualize Grb2, cells were fixed, permeabilized, and incubated with a rabbit anti-His₆ followed by a Cy3-conjugated secondary antibody (red). The GFP–EGFR fluorescence is shown in the left column (green). The right column presents the overlay of GFP and Cy3 fluorescence; a yellow color is seen in areas of co-localization.

The ability of Grb2 to enhance ligand-induced internalization of Y1045F was supported by fluorescence microscopy. As is shown in Figure 2, EGF and c-Cbl exerted only a small effect on endocytosis of GFP–Y1045F. However, expression of an ectopic Grb2 together with c-Cbl enhanced ligand-induced endocytosis of GFP–Y1045F (Figure 6C). Interestingly, we observed partial co-localization of Grb2 with the internalized EGFR (note the yellow figures in the merge panels of Figure 6C). Indeed, a recent report utilizing fluorescence energy transfer detected physical association between Grb2 and EGFR in endosomes (Sorkin et al., 2000). Together with the biochemical lines of evidence, our morphological analyses support the existence of a dual mechanism of c-Cbl-induced endocytosis: a major pathway mediated by Tyr1045 and c-Cbl, and a second route that involves recruitment of Grb2, presumably pre-complexed with c-Cbl.

The RING finger and the regulatory region of c-Cbl, but not the SH2 domain, are required for Grb2-mediated receptor ubiquitylation

We next analyzed the domains of c-Cbl that are necessary for interaction with Grb2 and for the associated increase in Y1045F ubiquitylation. Three mutants of c-Cbl were used: a protein whose SH2 domain is defective due to a point mutation [G306E (Bonita et al., 1997)] and two deletion mutants lacking either the N-terminal or the C-terminal half [Cbl-C and Cbl-N(RF), respectively]. Both deletion mutants carried an intact RING finger, but only Cbl-N(RF), whose SH2 domain is intact, could support ubiquitylation of wt-EGFR (data not shown; Lill et al., 2000). In contrast, this mutant displayed only weak co-immunoprecipitation with Grb2 (Figure 7A), and when co-expressed together with the adaptor it mediated no increase in ubiquitylation and degradation of Y1045F (Figure 7C). Thus, recruitment of Grb2 to the C-terminus of c-Cbl appears to be essential for the surrogate pathway of EGFR ubiquitylation, but the presence of the SH2 domain is not critical. Indeed, a mutant containing an intact poly-proline domain, the site of Grb2 binding (Meisner and Czech, 1995; Donovan et al., 1996), but lacking the SH2 domain (Cbl-C), retained binding to Grb2 (Figure 7A) and partially enhanced Y1045F ubiquitylation and degradation (Figure 7C). In experiments that are not presented, we examined the relative ability of native c-Cbl and Cbl-N(RF) to enhance ubiquitylation and degradation of wt-EGFR. As expected, the mutant was inferior to c-Cbl, probably due to evading the surrogate pathway. By analyzing a natural mutant of the RING finger, namely 70Z-Cbl, we learned that this domain is essential for the surrogate pathway of receptor ubiquitylation (Figure 7B). In contrast, by using the G306E mutant, we confirmed that the SH2 domain of c-Cbl is not involved in the surrogate pathway. In conclusion, unlike the major pathway of receptor ubiquitylation, which requires binding of the SH2 domain of c-Cbl to a specific phosphotyrosine of the receptor, the surrogate pathway is independent of the c-Cbl’s SH2 domain. In addition to the RING finger, this pathway involves the C-terminal half of c-Cbl, probably because this portion of the molecule contains several proline-rich sites that recruit Grb2.

Fig. 7. Subdomains of c-Cbl involved in receptor ubiquitylation. (A) CHO cells were transfected with plasmids encoding EGFR, Grb2, the indicated HA peptide-tagged mutants of c-Cbl or a control empty vector. Whole-cell extracts were analyzed by immunoblotting either directly or after immunoprecipitation of c-Cbl. The following mutants of c-Cbl were used: a protein the SH2 domain of which is defective (G306E); and deletion mutants containing either the N-terminal half [Cbl-N(RF), residues 1–429] or the C-terminal half (Cbl-C, residues 338 to end). (B) Monolayers of CHO cells were transiently transfected with the Y1045F mutant of EGFR, along with wild type (WT) or the 70Z mutant of c-Cbl, in the presence or absence of a Grb2 expression vector. Receptor ubiquitylation was detected using a HA-tagged ubiquitin expression vector. Forty-eight hours post-transfection, monolayers were incubated without or with EGF (100 ng/ml). Cell lysates were subjected to immunoprecipitation (I.P.) with an anti-EGFR antibody and immunoblotting (I.B.) with either an antibody to HA-ubiquitin or an antibody recognizing EGFR. (C) CHO cells were co-transfected with vectors driving expression of the indicated c-Cbl mutants [see (A)], along with a plasmid encoding the Y1045F mutant receptor. As indicated, transfections were performed with a plasmid encoding Grb2 or a control empty vector. All transfections were carried out in the presence of a plasmid directing expression of a HA-tagged ubiquitin. Cells were treated for 10 min at 37°C without or with EGF (100 ng/ml). Whole-cell lysates were analyzed by immunoblotting (I.B.) either directly (panels labeled NONE) or following immunoprecipitation (I.P.).

Discussion

Receptor endocytosis terminates signal transduction

Several previous attempts to block EGFR internalization, by either receptor mutagenesis (Wells et al., 1990) or a mutant of dynamin (Vieira et al., 1996), concluded that endocytosis terminates the mitogenic activity of EGF. Nevertheless, the effect of receptor endocytosis on signaling potency remained a matter of some controversy (Di Guglielmo et al., 1994; Emlet et al., 1997; Haugh et al., 1999). In part, this issue is complicated by the existence of at least two routes of receptor endocytosis: the ligand-activated route requires the intrinsic tyrosine kinase activity of EGFR. In contrast, the slower pathway is constitutive, and unlike the inducible pathway it primarily recycles receptors back to the cell surface (Wiley et al., 1991). By resolving the role of c-Cbl in receptor endocytosis and sorting to destruction, our results favor the notion that receptor endocytosis terminates mitogenic signaling. Thus, a c-Cbl’s site mutant receptor (Y1045F) with defective endocytosis (Figures 2 and and3A)3A) and increased recycling (Figure 3C) is endowed with enhanced signaling capacity (Figure 1).

Previous attempts to construct an internalization-defective mutant of EGFR led to the realization that the 213 C-terminal amino acids of EGFR include an inhibitory domain and an internalization region (amino acids 973–1022). It is interesting to note that Tyr1045 is located outside of the mapped internalization region. However, consistent with its role in endocytosis, genetic analyses of vulva formation in C.elegans attributed an inhibitory role to the respective residue (Yoon et al., 2000). In addition, mutagenesis of EGFR has identified residues 1022–1123 as a region required for lysosomal degradation (Kornilova et al., 1996). Consistent with the association of internalization with decreased mitogenic signaling, virulence of several strains of the oncogenic avian erythroblastosis virus, which encodes an ortholog of EGFR, share deletions of a region encompassing the c-Cbl-specific docking site (Shu et al., 1991). Likewise, this site is missing in oncogenic forms of the human EGFR, which are frequently found in brain tumors (Ekstrand et al., 1992). In a complementary way, the transforming activity of some ubiquitylation-defective forms of c-Cbl depends on binding to EGFR (Thien et al., 2001), and targeted inactivation of c-Cbl sensitizes macrophages to the colony stimulating factor-1 (Lee et al., 1999).

A linkage between receptor ubiquitylation and endocytosis

Previous attempts to resolve the relationships between receptor ubiquitylation and endocytosis reported no effect of an overexpressed c-Cbl on the rate of EGFR endocytosis (Levkowitz et al., 1998; Thien et al., 2001). This observation and the ligand-induced co-translocation of c-Cbl and EGFR to endosomes led us to suggest that the effect of c-Cbl on receptor sorting occurs at the early endosome (Levkowitz et al., 1998). By using a receptor mutant incapable of c-Cbl recruitment (Y1045F), we identified an earlier effect of c-Cbl. Apparently, ubiquitylation by c-Cbl occurs at the plasma membrane or very close to it, and it determines the rate of receptor endocytosis. It is conceivable that c-Cbl-mediated ubiquitylation of a cell surface-localized EGFR dictates sorting to the invaginating clathrin-coated pit. This conclusion is consistent with the ability of a mutant dynamin to block endocytosis, but not ubiquitylation of EGFR (Stang et al., 2000).

Collectively, the observations made with an overexpressed c-Cbl and the Y1045F mutant receptor lead us to conclude that sorting of internalized EGFRs is a progressive process. It probably initiates at the cell surface upon limited c-Cbl-mediated ubiquitylation, which accelerates endocytosis (de Melker et al., 2001). However, ubiquitylation seems to proceed en route to the late endosome/pre-lysosome, because c-Cbl and EGFR remain physically associated (Levkowitz et al., 1998), and blocking receptor internalization prevents maximal ubiquitylation (Stang et al., 2000). The emerging model is thus reminiscent of the mechanism regulating endocytosis in yeast (reviewed in Hicke, 2001). Mono-ubiquitylation seems sufficient to signal internalization of several yeast proteins into primary endocytic vesicles. Apparently, ubiquitin itself carries an internalization signal, because fusion of a lysine-less ubiquitin to several yeast and mammalian proteins enhances their rate of internalization (Nakatsu et al., 2000 and references therein). It is therefore possible that c-Cbl enhances the rate of EGFR endocytosis by appending an internalization signal intrinsic to ubiquitin.

The dual role of Grb2

The Grb2 adaptor protein is involved in many signal transduction pathways, including those initiated by growth factors, antigens and antibodies. Its single SH2 domain allows inducible binding to tyrosine-phosphorylated proteins, whereas the two flanking SH3 domains recruit signaling proteins, including the guanine nucleotide exchange protein Sos and the E3 ubiquitin ligase, c-Cbl (Fukazawa et al., 1995). The interaction with Sos is relatively well characterized; it recruits the exchange protein to the plasma membrane and activates the Ras pathway. However, although a complex of c-Cbl and Grb2 is abundant in many cell types and its dissociation may be regulated (Buday et al., 1996; Donovan et al., 1996), the functional consequences of recruiting a c-Cbl–Grb2 complex to activated receptors is currently unknown. The results presented in this study indicate that one function of Grb2–Cbl interaction is to negatively regulate signaling by mediating receptor degradation. Interestingly, genetic evidence raised the possibility that Grb2 recruits another negative regulator of EGFR in worms, namely Ark-1 (Hopper et al., 2000). However, unlike c-Cbl/Sli-1, the mechanism underlying the Ark-1 pathway of receptor desensitization remains unknown.

Although our conclusions rely on overexpression, and therefore they must be confirmed by additional approaches, it is conceivable that two distinct mechanisms underlie inducible degradation of EGFR. The major one is mediated by Tyr1045, which directly binds to the SH2 domain of c-Cbl. The secondary mechanism seems to involve one of the Grb2-binding sites of EGFR, and indirect recruitment of c-Cbl through its constitutive interactions with an SH3 domain of the adaptor. The relative contribution of the two pathways to EGFR desensitization will require further investigation; according to a previous report a c-Cbl mutant incapable of Grb2 binding is as effective as the wild-type protein (Lill et al., 2000), but the data we presented and additional preliminary results suggest that in some cell lines, coupling to Grb2 may contribute to the overall extent of receptor degradation.

Additional open questions relate to the possible involvement of the many other c-Cbl-associated proteins, like the adaptors Nck, Shc and Crk. The involvement of Grb2, however, was predictable because of two lines of evidence. First, microinjection of a recombinant SH2 domain of Grb2 inhibited endocytosis of EGFR (Wang and Moran, 1996). Secondly, deletion of the Grb2-binding poly-proline motif of SLI-1 significantly affected vulva formation in transgenic worms (Yoon et al., 2000). Together with the results we have presented, these lines of evidence attribute to Grb2 a dual role in signaling; along with the extensively characterized stimulatory activity of the Ras pathway through binding to SOS, the adaptor also initiates the process of receptor degradation by recruiting c-Cbl. Because c-Cbl and SOS do not co-immunoprecipitate (Meisner et al., 1995; Fukazawa et al., 1996), they seem to form exclusive complexes with Grb2. Thus, it is conceivable that by alternative interaction with SOS and c-Cbl, Grb2 integrates both positive and negative inputs to signaling pathways.

In summary, c-Cbl critically controls receptor fate: its close apposition to and phosphorylation by EGFR initiate receptor ubiquitylation. Apparently, ubiquitylation of EGFR identifies it for rapid endocytosis, which starts the process of homologous desensitization. However, c-Cbl remains associated with the receptor throughout the endocytic compartment, probably in order to allow effective sorting to degradation. Evidently, several mechanisms of c-Cbl recruitment exist in cells: the major route involves the SH2 domain of c-Cbl and autophosphorylation at Tyr1045 of EGFR, which is situated within a lysosome-targeting motif. In contrast, the surrogate route bypasses the lysosome-targeting motif. Apparently, c-Cbl is recruited to this route in a complex with the Grb2 adaptor, which engages with EGFR through several tyrosine autophosphorylation sites. Future studies will address functional characteristics of each route of receptor ubiquitylation, as well as the relationships to positive signaling through the adaptors Grb2 and Shc.

Materials and methods

Materials

Rabbit antibodies to c-Cbl (C-15), a His₆ tag and EGFR were from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal antibody (mAb) to Grb2 was from Transduction Laboratories (Lexington, KY) and an anti-hemagglutinin (HA) mAb was purchased from Boehringer Mannheim. A mAb to the active doubly phosphorylated form of Erk was from Sigma. The composition of buffers was as described previously (Waterman et al., 1999).

Plasmid construction and transfection of expression vectors

Mammalian expression plasmids for wild-type and mutant EGFR, c-Cbl, glutathione S-transferase (GST)–Cbl and Cbl-C have been described previously (Levkowitz et al., 1999). The Cbl-N(RF) deletion mutant was prepared by introducing a stop codon next to the codon encoding amino acid 429. GST–Grb2 mutants were obtained from Dr Jan Sap (New York University). A His₆ tag was inserted at the C-terminus of the human Grb2 cDNA by PCR amplification. Expression vectors were introduced to CHO cells by using the LipofectAMINE transfection method (Gibco-BRL). The total amount of DNA in each transfection was normalized with the pCDNA3 plasmid.

Cell proliferation and SRE transcription assays

Cells were washed free of IL-3, resuspended in RPMI 1640 medium at 5 × 10⁵ cells/ml, and treated without or with growth factors or IL-3. Cell survival was determined by using the MTT assay (Mosman, 1983). The SRE transcription assay was performed as described previously (Waterman et al., 1999).

Lysate preparation, immunoprecipitation and western blotting

Following stimulation, cells were extracted in solubilization buffer, and lysates cleared by centrifugation. The proteins in the lysate supernatants were immunoprecipitated for 2 h at 4°C. The immunoprecipitates were washed three times with HNTG buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Triton X-100 and 10% glycerol), resolved by electrophoresis and electrophoretically transferred to a nitrocellulose membrane. Membranes were blocked for 1 h in Tris–HCl-buffered saline containing 1% milk, blotted for 2 h with a primary antibody (1 µg/ml), followed by a secondary antibody (0.5 µg/ml) linked to horseradish peroxidase. Immunoreactive protein bands were detected using the enhanced chemiluminescence reagent (Pharmacia-Amersham).

Biochemical analyses of endocytosis

Receptor down-regulation was performed as described previously (Levkowitz et al., 1998). To follow receptor recycling and to monitor EGF internalization, we used the previously described protocols of Kornilova et al. (1996) and Sorkin et al. (1993), respectively. Ligand degradation was assayed as follows: conditioned media containing radiolabeled ligands were assayed by the addition of cold trichloroacetic acid (TCA) (10% final concentration). Samples were precipitated after 1 h at 2°C by centrifugation. Radioactivity present in the supernatants and pellets (TCA-insoluble fraction) was determined.

In vitro assay for receptor ubiquitylation

Receptors were immunoprecipitated from cleared lysates of transfected HEK-293T cells. Following isolation, agarose beads were extensively washed with HNTG followed by an additional wash with ubiquitin wash buffer (5 mM MgCl₂, 40 mM Tris–HCl pH 7.5). Agarose beads were then resuspended in buffer containing 40 mM Tris–HCl (pH 7.5), 5 mM MgCl₂, 2 mM dithiothreitol, 2 mM ATP-γ-S and 3 µg/ml radiolabeled ubiquitin supplemented with crude rabbit reticulocyte lysate (5 µl; Promega) and the indicated recombinant proteins (5 µg), and incubated for 1 h at 30°C. The beads were then washed extensively and EGFR eluted with gel sample buffer.

Immunofluorescence

Transfected cells grown on cover slips were incubated for 15 min at 37°C with or without EGF (100 ng/ml). The cells were then fixed for 15 min with 3% paraformaldehyde in phosphate-buffered saline (PBS). Fixed cells were washed, and permeabilized for 10 min at 22°C with PBS containing 1% albumin and 0.2% Triton X-100. For labeling, cover slips were incubated for 1 h at room temperature with an anti-HA antibody or a rabbit antibody to His₆ peptide tag. After extensive washing in PBS, the cover slips were incubated for an additional 1 h with a Cy3-conjugated secondary antibody. The cover slips were mounted in mowiol. Confocal microscopy was performed using a Zeiss Axiovert 100 TV microscope (Oberkochen, Germany) with a 63X/1.4 plan-Apochromat objective, attached to the Bio-Rad Radiance 2000 laser scanning system, operated by LaserSharp software. Figures were taken from middle sections of cells.

The C-terminus of the kinase-defective neuregulin receptor ErbB-3 confers mitogenic superiority and dictates endocytic routing.
Hadassa Waterman, Patent Attorney, Ehrlich & Fenster, 2001

Abstract:

Signaling by the epidermal growth factor (EGF) family and the neuregulin group of ligands is mediated by four ErbB receptor tyrosine kinases, that form homo- and heterodimeric complexes. Paradoxically, the neuregulin receptor ErbB-3 is devoid of catalytic activity, but its heterodimerization with other ErbBs, particularly the ligand-less ErbB-2 oncoprotein of carcinomas, reconstitutes superior mitogenic and transforming activities. To understand the underlying mechanism we constructed a chimeric EGF-receptor (ErbB-1) whose autophosphorylation C-terminal domain was replaced by the corresponding portion of ErbB-3. Consistent with the possibility that this domain recruits a relatively potent signaling pathway(s), the mitogenic signals generated by the recombinant fusion protein were superior to those generated by ErbB-1 homodimers and comparable to the proliferative activity of ErbB-2/ErbB-3 heterodimers. Upon ligand binding, the chimeric receptor recruited an ErbB-3-specific repertoire of signaling proteins, including Shc and the phosphatidylinositol 3-kinase, but excluding the ErbB-1-specific substrate, phospholipase Cgamma1. Unlike ErbB-1, which is destined to lysosomal degradation through a mechanism that includes recruitment of c-Cbl and receptor poly-ubiquitination, the C-terminal tail of ErbB-3 shunted the chimeric protein to the ErbB-3-characteristic recycling pathway. These observations attribute the mitogenic superiority of ErbB-3 to its C-terminal tail and imply that the flanking kinase domain has lost catalytic activity in order to restrain the relatively potent signaling capability of the C-terminus.

To view the full text please follow the link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1171415/pdf/003348.pdf

Differential endocytic routing of homo- and hetero-dimeric ErbB tyrosine kinases confers signaling superiority to receptor heterodimers.
Hadassa Waterman, Patent Attorney, Ehrlich & Fenster, 1998

Abstract: Both homo- and hetero-dimers of ErbB receptor tyrosine kinases mediate signaling by a large group of epidermal growth factor (EGF)-like ligands. However, some ligands are more potent than others, although they bind to the same direct receptor. In addition, signaling by receptor heterodimers is superior to homodimers. We addressed the mechanism underlying these two features of signal tuning by using three ligands: EGF; transforming growth factor alpha (TGFalpha); and their chimera, denoted E4T, which act on cells singly expressing ErbB-1 as a weak, a strong, and a very strong agonist, respectively. Co-expression of ErbB-2, a developmentally important co-receptor whose expression is frequently elevated in human cancers, specifically potentiated EGF signaling to the level achieved by TGFalpha, an effect that was partially mimicked by ErbB-3. Analysis of the mechanism underlying this trans-potentiation implied that EGF-driven homodimers of ErbB-1 are destined for intracellular degradation, whereas the corresponding heterodimers with ErbB-2 or with ErbB-3, dissociate in the early endosome. As a consequence, in the presence of either co-receptor, ErbB-1 is recycled to the cell surface and its signaling is enhanced. This latter route is followed by TGFalpha-driven homodimers of ErbB-1, and also by E4T-bound receptors, whose signaling is further enhanced by repeated cycles of binding and dissociation from the receptors. We conclude that alternative endocytic routes of homo- and hetero-dimeric receptor complexes may contribute to tuning and diversification of signal transduction. In addition, the ability of ErbB-2 to shunt ligand-activated receptors to recycling may explain, in part, its oncogenic

To view the full text please follow the link:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170676/pdf/003385.pdf

Structure of acetylcholinesterase complexed with E2020 ( Aricept ^®): implications for the disign of the new anti-Alzheimer drugs
Gitay Kryger, Patent Attorney, Ehrlich & Fenster, 1996

Abstract:

Background: Several cholinesterase inhibitors are either being utilized for symptomatic treatment of Alzheimer's disease or are in advanced clinical trials. E2020, marketed as Aricept^®, is a member of a large family of N-benzylpiperidine-based acetylcholinesterase (AChE) inhibitors developed, synthesized and evaluated by the Eisai Company in Japan. These inhibitors were designed on the basis of QSAR studies, prior to elucidation of the three-dimensional structure of Torpedo californica AChE (TcAChE). It significantly enhances performance in animal models of cholinergic hypofunction and has a high affinity for AChE, binding to both electric eel and mouse AChE in the nanomolar range.

Results: Our experimental structure of the E2020–TcAChE complex pinpoints specific interactions responsible for the high affinity and selectivity demonstrated previously. It shows that E2020 has a unique orientation along the active-site gorge, extending from the anionic subsite of the active site, at the bottom, to the peripheral anionic site, at the top, via aromatic stacking interactions with conserved aromatic acid residues. E2020 does not, however, interact directly with either the catalytic triad or the ‘oxyanion hole’, but only indirectly via solvent molecules.

Conclusions: Our study shows, a posteriori, that the design of E2020 took advantage of several important features of the active-site gorge of AChE to produce a drug with both high affinity for AChE and a high degree of selectivity for AChE versus butyrylcholinesterase (BChE). It also delineates voids within the gorge that are not occupied by E2020 and could provide sites for potential modification of E2020 to produce drugs with improved pharmacological profiles.

To view the full text please follow the link:
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VSR-3WM4NN5-B&_user=10&_coverDate=03%2F15%2F1999&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=e6456f67dbb2b8029d94c2418f6e8f81&searchtype=a

על הבקשה הזמנית: אליה וקוץ בה
הבקשה הזמנית ככלי אסטרטגי לניהול קניין רוחני

החל מ- 8 ביוני, 1995 מאפשר משרד הפטנטים האמריקאי לממציאים מכל העולם להגיש בקשה זמנית לפטנט (Provisional Application for Patent). הבקשה מיועדת לפתוח בפני הממציאים דרך יחסית זולה ופשוטה לדחות את הגשתה של בקשת פטנט רגילה (Application for Patent). המשרד אינו מעמיד דרישות הגשה מחמירות לשם הגשת הבקשה הזמנית. הממציאים נדרשים להגיש תיאור של עיקר המצאתם ללא פירוט התביעות ולשלם אגרת הגשה נמוכה (100$ לישות קטנה ו-200$ לישות גדולה נכון ליום פרסום הכתבה). מאחר והבקשות הזמניות אינן בקשות פטנט רגילות, לא ניתן לקבל פטנט על בסיסן. הבקשות אינן נבחנות על ידי משרד הפטנטים האמריקאי ומאבדות מתוקפן לאחר שנה. עם זאת, הבקשה הזמנית נותנת לממציאים שהות להחליט האם ברצונם להמשיך ולהשקיע בפרי פיתוחם או לזנוח אותו ללא הפסד כספי של ממש. חשוב לציין כי הגשת הבקשה הזמנית אינה פוגעת באורך חיי הפטנט מאחר ותוקף הפטנט נשמר לעשרים שנה מיום הגשת בקשת פטנט הרגילה.

עם השנים הפכה הבקשה הזמנית לכלי אסטרטגי המשמש לניהול השוטף של הנכסים הרוחניים של חברות יזמיות וותיקות כאחד. שימוש בכלי אסטרטגי זה טומן בחובו יתרונות וחסרונות אשר מוטב וישקלו טרם הגשת הבקשה הזמנית.

אחד היתרונות המשמעותיים נוגע לתשלום האגרות הקשורות בהליך קבלת הפטנט. הגשת הבקשה הזמנית מעכבת את הגשת הבקשה הרגילה ובכך דוחה את תשלום האגרות הקשורות להגשתה. הגשתה של בקשת פטנט בינלאומית (PCT) על בסיס הבקשה הזמנית יכולה אף להביא לדחיית תשלום האגרות למשך זמן של עד כשלושים ואחד חודשיים מיום גילוי ההמצאה. יתרון זה משמעותי במיוחד לגבי ממציאים התלויים בגיוס השקעות להמשך פיתוח המצאתם.

יתרון נוסף של הבקשה הזמנית הוא הבטחת תאריך הקדימה תוך כדי הליך הפיתוח ההמצאה. בתחומים כגון חומרה ותוכנה, בהם קצב ההתפתחות הטכנולוגית גבוה במיוחד, עלול ממציא המתמהמה בהגשת בקשת פטנט לאבד את זכותו לפטנט עקב רישום או פרסום בציבור של המצאה דומה או זהה על ידי ממציאים אחרים. התמהמהות זו הנה חזון נפרץ מאחר ופעמים רבות אין לממציאים הנמצאים בשלבים מוקדמים של הפיתוח אפיון מדויק של ההמצאה. הגשת בקשה זמנית על עיקרי ההמצאה, כבר בעת הגילוי, מונעת את אובדן קבילות ההמצאה לפטנט מאחר והיא מבטיחה לממציאים תאריך קדימה לעיקרי ההמצאה. חשוב להדגיש כי הבקשה הזמנית אינה מגבילה את רוחב הפטנט העתידי וניתן להמשיך לפתח, לשפר ולהרחיב את ההמצאה ללא הגבלה, כל עוד קיים פירוט לכך בבקשה הרגילה המוגשת על בסיס הבקשה הזמנית. אומנם, תאריך הקדימה של הפירוט המוסף הוא תאריך הגשתו ולא תאריך הגשת הבקשה הזמנית, אך כך לא מסתכן הממציא באיבוד הזכות לפטנט על בסיס המצאתו. יתרה מכך, העובדה שניתן להגיש בקשה רגילה על בסיס יותר מבקשה זמנית אחת, מאפשרת אסטרטגיה של הגשת בקשה זמנית נוספת לכל שיפור, פיתוח או הרחבה של ההמצאה. אסטרטגיה זו מבטיחה לממציא תאריך קדימה מוקדם ככל האפשר לכל תוספת להמצאה עליה נתבע הפטנט. נציין כי כבר מיום רישום הבקשה הזמנית ממציאים יכולים להציג את המצאתם כ PATENT PENDING בפני משקיעים וצרכנים.

הגשת הבקשה הזמנית פותחת בפני הממציא הזדמנות נוספת להעריך את סיכוייו לקבל פטנט ובכך יש בכוחה לחסוך הוצאות מיותרות. ברוב המקרים, בקשות פטנט מתפרסמות רק כשנה וחצי לאחר הגשתן. מצב דברים זה מאפשר לממציא לבחון רק בקשות שפורסמו כשנה וחצי לפני הגשת בקשת הפטנט. ברם, במידה והגיש הממציא בקשה זמנית הוא יוכל לבחון האם פורסמו בקשות פטנט העלולות לפגוע בסיכוייו לקבל פטנט בזמן שיגיש בקשה רגילה. אסטרטגיה זו מאפשרת לממציא לבחון בקשות שהוגשו עד כחצי שנה לפני שהגיש את הבקשה הזמנית ועוזרת לו להעריך נכונה את החדשנות שבהמצאתו.

עם זאת, בכדי ליהנות מפירות הבקשה הזמנית יש להביא בחשבון מספר קשיים.

דין התיאור בבקשה הזמנית כדין התיאור בבקשה הרגילה. בכדי להבטיח את היתרונות שבהגשת הבקשה הזמנית ולזכות בעתיד בפטנט רחב ומלא, חייבים הממציאים לנסח את הבקשה הזמנית בדיוק ובמקצועיות הראויה. כתיבה חובבנית ולא מדויקת עלולה לפגוע בסיכויי הממציא לבסס פטנט על הבקשה הזמנית.

כמו כן, מאחר והגשת בקשה זמנית דוחה את הגשת הבקשה הרגילה, הליכי בחינת הפטנט נדחים גם הם. זאת ומאחר והליכים אלו נפתחים רק לאחר הגשת הבקשה הרגילה. הואיל וגם כך עלולים ההליכים להמשך שנים רבות, יצא שכר מגיש הבקשה הזמנית, הממהר לממש את המצאתו, בהפסדו.

את החלטה להגיש בקשה זמנית יש לבסס על מטרותיו העסקיות של בעל ההמצאה ועל שלב הפיתוח בו ההמצאה נמצאת. בכדי להעריך נכונה האם יש להגיש בקשה זמנית או רגילה מומלץ להתייעץ עם עורך פטנטים אשר בכוחו לבחון את השיקולים הנזכרים לעיל.

הגלובליזציה בתחום סימני המסחר

בדומה לדיני פטנטים ודיני מדגמים, חל עיקרון הטריטוריאליות גם לגבי סימני מסחר. על כן, סימן הרשום בישראל יהיה מוגן בגבולות הארץ בלבד. מי שרוצה להגן על סימנו גם במדינות אחרות, חייב לרשום אותו בכל מדינה בנפרד. שיטת הרישום המקובלת הינה שיטה לאומית לפיה יש להגיש בקשות נפרדות באופן ישיר בכל מדינה ומדינה בה מעוניינים ברישום הסימן המסחרי. חסרונה הוא העלות הגבוהה למי שרוצה לרשום את סימניו במדינות רבות בעולם. בעקבות הגלובליזציה המתפתחת בשנים האחרונות, יוצרו שיטות רישום בינלאומיות, אשר מקלות על מבקשי סימני המסחר לרשום את סימנם במספר רב של מדינות על ידי הגשת בקשה אחת בלבד. המנגנונים העיקריים לרישום בינלאומי הינם הבקשה האירופית והבקשה 'הבינלאומית' במסגרת פרוטוקול מדריד. חשוב לציין כי המנגנונים האלה אינם באים להחליף את שיטת הרישום הלאומי אלא קיימים במקביל.

הגשת בקשה אירופאית (CTM)- זה הכל או כלום.

החל מאפריל 1996, האיחוד האירופאי יישם מנגנון לרישום סימן מסחר שמאפשר למבקשי סימני מסחר להגיש בקשה אחת בלבד לכל המדינות באיחוד (כיום 25 מדינות). הגוף אשר קולט את הבקשות לסימני מסחר באירופה הוא המשרד להרמוניזציה בשוק המקומי (OHIM).

מנגנון הרישום של האיחוד האירופאי הינו יעיל, פשוט ויחסית זול. מנגנון זה משמש כגוף אחד - קרי, אם הרשם האירופי מקבל את הסימן, הוא יירשם בכל 25 המדינות אבל אם חלילה הרשם אינו מקבל את הסימן (מכל סיבה שהיא), הסימן לא יירשם באף אחת מהמדינות. החלטת הרשם האירופי תחייב את כל המדינות לחיוב או לשלילה.

פרוטוקול מדריד - המהפכה קרובה

מנגנון הרישום ע"פ פרוטוקול מדריד הינו מהפכני עוד יותר בכך שהוא מאפשר למבקשי סימן מסחר לרשום את סימנם בכמעט 80 מדינות בו זמנית על ידי בקשה אחת ותשלום אחד לכל המדינות. הגוף האחראי לפרוטוקול מדריד הוא ארגון הקניין הרוחני העולמי (WIPO). ישראל אמורה להצטרף באופן רשמי לפרוטוקול מדריד לפני סוף שנת 2007 ואז בעלי סימן מסחר ישראליים יוכלו להגיש בקשות 'בינלאומיות' במסגרת פרוטוקול מדריד.

למרות היתרונות הרבים שיש למנגנונים הנ"ל, לעיתים עדיף להגיש בקשות נפרדות בכמה מדינות בודדות וכך להימנע מהתנגדויות מיותרות במדינות פחות חשובות לבעל סימן המסחר. יש לברר כל מקרה לגופו עם עו"ד או עו"פ המתמחה בסימני מסחר על מנת לבחור את האסטרטגיה הנכונה ביותר.

האמור לעיל אינו מהווה יעוץ משפטי. לצורך מיצוי זכויותיך יש להיוועץ בעו"פ ו/או עו"ד.

A REALISTIC, OBJECTIVE TEST FOR THE BEST MODE REQUIREMENT

By: Benjamin J. Barish, Adv., B.Sc. in E.E., 1946, Illinois Institute of Technology, Chicago;

J.D., 1950, LL.M. 1951, George Washington University, Washington, D.C.

(All opinions expressed herein are those solely of the author)

A woman went to the market to buy a chicken. She approached a stand covered with a pile of chickens for sale, grasped a chicken, held it up to the light, spread the wings and legs, one way, then the other, and carefully examined, probed and sniffed each part of the chicken, but eventually discarded the chicken and picked up another one for examination. After she had thus examined and discarded a number of chickens, the shopkeeper, witnessing all this with patient silence, could hold back no longer: "Lady", he asked, "could you yourself pass such an examination?"

How many of the issued patents in the field of high technology could confidently pass such an examination with respect to the best mode requirement of 35 USC 112, first paragraph, as interpreted by recent court decisions, particularly when the invention relates to a highly technical development, and the patent application was filed just before commerica1 production? A review of recent decisions on this issue, as set forth below, indicates that, at best, the answer to this question is unpredictable, and in many or most cases would be negative because the tests applied are both subjective and unrealistic in today's high technology world.

The Present Situation

Take the case of a highly technical development for an engine control system that you have been requested to protect by one or more patent applications. The engine control system includes the following main components: (a) a fuel feeding system for feeding fuel to the engine; (b) a plurality of sensors for sensing predetermined conditions in the engine; (c) an electrical processor for processing the outputs of the sensors according to certain algorithms to output control signals; and (d) control devices, such as actuators, valves, etc., for controlling the fuel to the engine in accordance with the output control signals.

Assume there is novelty in certain aspects of the overall system, as well as in one or more of the above components, so that it may be necessary to file more than one patent application to cover all the novel features. However, only one system has been developed and optimized for production. Each of the above four components involves considerable detailed information including information characterised as manufacturing data, customer requirements, and trade secrets. Such information may include, for example, dimensions, tolerances, materials, heat treatments, performance and design specifications, assembly methods, algorithms of processor operations, commercial name of outside-supplied components, etc. A written description of all such information of the optimized system developed for commercial production would require hundreds or thousands of pages or documents.

It is clear that such detailed information involved a tremendous investment on the part of the developer. Such information might be, and frequently is, included in a separate "know-how package" if the optimized form of the development is licensed to another for production.

The attorney charged with drafting the patent application or applications is first faced with the dilemma of determining exactly what information to include in the patent application to assure compliance with the best mode requirement, as well as the enabling requirement, of 35 USC 112, first paragraph. Since only one mode has been developed and optimized, the safe course of action would be to include in the patent application(s) all the information including the detailed manufacturing data, customer requirements and trade secrets embodied in the many hundreds or thousands of documents, particularly in view of the holding in the leading case on this issue, Chemcast Corp. v. Arco Industries Corp. 913 F.2d 923 (Fed. Cir.. 1990), at 930:

"--Whether characterizable as “manufacturing data” “customer requirements,” or even “trade secrets,” information necessary to practice the best mode simply must be disclosed".

Not only would this be impractical (or perhaps even impossible), but an attorney attempting to do this, in all probability, would be performing a disservice to the client, to the Patent Office, and to the public. Thus, the eventual publication of this valuable know-how information to possible competitors could seriously compromise the client’s investment in developing this information. Including this detailed information in the patent application would require the Patent Office to accommodate this great bulk of material in its files and the Examiner to consider it in the examination for novelty, thereby obscuring the really novel features sought to be patented. It would have little value to the public except to competitors who wished to manufacture the optimized system in all its details. Even then, the value of such information would be very limited since the system, if successfully patented, could not be legally manufactured without license until the patent expires; and by that time, much of the information would probably have become outdated.

If anything less than all the technical details, requiring hundreds or thousands of pages or documents involved in the optimized development, is included in the patent application, the attorney runs the risk of omitting something which may later be considered to be a feature of the patented development sufficiently significant to risk the patent being invalidated on the grounds of failure to comply with the best mode requirement.

In any event, this requirement, as set forth in the above quotation from the Chemcast decision, effectively invites everyone charged with infringing a patent to conduct an extensive “sniff and probe” examination in an attempt to identify any technical information which was known to the inventor or employer at the time the patent application was filed, which was not disclosed in the patent application, and which had some significant effect on the optimized implementation of the invention, in order to invalidate the patent for failure to comply with the best mode requirment. Even if all the details in these hundreds or thousands of documents are disclosed in the patent, there is still no certainty that the patent will be immune from such attack because of the impossibility of assuring that all relevant information known to all the individuals involved in the optimized development was properly considered or documented, particularly in a sophisticated invention in high technology.

The above Chemcast decision applied a two-step analysis for the “best mode requirement”: The first step is to determine whether the inventor, at the time the patent application was filed, knew of a mode of practicing the claimed invention considered by the inventor to be better than any other. If the inventor in fact contemplated such a preferred mode, the second step of the analysis is to compare what the inventor knew with what the inventor disclosed, and particularly to determine whether all such information “necessary to practice the best mode” was indeed disclosed.

The problem with the Chemcast two-step test is that it “begs the issue”.
Thus, the first inquiry – “whether the inventor in fact contemplated such a preferred mode” – presumably should always be answered in the affirmative since 35 USC 112 requires that the inventor:

“shall set forth the best mode contemplated by the inventor of carrying out his invention”.

It is hardly conceivable that an inventor can avoid this issue by merely refusing to admit that a best mode was indeed contemplated before the application was filed.

Therefore, we are left only with the second inquiry; but this second inquiry merely restates the ultimate issue involved in determining compliance or non-compliance with the best mode requirement, and provides no guidance, except in the all-inclusive statement quoted above, for making this determination.

Subsequent court decisions have variously applied the above two-step Chemcast test, and/or a "concealment" test, among many other tests. Some cases held that to invalidate the patent "concealment" must be based on knowledge coupled with a specific intent to conceal, whereas other cases held that no specific intent is necessary. However, the problem with using "concealment" as a test is that anything known but not disclosed may be considered as "concealed". Also, since every detail of the manufacturing data included in the hundreds or thousands of documents describing a highly-technical optimized development can be considered as having some affect on the manufacture or operation of the development, a "concealment" test is not only unrealistic, but is highly subjective, depending on the sympathies and inclinations of the decider (judge or jury), and produces unpredictable results. It also encourages one to “sniff and probe” without limitation to locate something that can be argued as concealed.

This dilemma presented by recent court decisions regarding the “best mode requirement” is well highlighted in an exhaustive analysis of the current situation by Stephen B. Walmsley; Best Mode: A Plea to Repair or Sacrifice This Broken Requirement of United States Patent Law; Walmsley12-9 Mich.Telecomm.Tech.L. Rev. 125 (2002). In this article, the author traced the evolution of this requirement and discussed the multitude of standards set forth in the cited cases for assessing the disclosure needed to comply with this requirement. The various standards were summarized into the following seven categories: (1) the “claims-only” standard; (2) the “essentiality” standard; (3) the “essentiality and novelty” standard; (4) the “necessity” standard; (5) the “necessary and nonobvious” standard; (6) the “material effect” standard; and (7) the “relation” standard. A diagram was attached (Diagram 2 of the Appendix) setting forth these seven standards, including the cases on which each is based. The article concludes with the following Summary (page 167):

“The definition of the best mode requirement of U.S. patent law is unclear and the scope unstable. Instability in the scope of the best mode requirement leads to great expenses incurred in over-complying with the statute and to even greater risks in under-complying with the statute. Such unnecessary expenses and risks are a great disservice to the patent system. Therefore, the best mode requirement desperately needs to be stabilized by an en banc ruling of the CAFC. In the alternative, the best mode requirement should be beneficially sacrificed as a bargaining chip during negotiations with other countries in furtherance of international patent law harmonization efforts. In the meantime, however, inventors are advised to disclose everything important enough to have been documented during the project development phase of their invention process.”

This dilemma, as to the information to be included by the US practitioner in a patent application in order to meet the best mode requirement, is no less for the foreign practitioner when preparing the first patent application to be filed in the foreign country, since the foreign application must also meet all the requirements of 35 USC 112, first paragraph, if it is to support a claim for its priority date under the International Convention.

Clearly, therefore, there is an urgent need for a realistic, objective test for the “best mode requirement”. Before formulating such a test, it is first necessary to consider the legislative intent in 35 USC 112, first paragraph.

The Legislative Intent

The first paragraph of 35 USC 112 provides:

“The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.”

The "enabling" requirement in the above-quoted passage appears first and involves primarily a quantitative test, namely sufficient information "to enable" an implementation of invention. The "best mode" requirement follows the "enabling" requirement in the above passage and appears to involve primarily a qualitative test qualifying the "enabling" requirement, namely by specifying the quality or type of mode to be described to meet the "enabling" requirement.

It is believed this indeed was the legislative intent in 35 USC 112, first paragraph. That is, it is not believed that the legislative intent was that the Applicant set forth all the information that may be involved in the “best mode contemplated by the inventor”, including all the know-how, technical details, etc. Rather, it is believed the legislative intent was that the Applicant set forth enough information (quantitatively speaking) to meet the "enabling requirement", and that such information (qualitatively speaking) relate to "the best mode contemplated" by the inventor at the time the patent application was filed, rather than to an inferior mode. In short, it is believed the legislative intent was:

(a) in the enabling requirement, to require that the applicant teach the public sufficient information to enable the practice of the invention (primarily a quantitative test), and

(b) in the best mode requirement (which follows the enabling requirement) to require that the information set forth to meet the enabling requirement relate to the best mode, and not to an inferior mode (primarily a qualitative test).

While issues concerning the "enabling requirement" arose from the start and were resolved without particular difficulty, it was only after Judge Rich's opinion in In re Nelson (126 USPQ 242, CCPA 1960) that difficult issues began to arise with respect to the best mode requirement, because of the statement:

“Hence Section 112 calls for description in 'full, clear, concise and exact terms' and the 'best mode' requirement does not permit an inventor to disclose only what he knows to be his second-best embodiment, retaining the best for himself”.

However, Judge Rich's opinion in the Nelson case itself suggests that the "best mode" requirement concerns primarily a qualitative test and not a quantitative test: namely, that the information disclosed to meet the enabling requirement must be directed, not to a second-best embodiment, but rather to the best embodiment known to the inventor at the time the application is filed.

The Nelson decision was followed by decisions in other courts applying a “concealment” test. Some decisions required both knowledge and special intent to conceal a best mode in order to invalidate on these grounds (Benger Laboratories. Ltd. v. R.K. Laros Co. 133 USPQ 693, third Cir. 1963); Carter-Wallace, Inc. v. Riverton Laboratories, Inc., 167 USPQ 656 (second Cir. 1970); whereas other cases held that specific intent to conceal is not necessary (Dale Electronics. Inc, v. R.C.L. Electronics. Inc. 180 USPQ 225, first Cir. It is believed these cases wrongly applied a primarily "quantitative" test, namely that all the information should be supplied regarding the best mode, rather than what is believed to be primarily a qualitative test in 35 USC 112, first paragraph, and in the Nelson case.

The "quantitative" test for the best mode requirement was particularly applied in Union Carbide Corp. v. Borq-Water Corp. 193 USPQ 1 (sixth Cir. 1977). This decision involved a patent directed to a process for moulding foamed thermoplastic products, and held that the inventor's failure to disclose a particular valve and a particular extruder used to perform the process violated the best mode requirement, even through the new valve was the subject of a subsequently filed U.S. patent application. The Court so held despite the fact that the patentee had signed up 54 licensees, each of which had paid for a "know-how package" containing extensive written specifications, including the specially designed extruder.

However, it is not believed the legislative intent, in 35 USC 112, first paragraph, was to require the patent application also to include clearly know-how information relating to an optimized form of the best mode of the invention. If this indeed had been the legislative intent, this would have effectively precluded inventors from protecting "know-how" and trade secrets with respect to patented inventions, as always permitted and as now quite common.

Also relevant to the issue of the “legislative intent” in 35 USC 112, first paragraph, is the scope of the relevant subject matter required to be described in the specification. Various courts on various occasions have applied many conflicting standards as to the scope of such subject matter, summarized in the above-cited Walmsley article into seven categories. It is believed, however, that the legislative intent with respect to the scope of the relevant subject matter is revealed by the actual words appearing in the first and second paragraphs of 35 USC 112. Thus, the first paragraph states:

“The specification shall contain a written description of the invention, …”

and the second paragraph of 35 USC 112 states:

“The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention. …”

It is believed that when both sentences are properly construed together, the legislative intent in defining the required description of the invention in the first paragraph refers to the invention defined in the claims specified in the second paragraph.

Determining the legislative intent with respect to the “best mode requirement” of 35 USC 112 should also take into consideration the legislative intent of other provisions of the same statute. For example, 35 USC 181 – 188 deal with “Secrecy of Certain Inventions and Filing Patent Applications in Foreign Countries”. These sections of the U. S. Patent Law have the purpose of preventing publication of information which may be detrimental to national security. Thus, the legislative intent of 35 USC 112, to require a full disclosure, should be reconciled with the legislative intent of 35 USC 181, et seq., to prevent public disclosure of information which may be detrimental to national security.

For example, a development may be made having both a military application and a commercial application. The preferred embodiment of the invention (the best mode) contemplated by the inventor may be the military application whose publication would be barred under the above national security provisions of 35 USC 181, et seq. Therefore if that best mode is indeed described in the specification as required under
35 USC 112, a “Secrecy Order” would be applied under 35 USC 181, et seq. barring publication of the invention. Such “a Secrecy Order” would thus prevent the applicant from exploiting the invention, would deprive the public of the information regarding the invention, and would subject the Government to a claim for reasonable compensation (35 USC 183).

On the other hand, if publication of the commercial, inferior embodiment would not be detrimental to national security, it would appear preferable, and consistent with the “legislative intent” with respect to the above two conflicting statutory provisions, to permit the inventor to describe the inferior (commercial) embodiment, rather than the preferred (military) embodiment. Such a legislative intent would benefit both the public and the inventor and would thus best implement the Constitutional basis for the patent laws (Article 1, Sec. 8):

“-- to promote the progress of science and useful arts, by securing for limited times to authors and inventors the exclusive right to their respective writings and discoveries”.

Such an approach would be consistent with the approach already taken by the courts in an analogous situation, illustrated by the “fun example” described in the Walmsley article. In this example, an inventor discovered that acid deposits from terminal posts of a car battery could be removed by using a carbonated soft drink, and that best results were obtained when using “Coca-Cola” (Registered Trade Mark of the Coca-Cola Company). Since the composition of a product identified by a trade mark may be kept secret (as indeed it is in this case), and may be changed at will by the manufacturer, the best mode disclosure requirement of 35 USC 112, first paragraph, if interpreted strictly and absolutely, would require a disclosure of the actual composition of “Coca-Cola”. However, as pointed out in the Walmsley article, it has been held that the disclosure of the trade name but not the formula, of a cleaning solution used in the claimed invention was sufficient for best mode purposes (see Randomex, Inc. v Scopus Corp., 849 f.2d 585 (Fed Cir. 1988)).

Summarizing the foregoing, it is believed that the legislative intent in
35 USC 112, first paragraph, was:

(a) to apply primarily a quantitative test to the "enabling requirement", i.e., to require sufficient information to enable a person skilled in the art to practice the invention;

(b) to apply primarily a qualitative test to the "best mode" requirement, i.e., to require that the information supplied in the specification be directed, or at least include, the "best mode" contemplated by the inventor for carrying out the invention;

(c) to apply a claims standard in determining the scope of the subject matter to be disclosed, i.e., to measure the information required to be supplied in the specification by the scope of the invention as defined in the claims; and

(d) to apply a public policy test to the “best mode” requirement, i.e., to permit an inferior mode to serve as the best mode where the actual best mode may not be legally permissible or practically possible, e.g., because of national security considerations, trade secret considerations, etc.

It is to be emphasized that the above tests apply only to the “best mode requirement” of 35 USC 112, first paragraph, and not to the “enabling requirement”. The “enabling requirement”, it is believed, was intended to be an independent requirement, separate and distinct from the “best mode” requirement, in determining compliance or non-compliance with 35 USC 112, first paragraph.

Proposed Four-Prong Approach

In the light of the foregoing analysis, a four-prong approach, believed to be realistic, objective and consistent with the legislative intent, is proposed below to test the specification disclosure with compliance with 35 USC 112, first paragraph:

1. Determine whether the specification contains sufficient information to meet the enabling requirement (primarily a quantitative test).

2. If so, determine whether the disclosed information relates to the “best mode” implementation of the invention, or is it restricted to an inferior mode (primarily a qualitative test). If it does not relate to or include the "best mode", then presumably there was an intent to conceal the best mode and to lead the public to an inferior mode, and therefore the best mode requirement is presumably violated.

3. In testing both the enabling requirement and the best mode requirement, consider that the scope of the subject matter required to be disclosed is that relating to the invention as defined in the claims. For example, if the claims recite a fuel feeding system including valves, controls, actuators, etc., and no novelty is claimed in the construction of the valves, controls, or actuators, it presumably would not be essential to disclose the details of such valves, controls, or actuators to meet the best mode requirement, assuming of course that the enabling requirement is met.

4. If the disclosed embodiment does not include or relate to the best mode, determine whether there is a legal justification, based on public policy, for not including a description of the best mode. As indicated above, one possible legal justification would be where the best mode involves a national security embodiment which may not be publicly disclosed and which, if described in the patent application, would subject the application to a Secrecy Order. As discussed above, public policy should justify permitting the disclosure of the best non-security implementation even though it was not the actual best mode. Another possible justification is illustrated by the “Coca Cola” example mentioned above, wherein the courts have already held that, in certain cases, failure to disclose a secret formulation identified by a trademark does not necessarily violate 35 USC 112, first paragraph.

By thus applying, per prongs 1 and 2 above, a primarily quantitative test to the enabling requirement, and a primarily qualitative test to the best mode requirement, Section 35 USC 112, first paragraph, would require: (1) that the description provide sufficient information to meet the enabling requirement, and (2) that the such supplied information relate to the best mode requirement. In other words, this provision would require that the specification include a description of the best mode, and that such a description be sufficient to meet the enabling requirement, but need not include all the know-how, trade-secret, etc., information known to the inventor for "optimizing" the best mode.

By applying, per prong 3 above, a “claims standard” to the scope of the subject matter required to be included in the description, numerous technical details not related to the claimed novelty would not be required to be included in the description, thereby relieving the inventor from the burden, and possible commercial implications, of disclosing technical details not directly related to the invention.
By applying, per prong 4 above, a “public policy” test permitting a “second best” mode in certain cases (e.g., because of a possible detriment to the national security), the constitutional purpose of the patent system, in promoting the progress of science and the useful arts, is best implemented, by making available to the public a description of the invention without compromising national security, while at the same time securing for limited times to the inventors the exclusive rights to their discoveries.

Replacing the present unrealistic, subjective tests for the best mode requirement as applied in recent decisions, by the above four-prong, realistic objective test, will provide a clear and stable definition of this requirement consistent with the apparent legislative intent. As a consequence, it will avoid, to a large extent, the many disadvantages in over-complying with this requirement, the risks in under-complying with this requirement, and last, but not least, the need to make the exhaustive “sniff and probe” type examination involved in the anecdote at the beginning of this article.

Benjamin J. Barish

Advocate, Patent Attorney

Ehrlich & Fenster

BSD

Genesis and Time

Rabbi, Dr. Yosef Freedland, Surgeon, Patent Attorney, Ehrlich & Fenster

Abstract

Close examination of the text of the Bible shows that the events of Genesis appear to be unrelated to the Big Bang and creation of the universe. More specifically, the 5,700 years since the events of Genesis do not pose a limitation on the age of the universe. This interpretation is supported by a focused examination of passages from Genesis, Psalms and prayer.

Introduction

The Bible and modern science present radically different outlooks on the creation of the earth, and the origins of man.

The Bible appears to state that some 5,700 years ago, the earth, all species of animals and man, were created. Scientific observation suggests that the earth is several billion years old; species on earth evolved over eons of time; and civilizations were extant long before 5,700 years.

The arguments presented by science, prima facie, appear to be cogent, to the extent that many modern books have been written by religious scientists to prove that the biblical time frame covers a much longer span of time, for example with each of the seven days of Genesis spanning eons of time.

The present article will present a different view on the events of Genesis, based extensively on the preeminent 12th century commentary, Rabbi Shlomo Yitzchaki, Rashi[i]. Further, support for this view will be cited in prayer and a Psalm.

In the Beginning – "Aw-retz"

Genesis 1:1 “In the Beginning of creating … Land”.

The first verse of the Bible refers to the creation of “Land”, "Aw-retz" in Hebrew. Land, throughout the Torah refers to a small geographic location, occupied by a group of people sharing common language or ancestry.

The “Land of Canaan”, for example, is an area of land initially occupied by the Canaanites, promised to the Patriarchs Abraham, Isaac and Jacob and bequeathed on their descendents. The descendants of the patriarchs were exiled in “Land of Egypt”. The Tigress River is associated with the “Land of Paras”, modern-day Persia.

The word "Aw-retz" in the very first verse that introduces Genesis, suggests that Genesis is not about creating a universe or even the planet earth, but something different entirely.

The Enigma of the Sun and Moon

Further support for this understanding that Genesis has nothing to do with the Creation of a universe, is provided by the verse at the end of the first day; “And there was sunset and there was sunrise, one day”.

Sunset and sunrise require man to observe the sun's position with respect to the earth, a universal definition throughout the Torah, Prophets Writings and Code of Law. In the Code of Jewish Law, for example, laws regarding the onset of the Sabbath are based solely upon sunset observed from the highest point in a given community.

On the first day, according to the commentary, the heavenly illuminators were created, but only "fixed" in place until the fourth day[ii]. The literal interpretation of this suggests that sunrise and sunset of days one through three was either non-existent or totally different than the sunset and sunrise of days four through seven.

In spite of this profound difference between what was observed on day one, versus what was observed on day four, the Bible invariably refers to sunrise and sunset at the end of each of the seven days of Creation; suggesting that sunset and sunrise on each of the seven days of creation were equivalent.

If something as fundamental as sunrise and sunset didn't change during the seven days of Creation, one could conclude that Creation had nothing to do with forming the sun and earth and that the sun and earth were fully extant, in place, on the first day of creation.

Moreover, the use of the words sunrise and sunset, suggests that man whose presence is required for the associated observations, was present on the planet from day one of Creation; suggesting that Creation occurred on a populated world.

What then is the creation about?

Genesis – The Divine Story of the Exodus

Genesis does not appear to be an account of how, or when, the universe was created, but an account of the miraculous events of the Exodus from Egypt.

Genesis 1:3 “And He said, Elokim, let there be light".

According to the commentary, as will be explained in more detail below, this verse refers to the creation of the pillar of fire that led the Israelite nation in the desert.

That Genesis is truly a description of Divine perspective of the holy events surrounding the Exodus should not be surprising as the relationship is specifically noted in the convocation of the wine on the Sabbath; Friday night. The convocation states that the onset of the seventh day of Creation constitutes "a remembrance to the Exodus from Egypt".

Unified Creator, Unified Redeemer

Genesis 1:5:”...and there was sunrise, one day”.

The commentary notes that the Torah chooses the words “one day” rather than “first day” because the word “one” signifies unity. This Divine unity parallels the Divine unity during the Exodus.

Exodus 12:12 “I will pass through the land of Egypt on this night, and I shall strike every firstborn... I the Almighty.”

The revelations of the first day of the Exodus, beginning with death of the first born Egyptians, were performed by the Almighty alone, without angels acting as emissaries of the Almighty.

In the short span of a article, it is virtually impossible to analyze every aspect of Genesis. However, we will trace the account of the creation of the "illuminators" to provide at least one example of how Genesis may actually be a description of the Divine Investiture and Divine love that lit up the world during the Exodus.

The Pillar of Fire – The Great Illuminator

Genesis 1:8 “and the Almighty...called the Rekiah, heaven (‘shawma-yim’)".

The commentary cites the word shawmayim (heaven) as being a contraction, inter alia, of the Hebrew words “Fire and water“[iii]. Water is an integral part of the clouds surrounding our plant. Lightning, a product of static electricity generated by cloud movement, is not present in every rain shower. Fire, referring to lightening that is expressed only during certain types rain cloud formation, is not integral to the heavens.

The commentary, however, is referring to the Exodus in which the Clouds of Glory lead the Hebrew nation during the Day and the pillar of fire leads the nation by night and later synergistically combined.

Exodus 13:21: “And the Almighty went before them during the day in a Pillar of cloud, to show them the way”.

The commentary relates that on the third day of the Exodus[iv], Pharaoh’s spies that accompanied the Hebrew nation thus far, went back to Egypt and informed Pharaoh that the Hebrews has stopped at the edge of the desert at Pi-hahiros and were lost. Concurrent with setting up of the Hebrew camp on the fourth day, the Almighty orders the Clouds of Glory and Pillar of Fire to remain fixed in place.

Exodus 14:5 “and it was told to the king of Egypt that the Holy nation had fled... And He harnessed his chariot and He took his people .." and Pharaoh starts out in pursuit of the Hebrews on the fourth day following the Exodus.

Day Four – Fixing the Position of the Illuminators

Genesis 1:14 “ “Moros”, lights, will be fixed “yehi” in the Rekiah ‘firmament’.

The commentary states that in Genesis, the lights were created on the first day, but fixed in place on the fourth day. The moros of Creation correspond to the pillar of fire. The "firmament", "rekiah", which was similarly fixed in place on the fourth day of Creation, corresponds to the Clouds of Glory during the Exodus.

Heavens of Fire and Water

Exodus 14:19-20”... and the Pillar of clouds traveled from in front of them to behind them. And it went between the Hebrew camp and the Egyptian camp..." along with the Pillar of Fire.

In anticipation of Pharaoh's attack, the clouds of Glory and the Pillar of Fire synergistically function together during both day and night. The Clouds create impenetrable darkness in the Egyptian camp and the Pillar of Fire lights up the Hebrew camp.

The simultaneous actions of the Pillar of Fire and the Clouds of Glory, is key to understanding the commentary that "Shawmayim" is a mixture of water and fire.

In addition, in Genesis, not Exodus, the commentary states that the "shawmayim" functioned to catch the “arrows of the night"[v]. What is generally read as "Chetzi", meaning "half", should apparently be read as "Chaytey", the word "arrow" in the construct form, referring to the "arrows of the night". During the Exodus, the commentary tells us that arrows and catapulted objects were slung on the Hebrew camp by Pharaoh's camp during the night. Only through the miraculous protection of the Clouds of Glory in combination with the Pillar of Fire, was the Hebrew Nation saved from injury.

Day Four, Week Two -- Diphtheria

Genesis 1:14 “And He, Elokim, said there will be (yehi) profane lights, "Moros”

This commandment that the Pillar of Fire must remain stationary over the Hebrew encampment on the fourth day of the Exodus, provides the commentary with another opportunity to equate the Exodus with Genesis.

According to the commentary, the word "yehi" is missing the "v" prefix that designates holiness.[vi] Most interpretations ignore the inclusion of the word "v'yehi" in the caption of the commentary and suggest that one of the two "v's" in "Moros" is missing. However, due to the various spellings of "Moros" during Genesis, there is disagreement as to which "v" of the two"v's" in Moros are missing. The solution appears to be the word "yehi" that earlier included a "v".

A "v" prefix signifies "holiness". The Divine command to the Pillar of Fire to stop in place should be introduced as “V-Yehi Moros”, “and let there be Holy lights” signifying the great miraculous events related to the Pillar. Instead, the pillar of fire is addressed with the more mundane command, “Yehi Moros”, “let there be profane lights”[vii].

The relegation of an outright miracle to the level of the mundane is quite unusual, but related to a specific occurrence; a terrible tragedy that occurred on the fourth day of the next week following the splitting of the Red Sea.

Exodus 15:23 “And they came to Moros-ah, and they could not drink the waters because they were bitter...”

On the fourth day of the week following the splitting of the Red Sea, the Hebrews stopped at a place called “Moros-ah” where the Hebrew nation complained about their thirst. The complaints represent a tremendous breach of faith that become the source for all further complaints and unholy actions throughout the Hebrew sojourn in the desert. The punishment for the complaints at “Moros-ah”, correspondingly were severe, with the Hebrew nation stricken with a plague similar[viii] to diphtheria[ix]. On this basis, the words "yehi Moros" of Genesis are missing the "v" prefix.

Additionally, the commentary makes the case that that[x] “Moros-ah”, the stopover on day eleven following the Exodus is grammatically equivalent to “Moros”[xi], the illuminator fixed in the heavens on the fourth day of Genesis; thereby providing further leads that connect Genesis and Exodus.

We actually have another source that the world is not limited to a 5,700 year span that can be explored in this article.

30,000+ Years of Civilization

Psalms 105:8: “He [the Creator] remembers his covenant…to the thousandth generation”

There were only 26 generations between Genesis and Exodus[xii]. From the creation of higher man in Genesis until the completion of higher man's sojourn on this planet, there is a maximum allotment of 7,000 years. Assuming that each of the 1,000 generations arises on average every 30 to 50 years[xiii], the history of man stretches some 30,000 to 50,000 years, with only the last 200 or less generations occurring during the final 7,000 year period that begins with the creation of higher man in Genesis.

The commentary[xiv] suggests two answers: A first answer posits that the 1000 generations may have been foreshortened due to the sins of man. This first answer, however, does not spell out that the 1000 generations were shortened specifically by 800 generations or more. If the 1000 generations were shorted by only 10 or even 100 generations, we have at least 700 generations, or about 21,000 to 35,000 unaccounted years during which the Torah was "remembered".

Further, the "shortening" may not refer to the number of generations, but to the lifespan of each generation. This foreshortening of man's lifespan to 120 years is specifically stated in the Bible in proximity to the account regarding the generation of Noah.

The second explanation by the commentary also does little to significantly limit the time span of the 1,000 generations. The commentary states that The Almighty guarded the Bible throughout 1000 generations. This "guarding" could have taken place both prior to and following the bestowal of the Bible at Mount Sinai, stretching about 21,000 plus years prior to the creation of higher man.

Based upon the above, it appears that a more primitive humanoid civilization, preceded Genesis. The creation of man during Genesis, appears to be an account of how primitive man was endowed, ex nehilo, with higher intellectual powers, in anticipation of receiving the Bible, and the higher moral code contained therein.

Commemorating the Generations of Man

The progression of civilization from primitive man to a single higher man; and from singular higher man present in each generation to a nation of Hebrews gathered at Mount Sinai, is commemorated in the prayer, "Blessed is the one who spoke". The prayer is translates as follows:

"Blessed is the one who spoke and brought the holy World into being, Blessed is he."

This appears to be a reference to the creation of a "Holy World" having the potential to express the Holiness of the Almighty, through adherence to a Holy moral code, the Bible. The elevation of the mundane world to a potentially "Holy World" occurred with the endowment for potential spiritual elevation in a physical plot of land, the Holy Land.

"Blessed is the one who spoke during holy procreation (oseh)".

In order to actuate this holiness, the Almighty created a "Holy Man", Adam, the first higher man.

The word "oseh", as noted above, refers to procreation and, specifically, the procreation of the first higher man that included Divine manifestation through creating a holy vessel in DNA[xv], the transcript of Holy Man's intellectual faculties.

"Blessed is the one who decreed and followed through",

The above stanza recounts how Adam failed to live up to the Divine commandments against eating of the fruit of the "Tree of Good and Evil", forcing the Almighty to mete out the required punishment.

"Blessed is the one who made Genesis".

The stanza above cannot be referring to the creation of higher man, Adam, which has already been commemorated above. The stanza commemorates the endowment of higher souls to the Israelite nation during the Exodus, an event integrally related to Genesis as noted in relation to the Sabbath convocation above.

The prayer, "Blessed is the one who spoke", then, commemorates the elevation of a mundane world with the Divine manifestation in the Holy Land, prior to the creation of Adam. This elevation of the Holy Land, however was only a potential that could not be actualized until Holy Adam was created.

With the creation of Holy Adam and his downfall, the actualization of the Holiness of the Land promised to our forefathers was delayed until the true "Genesis", creation of a holy nation, following the Exodus.

2,488 Years To Higher Man

According to the above understanding, the Bible accounts for only 2,488 years of the history of higher man and have little to do with the birth of the universe eons before.

This understanding is actually quite logical when one considers that the purpose of creating the world had nothing to do with the Big Bang, the rise of lower species, the appearance of primates or primitive man; all of which were probably "ho hum" events when viewed against the backdrop of our infinitely Holy and Unfathomably Intelligent Creator.

This is confirmed by the statement that the purpose of creating higher man world was to create a holy dwelling place in the lower worlds; the lower worlds representing the balance of the world outside the boundaries of the Bible, and its associated immorality.

To the Almighty, infinite and completely unbound by time, the billions of years prior to the Exodus, took no longer than the blink of a human eye. The billions of years during which the earth passed through eons of speciation have no significance to the Divine plan for the world and the universe. the only event of significance occurs in the last 7,000 years of world with the appearance of higher man and bestowal of free choice and higher reasoning does this mundane world, indeed this mundane universe, have the potential to be elevated to a new plane of holiness; a holiness that should be soon be revealed.

It is only with the Exodus, and the many associated miracles manifest with Divine love as recounted in the deep involvement of the Almighty in Genesis, does the Bible truly begin. Further, the true revelation of holiness will only culminate in the Messianic era upon us.

Supernal Holiness, Then, and In the Future

Genesis is a Divine perspective of how the vast power of the Almighty was lovingly concentrated in creating changes with the potential to elevate higher man, the Holy Land, the planet and the universe. Exodus exquisitely records man's perspective of the actual events that occur in preparation for receiving a new Moral framework for living in the Holy Land and throughout the world as a preparation to greeting the true and all encompassing Messianic Era, led by our righteous redeemer.

While the space allotted by a article, and Rabbinic interdiction, have curtailed a more in depth analysis of the Creation, it is hoped that this brief article will aid Jews and Gentiles in strengthening adherence to their respective Divine Codes and Obligations – the 613 Codes and the Noahite Guidelines, respectively – as presented in the Bible, the greatest moral and spiritual work of all time.

[i] Rashi Rabbi Shlomo Yitzchaki, known as Rashi, February 22, 1040 – July 17, 1105, who wrote the first and foremost comprehensive commentary on the Hebrew Bible and Talmud.

[ii] See Rashi Genesis 1:14

[iii] This is not a reference to rain clouds and lightning. Lightning may be produced together with rain but lightning does not have the same source as rain, nor is it a blessing. For example, Deuteronomy 11:14 “when you will serve with all your heart, I (the Almighty) will provide the rain for your land in its time. There is no mention of lightning as a blessing.

[iv] Exodus 14:5 See Rashi for accounting of the days of the Exodus

[v] Genesis 1:14 Rashi caption “For days “

[vi] Holiness is signified by a "v" prefix. See Rashi to Genesis 2:3, on the words “And He gave a Holy Blessing, And He gave a Holy Sanctification”. The “V” prefix clearly imparts additional holiness to these words, associating them with miracles related to the Exodus.

[vii] Without any prefix of "v" or "h" on either the word "lights" or "let there be", the word "profane" has been added to emphasize the lack of holiness in this event.

[viii] See Proverbs 3:9 where Rashi brings an interpretation from “Pesikta”.

[ix] diphtheria “Askufah”, a disease of the throat that causes suffocation

[x] Note that the caption to this Rashi is “yehi Moros”. Rashi goes on to state that the “V” is missing. While one could postulated that the missing “V” is in the word “Moros”, it is missing from the word “yehi”, let there be.

[xi] Exodus 15:23 Rashi: “marosah” is equivalent to “le morah”, and the “s” is equivalent to an “h”.

[xii] Rashi on Psalm 105:8 based upon Talmud Chagigah

[xiii] This figure is offered only to demonstrate a possible time frame for the Bible based upon observable events. During the first ten generations of the Bible, each generation is recorded as being many years longer.

[xiv] Ten generations were born from Adam to Noah, ten generations from Noah to Abraham and six generations from Abraham to Moses[xiv], a total of 26 generations

[xv] The word "oseh", “make” is used throughout the Holy Chariot and Creation Account as a reference to procreation.

Rights locker architecture - the next step?
Roy Melzer, Ehrlich & Fenster, Sep 2004

Potential and risks of a new approach to digital content delivery

Abstract
The growth of bandwidth leads to the integration of new content distribution technologies and models. One example is the possible integration of right locker architectures. The article addresses this technology from a legal point of view and analyzes the possible advantages and impediments that might result from the integration of this model.

Keywords: Right locker architectures, bandwidth, privacy, digital contracts

Introduction
The digital content delivery is facing tremendous changes since the advent of the Internet. Those changes are primarily led by the constant expansion of the net bandwidth. Broader bandwidth enables various new web-based applications with different methods to disperse digital content efficiently. Those new possibilities alter the content industry and change the way people use and enjoy consumer electronic products, media, and entertainment. One indication of change in the consumers' consumptive behaviour is the increasing demand to access digital content from portable devices like laptop, PDA and mobile phone, a tangible manner in the shade of the third generation of wireless services.

Enabling the user to access digital content either from his home stationary or from his mobile devices raises some challenges regarding the traditional DRM model. In the current technology, users' digital rights are annexed to the protected content that is fixated in a particular device and can be accessed either directly, or from any other device that stores another private copy of the content. However, the user cannot access the purchased content from any other device, though he already acquired the right to use the content. Rights locker architecture technology presents a model that circumvents this content fixation problem.

The "Rights Locker" model
In this model, the content resides only on the rightholder's data server memory. The user practically purchases only the right to access the content and not a physical copy of it. The user rights are stored on a server that was configured to hold authorization information. At the moment of purchase, the authorization server updates the user rights and stores them in accordance with the transaction contract.

Whenever the user desires to access the content, an adjusted application at the user device sends a request to the authorization server. After the server verified the request, the content is streamed to the user from the data server. The same procedure takes place irrespective of the used device, enabling the identified user to access content.

This is not a theoretical model. For example, Digital World Services (DWS 2004), a provider of software for secure delivery of digital content, implemented "rights locker" technology in its ADo2RA system, a content independent digital distribution infrastructure, that is designed to enable content providers and retailers to package, protect, and deliver digital content across multiple devices.

Customer advantages of "Rights Lockers"
Apart from the accessibility, rights locker architecture provides several additional advantages for the customers. The acquired data is backed-up safely, proofed from hardware failures or viruses hazards. Additionally, the user does not have to allocate new memory for the content, and therefore the hardware costs reduce.

Content supplier's can now offer new business models, based on access control: Different contracts regarding the same data can suit different customers better then the current prevailing download model by offering variant prices, based on time periods or number of access permissions. The right to access the content can be sold not only to individuals but also to groups of people, reducing the price per capita (i.e. access is bought for a group of employees).

Drawbacks and uncertainties
The adoption of the new technology still has to face several impediments. From the technological point of view, the current bandwidth is not sufficient to enable real time streaming in a quality that will satisfy the average customer. It seems that the "rights locker" model will only proliferate when bandwidth will allow streaming of content at the same quality of service known in today's apparatus (i.e. supplying real time streaming of songs and movies at the same quality as playing them from the memory of the used devise). However, reaching this quality threshold is just a matter of time. According to Edholm's Law (Cherry 2004), in about five years third-generation wireless will routinely deliver 1 Mb/s, allowing audio streaming directly to mobile phones. Wi-Fi technologies will deliver 10 Mb/s wireless access for PDA and laptops, allowing video and audio streaming simultaneously.

From the legal point of view, the current European copyright legal frame is phrased in terms of usage, not access. An authorization is needed from the right holder to carry through actions like reproduction, communication to the public and making available to the public. The copyright directive defines the lawful use of the content and the usage exemptions in terms of "private copy" or "fixation of the content". However, full integration of the rights locker architectures means that no physical copy of the content would be stored on the consumer devices. The user will "access" the content subjected to contract stipulations rather then "use" as in the sense of lawful use definitions and exemptions.

Even though, one can argue that the new technology is just a new way to handle DRM by mobilizing the digital rights rather than confining them to certain data files, with other words: a way to adjust to the new broad wireless bandwidth surrounding. However, if rights locker architectures will be adhered, a re-thinking of existing terms and definitions in copyright law is required: Sharing files with friends is not "space shifting" anymore but sharing access to the same content, Peer-to-Peer phenomena might transpose into password sharing and "private copying" will be subject to the contract terms.

Moreover, the technology facilitates copyright enforcement. Firstly, this is because the supplier can encrypt the transmitted signals, and thereby impede the fixation of the content. The supplier can digitally tag each transmission of the content, enabling easy tracing of the origin of the fixated copy. Thirdly, the content supplier can easily monitor the use of content, regarding the frequency of use and the IP address of the user devices to enforce the purchase contract.

Open questions
The integration of "right locker" technology might have substantial implications on the current legal frame and therefore should be examined by copyright legislators. In regard to the access agreements, the contract frame should raise questions regarding access control: can a database owner criminalize a user who stores "private copies" on his hard-drive when the contract terms prohibit this? Can the supplier control the access to the content eternally or is he obligated to enable free access to content after the expiration of the content copyright? And even if the release of the content to the public domain is obliged, what are the incentives for suppliers to enable access for content that is in the public domain. From the customers' point of view, basic rights should be secured. User's privacy might be endangered because of the ability of content owners to monitor the central repository server and to document user actions. Dilemmas for possible legislation can be what are the limits of access data compilations? Who should hold the ability to access this information? And what uses of the data is the supplier entitled to? It seems that this problem is inherited in the technology and will require a continuous monitoring mechanism to guard the users' human rights.

Bottom line
It is still early to estimate in the light of the current bandwidth potential if rights locker architectures will succeed to enter the content delivery market. The integration of the technology holds great advantages for customers especially by enabling various access possibilities from different devices. However, the impediments and dangers to customers’ privacy should be kept in mind.

Sources

Cherry, Steven: Edholm's Law of Bandwidth. Telecommunications data rates are as predictable as Moore's Law . IEEE Spectrum Online (15/07/2004), http://www.spectrum.ieee.org/WEBONLY/resource/jul04/f1
DWS (2004): The ADORA DRM Content Delivery Platform of Digital World Services is described at http://www.dwsco.com/ps_adora.html; the news section http://www.dwsco.com/news_events.html announces implementations of ADORA. Customers currently listed are Vodafone D2, AOL Germany, O2, MediaMarkt? and T-Online International.

LEGAL OBSERVATIONS OF THE EUROPEAN AUDIOVISUAL OBSERVATORY

The Legal Protection of Broadcast Signals

by Lucie Guibault*

Institute for Information Law (IViR), University of Amsterdam

Roy Melzer**

Ehrlich & Fenster - Patent Attorneys, Ramat-Gan (Israel)

1. Introduction

The history of neighboring rights is a turbulent one. Broadcasting organizations, together with performers and phonogram producers, were for the first time granted legal protection on a worldwide level through the International Convention for the Protection of Performers, Producers of Phonograms, and Broadcasting Organizations of 26 October 1961 (the Rome Convention).¹ Although it may have been a first step in the right direction, the text of the Rome Convention soon proved unsatisfactory to all three categories of rights holders, not least because of the subordinate place that neighboring rights seemed to occupy in relation to copyright. The technology-dependent wording of its provisions further ensured that the Convention would lose much significance over the years, as technology evolved. Several international and regional instruments have been adopted since then in order to strengthen the protection of broadcasting organizations and to take account of new technological developments, such as satellite broadcasting, cable distribution, and direct-broadcast satellites.² Calls for the elimination of the main causes of irritation arising from the Rome Convention are increasingly being heard from the broadcasting organizations, especially since the technological gap between the broadcasting techniques covered by the Convention and the current possibilities has only grown over the last forty years.

The protection of broadcasting organizations has, however, always been surrounded by controversy. Unlike authors’ rights, which reward the author for his creative effort and protect his personality rights, the grant of neighboring rights for broadcast signals merely reflects the acknowledgement of the organizational, technical, and economic effort invested in a program and its broadcast.³ The main rationale of this type of neighboring rights is to protect broadcasting organizations against piracy and unfair competition and, in general, against all acts whereby a third party derives unfair commercial profit from its investment.⁴
Another, more prosaic, explanation for the introduction of neighboring rights for broadcasting organizations is that, with the adoption of the principles of the Rome Convention, the broadcasters became the main source of royalty payments to performing artists and phonogram producers and so, as a form of compensation, they should receive a
neighboring right of their own.⁵ Some uneasiness towards this category of neighboring rights results from the fact that the regime sets no condition or investment threshold on the broadcasting organizations in order for them to benefit from the protection. Such an unconditional grant of rights is in contrast with every other type of intellectual property regime, where the grant of an exclusive right is predicated on the fulfillment of a condition of originality, novelty, inventiveness, or substantial investment. Other critics point to the fact that this category of neighboring rights is granted irrespective of whether the content of the broadcast is subject to the copyright protection of other rights holders or whether it constitutes material in the public domain.⁶

The question then arises whether the fight against signal piracy or the need to compensate broadcasters for the high licensing fees of performers and phonogram producers constitute sufficient grounds to confer on broadcasting organizations such a strong monopoly on their signals.

This article on the protection of broadcasting organizations is divided into two main parts: the first one describes the current state of the protection at the international and European levels; and the second one, examines the protection as currently proposed in the consolidated text of the WIPO Standing Committee on Copyright and Related Rights.⁷ The analysis of the present state of the protection of broadcasting organizations will highlight the most important shortcomings of the international instruments on the subject, as well as draw attention to the relatively high level of protection that broadcasting organizations enjoy at the European level. We will thereafter consider some of the issues that are hotly debated within the WIPO Standing Committee and, more specifically, analyze how the proposed changes would compare with the current situation. Finally, we will conclude by making some general remarks on the progress of the Standing Committee towards the adoption of a treaty on the
subject.

2. Current Status of Broadcasting Organizations
At the international level, the most important instrument remains the Rome Convention of 1961. The Convention on the Distribution of Programme Carrying Signals Transmitted by Satellite signed in Brussels in 1974 was specifically intended to adapt the protection of broadcasting organizations to the new technological development of satellite broadcasting. As we shall see below, apart from the Rome and Brussels Conventions, the regulation of broadcasting rights is further completed at the international level only by the very succinct provisions of the Agreement on the Trade-Related Aspects of intellectual Property (TRIPS). At the European level, the Council of Europe has also adopted two instruments and several recommendations dealing with the neighboring rights of broadcasting organizations. However, these instruments have had a very limited impact in practice. The core of the protection enjoyed by broadcasting organizations in Europe derives from a number of directives adopted by the European Council and the Parliament in the field of copyright and neighboring rights law.

2.1 Protection at the International Level
2.1.1. Rome Convention
The Rome Convention protects three categories of beneficiaries with radically diverging interests, namely performers, phonogram producers, and broadcasting organisations.8 This explains why the road towards the adoption of the Convention was a bumpy one and why the final text is, to a large extent, a reflection of significant compromises on the part of the three categories of rights holders. Its completion has been described as a remarkable tribute to the diplomatic talents of those who guided the countervailing forces into a workable solution. Its survival, despite the shortcomings, is almost miraculous.9 The Convention, which is currently binding upon 73 States, has served as a model for most of the existing national provisions on the protection of neighboring rights. With respect to broadcasting organizations, the object of the protection is the programme output, or broadcast
signal, as opposed to the content of the broadcast, upon which may rest copyright protection that may or may not belong to the broadcasting organisation.10 Article 13 of the Convention provides for a number of minimum rights to “broadcasting organizations” with respect to their “broadcasts”, two terms that are left undefined. As Rumphorst explains, it is the combined effort of the broadcasting organization to plan, produce and/or acquire, schedule and transmit programmes that deserves protection against unauthorized appropriation by third parties.11 “Broadcast” is therefore to be understood as the programme output as assembled and broadcast by or on behalf of the “broadcasting organization”, which in turn may be defined as the organization which engages in this activity.

The duration of the protection granted under the Convention lasts at least until the end of a period of twenty years computed from the end of the year in which the broadcast took place. Since the content of the broadcast is irrelevant, the period of protection must be established with regard to each individual broadcast. Thus, if a broadcasting organization transmitted a given programme in 2000 and repeats the broadcast ten years later, each individual transmission enjoys a separate period of protection of twenty years. The term “broadcasting” is defined at Article 3 (f) as a “transmission by wireless means for the public reception of sound or of images and of sounds”.12 Pursuant to Article 13, broadcasting organizations have the right to authorize or to prohibit the following acts:

1) The re-broadcasting of their broadcasts;
2) The fixation of their broadcasts;
3) The reproduction of unauthorized fixations; and
4) The communication to the public of their television broadcasts
if such communication is made in places accessible to the public against payment of an entrance fee.¹³

Clearly, a transmission via cable, or a communication to the public by fixed-service satellite whose signals cannot be directly picked up by the public is excluded from the scope of the definition. The fact that the Rome Convention does not take into account the technological developments that have occurred since its adoption in 1961 undeniably constitutes, from the point of view of broadcasting organizations, the Convention’s main shortcoming.¹⁴

2.1.2. Brussels Satellite Convention
The Convention on the Distribution of Programme Carrying Signals Transmitted by Satellite,15 which was adopted in 1974, deals with the protection of satellite transmission by which programmes are transmitted between different broadcasting organizations or between broadcasting organization and cable distributor. According to the provisions of this Convention, Contracting States are required to undertake adequate measures to prevent the distribution on or from their territories of any programme-carrying transmission by any distributor for whom the signal is not intended. In other words, the protection granted under this Convention relates exclusively to the transmissions of signals between broadcasting organizations. Article 3 of the Brussels Convention expressly excludes from the scope of protection “the signals emitted by or on behalf of the originating organization [that] are intended for direct reception from the satellite by the general public”. Arguably, this Convention was negotiated and signed before direct broadcast satellite services had become an economically viable means of exploitation. This exclusion did have as a direct consequence the removal of any practical significance from the Convention. As a result, only 26 States have ratified the Brussels Satellite Convention, in comparison to the 73 Contracting States of the Convention of 1971 for the Protection of Producers of Phonograms against Unauthorized Duplication of their Phonograms,16 which was also
intended to supplement the norms laid down in the Rome Convention.

2.1.3. TRIPS Agreement
The provision of the TRIPS Agreement on neighboring rights is, once again, the result of a compromise. Like the Rome Convention, it deals with all three traditional categories of beneficiaries of the protection.17 Unlike the solution adopted with regard to Articles 1 to 21 (except 6bis) of the Berne Convention, however, the TRIPS Agreement does not mandate the Contracting Parties to implement the material regulations of the Rome Convention as such. While the TRIPS Agreement reiterates most of the substantial regulations of the Rome Convention and even provides for some supplemental regulations with respect to the rights of performers and phonogram producers, it imposes no obligation on Contracting Parties to confer neighboring rights on broadcasting organizations. Article 14, paragraph 3 of TRIPS provides that:

“Broadcasting organizations shall have the right to prohibit the following acts when undertaken without their authorization: the fixation, the reproduction of fixations, and the rebroadcasting by wireless means of broadcasts, as well as the communication to the public of television broadcasts of the same. Where Members do not grant such rights to broadcasting organizations, they shall provide owners of copyright in the subject matter of broadcasts with the possibility of preventing the above acts, subject to the provisions of the Berne Convention (1971).”

In other words, as long as a Contracting Party complies with the relevant provisions of the Berne Convention, it does not have to grant special rights to broadcasting organizations. This option was introduced mainly to take account of the fact that, in common law countries, copyright protects not only literary and artistic works, but also all material products which may be reproduced or copied without benefiting from industrial property protection.18 On the other hand, if a Contracting Party chooses to grant protection for broadcast signals, it must meet the minimum standards of Article 13 of the Rome Convention.

Another noticeable difference in treatment between performers and phonogram producers, on the one hand, and broadcasting organizations, on the other hand, concerns the term of protection of the respective neighboring rights. Article 14, paragraph 5, of the TRIPS Agreement extends the term of protection offered to performers and phonogram producers from the 20 years provided under the Rome Convention, to a period of protection lasting 50 years computed from the end of the calendar year in which the fixation was made or the performance took place. By contrast, this same provision of the TRIPS Agreement merely confirms the term of protection set by the Rome Convention with respect to broadcasting organizations, protection that lasts for at least 20 years from the end of the calendar year in which the broadcast took place.

2.2 Protection at the European Level
2.2.1. Council of Europe
Over the years, the Council of Europe also made a few attempts to regulate the protection of broadcasting organizations. Like all other international instruments in the field of broadcasting rights, those of the Council of Europe have met the same limited success. Adopted in June 1960, the European Agreement on the Protection of Television Broadcasts (EAT)19 was in fact the first international instrument to provide for neighbouring rights protection for broadcasting organizations. Unlike the Rome Convention, the European Agreement deals exclusively with the protection of broadcasting organizations. In many respects, this Agreement is more modern than the Rome Convention. For example, it grants broadcasters the additional right to authorize or prohibit the diffusion of broadcasting by wire. However, for reasons of technical nature, it now has only a limited number of ratifications, some of which have been accompanied by important reservations, which relate exactly to those provisions that go further than the Rome Convention.²⁰

The European Convention Relating to Questions on Copyright Law and Neighboring Rights in the Framework of Transfrontier Broadcasting by Satellite (European Satellite Convention) was opened for signature in May 1994.21 This regional instrument specifically addresses the technical developments, in particular in the field of broadcasting by satellite, which have resulted in the blurring of the technical differences between direct broadcasting satellites and fixed service satellites. Whereas this Convention has a rather broad coverage, including copyright and neighboring rights, it contains only one provision on the rights of broadcasting organizations. Article 5, paragraph 1 of the Convention provides that ‘as far as trans-frontier broadcasting by satellite is concerned, (…) broadcasting organizations from States parties to this Convention shall be protected, as a minimum, in accordance with the provisions of the Rome Convention’. In essence, the European Satellite Convention adds nothing to the protection afforded to broadcasting organizations under the Rome Convention, whether it is in regard to the scope of the protection or its duration. The European Satellite Convention will enter into force upon ratification by 7 States. Since only two States, Cyprus and Norway, have ratified it so far, only time will tell whether the Convention will ever enter into force.

Both instruments are completed by quite a number of Recommendations of the Committee of Ministers to member States on the subject of copyright and neighboring rights.²² The latest of these Recommendations was adopted in 2002 and deals expressly with the
rights of broadcasting organizations in the digital environment.²³ If implemented, the principles laid down in this Recommendation would indeed bring quite significant changes to the current level of protection of the neighboring rights of broadcasting organizations. In addition to the rights granted under the Rome Convention, the Recommendation would extend the protection t
1) The right of retransmission of a broadcast by wire or wireless means, whether simultaneous or based on fixations,
2) The right of direct or indirect reproduction of the fixations of broadcasts in any manner or form;
3) The right of making fixations of broadcasts available to the public by wire or wireless means, in such a way that members of the public may access them from a place and at a time individually chosen by them; and
4) The right of distribution of the fixations and copies of fixations of broadcasts.

The Recommendation encourages Member States to take measures to ensure that broadcasting organizations enjoy adequate protection against any of the acts referred to above in relation to their pre-broadcast programme carrying signals.²⁴ Member States are also invited under the Recommendation to take adequate measures for the protection of technological measures that are used by broadcasting organizations in connection with the exercise of their neighboring rights, as well as for the protection of rights management information. Finally, the protection would be extended to a period of 50 years from the end of the year in which the broadcast took place. Be that as it may, the Recommendation, as its name indicates, is by nature not a binding legal instrument and we have no indication as to whether or to what extent it has led to any express legislative amendment within the Member States of the Council of Europe.

2.2.2. European Union
In comparison to the protection afforded at the international level, the protection granted to broadcasting organizations within the European Union is relatively high. The acquis communautaire with respect to the protection of neighboring rights of broadcasting organizations derives for a large part from a number of directives adopted over the past decade in the field of copyright and neighboring rights. For our purpose, the most important directives are the Rental and Lending Rights Directive²⁵ and the InfoSoc Directive.²⁶ In the course of this effort at harmonization, the European Commission has always striven, as much as possible, to bring the protection of neighboring rights up to par with that of copyright law. In effect, such an upgrade in protection has profited not only performers and phonogram producers, but also filmmakers and broadcasting organisations.²⁷Today, the protection afforded within the European Union far exceeds the norms of the Rome Convention and is almost equivalent to that of Recommendation (2002)7 of the Council of Europe.

Pursuant to article 7 of the Rental and Lending Rights Directive, broadcasting organizations have the exclusive right to authorize or prohibit the direct or indirect reproduction of fixations of their broadcasts. Article 6(2) confers on broadcasting organizations the right to authorize or prohibit the fixation of their broadcasts, whether these broadcasts are transmitted by wire or over the air, including by satellite.28 Paragraph 3 specifies, however, that “a cable distributor shall not have the right provided for in paragraph 2 where it merely retransmits by cable the broadcasts of broadcasting organizations”. This last provision takes account of the opinion of some Member States that it is not appropriate to grant a proper neighboring right for cable distributors that only make simultaneous retransmissions of received broadcasts. Articles 6(2) and article 6(3) would seem to give an indirect definition of the ‘broadcasting organization’, whereby the fixation right is vested in traditional broadcasting organizations, satellite broadcasters, and cable distributors. According to Reinbothe and von Lewinski, this definition applies to the entire Directive, and the fact that Article 8(3)²⁹ refers exclusively to the rebroadcasting by wireless means does not mean that transmission by satellite or by cable is not included.³⁰ The rights conferred under article 8 of the Rental and Lending Rights Directive are modeled after the provisions of the Rome Convention and constitute a minimum level of protection. As article 6 of the Satellite and Cable Retransmission Directive expressly indicates, Member States may provide for more far-reaching protection.³¹ Finally, Article 9(1) of the Rental and Lending Rights Directive
requires Member States to grant broadcasting organizations the exclusive right to distribute fixations of their broadcasts, including copies thereof, by sale or otherwise.

According to Article 3(4) of the Directive harmonizing the term of protection of copyright and certain related rights,³² the rights of broadcasting organizations expire 50 years after the first transmission of a broadcast, whether this broadcast is transmitted by wire or over the air, including by cable or satellite. Contrary to the Rome Convention however, the duration is calculated from the first broadcast only, so that the repeat of a broadcast does not give rise to a separate term of protection.³³

Like Recommendation (2002)7 of the Council of Europe, the InfoSoc Directive is intended to adapt the protection granted to authors and neighboring rightsholders, such as broadcasting organizations, to the digital environment. Among other things, the Directive clarifies that broadcasting organizations have the exclusive right to authorize or prohibit direct or indirect, temporary or permanent reproduction by any means and in any form, in whole or in part of fixations of their broadcasts, whether those broadcasts are transmitted by wire or over the air, including by cable or satellite.³⁴The Directive grants an exclusive right to communicate a work to the public only to authors and not to neighboring rightsholders, such as broadcasting organizations, since this right is already provided for under the Rental and Lending Right Directive. Broadcasting organizations enjoy, under the InfoSoc Directive, the exclusive right to authorize or prohibit the making available to the public, by wire or wireless means, in such a way that members of the public may access them from a place and at a time individually chosen by them of fixations of their broadcasts, whether those broadcasts are transmitted by wire or over the air, including by cable or satellite.³⁵ Generally speaking, this provision is designed to cover on-demand services over the Internet. It is unclear whether webcasting and other similar types of on-line transmission techniques are protected under the InfoSoc Directive as, although they do not constitute on-demand delivery services, they do constitute a transmission “by wire”. In addition to the creation of new rights, Member States are required to implement the provisions of the InfoSoc Directive with respect to the legal protection of technological measures and of rights management information. Unlike Recommendation (2002)7 of the Council of Europe, however, no protection is afforded at Community level with respect to pre-broadcast programme carrying signals.

3. Issues Raised by the Possible Treaty
Since the adoption of the WIPO Copyright Treaty (WCT) and the WIPO Performers and Phonograms Treaty (WPPT) of 1996,36 stakeholders and lawmakers have been committed to bringing the international protection of broadcast signals up to par with that of performers and phonogram producers and to adapt it to the digital network environment. The topic of modernisation of the principles of the Rome Convention with respect to the protection of broadcasting organizations has been on the agenda of the WIPO Standing Committee on Copyright and Related Rights since November 1998. At that time, most delegations agreed on the principle that the existing international framework should be updated and improved and were confident that a new instrument would see the light in 2000-2001.37 Today, more than six years after the launch of the discussion process, the
protection of broadcasting organizations remains the main topic on the agenda of the Standing Committee and a new instrument has yet to be adopted. Although the Chairman of the Standing Committee recently reported some progress towards a consensus, several important issues still generate intense debate: the object of protection and definitions, beneficiaries of protection and national treatment, rights of broadcasting organizations, obligations on technological measures, application in time and relationship with other treaties. Before the latest meeting of the Standing Committee, held in June 2004, the delegations had once again been invited to submit proposals in treaty language, which have been compiled into a consolidated text.³⁸ Time will tell if and when a new treaty on the protection of broadcasting organizations will be adopted and how far the protection will reach.

For now, let us concentrate on two major stumbling blocks encountered during the discussions of the Standing Committee: first, what is the scope of protection foreseen under the possible treaty. In other words, who are the beneficiaries of the protection: should it be granted only to traditional broadcasting entities or should it also extend to datacasters, cablecasters, simulcasters and webcasters? Second, which new rights should be conferred on broadcasting organizations to allow them to combat piracy? What would be the status of pre-broadcast programme carrying signals?

3.1 Scope of Protection
When the Rome Convention was signed, FM radio barely existed, digital audio broadcasting was hardly imaginable, and satellite and computer networks belonged to the realm of science fiction. Technological developments change the broadcasting market by adding new channels of mass communication. In the current technological environment, a broadcast signal may still originate from a terrestrial transmitter, but it is more likely to be delivered via satellite or via cable. A drastic increase in bandwidth and computer networking further accounts for the emergence of streaming video and audio services, as well as for datacasting, cablecasting, simulcasting or webcasting technologies. The new digital transmission technologies allow the creation and distribution of new kinds of services. This is for instance the case of multi-channel TV, which implies a greater choice of programmes and video-on-demand. Estimates predict that within the next few years, computer networks will allow average home users to listen to music and to watch live shows, movies and series of the same technological quality as today’s TV and Radio broadcasting services.

The question arises today of whether these new techniques fall under the definition of a “broadcasting entity” in the sense of a possible international instrument on neighboring rights of broadcasting organizations. Does the expression “broadcasting entity” include datacasters, cablecasters, simulcasters and webcasters? Or should these new categories of “broadcasters” be the subject of a separate definition? Although the definition of what constitute “broadcasting” and a “broadcasting organization” has always been a thorny issue, it has proven particularly difficult throughout the discussions of the Standing Committee. Delegations are indeed strongly divided on the question of webcasting. The elaboration of workable definitions is paramount for the proper determination of the scope of protection of any upcoming treaty on the broadcasters’ rights, as well as to avoid any inconsistency with existing international instruments.

In the course of the work of the Committee, Governments and the European Community were invited to submit proposals on this issue. Several proposals for a new instrument on the protection of broadcasting organizations have been received by the Secretariat of the World Intellectual Property Organization (WIPO) and made available to all participating Delegations. The Secretariat has prepared at different times several documents containing comparisons of the proposals, the latest updated version being dated 15 September 2003 (SCCR/10/3) and prepared for the tenth session of the Standing Committee. The discussions of the Standing Committee from its second session until the tenth session were based on the above-mentioned proposals and facilitated by the comparative documents prepared by the Secretariat. The consolidated text covers all the necessary articles for a new treaty, both substantive provisions and administrative and final clauses. The consolidated text provides a facilitating tool for the Standing Committee, which represents a simplifying step forward from the comparative document referred to above. The function of the consolidated text is to indicate clearly areas where there is a high degree of agreement in substance in the proposals and areas where there are important divergences in the proposals. In areas of agreement single proposals of articles are presented, sometimes in a combined, reorganized or reformulated format. In areas of divergence varying solutions have been presented.

The current version of the consolidated text prepared by the Standing Committee³⁹ contains a number of definitions. Among them is the definition of “broadcasting”, which now reads as follows:

““Broadcasting” means the transmission by wireless means for public reception of sounds or of images or of images and sounds or of the representations thereof; such transmission by satellite is also “broadcasting”. Wireless transmission of encrypted signals is “broadcasting” where the means for decrypting are provided to the public by the broadcasting organization or with its consent. “Broadcasting” shall not be understood as including transmissions over computer networks.”

This definition is inspired from Article 2 of the WPPT, whereby “broadcasting” is confined to transmissions by wireless means. Note that such a definition would diverge from the definition of “broadcasting” as it is presently applied at the European Union level that also includes broadcasts by wire. Contrary to this same provision of the WPPT, which is silent on the issue, Article 2(a) of the consolidated text expressly excludes computer network transmissions from the definition of broadcasting, in such a way that it is reasonable to infer that webcasting or any other use of the Internet to transmit content is excluded from the definition. With respect to the reference to “sounds and images”, the new convoluted wording differentiates “sounds and images” from written text and data, conferring protection only on the first. This distinction could be particularly problematic in the light of digital signals, where the technology allows for the combination of ancillary data as texts, graphics, moving pictures and subtitles alongside the “traditional” image and sound. These additional forms of transmission can be used, for example by webcasters, to provide added value for consumers with the supply of programme related background information, interviews, links, biographies of the ors, different language versions or subtitles etc. At this time, it would seem that such signals are not entitled to any neighboring rights protection under the consolidated text.

In contrast with all other international instruments on the subject, the consolidated text provides for a definition of “broadcasting organizations”. In the discussions in the Standing Committee, it was felt that some limits should be set concerning the persons benefiting from the protection of the new Instrument. Not everybody transmitting program-carrying signals shall be regarded as a “broadcasting organization.” The definition proposed in item (b) consists of three main elements: (1) the person shall be a “legal entity”, (2) taking “the initiative” and having “the responsibility,” for “the transmission”, and (3) for “the assembly and scheduling of the content of the transmission.”⁴⁰ It is suggested that the definition of “broadcasting organization” would be applied mutatis mutandis to the legal entities engaged in cablecasting and, subject to the final scope of the new Instrument, in webcasting.⁴¹

Article 2(c) of the current version of the consolidated text provides for a separate definition of “cablecasting”, which reads as follows:

““Cablecasting” means the transmission by wire for public reception of sounds or of images or of images and sounds or of the representations thereof. Transmission by wire of encrypted signals is “cablecasting” where the means for decrypting are provided to
the public by the cablecasting organization or with its consent. “Cablecasting” shall not be understood as including transmissions over computer networks;”

The definition follows mutatis mutandis the definition of “broadcasting” in item (a), and also in the WPPT. The notion of “cablecasting” is confined to transmissions by wire in line with the proposals of Argentina (using the term “cable distribution”), Egypt, Singapore, and the United States of America. No wireless transmissions, including by satellite, are included in “cablecasting.” In the definition, the interpretative clause referring to encrypted signals is maintained. For the same reason as in the case of the definition of “broadcasting”, “transmissions over computer networks” are excluded from the notion of “cablecasting”. The definition of “cablecasting” is needed if the notion of traditional broadcasting is adopted in the new Instrument as proposed, but would be superfluous if the new Instrument were
based on a broader notion.

Proposed Article 2(d) contains a definition of “retransmission”.⁴² The term “retransmission” encompasses all forms of retransmission by any means, i.e. by wire or wireless means, including combined means. It covers rebroadcasting, retransmission by wire or cable, and retransmission over computer networks. All proposals contained suggestions on retransmission in narrower or broader form, either in the definitions or in the clauses on rights. In the open-ended form of the definition, “retransmission” covers the substance of all proposals. Language has been added to make it clear that protection should extend to subsequent retransmissions. The definition is confined to simultaneous retransmissions only.

As mentioned above, webcasting currently represents the most contentious issue among the delegations. Webcasting is the delivery of content as real-time and recorded audio and video signals by broadcasting them over the Internet.⁴³ The technology involves the digital compression of audio, video, and text, which is then transmitted without delay. In practice, the streaming software creates a “buffer” in a computer’s RAM memory allowing its user to download video or audio in packets, a few seconds at a time, creating the effect of a continuous flow of transmission. The streaming technology enables suppliers to opt for a few models of transmission over the Internet, among them simulcasting and fixed or on-demand webcasting.⁴⁴ In fixed web-casting, the schedule and appearance of the webcast is determined in advance without allowing the user to change or control the streamed content. This model, mutatis mutandis, resembles any other form of traditional television or radio broadcasting. On-demand webcasting involves the transmission of compressed audio or video signals most commonly used over the Internet today. This model allows users to fast-forward, rewind, pause, stop, record and replay the event being viewed on their personal user-end device.⁴⁵ The key feature of on-demand webcasting is the ability of the user to control the scheduling and transmission of the webcast.⁴⁶ On-demand webcasting is similar to the cablecasters’ “on-demand services”.⁴⁷ Simulcasting on the Internet involves the transmission of signals that hold an identical representation of images and sound that are broadcast at the same time through traditional television or radio broadcasting media.

Until now, broadcasting has always been understood as a transmission intended for “public reception”, whereas webcasting and “video-on-demand” services are sent through computer networks from the server of the content provider directly to the end-user’s device. In “video-on-demand” services, the transmitter streams the requested content directly to the customer’s decoder. One can argue that a particular webcast or “on-emand” service cannot fall within the scope of “broadcasting”, since the transmission is not preformed “for the public”, but rather exclusively for a user who explicitly requested the transmission. In traditional broadcasting, which uses a “point to multi-point” technology, a single process is involved that transfers content from a single origin to multiple consumers. On the other hand, webcasting or “video-on-demand” services are accessible to a group of anonymous individuals. One could argue that a webcast transmission is available for “public reception” and therefore, could be defined as a transmission to the public. It is also a service intended for the public as long as any “member of the public” has at least the opportunity to access the service. Opinions are divided regarding the proper interpretation of “broadcasting”.

In this context, delegations have been discussing the need for and possibility of introducing a separate definition of “webcasting” in the text. It is essential to realize that a broad interpretation of the notion of “broadcasting organization” has a direct influence on the scope of application of the treaty. Two alternatives have been included in the consolidated text. Alternative C, which was put forward by the delegation from the United States of America, proposes the inclusion in the text of the following definition of “webcasting”:

““Webcasting” means the making accessible to the public of transmissions of sounds or of images or of images and sounds or of the representations thereof, by wire or wireless means over a computer network at substantially the same time. Such transmissions, when encrypted, shall be considered as “webcasting” where the means for decrypting are provided to the public by the webcasting organization or with its consent.“

In Alternative C, the structure of the definition of “webcasting” follows the definitions of “broadcasting” and “cablecasting”. The operative term of the definition is not “transmission” but “making accessible to the public of transmissions.” This expression implies the modicum of interactivity in today’s technological environment that is necessary to access the streaming of a program-carrying signal. It is the receiver who activates or instigates the transmission over a telecommunications path. The elements “to the public” and “at substantially the same time” serve to limit the definition to accessibility of real-time streaming that may be received by several receivers at the same time. The receiver may log on to the program flow at a given point of time and receive what follows but cannot influence the program flow otherwise. The definition confines the making accessible of transmissions to such activity over computer networks, which by nature may take place by wire or wireless means.

Alternative D consists in not including any definition of “webcasting” at all in the text. This Alternative recognizes the fact that a great majority of delegations at the debates in the Standing Committee opposed the extension of the protection to webcasts. Many delegations have indicated that further study is needed and have suggested that the issue of webcasting deserves to be dealt with in future discussions and not in the present framework.

These two Alternatives on the definition of “webcasting” are completed by those of Article 3 of the consolidated text on the scope of application of the Treaty. According to Article 3(1), the protection granted under this Treaty would apply to the rights of broadcasting organizations in respect of their broadcasts. Article 3(2) states that the provisions of this Treaty would apply mutatis mutandis to the rights of cablecasting organizations in respect of their cablecasts. However, with respect to webcasting entities, three alternatives have been put forward. Alternative E offers the possibility of extending, by mutatis mutandis application, the rights of broadcasting organizations to the simultaneous and unchanged webcasting by them of their own broadcasts (“simulcasting”). It corresponds to the proposal of the European Community and its Member States, which was based on the legal techniques of assimilating such simulcasting to broadcasting (“as if it were broadcasting”). Alternative F offers, in line with the proposal of the United States of America, the possibility of extending, by mutatis mutandis application, to webcasting organizations the same protection that will be accorded to broadcasting and cablecasting organizations. Finally, Alternative G recognizes the limited support at this stage of international debate for protection beyond the areas of broadcasting and cablecasting. This alternative would lead to the adoption of Alternative D in Article 2(g), as the definition of
“webcasting” would not be needed.

3.2 Substance of Protection
The rights enumerated in the consolidated text of the Standing Committee are very similar to those listed in Recommendation (2002)7 of the Council of Europe. Despite some minor doctrinal differences, there appears to be general agreement that any new treaty should clearly protect broadcasts transmitted for public reception via wire or wireless means, including by cable or satellite. Some of the rights proposed in the consolidated text⁴⁸ include rights with respect t
a) The retransmission, by any means of their broadcasts (article6);
b) The communication to the public of broadcasts, if such communication is made in places accessible to the public against payment of an entrance fee (article 7);
c) The fixation of their broadcasts (article 8);
d) The direct or indirect reproduction of fixations of broadcasts (article 9);
e) The making available to the public of the original and copies of fixations of their broadcasts, through sale or other transfer of ownership (article 10);
f) The transmission of their broadcasts following fixation of such broadcasts (article 11); and g) The making available to the public of their broadcasts from fixations, by wire or wireless means, in such a way that members of the public may access them from a place and a time individually chosen by them (article 12).

With respect to the right of reproduction of fixations of broadcasts under Article 9 of the consolidated text, two alternatives were put forward. The first alternative confers on broadcasting organizations the exclusive right of authorizing the direct and indirect reproduction, in any manner or form, of fixations of their broadcasts. The second alternative gives broadcasting organizations two distinct rights: (1) the right to prohibit the reproduction of fixations of their broadcasts; and (2), the right to authorize the reproduction of their broadcasts from fixations made pursuant to the fair use doctrine or other national
legislation exceptions, when such reproduction would not be permitted by law or otherwise made without their authorization. This second paragraph corresponds to Article 13(c)(i) and (ii) of the Rome Convention.

With regard to article 10 of the text, the United States of America suggested, instead of a right of distribution as worded above, a second alternative: to grant a more restricted right to prohibit the distribution to the public and the importation of reproductions of unauthorized fixations of their broadcasts. Note that this solution, confined to the “outlawing” of bootlegged copies of broadcasts, would derogate from the one adopted by the WPPT and by the European Union.

Apart from the problems raised by webcasting, another long-debated issue within the Standing Committee concerns the protection that should be afforded to pre-broadcast programme carrying signals. Generally speaking, “programme-carrying signal transmission” can be divided into two types: pre-broadcast and post-broadcast transmission. In post-broadcast transmissions, the signals that are transmitted to the public can be perfectly reproduced, whereby perfect digital copies of broadcast programs can easily be copied and offered as Internet downloadable copies that can be redistributed. Transmissions of broadcasts over the Internet are vulnerable to piracy because of the ease with which contents can be accessed and copied. Pre-broadcast signals are signals that are not intended for direct reception by the public. Such signals are used by broadcasting organizations to transfer program material from a studio or e.g. from the site of an event to the place where a transmitter is situated. Such signals may also be used for transfer of program material between broadcasting organizations, as may be used for broadcast after a delay or after some editing of the material.⁴⁹ They do not constitute “broadcasting”, since they are not intended for the public, but form rather a point-to-point transmission by telecommunications links. Since pre-broadcast signals are often transmitted in digital form, perfect digital copies can be obtained from the programme-carrying signals and copies, and downloads or re-broadcasting can be made. The pre-broadcast signals can be disseminated simultaneously with the official transmissions or even before the scheduled time for those transmissions.

The issue of pre-broadcast signals had already been addressed during the negotiation process leading to the adoption of the Brussels Satellite Convention, as a result of which Contracting States were required to undertake adequate measures against unauthorized distribution. However, the question of whether the measures to control this phenomenon should be regulated by public or private law stayed open. After long discussions, Article 13 of the consolidated text now provides that “broadcasting organizations shall enjoy adequate and effective legal protection against any acts referred to in Article 6 to 12 of this Treaty in relation to their signals prior to broadcasting”. The open wording of this provision leaves Contracting Parties the necessary room to decide how best to implement this obligation.

Conclusion

In November 2004 the WIPO Standing Committee on Copyright and Related Rights will hold its twelfth session dedicated to the elaboration of an instrument on the protection of the rights of broadcasting organizations. Although the agenda for the meeting is not yet available, it is safe to assume that the delegations will attempt to consolidate the common ground achieved so far, with a view to convening a Diplomatic Conference at a later date for the adoption of a treaty on the subject. According to the current version of the consolidated text drawn up by the Standing Committee, the protection afforded by any new treaty would show distinct similarities with the protection actually granted at the European Union level, as well as with Recommendation (2002)7 of the Council of Europe. In addition to creating new rights that are arguably better adapted to the state of technology, the consolidated text would, like the WPPT and the European instruments, contain provisions with respect to the legal protection of technological measures and of rights management information. A new treaty based on this consolidated text would follow the trend established in Europe and increase the duration of protection to 50 years after the first transmission of a broadcast, whether this broadcast is transmitted by wire or over the air, including by cable or satellite, instead of the 20 years provided for under the Rome Convention. For the rest, it remains to be seen whether the discussions of the upcoming meeting of the Standing Committee will lead to the adoption of a treaty and whether the protection of such a treaty will extend to webcasters.

___________________________________________

*) Assistant professor, Faculty of Law, University of Amsterdam, and researcher at the Institute
for Information Law (IViR).
**) Currently Technical Advisor at Ehrlich & Partners - Patent Attorneys, Ramat-Gan (Israel), ex- law clerk at the law firm Gilat, Bareket & Co., Tel-Aviv (Israel) and a trainee at IViR.
1) Text available at: http://www.wipo.int/clea/docs/en/wo/wo024en.htm
2) See: European Agreement on the Protection of Television Broadcasts (EAT), Council of Europe, Strasbourg, 22 June 1960, text available at:
http://conventions.coe.int/Treaty/en/Treaties/
Html/034.htm
Convention Relating to the Distribution of Programme-Carrying Signals Transmitted by Satellite (Brussels Convention), signed in Brussels, 21 May 1974, text available at:
http://www.wipo.int/clea/docs/en/wo/wo025en.htm
European Convention Relating to Questions on Copyright Law and Neighbouring Rights in the Framework of Transfrontier Broadcasting by Satellite (European Satellite Convention), Council of Europe, Strasbourg, 11 May 1994, text available at:
http://conventions.coe.int/Treaty/en/Treaties/
Html/153.htm
3) Natali Helberger, Neighbouring rights protection of broadcasting organisation: Current
problems and possible lines of action, Council of Europe, Strasbourg, 15 November 1999, Doc. No. MM-S-PR(1999)009 def., p. 3. Text available at:
http://www.coe.int/t/e/human_rights/media/
5_Documentary_Resources/2_Thematic_
documentation/Copyright_&_neighbouring_rights/MM-S-PR(1999)009%20def%20E%20Hellberger.asp#TopOfPage
4) Andr. Kerever, Should the Rome Convention be revised and, if so, is this the right moment?, Copyright Bulletin, vol. XXV, No. 4, 1991, p. 9.
5) Sam Ricketson, The Berne Convention for the protection of literary and artistic work: 18861986, (1987) Kluwer Queen Mary College, University London, p3
6) Ian D. Thomas, Revision of the Rome Convention: is it necessary and timely?, Copyright Bulletin, vol. XXV, No. 4, 1991, pp. 32-35, p. 32.
7) Consolidated Text For A Treaty On The Protection Of Broadcasting Organisations, Geneva, 29 February 2004, SCCR/11/3. Text available at:
http://www.wipo.int/documents/en/meetings/2004/
sccr/doc/ sccr_11_3.doc
8) Kerever (1991), p. 5.
9) Thomas (1991), p. 35.
10) Helberger (1999), p. 4.
11) Werner Rumphorst, Protection of broadcasting organisations under the Rome Convention, Copyright Bulletin, Vol. 27, 1993, p. 11.
12) Rome Convention, Art 3(f).
13) Andr. Kerever, Should the Rome Convention be revised and, if so, is this the right moment?, Copyright Bulletin, Vol. XXV, No. 4, 1991, p. 13.
14) Andr. Fran.on, Should the Rome Convention on neighbouring rights be revised?, Copyright Bulletin, Vol. XXV, No. 4, 1991, p. 21.
15) Convention on the Distribution of Programme Carrying Signals Transmitted by Satellite, signed in Brussels, 21 May 1974. Treaty available at:
http://www.wipo.int/clea/docs/en/wo/wo025en.htm
16) Phonograms Convention, WIPO, Geneva, 1971. Text available at:
http://www.wipo.int/clea/docs/en/wo/ wo023en.htm
17) D. Gervais, The TRIPS Agreement: Drafting History and Analysis, 2sd ed., London, Sweet & Maxwell, 2003, p. 160.
18) Kerever (1991), p. 9.
19) Text available at: http://conventions.coe.int/Treaty/en/Treaties/
Html/034.htm
20) Helberger (1999), p. 4.
21) Text available at: http://conventions.coe.int/Treaty/en/Treaties/
Html/153.htm
22) Recommendation No. R (86)2 of the Committee of Ministers to member States – On Principles Relating To Copyright Law Questions In the Field of Television By Satellite and Cable, 14 February 1985; Recommendation No. R (86)9 of the Committee of Ministers to member States – On Copyright and Cultural Policy, 22 May 1986; Recommendation No. R (88)1 of the Committee of Ministers to member States - On Sound and Audiovisual Private Copying, 18 January 1988; Recommendation No. R (88)2 of the Committee of Ministers to member States - On Measures to Combat Piracy in the Field of Copyright and Neighbouring Rights, 18 January 1988; Recommendation No. R (91)5 of the Committee of Ministers to member States on the Right to Short Reporting on Major Events Where Exclusive Rights for their Television Broadcast have been Acquired in a Transfrontier Context, 11 April 1991; Recommendation No. R (91)14 of the Committee of Ministers to member States on the Legal Protection of Encrypted Television Services, 27 September 1991; Declaration on Neighbouring Rights, Committee of Ministers, 17 February 1994; Recommendation No. R(94)3 of the Committee of Ministers on the Promotion of Education and Awareness in the Area of Copyright and Neighbouring Rights Concerning Creativity, 5 April 1994; Recommendation No. R (95)1 of the Committee of Ministers to member States on Measures against Sound and Audiovisual Piracy, 11 January 1995. The text of these Recommendations can be found by date at: https://wcm.coe.int/rsi/CM/index.jsp
23) Recommendation Rec(2002)7 of the Committee of Ministers to member States on measures to enhance the protection of the neighbouring rights to broadcasting organisations, Strasbourg, 11 September 2002. Text available at:
https://wcm.coe.int/ViewDoc.jsp?id=303043&Lang=en
24) For an explanation of the different types of programme carrying signals, see infra at 3.2. 25) Council Directive 92/100/EEC of 19 November 1992 on rental right and lending right and on certain rights related to copyright in the field of intellectual property, OJ L 346 27 November 1992 p. 61. Text available at:
http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexapi!prod!CELEXnumdoc&lg=EN&n umdoc=31992L0100&model=guichett
26) Directive 2001/29/EC of the European Parliament and of the Council of 22 May 2001 on the harmonization of certain aspects of copyright and related rights in the information society, Official Journal L 167, 22 June 2001 p. 10. Text available at:
http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!celexapi!prod!CELEXnumdoc&lg=EN& numdoc=32001L0029&model=guichett
27) J. Reinbothe, S. von Lewinski, The EC Directive on Rental and Lending Rights and on Piracy, London, Sweet & Maxwell, 1993, p. 85.
28) Rental and Lending Rights Directive, Art. 6(2).
29) Id., Art. 8 (3) which reads as follows: “Member States shall provide for broadcasting organisations the exclusive right to authorise or prohibit the rebroadcasting of their broadcasts by wireless means, as well as the communication to the public of their broadcasts if such communication is made in places accessible to the public against payment of an entrance fee.”
30) J. Reinbothe, S. von Lewinski (1993), pp. 88 and 99, 100. See als M. Walter et al. (ed.), Europ.isches Urheberrecht, Springer Verlag, Vienna, 2001, p. 1041.
31) Council Directive 93/83/EEC of 27 September 1993 on the coordination of certain rules concerning copyright and rights related to copyright applicable to satellite broadcasting and cable retransmission, OJ L 248 6 October 1993 p.15. Text available at:
http://europa.eu.int/smartapi/cgi/ sga_doc?smartapi!celexapi!prod!CELEXnumdoc&lg=EN& numdoc=31993L0083&model=guichett
32) Council Directive 93/98/EEC of 29 October 1993 harmonizing the term of protection of copyright and certain related rights, OJ L 290 24 November 1993, p. 9. Text available at: http://europa.eu.int/smartapi/cgi/sga_doc?smartapi!
celexapi!prod!CELEXnumdoc&lg=EN&numdoc=
31993L0098&model=guichett
33) M. Walter, The relationship of, and comparison between, the Rome Convention, the WIPO Performances and Phonograms Treaty (WPPT) and the Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS Agreement; the evolution and possible improvement of the protection of the neighbouring rights recognized by the Rome Convention, Copyright Bulletin, Vol. XXXIV, No. 3, 2000, p. 31.
34) InfoSoc Directive, Art. 2(e)
35) Id., Art. 3(1)(d).
36) WIPO Copyright Treaty (WCT) and WIPO Performers and Phonograms Treaty (WPPT) both signed in Geneva, 20 December 1996.
Text of the WCT available at: http://www.wipo.int/clea/docs/en/wo/wo033en.htm
and text of the WPPT available at: http://www.wipo.int/clea/docs/en/wo/wo034en.htm
37) Helberger (1999), p. 6.
38) Standing Committee on Copyright and Related Rights, Report, WIPO, Geneva, 23 June 2004, Doc. SCCR/11/4 Prov., p. 11. Text available at:
http://www.wipo.int/documents/en/meetings/2004/
sccr/doc/sccr_11_4_prov.doc
39) Standing Committee On Copyright And Related Rights, Consolidated Text For A Treaty On The Protection Of Broadcasting Organisations, Geneva, 29 February 2004, SCCR/11/3, Art. 2(a), text available at: http://www.wipo.int/documents/en/meetings/
2004/sccr/doc/sccr_11_3.doc
40) Id., Art. 2(b).
41) Id., comment 2.04 of the Explanatory notes.
42) Id., Art. 2(d) which reads as follows: ““retransmission” means the simultaneous transmission to the public by any means of any transmission referred to in provisions (a), (c) or (g) of this Article; simultaneous transmission of a retransmission shall be understood as well to be a retransmission.”
43) D. Wittenstein and M. Lorrane Ford, The Webcasting Wars, [1999] 2 J.I.L., p. 1.
44) A. Morrison and L. E. Gillies, Securing Webcast Content in the European Union, Copyright Technical Protection and Problems of Jurisdiction on the Internet, [2002] E.I.P.R. 74-80, at p.74.
45) B. Michaux, Webcasting ou diffusion musicale sur internet: licence obligatoire ou droit
exclusif des titulaires de droits voisins?, (2002) vol. 6 Auteurs et Media pp. 479-484, p.481.
46) Peggy Miles, Internet World Guide to Webcasting, John Wiley Computer Publishing, 1998, page 4.
47) For example: Time Warner on demand channel available at:
http://www.twcnebraska.com/cable/vod.shtml
48) SCCR, Consolidated Text for a Treaty on the Protection Of Broadcasting Organisations, WIPO, Geneva, 29 February 2004, SCCR/11/3.
49) SCCR, Consolidated Text for a Treaty on the Protection of Broadcasting Organisations, WIPO, Geneva, 29 February 2004, SCCR/11/3, Art. 13, comment 13.02.

פארק היורה : מציאות או דמיון
מחבר: ד"ר גל ארליך
טבע הדברים, אוקטובר-נובמבר 1994, גליון 6

לאחרונה, בספר רב המכר "פארק היורה" ובסרט שהופק בעקבותיו, הועלתה האפשרות לתרגם בשיטות ההנדסה הגנטית את המידע הגנטי המוצפן במולקולות דנ"א של דינוזאורים, אשר נשתמרו כלואות באיבר המציצה של חרקים עוקצים, שנלכדו בענבר (שרף עצים מאובן), לכדי דינוזאורים חיים.

כבר בשנות ה-30 וה-40 במאה הזאת השתעשעו שליטים עריצים ברעיון לשכפל תא מגופם כדי ליצור אנשים הזהים להם מבחינה גנטית. להבדיל מניסיונות המבוצעים בימינו בכיוונים, העשויים להוביל לפיתוחה של טכנולוגיה שתאפשר להכפיל בני אדם, מטרתם של מדעניו של היטלר ובראשם ד"ר מנגלה היתה לשכפל את הפיהרר, ובהמשך אנשים בעלי תכונות המוגדרות כאריות על מנת להגדיל את ייצוגו היחסי של הגזע הארי, שנחשב בעיניהם לעליון בין גזעי העולם. הנתון המדעי שמאחורי התוכנית התבסס על קיומם של תאומים זהים מדרך הטבע, והרעיון היה לשחזר במבחנה את התהליך, המתרחש באופן טבעי בגוף האשה. לשם כך החלו לערוך ניסיונות זוועה על תאומים, שנודעו לימים כ"תאומי מנגלה". ניסיונות שנועדו, בין השאר, לפצח את סוד הזהות ביניהם.

הדנ"א, החומר התורשתי, זהה לחלוטין ברצף הבסיסים שלו בכל תא מתאי גופם של תאומים זהים (מונוזגוטיים), להבדיל מתאומים שאינם זהים (דיזיגוטיים), ומאחים שאינם תאומים. תאומים זהים נוצרים כאשר בשלבי החלוקה הראשונים של הביצית המופרית (זיגוטה) חלה הפרעה, שכתוצאה ממנה נבקע לשניים גוש התאים המתחלקים, המכונה תותית (בלאסטולה), וכל אחד מהם ממשיך להתפתח התפתחות עוברית מושלמת עד ליצירת תאומים זהים גנטית. להבדיל, אחים ותאומים שאינם זהים, נוצרים כתוצאה מהפריה כפולה, שבה שתי ביציות מופרות כל אחת על ידי תא זרע אחר. ההבדל בין אחים ובין תאומים שאינם זהים, מסתכם במועדי ההפריות. במקרה של תאומים ההפריה של שתי הביציות מתרחשת באותו מועד עקב ביוץ כפול, ובמקרה של אחים במועדים שונים. לכן, אחים ותאומים שאינם זהים גנטית שונים כיד המקרה זה מזה אף במינם בכמחצית מהמקרים. נמצאנו למדים, אם כן, שתאומים זהים אינם רק דומים זה לזה דמיון מלא אלא הם גם זהים גנטית. תהליך שבו נוצרת ישות גנטית אחת זהה לישות גנטית אחרת, מכונה שיבוט (מלשון שבט - Cloning)

הדמיון הגנטי בין תאומים זהים הוא כלי מחקר חשוב ויעיל לקביעת השפעתם של גורמים מורשים וגורמים סביבתיים שאינם מורשים, על תכונות שונות כגון משכל, קווי אופי וכן נטייה לחלות במחלות שונות, שאינן בעלות הורשה ברורה כמו סכרת, אסטמה, אלרגיות ועוד.

תאומים זהים שהופרדו ונמסרו לאימוץ לאחר לידתם, והיו חשופים להשפעות סביבתיות שונות במשך חייהם, מקור כל ההבדלים ביניהם בהשפעות הסביבה אליה נחשפו בעת גידולם,שכן, כאמור, הם זהים גנטית. ניתוח סטטיסטי של ההבדלים האלה מאפשר לקבוע את השפעת הרכיב ההורשתי יחסית לרכיב הסביבתי לגבי התכונות הנבדקות. כך, למשל, התגלה כי פוטנציאל המשכל (I.Q.) המרבי של בני אדם תלוי מעיקרו בגורמים גנטיים, אולם, המידה אליו יתפתח המשכל באדם מסוים תלויה רבות גם בגירויים סביבתיים, בעיקר בתקופת הילדות. כלומר המשכל המאפיין אנשים שונים תלוי הן בגורמים מורשים והן בגורמים סביבתיים. באופן דומה, התגלה כי הגובה המרבי אליו עשוי אדם לצמוח נקבע גנטית, אולם מימוש פוטנציאל הגדילה תלוי בטיב הזנתו בעיקר בתקופת הגדילה.

ממצא זה עשוי אולי להסביר את הבדלי הגובה בין ילדים בני ימינו להוריהם בני הדור הקודם, שכנראה גדלו על דיאטה מזינה פחות.

מדעניו של היטלר ניסו לפתח שיטה, שתאפשר לקחת תא מגופו של אדם אותו מעוניינים לשכפל גנטית, ולהפוך אותו בצורה מבוקרת לאדם חי ונושם, שיהיה זהה בתכונותיו המורשות לאדם ממנו נלקח התא המקורי, בדומה לתאומים זהים גנטית. למרבית המזל לא צלח הדבר בידיהם, והעלילה שנכתבה על ידי Iralezin Levin בספרו הדמיוני "The Boys from Brazil" (הנערים מברזיל) מעולם לא התרחשה. בתקופה שבה נערכו ניסיונות הזוועה האלה עדיין לא היה מוכר מבנה הדנ"א, ולכן גם לא הבסיס המולקולרי של התורשה, וזו אחת הסיבות לחוסר הצלחתם. יתר על כן, היו אז מדענים שפקפקו בהיותו של הדנ"א החומר התורשתי, שכן לטענתם חומר כה פשוט לא כשיר לשמש למטרה כה מורכבת כמו הורשה. היום מבנה הדנ"א מוכר, ומבנהו המולקולרי המאפשר הורשה, פוענח. ניסיונות הנעשים לשינוי מולקולות דנ"א והתכונות המורשות על ידיהן, מהווים את הענף המדעי המכונה "הנדסה גנטית".

לאחרונה, בספר רב המכר "פארק היורה" ובסרט שהופק בעקבותיו, הועלתה האפשרות כי בשיטות ההנדסה הגנטית יתורגם המידע הגנטי המוצפן במולקולות דנ"א של דינוזאורים, אשר נשתמרו כלואות מאז תקופת היורה (לפני 150 מיליון שנה) והתקופה הקריטונית (לפני 60 מיליון שנה) באיבר המציצה של חרקים עוקצים, שנלכדו בענבר (שרף עצים מאובן), לכדי דינוזאורים חיים. יש לאבחן אפשרות זו מן האפשרות של שיבוט יצורים חיים, שכן, להבדיל מתא חי שמתחלק ומתרבה, מולקולות דנ"א הן חומר כימי לא מורכב יחסית, והן ניתנות לייצור בתהליך פשוט מהחומרים המרכיבים אותן. לכן קשה לדמיין אינטואיטיבית כיצד ניתן במבחנה להביא דנ"א לכדי יצירת תא חי, כל שכן - יצור חי.

התרחיש המתואר בסרט "פארק היורה", אם כן, מסובך עוד יותר מהמתואר כאן בדבר שכפול גנטי של בני אדם, שכן על פיו צריך ליצור תא יש מאין, ורק לאחר מכן, באופן דומה לשכפול תא אדם כדי ליצור אדם שלם, להביא להפיכתו לדינוזאור חי.

על מנת ליצר דינוזאורים בשיטות ההנדסה הגנטית יהא עלינו: 1. לאתר דנ"א של דינוזאורים ולבודדו 2. לקבוע את רצפו 3. לסווג את הרצפים לכרומוזומים 4. ליצר כרומוזומים של דינוזאור 5. להחדיר את הכרומוזומים לתא חי 6. להביא להתפתחות התא לדינוזאור חי. בסעיפים הבאים אעמוד על האפשרויות והמגבלות הקיימות בדרך להשגת המטרה הזאת, והאם רצוי כלל להשיגה.

בעוד שמרקמה חיה ואף מתאים בודדים הגדלים במבחנה ניתן בנקל להפיק כמויות בלתי נדלות של דנ"א, יש שני מקורות בלבד לבידוד דנ"א מחיות שנכחדו. אלו הם עצמות מאובנות ותאי דם המכילים דנ"א, המצויים באיבר המציצה של חרקים, שנלכדו ונשתמרו בענבר.

עצמות הירך של בעלי-חוליות מכילות את בית החרושת העיקרי לייצור תאי דם. במח העצם מצויים תאי נבט (תאי מקור) המתחלקים ללא הרף, ומהם נוצרים תאי הדם החדשים, המצטרפים לזרם הדם ומחליפים תאי דם ישנים, שהושמדו בתהליך מבוקר שחל בטחול. מח העצם מוגן היטב על ידי רקמת העצם הקשה, רקמה לא תאית העשויה חלבונים וסידן, ומכאן קשיותה הרבה. הגנה זו מאפשרת את שמירתן של מולקולות הדנ"א העשויות להימצא בתוך עצמות מאובנות. כלל הוא בטבע שכל חומר אורגני (חומר שמייצרים יצורים חיים) סופו להתפרק ולהיות ממוחזר, שכן לולא היה כך, בסיומו של דבר היו האטומים המרכיבים חומרים אורגניים (בעיקר פחמן, חמצן, מימן, חנקן, וזרחן) מתרכזים באותו חומר לא ממוחזר, שהיה מצטבר על פני כדור הארץ בכמויות אדירות. קצב הפירוק של חומרים אורגניים שונים תלוי בסוג החומר המתפרק ובתנאי הסביבה. חיידקי הריקבון ממלאים את אחד הגורמים החשובים ביותר בפירוקם ובמחזורם של חומרים אורגניים. בפעילותם מזרזים החיידקים האלה את מחזורם של חומרים, המצויים בגוויותיהם של יצורים שמתו. פעילות מהירה זו יוצאת לפועל עקב ייצורם והפרשתם של אנזימים ייחודיים, שהם קטליזטורים ביולוגיים, וכל אחד מהם מזרז את פירוקו של חומר אורגני או של קבוצת חומרים אורגניים שונה. לפעילות זו, שתוצאותיה הן ריקבון, יש שימוש מעשי, למשל, יצירת "זבל אורגני" המפרה את האדמה בחומרים אורגניים ממוחזרים לצורכי חקלאות.

תהליכים נוספים המביאים לפירוקם ולמחזורם של חומרים אורגניים הם תהליכים כימיים שמטבעם, בלחץ ובטמפרטורה המאפיינים את כדור הארץ, איטיים הרבה יותר. למשל, תהליך פירוקם של חלבונים לחומצות אמיניות, שהן אבני הבניין המרכיבות אותם, עשוי לארוך כדי 24 שעות בתנאי לחץ, טמפרטורה וחומציות (PH) גבוהים, בעוד שאנזימים מהסוג המכונה פרוטאזות, שהם זרזי פירוק של חלבונים, ממלאים משימה זו תוך מספר דקות בטמפרטורה ולחץ רגילים ובדרגת חומציות ניטראלית.

חומרים אורגניים שונים, כאמור, נבדלים ביניהם בקצב הפירוק המאפיין אותם. קצב התלוי גם בתנאי הסביבה שבה הם מצויים. חיידקי הריקבון, כשלעצמם יצורים חיים, לא יכולים לפעול בסביבה יבשה או חמה מדיי, למשל קצב הפירוק הכימי תלוי אף הוא בתנאים סביבתיים כגון לחות. לכן, עשויים להתקיים תנאים, שבהם פירוקו של חומר אורגני מסוים יארך זמן רב. תנאים כאלה מתקיימים לעתים לגבי עצמות מאובנות. בעצמות האלה התפרק רוב החומר האורגני ונותר המבנה הלא אורגני של העצם, המורכב רובו מסידן. עם זאת, שרידי החומר האורגני נותרים בעצם גם לאחר זמן רב בתנאי שנמצאה בתנאי יובש מתאימים, שמנעו את פעילותם המואצת של החיידקים המפרקים את החומר האורגני, ובכלל זה את הדנ"א שמקורו בתאי מח העצם. העצמות העתיקות ביותר מהן הצליחו להפיק דנ"א הן בנות 20,000 שנה. כה, ניסיונות שנעשו להפיק דנ"א מעצמות עתיקות מאלה לא צלחו. פירושו, כי ככל הנראה, לא ניתן להפיק דנ"א מעצמות מאובנות של דינוזאורים. ואולם ייתכן ותימצאנה עצמות דינוזאור, שהשתמרו בצורה יוצאת דופן טובה עקב תנאים סביבתיים ייחודיים, המונעים פעילות חיידקית, למשל, עטופות זפת או קפואות. אז, אולי אפשר יהיה להפיק מהן דנ"א של דינוזאור.

חרקים עוקצים שמצצו דם דינוזאורים, ולאחר מכן נלכדו בשרף עצים, שהתאבן והפך ענבר, הם המקור האפשרי השני להפקת דנ"א של דינוזאור. השרף יצר סביבה מגנה כנגד תהליכי פירוק של חיידקים ותהליכי פירוק כימיים, והחומר האורגני הכלוא בתוכו נותר שמור היטב. יתרה מזאת, גם מבנה החרק נותר במקרים רבים מושלם עד שניתן לזהות את חלקיו השונים. בצורה זו הצליחו קאנו וחבריו מהאוניברסיטה הפוליטכנית של קליפורניה להפיק דנ"א מוזרק, שגילו הוערך ב-120-135 מיליון שנה, התקופה שבה שלטו הדינוזאורים ביבשות ובימים.

לשתי השיטות האפשריות להשגת דנ"א של דינוזאורים יתרונות וחסרונות. בשיטה הראשונה, על פיה מפיקים דנ"א מעצמות דינוזאור מאובנות, ניתן לדעת מה מינה המדויק של החיה ממנה מופק הדנ"א. ואולם החומר האורגני הוא דל מאוד עד כדי כך שייתכן שגישה זו אינה ניתנת ליישום כלל. באמצעות השיטה השנייה, שבה מפיקים דנ"א מחרקים לכודים בענבר, ניתן להשיג דנ"א באיכות יחסית טובה, אך אי אפשר לדעת את מי החיה אותה עקץ החרק לפני מותו, אם בכלל. כמו כן, ייתכן שבאיבר המציצה כלוא דם משתי חיות שונות, דבר שיקשה על הניסיונות שיתוארו להלן, שמטרתם להחזיר את הדינוזאורים לחיים. כפי הנראה, הפקת דנ"א של דינוזאורים מחרקים לכודים בענבר, השיטה המתוארת בסרט "פארק היורה", טובה יותר למרות חסרונותיה.

ב-1953 הציעו שני המדענים המבריקים ווטסון וקרייג מודל לתיאור מבנה הדנ"א, ששנים מועטות לאחר מכן הוכחה נכונותו. על הגילויים האלה זכו ווטסון, קרייג ווילקינס בפרס נובל בשנת 1962. הדרך שהובילה לפענוח מבנה הדנ"א, מתוארת בספרו המרתק של James Dewey Watson - "Double Helix" (הסליל הכפול). הדנ"א, אם כן, בנוי כסליל כפול, שבו כל גדיל הוא שרשרת בסיסים חנקניים (דאוקסינוקלאוטידים) הקשורים זה לזה בקשר כימי אופייני, המכונה קשר פוספודיאסטרי. בדנ"א קיימים ארבעה סוגים של בסיסים חנקניים: אדנין, ציטוזין, גואנין ותימין. כיווניהם של הגדילים הכרוכים אחד סביב השני במבנה סלילי, הפוכים זה לזה, וקשורים בקשרים אופייניים המכונים קשרי מימן. אלה נוצרים בין זוגות בסיסים המצויים זה מול זה, כל אחד על גדיל אחר, ותמיד בין ציטוזין (C) לגואנין (G), ובין אדנין (A) לתימין (T) לכן, מקביעת הרצף של אחד הגדילים של מולקולת דנ"א דו-גדילית ניתן להסיק את רצפו של הגדיל המשלים לו. קשרי המימן תורמים ליציבות המבנה הסלילי של מולקולת הדנ"א.

מבנה הדנ"א: הדנ"א בנוי מ-4 אבני בניין שהם בסיסים חנקניים המכונים: אדנין A (חום), תימיןT (כתום), גואנין G (אפור) וציטוזין C (אדום). אבני הבניין ערוכות בשני גדילים אנטי-מקבילים כפי שניתן ללמוד מכיווני החצים באיור. בין הבסיסים החנקניים האמונים על שני הגדילים קיימים קשרים כימיים אופייניים. קשרים אלה, המכונים קשרי מימן, נוצרים בין זוגות בסיסים המצויים זה מול זה, כל אחד על גדיל אחר. קשרי המימן נוצרים תמיד בין אדנין לתימין ובין גואנין לציטוזין, ולכן משניתן רצף הבסיסים של אחד הגדילים, לא נותרת אלא ברירת המחדל לרצפו של הגדיל השני, המשלים לו. היות ששני גדילי הדנ"א משלימים זה לזה ברצפם, תכונה זו משמשת כבסיס למנגנון הכפלת הדנ"א, המתואר בחלק התחתון של האיור, שבו נפרמת מולקולת הדנ"א המקורית לשני הגדילים המשלימים המרכיבים אותה, ובתהליך המזורז על ידי אנזימים ייחודיים, נבנה על פי רצפו של כל גדיל מקורי גדיל המשלים לו (כלומר, מול A תמיד יבוא T וכו') ברצף הבסיסים. תהליך זה מכונה הכפלה "שמרנית-למחצה", שכן בסופו מתקבלות שתי מולקולות דנ"א המכונות מולקולות בת. בכל אחת שני גדילים משלימים, האחד "ישן" והשני "חדש".
איירה: אנה נזננסקי

המבנה המרחבי של דנ"א: קשרי המימן (הקווים הדקים) הנוצרים בין זוגות בסיסים, האמונים על גדילים משלימים (אדום ואפור) במולקולת הדנ"א, מאפשרים את קיומו של המבנה הסלילי האופייני לה.
איירה: אנה נזננסקי

סדר הבסיסים הערוכים ברצף מאפיין את סך כל הדנ"א של יצור מסוים, והוא קבוע וזהה בכל תאי גופו של אותו יצור. ברצף הדנ"א מקודד (גלום) המידע לייצורם של כל החלבונים, ובכלל זה, האנזימים, אף הם מולקולות חלבון המנתבות, בין השאר, את ייצורם של שאר החומרים, המאפיינים את אותו יצור. לאמיתו של דבר, ניתן לקבוע כי רצף הדנ"א המאפיין כל יצור, ובכלל זה בן אנוש, מהווה את תעודת הזיהוי הטובה ביותר שלו. לכן קביעת רצפי דנ"א מדגימות ביולוגיות של דם, זרע, שערות, פיסות עור ועוד, שנותרו בזירת פשע, משמשת את חוקרי המשטרה לזיהוי ודאי של חשודים במעשה. יתר על כן, מאחר שרצפי דנ"א מורשים מהורים לילדיהם, ניתן על ידי השוואתם של רצפים מפרטים שונים לקבוע אם קיימים קשרי הורשה ביניהם. הליך המשמש רבות בבדיקות לקביעת אבהות אצל בני אדם, לשיוכם של בעלי חיים מיוחסים כגון כלבים וסוסים גזעיים, לבית הגידול בו נולדו, ולאחרונה גם לשיוך לווייתנים שגודלו בשבי, לקבוצת הלווייתנים שבה נולדו לשם שחרורם חזרה לחופשי.

במקביל לפענוח מבנה הדנ"א הוצע גם המנגנון המולקולרי, באמצעותו מתאפשרת הורשת התכונות. הדנ"א בנוי, כאמור, משני גדילים המשלימים זה את זה ברצף בסיסיהם. ניתוק הקשרים בין שני הגדילים הקיימים, ובניית גדילים משלימים לכל אחד מהם מכפילה את הדנ"א. הכפלה זו מכונה "שמרנית למחצה" (סמי-קונסרבטיבית), שכן, בשתי מולקולות הדנ"א החדשות, מקורו של גדיל אחד במולקולה הראשונית, ואילו הגדיל השני נבנה מחדש על פי הרצף של הראשון.

במהרה פוענח גם הקוד הגנטי. הסתבר כי כל שלישיית בסיסים סמוכים ברצף דנ"א המהווה גן, מגלמת מידע לשיבוצה של חומצה אמינית אחת בחלבון המקודד על ידי אותו גן. כך, באמצעות קביעת הרצף של הדנ"א ניתן לנבא את רצפו של החלבון המקודד על ידו, שכן, הרצף הקווי של הדנ"א ושל החלבון הם קו-לינאריים. לאחר שפוענח הקוד הגנטי, התפתחו גם טכניקות המאפשרות שינויי רצפי הדנ"א כך שיקודדו לחלבונים מעט שונים מהחלבונים הטבעיים.

התופעה הזאת קיימת גם בטבע ומכונה מוטציה. גידולים סרטניים במקרים רבים מאופיינים במוטציות החלות בגנים המקודדים לחלבונים, שתפקידם לפקח על חלוקת התא. כתוצאה ממוטציות החלבונים האלה חדלים למלא את תפקידם כראוי, וקצב החלוקה של התאים מתגבר עד כדי התפתחות גידול סרטני. מוטציות נוספות בגנים המקודדים חלבונים נוספים באותם תאים עלולות להוביל להתפתחות תת-אוכלוסייה של תאים, המתחלקים מהר עוד יותר מהתאים הראשונים, או אף לתאים בעלי כושר לנדוד ברחבי הגוף, ולייסד מושבות תאים או גידולים חדשים, המכונים גרורות סרטניות. במקרה האחרון הסרטן מוגדר כממאיר.

מוטציות נגרמות באופן טבעי עקב טעויות בהכפלת הדנ"א או בעת תיקונו. ישנם חומרים שונים המוכרים בשם כללי חומרים מסרטנים, הגורמים לריבוי טעויות בהכפלת דנ"א או בעת תיקונו, ולכן גורמים לתדירות גבוהה יותר של מוטציות בדנ"א של אנשים החשופים להשפעתם, ועלולים לגרום להתפתחות סרטן. רשימת החומרים המוגדרים כמסרטנים הולכת וגדלה משנה לשנה עם פיתוחם של החומרים חדשים, ועם צירופם לרשימה של חומרים ישנים, שהתגלה שהם מסרטנים. למשל, מספר חומרים המצויים בעשן סיגריות הם מסרטנים. כתוצאה מכך תדירות המקרים של סרטן ריאות בקרב אוכלוסיית המעשנים גדולה פי כמה מזו המאפיינת את אוכלוסיית הלא מעשנים.

אף על פי שרוב המוטציות הן הרסניות מטבען, ולאחר שהן חלות, החלבון המקודד על ידי הגן ששונה אינו פעיל ביולוגית, הרי לעתים נדירות מוטציות עשויות להיות חיוביות במובן שהן גורמות לשינוי בחלבון, ההופך אותו מותאם טוב יותר לתפקודו הפיסיולוגי. למשל, אם מדובר בגן המקודד חלבון בעל פעילות אנזימטית, מוטציה מסוימת עשויה לגרום לו לפעול מהר יותר. אם מהירות הפעילות של האנזים מהווה את הגורם המגביל פעילות פיסיולוגית כלשהי, הרי שפעילות מהירה יותר שלו, מגבירה את אותה פעילות פיסיולוגית, והופכת את החיה נושאת השינוי מותאמת יותר לסביבתה. חיה כזו תחיה זמן ארוך יותר ותהיה חיונית יותר, ועל כן תעמיד בממוצע מספר רב יותר של צאצאים יחסית לאחיותיה בעלות החלבון המקורי. צאצאים שירשו ממנה את התכונה החדשה, יהיו אף הם מותאמים לסביבתם טוב מן החיות המקוריות וצאצאיהן. כתוצאה מכך, לאחר תקופה שבה חל תהליך ברירה טבעית, יהיו כל החיות מן הסוג החדש, ואילו חיות מהסוג הישן ייכחדו. קצב התהליך תלוי, כמובן, ביתרון היחסי המוקנה לחיות הנושאות את המוטציה. קרי, תלוי במהות המוטציה ובלחץ הברירה הסביבתית. דרווין בספרו הידוע "מוצא המינים", תיאר את מהלך הדברים האלה לאחר שצפה בבעלי חיים באזורים שונים בעולם, למרות שלא ידע על קיומו של הדנ"א, כל שכן, על היותו החומר התורשתי. מוטציות או שינויים בדנ"א יוצרים, אם כן, מגוון שהינו כר הפעולה לברירה הטבעית או לקיומה של אבולוציה.

מאז ימי קדם ועד ימינו אנו משמש המגוון הגנטי הטבעי לתועלתו של המין האנושי. על ידי קיומם של זיווגים נבחרים, המבוססים על בחירה של פרטים בעלי תכונה מועדפת מהאוכלוסייה הכללית, ניתן לשפר את התכונה באוכלוסייה כולה. למשל, גידולים הגדלים מהר יותר, לגודל רב יותר, ועמידים מאחרים בפני פגעי מזג האוויר, בפני מחלות ובפני מזיקים, פותחו בשיטות של ברירה מלאכותית, ככל הנראה, עוד בתקופה הפרהיסטורית. כתוצאה מכך, זני השעורים המשמשים להכנת קמח, שונים לטובה מבחינות רבות מזני שעורת הבר. יתר על כן, למעשה כל עולם החי והצומח המכונה "מתורבת", מכלבים וחתולים ועד עגבניות ופלפלים, הוא תוצאה של זיווגים בין פרטים נבחרים, שמטרתם להשביח תכונות שהוגדרו על ידי האדם כרצויות.

בימינו, לאחר פיתוח שיטות ההנדסה הגנטית, יכול המדען לשנות רצפי דנ"א כרצונו, ולשלוט על טיבם וסוגם של החלבונים המיוצרים מאותו דנ"א באותו יצור, ולכן למעשה, על כל תכונותיו המורשות. טכנולוגיה זו ניתנת ליישום הן לצרכים מועילים, למשל, ריפוין של מחלות גנטיות תורשתיות, והן למטרות שנויות במחלוקת מוסרית ואתית, למשל, ייצור אנשים חכמים יותר, יפים יותר ועוד, כיד הדמיון.
בבעלי חיים רב תאיים הדנ"א מצוי בגרעין התא שהוא מבנה תוך תאי מוקף קרום, המבודדות משאר נפח התא. הדנ"א מאורגן במבנים שניתנים לצפייה במיקרוסקופ לאחר צביעה מתאימה, ולכן מכונים כרומוזומים (כרומו=צבע). מספר הכרומוזומים המאפיין מין מסוים הוא קבוע. תאי אדם למשל, מאופיינים ב-46 כרומוזומים. הכרומוזומים השונים נבדלים זה מזה בגודלם ובצורתם, ולכן ניתנים לזיהוי ולאפיון. בבעלי חיים רב-תאיים, להבדיל מחד-תאיים כגון חיידקים, כל סוג כרומוזום קיים בכל תא בשני עותקים המכונים זוג כרומוזומים. לפיכך, תאיהם של בני אדם, מאופיינים ב-23 זוגות כרומוזומים. כל כרומוזום מורכב ממולקולת דנ"א יחידה (שני גדילים) וחלבונים ייחודיים, המכונים חלבוני היסטון, המאפשרים את ארגון הדנ"א במבנה הכרומוזומלי. מספר הבסיסים החנקניים בכל אחד מגדיליה של מולקולת דנ"א הכלולה בכל כרומוזום עשוי לנוע בין עשרה למאה מיליון. אצל האדם, מספרם הממוצע של הגנים השונים המקודדים על ידי כל כרומוזום הוא כ-5000. מספרם של הבסיסים החנקניים המרכיבים את הגנום כולו (תכולת הדנ"א בכל תא מתאי יצור מסוים) של בעלי חוליות מוערך בארבעה מיליארד זוגות בסיסים, והם מקודדים לכ-100,000 גנים. בכרומוזומים הבודדים ניתן לצפות רק כאשר התא מצוי בשלב של טרום חלוקה. בשלב הזה, כהכנה לשלב החלוקה עצמו, מתכווצים הכרומוזומים ליצירת המבנה הכרומוזומלי המאפיין אותם בצפייה במיקרוסקופ.

בשנת 1976 הציג סנגר שיטה נוחה לקביעת רצפי דנ"א, ועל פיתוחה זכה יחד עם מקסם וגילברט בפרס נובל ב-1980. היה זה פרס הנובל השני, שבו זכה סנגר, בפרס הראשון זכה על פיתוח שיטה לקביעת רצפם של חלבונים. אוטומטיזציה של השיטה הזאת עשתה אותה יעילה, מדויקת ושימושית ביותר לקביעת רצפי דנ"א. בתנאים המרביים מכשיר קביעת רצף יחיד, שעלותו כ-100,000 דולר, עשוי לקבוע רצפם של כ-25,000 בסיסים חנקניים ביממה. עם זאת, חישוב פשוט מראה כי על מנת לקבוע את רצפו של גנום שלם של חולייתן מסוים באמצעות מכשיר יחיד, תהיינה דרושות כ-440 שנים.

לפני מספר שנים הוחל בפרויקט בינלאומי, שמטרתו למפות את הגנים ולקבוע את רצף הגנום של האדם. פירושו, חלוקת הגנים לכרומוזומים, קביעת מיקומם היחסי של הגנים בתוך כל כרומוזום, קביעת רצפם של הגנים, וקביעת רצפו של כל הגנום כולל רצפים שאינם מקודדים, קרי, אינם מהווים גנים. לפרויקט הזה השלכות מדעיות ורפואיות ממדרגה ראשונה, בין השאר, כיוון שלאחר השלמתו יתאפשר זיהויים, ובהמשך אולי תיקונם של הגורמים הגנטיים האחראיים לקיומן של כ-4,000 המחלות התורשתיות המוכרות באדם. השלמתו של הפרויקט, שבו מעורבות מאות מעבדות מחקר מכל האולם צפויה להסתיים בעוד כ-20 שנים. תקציבו הנוכחי של הפרויקט 10 מיליארד דולר שעיקרם מכספי משלם המסים האמריקני. הגישה המנחה את פרויקט מיפוי הגנים וקביעת הרצף של גנום האדם, מבוססת על קיומן של שיטות גנטיות ופיסיקליות לקביעת מיקומו של רצף דנ"א נבחן על גבי הכרומוזומים. כך, תוך שימוש בשיטות האלה, לאחר שנקבע הרצף של מקטע דנ"א מסוים הוא ממוקם יחסית למקטעי דנ"א נוספים, שרצפם מוכר זה מכבר והאמונים על אותו כרומוזום.

ואולם, השיטות האלה לא תצלחנה ליישום בפרויקט שייעודו יצירת כרומוזומי דינוזאור, שכן הן מותנות באנאליזה גנטית של משפחות לדורותיהן בשיטה הראשונה, ובאנאליזה פיסיקלית של כרומוזומים שלמים כשהם בשלב של טרום חלוקת התא וניתנים לצפייה במיקרוסקופ, בשיטה השנייה. ברור שלא ניתן יהיה למצוא משפחות של דינוזאורים, שכן הם נכחדו מעל פני כדור הארץ לפני מיליוני שנים. כמו כן, קשה להאמין כי ניתן יהיה למצוא כרומוזומים שלמים ששמרו על צורתם. אף לא בתאי דם מאיבר המציצה של חרקים עוקצים, שנכלאו ונשתמרו בענבר.

בשיטות המקובלות להפקת דנ"א מתקבלות מולקולות הדנ"א שבורות לחלקים הקטנים במספר סדרי גודל מכרומוזום שלם. כל שכן, בטרם הפקתו דנ"א עתיק הוא מקוטע ושבור מלכתחילה. על מנת ליצור כרומוזומי דינוזאור, ראשית יש לקבוע את רצף המקטעים ולארגן אותם בכרומוזומים, כפי שהיו מסודרים מלכתחילה. מהאמור לעיל, עולה כי הגישה באמצעותה ניתן יהיה לקבוע את רצפם המלא של כרומוזומי דינוזאור תהיה קביעת רצף של מקטעים בודדים בתקווה שתהיה חפיפת רצפים בין מקטעים שמקורם מתאים שונים, כך שניתן יהיה לחפוף את קצותיהם על מנת לקבוע את סידורם אחד יחסית לשני, ועל ידי כך את רצפם של הכרומוזומים השלמים.

לאחר שייקבעו רצפיהם של כל הכרומוזומים, יש ליצרם. שתי גישות הניתנות לשילוב ביניהן עשויות לצלוח לשם כך. בגישה אחת מחברים את המקטעים ששימשו לקביעת הרצף, זה לזה. לגישה הזאת חיסרון מסוים כיוון שעל החיבור להיות מדויק ביותר, ואל לו לכלול מקטעים חופפים, לחילופין חסרים, שכן הדבר עלול לגרום לביטוי לא מדויק של הגנים, ולאי הצלחת התהליך הסופי בכללותו. יש לזכור שיש לחבר לפחות 10,000 מקטעים שונים כדי לשחזר כרומוזום יחיד, וכי על כל החיבורים, ללא יוצא מן הכלל, להיות מדויקים עד כדי בסיס חנקני יחיד. על פי הגישה השנייה מייצרים את הכרומוזומים בצורה כימית מלאכותית. לשם כך פותחה שיטה כימית ופותחו מכשירים אוטומטיים ליישומה, המסוגלים ליצר מולקולות דנ"א מנגזרות של הבסיסים החנקניים המרכבים אותן. כיום המכשירים האלה יקרים ביותר ויעילות פעולתם נמוכה. כל מכשיר כשיר ליצור גדיל דנ"א יחיד בן כ-100 בסיסים ביממה. חישוב פשוט מראה כי על מנת ליצור בצורה רציפה כרומוזום באורך עשרה מיליון זוגות בסיסים, נדרשות כ-2,200 שנים.

נמצאנו למדים, אם כן, כי לפני שנוכל להיכנס למשימת "החייאתם" של הדינוזאורים, יש לשפר את היכולות העומדות לרשותנו לקביעת רצפים וייצורם, ולפתח גישות חדשות כדי להתמודד עם משימה בסדר גודל המתואר.

בתום השלב הראשון של יצירת הכרומוזומים יש בידינו מולקולות דנ"א ארוכות, כל מולקולה כוללת את רצפיו של כרומוזום שלם. כעת, נרצה "לעטוף" את הכרומוזומים בקרום גרעין ובתא. יתר על כן, נרצה שפעילויותיו של התא שניצור, ינותבו על ידי המידע הדינוזאורי המקודד על ידי הכרומוזומים שיצרנו. ברשות המדע עדיין לא קיים המידע לגבי תכולתו המדויקת של תא. כלומר, עדיין לא מוכרים כל החלבונים ושאר החומרים המרכיבים תא, וכן לא מוכרת דרך לארגונם במסגרת תאית. לכן לא נוכל להשיג את המטרה על ידי ערבובם של החומרים המתאימים במבחנה. עם זאת, ניתן לדון באפשרות של החלפת סט הכרומוזומים של תא קיים בכרומוזומים המלאכותיים שיצרנו.

ניסיונות ראשוניים בכיוון הזה נעשו על ידי גורדון מאוניברסיטת קיימברידג באנגליה, שזכה על כך בפרס קרן וולף הישראלית. גורדון התעניין בתהליכי התמיינות של תאים: תהליכים המביאים להבשלתו או להתבגרותו של תא, במהלכם הוא רוכש את תכונותיו האופייניות. התהליכים האלה הם בלתי הפיכים משלב מסוים ופירושו שתא ממוין, למשל, תא עור בוגר, סופו מיתה. בניסיונותיו ביקש גורדון לבדוק האם תהליך ההתמיינות בלתי הפיך עקב שינויים בלתי הפיכים בגנום. לשם כך נטל ביצית (תא מין נקבי) של צפרדע ירוקה והרס את גרעינה, המכיל את הגנום המקורי, על ידי חשיפתה לקרינה באורך גל אולטרא סגול. התקבל תא ביצית שתפקידו המקורי לתמוך ולהזין את העובר, נטול גנום משל עצמו. משול הדבר לבית חרושת (על מכונות הייצור שלו) נטול תוכניות עבודה. לאחר מכן החדיר גורדון לתוך הביצית נטולת הגנום המקורי, גרעין שבודד מתא ממוין שמקורו באפיתל המעי ונלקח מצפרדע מזן אלבינו (לבנה), ובדק האם חלה התפתחות עוברית. התוצאות היו מפתיעות. ברוב המקרים לא היתה כלל התפתחות עוברית, אך במספר מועט של מקרים התפתחו הביציות לעוברי צפרדע מזן אלבינו. בניסיונות האלה הודגם כי לפחות לגבי דו-חיים, גנום של תא ממוין עשוי להוביל להתפתחות עוברית מחודשת, ולהתמיינות כל סוגי התאים המרכיבים עובר. ניסיונות מסוג דומה שנעשו בחולייתנים אחרים, למשל, יונקים, לא צלחו, ועדיין לא ברור אם סיבת הכישלון טמונה בהבדל בסיסי בין גרעיני דו-חיים ליונקים או בתנאי ניסיון לא מתאימים. כמו כן, בניסיונות שבהם ניסו להביא להתמיינות מחודשת של תאים ממוינים לכדי יצור שלם מבלי להעביר את גרעינם לסביבה, התומכת בהתפתחות עוברית (ביצית), עדיין לא צלחו בבעלי חיים. להבדיל, בצמחים ניתן בקלות רבה יחסית, בתנאים לא מורכבים, לגרום לתאים ממוינים (למשל, תא שורש יחיד של גזר, או תא קליפה יחיד של תפוח אדמה), להפוך לירק הכולל את כל מאפייניו של הירק השלם ואף כשיר לרבייה.

ביצית (1) של צפרדע ירוקה (2) מבודדת מאחיותיה (3) המורחקות בניתוח (4) מהצפרדע, מוקרנת בקרינת אור אולטרא-סגול (5) על מנת להרוס את גרעינה המקורי (6). גרעין (7) של תא אפיתל (8) שנלקח מהמעי (9) של צפרדע מזן אלבינו (10) מוחדר אל הביצית המוקרנת (11) אשר ממנה מתפתחת צפרדע מזן אלבינו (12).
בניסיונות אלה הודגם כי לפחות לגבי דו-חיים, גנום של תא ממוין עשוי להוביל להתפתחות עוברית מחודשת, ולהתמיינות כל סוגי התאים המרכיבים עובר. איירה: הדס מרכוס.

מכל מקום, המשימה הניצבת בפנינו מורכבת הרבה יותר. הדנ"א המלאכותי שיצרנו, ובו גלום המידע להחייאתם של הדינוזאורים, אינו מאורגן במבנה גרעין אותו ניתן להחדיר לתא נטול גרעין, באופן דומה לניסיונותיו של גורדון. משול הדבר לקיומן של תוכניות ייצור בלי מפעל שייצר על פיהן. לכן, גישה מעשית יותר תנסה להחליף כרומוזומים של תא קיים בכרומוזומים המלאכותיים. ייתכן שניתן יהיה לעשות זאת בהדרגה.

מאז בידוד הגן הראשון וזיהויו, הגן המקודד עבור בטה-גלובין, לפני למעלה משני עשורים, חיפשו המדענים אחר גישות שתאפשרנה ההחדרה וביטוי של רצפי דנ"א נבחרים בתאים חיים. במהלך השנים פותחו גישות יעילות לביצוע המשימה הזאת. כיום ניתן להחדיר גן נבחר לתאים הגדלים במבחנה, ולגרום להם ליצר את החלבון עבורו הוא מקודד. בדרך זו מיוצרים כיום בצורה תעשייתית חלבונים רבים כגון אינטרפרון ואינסולין המשמשים לצורכי הרפואה. יתר על כן, בידי המדע האמצעים להחדיר גנים ליצורים חיים ולשפרם לצורכי האדם. החיות האלה מכונות טרנסגניות. למשל, בפקולטה לחקלאות ברחובות הצליחו להחדיר גן המקנה עמידות לגידולי שדה שונים נגד מזיקים, או להחדיר גן אינסולין מאדם לעזים, המפרישות את חלבון האינסולין לחלב, חלב אותו ישתו חולי סכרת, התלויים באינסולין כדי לאזן את משק הסוכר בגופם.

לאחרונה, במרכז הבריאות הלאומי בארצות הברית (NIH) הוחדר הגן המקודד לאנזים אדנין-דה-אמינאז, לתאי דם לבנים, שנלקחו מילדה הסובלת ממחלת כשל חיסוני מולד כתוצאה מפגם גנטי בגן האמור, שהורש לה מהוריה. כתוצאה מהפגם הגנטי, תאי הדם הלבנים של הילדה לא כשירים ליצור נוגדנים, והיא חשופה להידבקויות חוזרות ונשנות במחלות זיהומיות, וצפויה לתוחלת חיים נמוכה כדי עשר שנים בלבד. תאי הדם הלבנים שהונדסו גנטית, הוחזרו למערכת הדם של הילדה, והם מייצרים נוגדנים המאפשרים לה להתמודד עם זיהומים. עד כה הילדה בריאה, ונראה שנרפאה ממחלתה. הניסוי שנערך רק לאחרונה, נעשה עד היום בשני חולים בלבד, ולכן עדיין לא ניתן לאמוד את טיבן של ההשלכות, ולבחון תוצאות ארוכות טווח של טיפולים מעין אלה.

בגישות הקיימות להחדרת דנ"א לתאים או ליצורים חיים מופעלת ברירה על מנת שתהליך החדרה יצא אל הפועל ביעילות. ברירה זו כוללת החדרה של רצף דנ"א או גן נוסף, המחובר לרצף הדנ"א המקודד לחלבון הרצוי, והמקנה עמידות בפני חומרים שרעילים לתאים. למשל, גן המקודד לחלבון, המקנה לתאים עמידות נגד האנטיביוטיקה נאומיצין. על ידי גידול התאים המטופלים במצע מזון המכיל נאומיצין, ניתן בנקל לאתר את התאים שקלטו את הדנ"א הזר ומבטאים אותו, שכן כל התאים שלא מבטאים את המידע הגלום בדנ"א המוחדר, ימותו.

בדרך דומה ניתן יהיה לכלול בכל רצף דנ"א של כרומוזום של דינוזאור גן המקנה עמידות בפני אנטיביוטיקה אחרת, ובהדרגתיות להחדיר את מולקולות הדנ"א אחת אחרי השנייה לתאים הגדלים במבחנה. בכל שלב שבו מוסף עוד כרומוזום, יש להוסיף למצע המזון של התאים אנטיביוטיקה מהסוג עבורה מוקנית עמידות על ידי הכרומוזום הנוסף. בצורה זו, לאחר מספר מחזורים כמניין הכרומוזומים הדרוש, יתקבל תא המכיל את כל גנום הדינוזאור. ההנחה היא כי תא כזה יאבד בהמשך את כל הכרומוזומים המקוריים, שהיו בו לפני תחילת מחזורי ההחדרה. להנחה זו עיגון מדעי, שכן בניסיונות שבהם גורמים לאיחוי גרעינים של שני תאים שונים לכדי גרעין יחיד עם תכולה כפולה של כרומוזומים, התאים נוטים לאבד במשך הזמן כרומוזומים שאמונים היו במקור על אחד התאים. במקרה שלפנינו קיומה של הברירה יאפשר חיות רק לתאים שיאבדו את כרומוזומיהם המקוריים אך לא את אלה החדשים, האחראיים להקניית העמידויות. במידה שגנום הדינוזאור גם יבטא את החלבונים המקודדים בו, למעשה, נקבל בתום התהליך תא של דינוזאור.

לעניין זה יש לציין כי ניסיונות דומים להעביר דנ"א לתוך תא זר מעולם לא בוצעו, ולא ברור כלל אם הם אפשריים. להבדיל מהשלבים הקודמים, שבהם הבעיה העיקרית היא של סדרי גודל גרידא, הבעייתיות בשלב זה עשויה להיות מהותית. עם זה, אין כרגע נתונים לקבוע כי היא בלתי אפשרית. גם בספר "פארק היורה" וגם בסרט התעלמו במכוון מן הנקודה הזאת, ופסחו על תיאור השלב הזה בייצור דינוזאורים.

לטכנולוגיה המאפשרת מניפולציה כזו בכרומוזומים עשויות להיות השלכות מרחיקות לכת. שליטה בטכנולוגיה זו תאפשר התערבות בסדרי בראשית. ייצור תאי מין מאופיין בחלוקת הפחתה שלאחריה בכל תא מין קיים רק כרומוזום יחיד מכל זוג כרומוזומים, המאפיינים את שאר תאי הגוף. פירושו, בתאי מין מחצית ממספר הכרומוזומים המאפיין תאים אחרים. לבני אדם 23 כרומוזומים יחידאיים, במקום 23 זוגות. מנגנון זה התפתח במהלך האבולוציה על מנת שלא יוכפל מספר הכרומוזומים ועמו כמות הדנ"א בכל דור, שכן עובר הנוצר ברבייה המינית המאפיינת את בעלי החיים העילאיים מתחיל מאיחוים של שני תא מין, זכרי ונקבי. מפענוח מנגנון חלוקת ההפחתה עולה כי במהלכה מתקבלים מיליוני צירופים שונים של כרומוזומים (2 בחזקת 23) שכן התהליך בו מתחלקים זוגות הכרומוזומים לתאי המין, אקראי. לאקראיות זו חשיבות אבולוציונית רבה שכן בעטיה גדלה הרבגוניות הגנטית של פרטים באוכלוסייה. תהליך אקראי נוסף המכונה שיחלוף (רקומבינציה), שלא כאן המקום לתארו, מגדיל את המגוון כדי עשרות מונים נוספים. קיומו של המגוון מבטיח כי חלק מן הפרטים האמונים על מין מסוים, עשויים להתאים טוב יותר לחיים בתנאים מסוימים מאשר פרטים אחרים. לכן, בעיקר בסביבה שבה השינויים בתנאי הסביבה תכופים וקיצוניים, מתגלה חשיבות רבה לרבגוניות, המאפשרת המשכת קיומו של המין, שכן תמיד יימצאו פרטים המותאמים דיים על מנת לשרוד בתנאים הסביבתיים החדשים שנוצרו. גם חלוקות הפחתה ליצירת תאי מין בבני אדם הן אקראיות, וכן אקראית היא פגישת תא המין הזכרי עם הנקבי. לכן, פרט לתכונות המאפיינות את המין כולו (למשל, לכל תינוק שתי אוזניים), ותכונות שניתן לזהות כי לגביהן לא קיימת שונות אצל ההורים (למשל, לשניהם עיניים כחולות), לעולם אין לדעת מה תהיינה תכונותיו של תינוק טרם ללידתו.

הטכנולוגיה החדישה שתוארה תאפשר לאחר השלמת פרויקט המיפוי וקביעת רצף גנום האדם, לבחור מקבצי כרומוזומים רצויים ולהחליפם בכרומוזומים לא רצויים בבני אדם בצורה מושכלת, ויש לכך השלכות מרחיקות לכת. מחד גיסא ניתן יהיה להחליף כרומוזומים, הנושאים גנים הגורמים למחלות גנטיות, בכרומוזומים הנושאים גנים שאינם פגומים. מאידך גיסא, ניתן יהיה לתכנן אנשים בעלי תכונות כאלה ולא אחרות, המתאימים טוב יותר לצרכים מסוימים ולא לאחרים, זאת על פי רצון ההורים, או אף גרוע מכך, על פי רצון השלטונות. מטרה דומה בחוסר מוסריותה ניסו להשיג מדעניו של היטלר, לשמחתנו ללא הצלחה.

במידה שיצליחו הניסיונות המתוארים להחלפת כרומוזומים הרי שהתאים שיתקבלו יכילו גנום של דינוזאור, יבטאו חלבוני דינוזאור, ולמעשה על פי כל הגדרה יהיו תאי דינוזאור. כעת משבידינו תאי דינוזאור הגדלים במבחנה, עלינו להביאם לידי התפתחות לחיה שלמה. אציע שתי חלופות אפשריות ליישום משימה מורכבת זו. בשני המקרים תלויות תוצאות הניסיונות בהנחה המוקדמת, שהגנום הדינוזאורי המאכלס את התאים אותם יצרנו, כשיר ליצירתם של כל תאי הדינוזאור הבוגר, על סוגיהם השונים, בדומה לגנום תא אפיתל המעי של הצפרדע בניסיונותיו המתוארים של גורדון.

על מנת לגרום להתפתחות דינוזאור מתא יחיד ניתן לנסות לבצע ניסיונות דומים לאלה שביצע גורדון. ואולם, יש לשים לב כי הביצית שתשמש כמאגר המזון וההשריה להתפתחות העוברית מן צד האחד, והגרעין המועבר אליה, מן הצד האחר, לא ייתכן שיהיו אמונים על אותו מין, שכן, כדי שתהיה בידינו ביצית דינוזאור עלינו ראשית ליצור דינוזאור שייצר אותה. עולה, אם כן, השאלה המעגלית המעניינת: מה קדם למה, הביצה או התרנגולת?

כאשר אנו שוקלים באיזו ביצית להשתמש, עלינו לקחת בחשבון את גורלו של עובר הדינוזאור. הביצית הגדולה ביותר של בעל החיים החי בימינו היא ביצת היען. ביצית זו היא בחירה טובה לא רק משום גודלה. לאחרונה הגיעו החוקרים למסקנה כי דינוזאורים היו בעלי דם חם, ולכן דומים יותר מבחינת מוצאם למחלקת העופות על אף דמיונם החיצוני הרב לזוחלים בעלי דם קר, החיים בימינו. לכן ביצית עוף כגון ביצת היען, כפי הנראה, מתאימה ביותר למטרה. עלינו, אם כך, להוציא מביצית בת-היענה את גרעינה, המכיל דנ"א של יען, ולהחדיר אליה גרעין מאחד מתאי הדינוזאור שיצרנו בשלב הקודם. לאחר מכן נדגיר את ביצת היען בתנאים מתאימים בתקווה שלבסוף יבקע ממנה דינוזאור חי. ייתכן וביצת היען לא מכילה את כל החומרים הדרושים לתמוך בהתפתחות עוברית של דינוזאור, או לחילופין, מכילה חומרים העלולים לעכב התפתחות כזו. לכן, אפשר שלפני השלמת התהליך נאלץ ליצר יענים טראנסגניים אליהם יוחדרו גנים ייחודיים של דינוזאור, שביטוים משפיע על תכולת הביצים אותן מטילה בת-היענה. בצורה זו ניצור ביצי יען המתאימות יותר לתמיכה בהתפתחות עוברית של דינוזאור.

קיימת אפשרות נוספת לגרום לתאי הדינוזאור שייצרנו, להפוך לחיה שלמה. כבר כיום נעשים ניסיונות בהם נלקחים תאי חיה נבחרת, ומשנים אותם גנטית על ידי החדרה של רצפי דנ"א או הוצאתם מהם, והשתלתם לאחר מכן בתוך עובר בשלביו המוקדמים. תאים כאלה עשויים להשתלב בכל אחת מרקמות החיה הבוגרת, בכלל זה גם בבלוטות הרבייה (גונדות), האחראיות על ייצור תאי המין; ביציות המיוצרות בשחלה אצל הנקבה, וזרעים המיוצרים באשכים אצל הזכר. לאחר זיווגם, זכרים ונקבות כאלה מביאים לעולם צאצאים שמקורם באותם תאי מין, אשר כל תאי גופם מכילים את השינוי הגנטי שהוצג מלכתחילה. בדרך דומה, ייתכן שניתן יהיה להשתיל בתוך עוברי יען את תאי הדינוזאור על מנת ליצור יצור בן-כלאיים (היבריד), שחלק מתאי גופו תאי דינוזאור הם, וחלקם האחר תאי יען. הפריה בין שני יצורים כאלה, בהנחה שתאי המין אותם הם מייצרים מקורם בתאי הדינוזאור המושתלים, תוביל להטלת ביצה ממנה יתפתח דינוזאור.

בעמודים הקודמים תוארה האפשרות להחזיר לחיים את הדינוזאורים, ששלטו עד לפני שישים מיליון שנה במרחבי כדור הארץ, בתקופה שבה היונקים, עליהם נמנה גם המין האנושי, היו עדיין פרימיטיביים ומאוד לא מפותחים. לא ברור מה תהיה השפעת חזרתם של היצורים הענקיים האלה לחיים היום. ידוע כי גנומים של יצורים מיצורים שונים מכילים רצפי דנ"א, המקודדים לווירוסים מסוגים שונים. מי יודע מה תהא השפעתם של הוירוסים הכלואים בגנום הדינוזאורים, שאולי נכחדו עמם לפני מיליוני שנים, על בעלי החיים ובני האדם החיים היום. כמו כן לא ברור אם תוכל המערכת החיסונית של הדינוזאורים להתמודד עם מגוון גורמי המחלות (וירוסים, חיידקים, טפילים ועוד) שהתפתחו בהם במהלך מיליוני השנים שנעדרו מכדור הארץ.

פיתוח הטכנולוגיה העומדת בבסיסו של "פארק היורה" תאפשר באופן דומה להחזיר לחיים בני אדם שהלכו לעולמם ואת שיבוטם, הכפלתם והנצחתם של בני אדם, שחיים עמנו כיום, ואת יצירתם של אנשים בעלי תכונות מוזמנות מראש. האם האנושות רוצה בכך? האם עליה לרצות בכך? כל אלה שאלות אתיות ממדרגה ראשונה, שעליהן יש להשיב לפני שניגשים לביצועם של ניסיונות מהסוג הזה. לדעתי אין האנושות רוצה בכך, ואף אין היא צריכה לרצות בכך. עם זאת, לטכנולוגיה המוצעת, כמו גם לטכנולוגיות רבות אחרות, שימושים מועילים רבים לאדם, למשל,בתחום הרפואי ובתחום המחקר הטהור, וייתכן גם בתחומים נוספים, עליהם אין לנו מושג כעת. לעניין הזה ראוי להביא כדוגמה מתחום הפיסיקה את פיתוח טכנולוגית האטום. כשבשלהי מלחמת העולם השנייה פותחה השליטה על אנרגית האטום על ידי אופנהיימר וחבריו, לא העלו המדענים על דעתם את תוצאותיה ההרסניות של השימוש בה כפצצה. החיים בצל איום גרעיני נוראיים, אך עם זאת, לאנרגיית האטום שימושים אזרחיים מועילים ביותר בתחום ייצור החשמל ואספקת אנרגיה. השימוש הזה יהפוך חיוני ביותר, אולי ללא תחליף, עם הידלדלותם של מרבצי הנפט והפחם העולמיים, הצפויים בעוד כמה עשרות שנים. על המדענים, אם כך, לפתח את הטכנולוגיות המתקדמות ביותר בכל תחומי המחקר ובכלל זה גם המחקר הביולוגי. בכך ממלאים המדענים את שליחותם וחובתם לחברה. על החברה כולה, ובעיקר על המנהיגים העומדים בראשה, מוטלת החובה והאחריות לדאוג כי יפותחו מנגנונים לשליטה על הטכנולוגיות האלה. במעשי החברה תלויה, אם כך, שאלת המפתח אודות ניצול הטכנולוגיות לטובת המין האנושי או לרעתו.

* * *

Universal Inheritable Barcodes for Identifying Organisms
Jonathan Gressel and Gal Ehrlich
Trends in Plant Science 2002, 7:542-544

Abstract

The needs for recognition of novel conventional or transgenic organisms include protection of patented or Identity Preserved lines, detecting transgenics and tracing dispersal. We propose simple 'Biobarcodes' using universal PCR primers to recognize the universal 'nonsense' recognition site of all biobarcodes, followed by a variable nonsense sequence. The proposed sequences are long enough to allow recognition in spite of mutations, have stop codons to prevent coding, and will not self anneal. Sequences of PCR-amplified biobarcodes can be compared to a universal database.

These are a variety of needs for devising simpler recognition methods for organisms marketed in commerce or released in the environment; whether they are conventionally selected, mutant or transgenic bacteria, fungi, plants or animals. The needs include: (1) the need for protection for patented or other IP lines, where IP takes on the designation of either 'Intellectual Property' or 'Identity Preserved'. It is often hard to prove that a line has been 'miss-appropriated' by a competitor or illegally grown. (2) Labeling regulatory authorities and various consumer groups are demanding labeling of transgenic commodities. This is accomplished by segregating plants or their products and externally following them throughout their commercial life. Internal markers are an adjunct to such external markings. (3) The need to trace organisms in the environment. The use of biocontrol agents to control weed, bacterial, fungal or insect pests and the use of other live organisms as inoculants is increasing. Knowledge about their dispersal in plants and other organisms as well as in the environment is necessary, irrespective of whether the organisms are indigenous or transgenic. Many of the agents are closely related to known pathogens or pests and there are claims that an organism can change its host range and attack valuable species (with consequences of liability). There are also fears that organisms will introgress into other organisms, and there are needs to ascertain whether the new organism changed host range or whether an epidemic was the result of wild strains [1]. These issues will become more acute with transgenically enhanced biocontrol agents [2]. This can still be a problem even if failsafe mechanisms are transformed into biocontrol agents along with hypervirulence genes [3]. Labeling biocontrol agents with selectable markers (such as gus, gfp or antibiotic resistance) has been used for following the movement of biocontrol agents in the environment [4,5] , but will not differentiate between them if the same few markers are continually used. RFLP has been used to follow and differentiate between one organism and closely related strains of the same pathogen [6].

There has been a considerable expenditure on identifying valuable organisms – transgenic and non-transgenic. Seed companies use AFLP and other molecular techniques to ascertain whether competitors have incorporated their genetic material, and the seed industry uses it to ascertain whether farmers have been storing and reusing transgenic seed off license. Consumer advocacy groups and regulators have been using similar techniques to ascertain whether transgenic products have been mixed with non-transgenic organisms. PCR amplification of the DNA in question, using a series of primers for typically used promoters, terminators, marker genes and the genes of importance, is often used to find trace amounts of transgenes in crops and commodities [7–11] . Some processed products might need tens of PCR reactions because of the mixing of crops and the numbers of possible transgenes that they might contain. This problem will be exacerbated as more transgenic crops and organisms reach the market. The multiple sampling and PCR reactions are time consuming and fraught with problems. For example, the commonly used 35S promoter can be found in almost any non-transgenic commodity containing DNA from a cole (Brassica) crop because a proportion of such crops is invariably infected by the ubiquitous cauliflower mosaic virus, the source of that promoter. The PCR results must be further verified by using either restriction endonucleases, Southern blotting or direct sequencing, or using nested PCR or quantitative competitive PCR reactions. Highly processed foods have their DNA fragmented to <400 bp. Detection can be hampered by a lack of availability of DNA reference material and of sequence information [7–11].

Immunodiagnostic methods have been developed for many of the gene products. Again, each sample would have to be reacted with a series of antibodies when there is a possibility of many different gene products. Even one product can be hard to detect when a single mutation is introduced into a commonly occurring plant gene, such as those encoding many herbicide resistances. It was easier to develop an immunoassay to detect bacterial 5-enolpyruvylshikimate-phosphate synthase encoding resistance to the herbicide glyphosate that could distinguish it from the susceptible plant enzyme [12]. Even then, a triple sandwich technique was required.

Even when transgenics are discovered by such procedures or 'kits', there is no information as to source. Thus, regulatory authorities might wish to consider simple, common recognition sequences for detecting transgenic or other organisms in the market place. It is depressing to contemplate how much is being invested in detecting transgenic DNA in commodities (and no one has died from eating it) against how little is being expended towards detecting commonly occurring 'natural' bacterial contamination and mycotoxins in foodstuffs (which have negatively affected many lives). We propose a new recognition system, which, if imposed, would require far less expenditure in detecting DNA, allowing resources to be used for detecting truly dangerous contaminants in foodstuffs.

Barcodes and biobarcodes

The barcode system was developed to identify individuals rapidly, whether articles in a store, automobiles or test tubes. A simple barcode reader first identifies that there is a barcode to be read, and in what orientation, by seeing a pattern of bars that is otherwise too rare to be expected randomly. It does so by recognizing a universal sequence of spaced bars in an orientation-dependent manner that appear as universal recognition sites on either end of the barcode ( Fig. 1a). In between the universal recognition sites is a variable series of bars of differing width and spacing. This assigned variable region is read by the reader into a computer and the database then identifies the object as being someone's car, candy bar or blood sample.

Fig. 1.
(a) A standard barcode with universal recognition sites at either end defining orientation, and a variable sequence of bars of various widths and spacing that can be accessed from a database. (b) A schematic representation of a Biobarcode with fixed universal nonsense DNA sequences defining universal recognition sites at either end defining orientation, and a unique defined variable nonsense DNA sequence that can be accessed from a database containing a stop codon (purple) to prevent frameshifts from defining peptide coding. A special assigned sequence can be added for those groups that are interested in having further definition of typed products. Abbreviations: L, left; R, right.

An analogous DNA-encoded system with universal recognition sites binding a variable region is proposed akin to the barcode system. This biobarcode system would enable material to be assayed in one PCR-sequencing run using universal primers that identify all 'biobarcoded' biological materials. In this system, open codes are to be designed and assigned by a single repository. They begin and end with the common universal 'barcode analogous' orientation-dependent nucleotide sequences that are recognized by the pair of universal recognition PCR primers, which, under PCR conditions, duplicate the whole intervening sequence ( Fig. 1b). The assigned code is transformed into the target organism, as part of a transgenic construct, or it can be transformed directly into an otherwise non-transgenic organism.

The universal 'bar code recognition' nonsense (non-coding) nucleotide sequence is designed to be long enough that a few mutational changes will still allow it to be recognized by a PCR primer set. The initial universal recognition code is followed by a designed nonsense (non-coding) nucleotide sequence that is long enough to allow tens of millions of different such sequences to be generated, and again allow for some mutational changes. Neither the initial common recognition sequence, nor the particular individual strain sequence, should even vaguely resemble nonsense sequences reported in any sequence database. Frame-shift mutations should not render any part of the barcode sequence as an open reading frame long enough to allow significant polypeptides to be made. Stop codons are thus inserted into the assigned sequences in all reading frames to prevent a frame-shift mutation from becoming a genetic coding sequence.

Some people might not care whether there is any biobarcode in an organism, just whether their propriety DNA is found in it. Such people could have a special sequence pair assigned to them that would follow the universal recognition sequence in a biobarcode. Thus, they could use a primer that recognizes this sequence, and start the PCR reaction from there. Others wishing to know whose and what DNA is in the same organism or sample can probe with the universal primer pair.

A considerable amount of computational power with appropriate algorithms is needed to generate the common universal code sequence and the following variable individual strain sequence. The algorithms used to generate the sequences are being designed to exclude sequences that could self anneal, preventing the taq polymerase from amplifying the biobarcoded DNA. The biobarcode can be inserted in tandem with the genes of choice for transformation, or it can be co-transformed with the gene of choice in cases where the organism is transformed with a 'sense' transformation. In other cases, an excisable selectable marker will be needed.

If a product contains more than one biobarcode (i.e. a foodstuff concocted from different transgenic crops bearing different transgenes), there will be more than one band on the PCR gel to be sequenced. The sequenced bands are then compared to the public database. This is a positive method, with results from all biobarcode-labeled species, versus the guessing at what transgenes might be present, as occurs using the present technologies.

Regulation

It is envisaged that a single body would assign the biobarcodes and maintain a public database listing the biobarcode sequences of organisms that have been released. It is clearly advantageous to industry, regulators and taxpayers that such a system be instituted because of the amount of resources saved and the protection provided. Because of the savings, it is in the public interest that such a system be universally instituted.

In Canada, for example, the insertion of a biobarcode would probably not come under regulatory scrutiny if biobarcodes are introduced into plants because they are not considered to be 'plants with novel traits' if they are non-protein producing. In Europe, any introduced sequence (except antisense) seems to come under regulatory scrutiny, but after due risk assessment it is hoped that a blanket approval could be obtained for biobarcodes that meet the specific criteria listed above (and possibly others).

Acknowledgements

Useful discussions with Eithan Rubin of the Weizmann Bioinformatics Group are gratefully acknowledged. This research is supported in part by the Levin Fund. J.G. is the Gilbert de Botton Professor of Plant Sciences.

References

[1] Gressel J. (2002) Molecular Biology of Weed Control. : Taylor & Francis

[2] Vurro M., et al. (Ed) (2001) Enhancing Biocontrol Agents and Handling Risks. Amsterdam: IOS Press

[3] Gressel J. (2001) Potential failsafe mechanisms against the spread and introgression of transgenic hypervirulent biocontrol fungi.
Trends Biotechnol., 19:149-154.

[4] Eparvier A. and Alabouvette C. (1994) Use of ELISA and GUS transformed strains to study competition between pathogenic and nonpathogenic Fusarium oxysporum for root colonization.
Biocontrol Sci. Technol., 4:35-47.

[5] Cohen B. et al. (2002) Transgenically enhanced expression of indole-3-acetic acid (IAA) confers hypervirulence to plant pathogens.
Phytopathology, 92:590-596.

[6] Hintz W.E. et al. (2001) Development of genetic markers for risk assessment of biological control agents.
Can. J. Plant Pathol., 23:13-18.

[7] Meyer R. (1999) Development and application of DNA analytical methods for the detection of GMOs in food.
Food Control, 10:391-399.

[8] Huebner P. et al. (1999) Quantitative competitive PCR for the detection of genetically modified organisms in food.
Food Control, 10:353-358.

[9] Wurz A. et al. (1999) Quantitative analysis of genetically modified organisms (GMO) in processed food by PCR-based methods.
Food Control, 10:385-389.

[10] Lipp M. et al. (2001) Validation of a method based on polymerase chain reaction for the detection of genetically modified organisms in various processed foodstuffs.
Eur. Food Res. Technol., 212:497-504.

[11] Anklam E. et al. (2002) Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant derived food products.
Eur. Food Res. Technol., 214:3-26.

[12] Rogan G.J. et al. (1999) Immunodiagnostic methods for detection of 5-enolpyruvylshikimate-phosphate synthase in Roundup-Ready soybeans.
Food Control, 10:407-414.

Articles and Publications:

ICLG Publication

Molecular mechanisms underlying endocytosis and sorting ERbB recptor tryosine kinasis Hasassa Waterman, Patent Attorney, Ehrlich & Fenster, 2001

A mutant EGF-receptor defective in ubiquitylation and endocytosis unveils a role for Grb2 in negative signaling Hadassa Waterman,Patent Attorney, Ehrlich & Fenster 2001

The C-terminus of the kinase-defective neuregulin receptor ErbB-3 confers mitogenic superiority and dictates endocytic routing. Hadassa Waterman, Patent Attorney, Ehrlich & Fenster, 2001

c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor Hadassa Waterman, Patent Attorney, Ehrlich & Fenster, 2001

Pathogenic poxviruses reveal viral strategies to exploit the ErbB signaling network. Hadassa Waterman, Patent Attorney, Ehrlich & Fenster, 1998

Differential endocytic routing of homo- and hetero-dimeric ErbB tyrosine kinases confers signaling superiority to receptor heterodimers. Hadassa Waterman, Patent Attorney, Ehrlich & Fenster, 1998

A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Hadassa Waterman, Patent Attorney, Ehrlich & Fenster, 1996

Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. Hadassa Waterman, Patent Attorney, Ehrlich & Fenster,1996

Structure of acetylcholinesterase complexed with E2020 ( Aricept ^®): implications for the disign of the new anti-Alzheimer drugs Gitay Kryger, Patent Attorney, Ehrlich & Fenster, 1996

על הבקשה הזמנית: אליה וקוץ בה רועי מלצר, עו“ד ועורך פטנטים, ארליך אנד פנסטר, 2006

הגלובליזציה בתחום סימני המסחר יואל שטיבל, עו“ד, ארליך אנד פנסטר, 2006

A REALISTIC, OBJECTIVE TEST FOR THE BEST MODE REQUIREMENT Benjamin J. Barish Advocate, Patent Attorney, Ehrlich & Fenster

Genesis and Time
Rabbi, Dr. Yosef Freedland, Surgeon, Patent Attorney, Ehrlich & Fenster

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The Legal Protection of Broadcast Signals Lucie Guibault and Roy Melzer

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Abstract