Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization.

Vascular endothelial growth factor (VEGF) is important in pathological neovascularization, which is a key component of diseases such as the wet form of age-related macular degeneration, proliferative diabetic retinopathy and cancer. One of the most potent naturally occurring VEGF binders is VEGF receptor Flt-1. We have generated two novel chimeric VEGF-binding molecules, sFLT01 and sFLT02, which consist of the second immunoglobulin (IgG)-like domain of Flt-1 fused either to a human IgG1 Fc or solely to the CH3 domain of IgG1 Fc through a polyglycine linker 9Gly. In vitro analysis showed that these novel molecules are high-affinity VEGF binders. We have demonstrated that adeno-associated virus serotype 2 (AAV2)-mediated intravitreal gene delivery of sFLT01 efficiently inhibits angiogenesis in the mouse oxygen-induced retinopathy model. There were no histological observations of toxicity upon persistent ocular expression of sFLT01 for up to 12 months following intravitreal AAV2-based delivery in the rodent eye. Our data suggest that AAV2-mediated intravitreal gene delivery of our novel molecules may be a safe and effective treatment for retinal neovascularization.

A family of related proteins is encoded by the major Drosophila heat shock gene family.

At least four proteins of 70,000 to 75,000 molecular weight (70-75K) were synthesized from mRNA which hybridized with a cloned heat shock gene previously shown to be localized to the 87A and 87C heat shock puff sites. These in vitro-synthesized proteins were indistinguishable from in vivo-synthesized heat shock-induced proteins when analyzed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of the pattern of this group of proteins synthesized in vivo during a 5-min pulse or during continuous labeling indicates that the 72-75K proteins are probably not kinetic precursors to the major 70K heat shock protein. Partial digestion products generated with V8 protease indicated that the 70-75K heat shock proteins are closely related, but that there are clear differences between them. The partial digestion patterns obtained from heat shock proteins from the Kc cell line and from the Oregon R strain of Drosophila melanogaster are very similar. Genetic analysis of the patterns of 70-75K heat shock protein synthesis indicated that the genes encoding at least two of the three 72-75K heat shock proteins are located outside of the major 87A and 87C puff sites.

Family of developmentally regulated, maternally expressed Drosophila RNA species detected by a v-myc probe.

Drosophila melanogaster genomic sequences that hybridize with v-myc have been reported (B.-Z. Shilo and R. A. Weinberg, Proc. Natl. Acad. Sci. U.S.A. 78:6789-6792, 1981). We have detected Drosophila RNA sequences that also hybridize with v-myc. In an attempt to characterize these RNA sequences, we used v-myc hybridization probes to isolate Drosophila genomic segments. None of the Drosophila genomic or cDNA clones that we have isolated hybridize with the 3' exon of v-myc. Preliminary nucleotide sequence analyses have revealed sufficient homology to account for the observed hybridization between v-myc and the Drosophila clones but have failed to detect significant amino acid sequence homology. Thus is seems unlikely that the mRNA sequences or the genomic sequences that we have isolated by hybridization with v-myc represent homologs of the vertebrate myc gene. Despite the lack of structural homology between the cloned Drosophila sequences and v-myc, we have investigated the pattern of expression of those RNA species that hybridize with v-myc. Polyadenylic acid-containing transcripts of 2.7, 2.2, and 1.7 kilobases (kb) in embryos, pupae, adults, and Kc cells and an additional 1.4-kb transcript in adults were complementary to the Drosophila genomic clones and to v-myc. The 1.7- and 2.2-kb transcripts were localized on polyribosomes in Kc cells. The 1.7- and 2.2-kb transcripts were present after 45 min, 2 h, and 4 h of embryonic development, but by 16 h of development their levels had decreased by more than sixfold. During metamorphosis, two peaks of expression of the 1.7- and 2.2-kb transcripts were observed, at 6 and 72 h postpupariation. The 1.4-kb RNA species was first detected at 72 h postpupariation. In adults, the 1.7- and 2.2-kb transcripts were detected only in ovaries in females, whereas the 1.4-kb transcript was present in female nonovarian RNA and in males. These results suggest that the transcripts in early embryos are of maternal origin.

Expression of a Drosophila melanogaster acetylcholine receptor-related gene in the central nervous system.

We isolated Drosophila melanogaster genomic sequences with nucleotide and amino acid sequence homology to subunits of vertebrate acetylcholine receptor by hybridization with a Torpedo acetylcholine receptor subunit cDNA probe. Five introns are present in the portion of the Drosophila gene encoding the unprocessed protein and are positionally conserved relative to the human acetylcholine receptor alpha-subunit gene. The Drosophila genomic clone hybridized to salivary gland polytene chromosome 3L within region 64B and was termed AChR64B. A 3-kilobase poly(A)-containing transcript complementary to the AChR64B clone was readily detectable by RNA blot hybridizations during midembryogenesis, during metamorphosis, and in newly enclosed adults. AChR64B transcripts were localized to the cellular regions of the central nervous system during embryonic, larval, pupal, and adult stages of development. During metamorphosis, a temporal relationship between the morphogenesis of the optic lobe and expression of AChR64B transcripts was observed.

Tissue-specific expression of human CD4 in transgenic mice.

The gene for the human CD4 glycoprotein, which serves as the receptor for human immunodeficiency virus type 1, along with approximately 23 kb of sequence upstream of the translational start site, was cloned. The ability of 5' flanking sequences to direct tissue-specific expression was tested in cell culture and in transgenic mice. A 5' flanking region of 6 kb was able to direct transcription of the CD4 gene in NIH 3T3 cells but did not result in detectable expression in the murine T-cell line EL4 or in four lines of transgenic mice. A larger 5' flanking region of approximately 23 kb directed high-level CD4 transcription in the murine T-cell line EL4 and in three independent lines of transgenic mice. Human CD4 expression in all tissues analyzed was tightly correlated with murine CD4 expression; the highest levels of human CD4 RNA expression were found in the thymus and spleen, with relatively low levels detected in other tissues. Expression of human CD4 protein in peripheral blood mononuclear cells was examined by flow cytometry in these transgenic animals and found to be restricted to the murine CD4+ subset of lymphocytes. Human CD4 protein, detected with an anti-human CD4 monoclonal antibody, was present on the surface of 45 to 50% of the peripheral blood mononuclear cells from all transgenic lines.

Modification of an adenoviral vector with biologically selected peptides: a novel strategy for gene delivery to cells of choice.

Recombinant adenoviruses are currently being used as vectors for gene delivery to a wide variety of cells and tissues. Although generally efficacious for gene transfer in vitro, improvement in the efficiency of vector delivery in vivo may aid several gene therapy applications. One major obstacle is the lack of high-affinity viral receptors on the surface of certain cells that are targets for gene therapy. In principle, incorporation of avid, cell-specific ligands into the virion could markedly improve vector entry into the desired tissues. We have developed a strategy for addressing this issue in the lung by biopanning differentiated, ciliated airway epithelial cells against a phage display library. The peptide with the most effective binding was coupled to the surface of an adenovirus using bifunctional polyethylene glycol (PEG) molecules. The chemically modified adenoviral vector was able to effect gene transfer to well-differentiated human airway epithelial cells by an alternative pathway dependent on the incorporated peptide. Coupling of PEG to the surface of the virus also served to partially protect the virus from neutralizing antibodies in vitro. These experiments will aid in the design of improved adenoviral vectors with the capacity for more specific and efficient delivery of therapeutic genes to desired target tissues. We have used a novel method for enhancing gene delivery to target cells by coupling a biologically selected peptide to the surface of an adenovirus with bifunctional PEG molecules. Modification of the viral capsid by the addition of a peptide with binding preference for differentiated ciliated airway epithelia allowed gene delivery to those cells by a novel entry pathway. Incorporation of the CFTR gene in a similarly modified vector resulted in correction of defective Cl- transport in well-differentiated epithelial cultures established from human cystic fibrosis (CF) donors. The presence of PEG molecules on the surface of the virus served, in addition, to reduce antibody neutralization. Modification of adenoviruses with PEG/peptide complexes can serve to partially overcome the barrier of inefficient gene transfer in some cell types and some of the adverse immunological responses associated with gene delivery by these vectors.

Safety of airway gene transfer with Ad2/CFTR2: aerosol administration in the nonhuman primate.

In this study, the safety and efficacy of aerosol delivery to non-human primates of an adenoviral vector encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) were evaluated. The technique of concurrent flow spirometry was used to determine the deposited dose of Ad2/CFTR-2, which ranged from 3 to 8 x 10(10) I.U. Transgene DNA was detected by the polymerase chain reaction (PCR) in lung tissue from all treated animals, and human CFTR mRNA was detected on days 3, 7, and 21 post-exposure. The treatment was well tolerated, with no evidence of respiratory distress. Histologic changes in the lungs from Ad2/CFTR-2-treated animals were mild and, overall, indistinguishable from animals exposed to aerosolized vehicle. One vector-treated animal demonstrated an increase in lavage lymphocyte numbers 3 days after treatment and another had an abnormal chest radiograph 14 days after treatment. A third vector-treated animal had histologic evidence of a bronchointerstitial pneumonia 7 days after aerosol treatment that resolved by day 21. This study demonstrated that Ad2/CFTR-2 can effectively be delivered to the lungs of nonhuman primates and result in minimal adverse effects.

PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo.

Replication-defective recombinant adenovirus (Ad) vectors are under development for a wide variety of gene therapy indications. A potential limiting factor associated with virus gene therapy requiring repeated treatment is the development of a humoral immune response to the vector by the host. In animal models, there is a dose-dependent rise in neutralizing antibodies after primary vector administration, which can preclude effective repeat administration. The strategy we have developed to circumvent the neutralization of adenovirus vectors by antibodies is to mask their surface by covalent attachment of the polymer polyethylene glycol (PEG). Covalent attachment of PEG to the surface of the adenovirus was achieved primarily by using activated PEG tresylmonomethoxypolyethylene glycol (TMPEG), which reacts preferentially with the epsilon-amino terminal of lysine residues. We show that the components of the capsid that elicit a neutralizing immune response, i.e., hexon, fiber, and penton base, are also the main targets for PEGylation. Several protocols for PEGylation of an adenovirus vector were evaluated with respect to retention of virus infectivity and masking from antibody neutralization. We show that covalent attachment of polymer to the surface of the adenovirus can be achieved with retention of infectivity. We show further that PEG-modified adenovirus can be protected from antibody neutralization in the lungs of mice with high antibody titers to adenovirus, suggesting that PEGylation will improve the ability to administer Ad vectors on a repeated basis.

Characterization of an adenovirus gene transfer vector containing an E4 deletion.

We describe the construction and characterization of an adenovirus type 2 vector, Ad2E4ORF6, which has been modified in the E4 region to contain only open reading frame 6. When assayed in cultured cells, Ad2E4ORF6 virus replication is slightly delayed but viral DNA synthesis, host-cell protein synthesis shut-off, and virus yield are indistinguishable from wild type. Late protein synthesis is normal with the exception of fiber synthesis, which is reduced approximately 10-fold. Despite the reduced fiber synthesis, Ad2E4ORF6 viral particles appear to contain a full complement of fiber protein. Virus replication in cotton rats indicates that Ad2E4ORF6 is replication defective in vivo. This may have safety implications for the development adenovirus vectors in that virus arising by recombination in the E1 region of an Ad2E4ORF6-based vector would be defective for growth in vivo. The deletion of E4 open reading frames that are not required for virus growth in vitro increases the cloning capacity of adenovirus vectors by 1.9 kb and may be generally useful for the construction of adenovirus vectors containing large cDNA inserts and/or regulatory elements. We describe the inclusion of the A2E4ORF6 modification in a recombinant adenovirus vector, Ad2/CFTR-2, for gene transfer of the human cystic fibrosis transmembrane regulator (CFTR).

Characterization of factors involved in modulating persistence of transgene expression from recombinant adenovirus in the mouse lung.

One potential limitation of adenovirus (Ad)-based vectors for the gene therapy of cystic fibrosis (CF) and other genetic diseases is the transience of expression observed in most in vivo systems. In this study, the influence of various factors on persistence of transgene expression in the lung was investigated. In the absence of immune pressure, such as in the nude mouse, the genomic structure of the vector was found to be predominant in determining the persistence of expression; Ad vector constructs with an E1-E3+E4ORF6+ backbone encoding beta-galactosidase (beta-Gal) or the cystic fibrosis transmembrane conductance regulator (CFTR) produced declining levels of expression while an Ad/CMV beta Gal vector with an E1-E3+E4+ backbone gave rise to sustained, long-term reporter gene expression. The ability of the latter vector to persist was in turn limited in part by the presence of cytotoxic T lymphocytes (CTLs). Adoptive transfer experiments indicated that CTLs directed against either viral proteins or the beta-Gal reporter gene product were able to reduce expression in nude C57BL/6 mice stably expressing beta-Gal from the E4+ vector. Finally, the specificity and strength of the CTL response elicited by Ad vector was found to vary considerably depending on mouse strain haplotype. These results indicate that persistence of transgene expression in a given system is determined by the interplay between several factors including genomic structure of the vector, host background, and immune response.

Increased contact time improves adenovirus-mediated CFTR gene transfer to nasal epithelium of CF mice.

Multiple dosing with recombinant adenoviral vectors containing the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the nasal mucosa of cystic fibrosis (CF) transgenic mice reportedly results in only partial correction of the CF defect in chloride (Cl-) secretion without normalizing sodium (Na+) hyperabsorption, perhaps indicating inefficient gene transfer into the nasal airway epithelium in vivo. In this study, we have examined whether optimizing vector administration such as contact time could improve gene transfer efficiency. Changes in basal nasal potential difference (PD), and in PD (delta PD) following addition of amiloride and subsequent removal of Cl- from the luminal perfusate were assayed. As reported previously, the basal nasal PD was significantly more negative in CF mice (-24.9 +/- 2.1 mV) than in normal mice (-6.3 +/- 1.2 mV). Normal mouse nasal mucosa exhibited a large hyperpolarization in response to low Cl- substitution (delta PD of 8.5 +/- 1.9 mV), whereas the nasal mucosa of the CF mouse depolarized in response to this treatment. No correction of either the Cl- or Na+ transport defects were observed when 5 x 10(9) IU of Ad2/CFTR-5 were administered to the nasal passage of CF mice over a period of 5-20 min. However, when CF mice were perfused over a period of 60 min with the same dose of vector, a significant response (delta PD of 5.9 +/- 1.1 mV) to low Cl- substitution was detected 2 days later. In these mice, the basal nasal PD (-10.5 +/- 1.4 mV) and the response to amiloride were also reduced, indicating a partial correction of the Na+ transport defect. Expression of functional CFTR activity was transient with no measurable delta PD signals observed by day 7 post-treatment. These results suggest that prolonging the contact between an adenoviral vector and the respiratory epithelium enhances the efficiency of gene transfer and can result in improved correction of the CF Na+ and Cl- ion transport defects. Therefore, strategies that improve internalization of viral vectors and that prolong their contact time with target cells may result in the improved clinical efficacy of such vectors.

Potentiation of gene transfer to the mouse lung by complexes of adenovirus vector and polycations improves therapeutic potential.

Recombinant adenovirus (Ad) vectors are being considered for in vivo delivery of various therapeutic genes. One limiting factor in the development of Ad-based gene therapy is the low efficiency of gene transfer to target tissues such as vascular endothelium, smooth muscle, and airway epithelium. Complexing Ad vector with various polycations has been shown to enhance transduction of cell lines otherwise resistant to Ad infection in vitro. On the basis of this observation, the activity of Ad/polycation complexes was tested in vivo in the mouse lung. The results indicated that several polycations were capable of enhancing transduction of mouse respiratory epithelium, leading to a 1-2 log increase in levels of transgene expression. Poly-L-lysine (PLL) and DEAE-dextran were examined further and were found to increase Ad-mediated gene transfer without any additional toxicity as assessed histologically or through the measurement of inflammatory cytokines in bronchoalveolar lavages. The two polycations also failed to affect the humoral response against Ad vector and were themselves nonimmunogenic under conditions leading to enhanced gene transfer. Moreover, the ability to use reduced doses of vector complexed with polycations resulted in lower levels of Ad-specific antibodies and, thereby, improved readministration of vector. These results suggest that complexing Ad vectors with polycations has the potential to improve the therapeutic index by increasing transgene expression while reducing unwanted responses associated with high doses of vector.

Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. I. Methods, safety, and clinical implications.

Cystic fibrosis (CF), an autosomal recessive disorder resulting from mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene, is the most common lethal genetic illness in the Caucasian population. Gene transfer to airway epithelium, using adenoviruses containing normal CFTR cDNA, leads to transient production of CFTR mRNA and, in some studies, to correction of the airway epithelial ion transport defect caused by dysfunctional CFTR. Inflammatory responses to the adenoviral vector have been reported, particularly at high viral titers. We evaluated the effects of adenovirus-mediated CFTR gene transfer to airway epithelium in 36 subjects with CF (34 individuals, 2 of whom received two separate doses of vector), 20 by lobar instillation and 16 by aerosol administration. Doses ranged from 8 x 10(6) to 2.5 x 10(10) infective units (IU), in 0.5-log increments. After lobar administration of low doses there were occasional reports of cough, low-grade temperature, and myalgias. At the highest lobar dose (2.5 x 10(9) IU) two of three patients had transient myalgias, fever, and increased sputum production with obvious infiltrates on CT scan. After aerosol administration there were no significant systemic symptoms until the 2.5 x 10(10) IU dose, when both patients experienced myalgias and fever that resolved within 24 hr. There were no infiltrates seen on chest CT scans in any of the patients in the aerosol administration group. There were no consistent changes in pulmonary function tests or any significant rise in serum IgG or neutralizing antibodies in patients from either group. Serum, sputum, and nasal cytokines, measured before and after vector administration, showed no correlation with adenoviral dose. Gene transfer to lung cells was inefficient and expression was transient. Cells infected with the vector included mononuclear inflammatory cells as well as cuboidal and columnar epithelial cells. In summary, we found no consistent immune response, no evidence of viral shedding, and no consistent change in pulmonary function in response to adenovirus-mediated CFTR gene transfer. At higher doses there was a mild, nonspecific inflammatory response, as evidenced by fevers and myalgias. Overall, vector administration was tolerated but transfer of CFTR cDNA was inefficient and transgene expression was transient for the doses and method of administration used here.

Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. II. Transfection efficiency in airway epithelium.

A phase I clinical trial was conducted in which recombinant adenovirus containing the cystic fibrosis trans-membrane regulator (CFTR) (Ad2/CFTR) was administered by bronchoscopic instillation or aerosolization to the lungs of cystic fibrosis (CF) patients. In this paper, we evaluate the efficiency of Ad2/CFTR-mediated transduction of bronchial airway cells. The ability of an Ad2/CFTR vector to transduce airway cells was first evaluated in patients to whom the vector was administered by bronchoscopic instillation. Cells at the administration site were collected 2 days after treatment by bronchoscopic brushing. Ad2-specific CFTR DNA was detected in four of five individuals by PCR, and Ad2-specific CFTR RNA was detected in three of five individuals by RT-PCR. Ad2/CFTR-mediated transduction of airway epithelial cells was then determined in CF individuals receiving this vector by aerosol inhalation. Ad2-specific CFTR DNA was detected in 13 of 13 individuals 2 days after aerosolization, and in 3 of 5 individuals 7 days after aerosolization. Ad2-specific RNA was detected in 4 of 13 individuals on day 2, but was not detected in the 5 individuals tested on day 7. The percentage of airway epithelial cells containing nuclear-localized vector DNA was < or =2.4% as determined by fluorescence in situ hybridization (FISH). However, in some cases, a high percentage of nonepithelial mononuclear cells or squamous metaplastic epithelial cells was infected with the adenoviral vector. In conclusion, aerosol administration is a feasible means to distribute adenoviral vectors throughout the conducting airways, but improvements in adenovirus-mediated transduction of airway epithelial cells are necessary before gene therapy for CF will be effective.

Protein-tyrosine kinase activity of alternate protein products of the Drosophila Dsrc28C locus.

The Dsrc28C gene is a unique member of the extensive tyrosine kinase family. Two proteins, p66Dsrc28C and p55Dsrc28C, are encoded by the gene. Each contains a highly conserved tyrosine kinase domain and each lacks the usual amino-terminal myristylation signal. The protein-tyrosine kinase activity of the two proteins was investigated through a recombinant baculovirus expression system. p66Dsrc28C expressed from a recombinant baculovirus phosphorylated a large number of Sf9 cell proteins on tyrosine. A group of proteins of approximately 100 kDa were the preferred substrates. No evidence of p66Dsrc28C autophosphorylation was found. In contrast to p66Dsrc28C, p55Dsrc28C did not exhibit protein-tyrosine kinase activity when expressed from a recombinant baculovirus. A deletion derivative of p66Dsrc28C lacking the SH3 and SH2 domains also failed to phosphorylate Sf9 cell proteins. These results suggest that the protein-tyrosine kinase activity of Dsrc28C proteins is tightly regulated.

Expression of Drosophila epidermal growth factor receptor homologue in mitotic cell populations.

Expression of the Drosophila homologue (DER) of the human epidermal growth factor (EGF) receptor has been studied during development by RNA blot hybridizations and in situ hybridizations. One of the hypothetical functions of the protein encoded by this gene is mitotic signal transduction and, therefore, we have searched for evidence of its expression in mitotic cell populations. Increased DER transcript levels were detected in virtually all cells in cellular blastoderm embryos, indicative of the onset of transcription of the gene at this stage of development. These transcripts persist at least until the formation of the ventral furrow at the beginning of gastrulation. Expression of DER has been investigated in populations of nondividing cells, imaginal cells and nervous tissue in third instar larvae. By RNA blot hybridizations, we have shown that imaginal discs contain the bulk of the DER transcripts present in the whole third instar larvae. This result has been confirmed by in situ hybridizations. All imaginal discs, representing dividing cells, contain DER transcripts while salivary glands, representing nondividing cells, do not. Within the central nervous system, hybridization of the DER probe is confined to small clusters of cells in the brain cortex. During oogenesis, cell division programs are highly ordered temporally and spatially. Follicle cells express high levels of DER RNA during their mitotic phase of growth but lose these transcripts as they cease division. Nurse cells also express DER transcripts at lower levels even though they are not dividing. However, nurse cell transcripts represent stored maternal RNA species destined for use during preblastoderm embryonic development. These results indicate that the DER gene is expressed in mitotic cell populations during several stages of development and is not expressed in certain populations of nonmitotic cells.

Embryonic expression of a Drosophila src gene: alternate forms of the protein are expressed in segmental stripes and in the nervous system.

Expression of the Drosophila src-related gene, Dsrc28C has been investigated at the protein level using monoclonal antibodies. This analysis has revealed that the Dsrc28C gene encodes two protein forms: a 66-kD doublet predicted from the sequence of a cDNA clone and an additional 55-kD form. The 66-kD protein doublet is observed first at the cellular blastoderm stage and is not detectable in embryos after 12 hr of development. Expression of the 55-kD protein lags behind that of the 66-kD doublet and then persists throughout embryogenesis. Indirect immunofluorescence microscopy reveals that Dsrc28C protein is localized to the cell periphery during cellular blastoderm and gastrulation. The cell periphery-associated staining is then resolved into ectodermal stripes along the fully extended germ band. After the stripes fade, cytoplasmic staining of the majority of cells within the central and peripheral nervous system is observed. The 66-kD protein was shown to represent the cell periphery-associated form of the protein through antibody staining of larval salivary glands expressing a heat shock promoter-driven, full-length Dsrc28C cDNA. Staining of embryos with a monoclonal antibody specific for the 66-kD protein indicates that the 55-kD protein is the nervous system form. Thus, the 66- and 55-kD proteins are products of the Dsrc28C gene, which exhibit different temporal and spatial patterns of expression in the embryo.

A Drosophila genomic sequence with homology to human epidermal growth factor receptor.

Vertebrate genomes contain an extensive family of genes possessing varying degrees of homology to the v-src oncogene. Most src-related proteins identified to date are intracellular and membrane-associated, although some are transmembrane proteins and function as receptors for peptide growth factors. Three Drosophila gene sequences related to the v-src gene have been identified, each exhibiting a high degree of homology to one or more of the src-family members encoding an intracellular protein. We have isolated a panel of cloned Drosophila sequences exhibiting weak v-src hybridization and were interested to determine whether any members of this group represented homologues of additional known src-family genes, especially those functioning as growth factor receptors. As we report here, four of these clones, representing overlapping portions of the same genomic segment, hybridized preferentially with the v-erb-B oncogene and were further characterized. The deduced amino-acid sequence from a portion of this Drosophila genomic segment is 77% homologous to the kinase domain of human epidermal growth factor (EGF) receptor, a substantially greater degree of homology than was observed with any other known src-family member. By hybridization with a human EGF receptor complementary DNA clone probe, we demonstrate that the same genomic segment showing homology with the kinase domain also contains sequences related to the extracellular domain of the EGF receptor gene.

Maternal inheritance of transcripts from three Drosophila src-related genes.

The Drosophila genome contains three major sequences related to the v-src gene. Previously published molecular studies have confirmed the structural homology between v-src and two of the Drosophila sequences. We have sequenced a portion of the third v-src-related Drosophila gene and found that it also shares structural homology with vertebrate and Drosophila src-family genes. RNA sequences from each of the src genes are present in pre-blastoderm embryos indicating that they are of maternal origin. As embryogenesis proceeds, the levels of each of the src RNA sequences decline. The pre-blastoderm src gene transcripts contain poly(A) and are present on polyribosomes suggesting that they are functional mRNAs. Since the Drosophila src transcripts were maternally inherited, we also investigated their distribution in adult females. The majority of the src transcripts in adult females were contained in ovaries. Only low levels of the transcripts were detected in males. These results strongly suggest that an abundant supply of src protein is required during early embryogenesis, perhaps at the time of cellularization of the blastoderm nuclei.