Critical role of c-Kit in beta cell function: increased insulin secretion and protection against diabetes in a mouse model.

AIMS/HYPOTHESIS: The receptor tyrosine kinase, c-Kit, and its ligand, stem cell factor, control a variety of cellular processes, including pancreatic beta cell survival and differentiation as revealed in c-Kit ( Wv ) mice, which have a point mutation in the c-Kit allele leading to loss of kinase activity and develop diabetes. The present study further investigated the intrinsic role of c-Kit in beta cells, especially the underlying mechanisms that influence beta cell function. METHODS: We generated a novel transgenic mouse model with c-KIT overexpression specifically in beta cells (c-KitßTg) to further examine the physiological and functional roles of c-Kit in beta cells. Isolated islets from these mice were used to investigate the underlying molecular pathway of c-Kit in beta cells. We also characterised the ability of c-Kit to protect animals from high-fat-diet-induced diabetes, as well as to rescue c-Kit ( Wv ) mice from early onset of diabetes. RESULTS: c-KitßTg mice exhibited improved beta cell function, with significantly improved insulin secretion, and increased beta cell mass and proliferation in response to high-fat-diet-induced diabetes. c-KitßTg islets exhibited upregulation of: (1) insulin receptor and IRSs; (2) Akt and glycogen synthase kinase 3ß phosphorylation; and (3) transcription factors important for islet function. c-KIT overexpression in beta cells also rescued diabetes observed in c-Kit ( Wv ) mice. CONCLUSIONS/INTERPRETATION: These findings demonstrate that c-Kit plays a direct protective role in beta cells, by regulating glucose metabolism and beta cell function. c-Kit may therefore represent a novel target for treating diabetes.

Induction of a NOTCH3 Lehman syndrome mutation in osteocytes causes osteopenia in male C57BL/6J mice.

Lateral Meningocele or Lehman Syndrome (LMS) is associated with NOTCH3 mutations causing deletions of the PEST domain and a gain-of-NOTCH3 function. We demonstrated that Notch3em1Ecan mice harboring Notch3 mutations analogous to those found in LMS are osteopenic because of enhanced bone resorption. To determine the contribution of specific cell lineages to the phenotype, we created a conditional-by-inversion (Notch3COIN) model termed Notch3em2Ecan in which Cre recombination generates a Notch3INV allele expressing a NOTCH3 mutant lacking the PEST domain. Germ line Notch3COIN inversion caused osteopenia and phenocopied the Notch3em1Ecan mutant, validating the model. To induce the mutation in osteocytes, smooth muscle and endothelial cells, Notch3COIN mice were bred with mice expressing Cre from the Dmp1, Sm22a and Cdh5 promoters, respectively, creating experimental mice harboring Notch3INV alleles in Cre-expressing cells and control littermates harboring Notch3COIN alleles. Notch3COIN inversion in osteocytes led to femoral and vertebral cancellous bone osteopenia, whereas Notch3COIN inversion in mural Sm22a or endothelial Cdh5-expressing cells did not result in a skeletal phenotype. In conclusion, introduction of the LMS mutation in osteocytes but not in vascular cells causes osteopenia and phenocopies Notch3em1Ecan global mutant mice.

Lymphoid and mesenchymal tumors in transgenic mice expressing the v-fps protein-tyrosine kinase.

src, abl, and fps/fes are prototypes for a family of genes encoding nonreceptor protein-tyrosine kinases. The oncogenic potential of the v-fps protein-tyrosine kinase was investigated by introduction of the gag-fps coding sequence of Fujinami sarcoma virus into the mouse germ line. Transgenic mice with v-fps under the transcriptional control of a 5' human beta-globin promoter (GF) or with both 5' and 3' beta-globin regulatory sequences (GEF) were viable. Unexpectedly, both GF and GEF transgenes were expressed in a wide variety of tissues and induced a spectrum of benign and malignant tumors. These tumors, which included lymphomas, thymomas, fibrosarcomas, angiosarcomas, hemangiomas, and neurofibrosarcomas, developed with various frequencies after latent periods of 2 to 12 months. The majority of lymphoid neoplasms appeared to be of T-cell origin and were monoclonal, as judged by rearrangements of the T-cell receptor beta or immunoglobulin genes. Some tissues that expressed the v-fps oncogene, such as heart, brain, lung, and testes, developed no malignant tumors. The v-fps protein-tyrosine kinase therefore has a broad but not unrestricted range of oncogenic activity in cells of lymphoid and mesenchymal origin. The incomplete penetrance of the neoplastic phenotype and the monoclonality of lymphoid tumors suggest that tumor formation in v-fps mice requires genetic or epigenetic events in addition to expression of the P130gag-fps protein-tyrosine kinase.

Novel protein-tyrosine kinase cDNAs related to fps/fes and eph cloned using anti-phosphotyrosine antibody.

A rat brain lambda gt11 cDNA expression library was screened with anti-phosphotyrosine antibody to identify recombinant clones that encode enzymatically active protein-tyrosine kinases. The inserts of two bacteriophage that gave positive signals were sequenced. Both translation products possess sequence motifs characteristic of protein-tyrosine kinases. However, each polypeptide is distinct from previously described members of the tyrosine kinase family. The predicted product of the lambda B1 clone contains a catalytic domain and C-terminal tail most closely related to the eph gene product, a presumed transmembrane receptor-like protein-tyrosine kinase. The clone lambda B2 encodes a partial SH2 domain and a kinase domain similar in organization and sequence to the fps/fes cytoplasmic protein-tyrosine kinase. These new protein-tyrosine kinases (elk and flk) are apparently members of subfamilies for which eph and fps/fes are prototypes. elk is predominantly expressed in brain, while flk RNA is widely distributed and most abundant in testes. The preferential isolation of cDNAs for previously uncharacterized protein-tyrosine kinases in a screen based on catalytic activity suggests that additional members of the protein-tyrosine kinase family remain to be identified.

A retrovirus encoding the v-fps protein-tyrosine kinase induces factor-independent growth and tumorigenicity in FDC-P1 cells.

There is increasing evidence that protein-tyrosine kinases play pivotal roles in the response to growth-factor signals. The cytoplasmic tyrosine kinase c-fps/fes, due to its restricted expression in hematopoietic tissue, is likely to participate in hematopoietic growth-factor signalling. We have introduced a retrovirus containing an activated fps gene (encoding P130gag-fps) into the growth factor-dependent myeloid cell line FDC-P1. Clonal cell lines were derived by selection for a marker gene coding for G418 resistance in the absence or presence of the hematopoietic growth factor IL-3. G418 resistant clones expressed P130gag-fps and its associated protein-tyrosine kinase activity and displayed either a factor-independent or IL-3 hypersensitive phenotype and were tumorigenic in syngeneic recipients. Thus, introduction of the activated v-fps gene was able to circumvent the requirement for exogenous growth factors by FDC-P1 cells. Bioassay of conditioned medium from the various clones did not detect hematopoietic growth factor activity and PCR analysis for IL-3 transcripts were negative, suggesting that growth-factor independence was achieved by a mechanism other than autocrine production of a growth factor. We suggest that P130gag-fps is acting to directly stimulate a hematopoietic growth-factor signalling pathway, perhaps one that normally involves the endogenous c-fps/fes protein-tyrosine kinase of FDC-P1 cells.

Human sex hormone-binding globulin gene expression in transgenic mice.

The sex hormone-binding globulin gene (shbg) is expressed in the liver and testis as well as in several other tissues that play important roles in reproduction. Expression of shbg in the human liver results in the production of plasma sex hormone-binding globulin (SHBG), which regulates the bioavailability of sex steroids in the blood. Although shbg is not expressed in rodent livers postnatally, it gives rise to the androgen-binding protein in their testes upon sexual maturation. Human shbg is also expressed in the testis, but its products and their function are less well characterized. To study the expression of human shbg in different tissues and the consequences of overexpressing this gene in vivo, we have produced several lines of mice containing approximately 11-kilobase (kb; shbg11) or 4.3-kb (shbg4) human shbg genomic fragments that comprise all eight exons encoding SHBG as well as approximately 6 kb or approximately 0.9 kb of 5'-flanking DNA, respectively. Northern blots indicated that human shbg transcripts were most abundant in liver, kidney, and testis of the shbg11 mice. The 4.3-kb shbg transgenes were expressed at similar levels in liver and kidney, but the abundance of human shbg transcripts in their testes was much lower than that in shbg11 mice. Primer extension analysis indicated that transcription starts 60 bp from the translation initiation codon for SHBG in liver and kidney of shbg11 mice, and that the shbg transcripts in their testis are derived from a separate promoter flanking an alternative exon that replaces the exon containing the translation initiation codon for SHBG or androgen-binding protein. At the cellular level, the human shbg transgenes are expressed in clusters of hepatocytes located mainly within the periportal region of hepatic lobules and in the epithelial cells lining the proximal convoluted tubules of the kidney. This results in high levels of human SHBG in serum (1.45-1.72 nmol/ml) and urine (6-16 pmol/ml) of mature male shbg mice. The abundance and distribution of human shbg transcripts in the Sertoli cells of shbg11 mice vary throughout the spermatogenic cycle, with levels increasing in the Sertoli cell cytoplasm until stage VII of spermatogenesis and declining after stage IX. At stages X-XII of spermatogenesis, these transcripts concentrate at the adluminal compartment of the Sertoli cells, and this suggests that they have a role in the elongation phase of spermiogenesis. The presence of human SHBG in the blood of shbg transgenic mice may result in serum levels of testosterone that are 10-100 times higher than those in wild-type littermates. Despite this, their reproductive performance is normal, and there is no obvious phenotypic abnormalities even in animals homozygous for the transgenes.

Analysis of multiple forms of human adenovirus type 5 E1A polypeptides using an anti-peptide antiserum specific for the amino terminus.

Studies were carried out to further characterize the proteins coded for by the early 1A region (E1A) of human adenovirus type 5 (Ad5) in lytically infected cells using an antiserum prepared against a synthetic octapeptide corresponding to the amino termini of E1A products as well as another anti-peptide serum specific for the carboxy termini. Both sera precipitated the same collection of four major and two minor acidic phosphoproteins and it was confirmed that each of the 1.1- and 0.9-kb E1A mRNAs code for two major and one minor species. These data also indicated that none of the E1A species was produced by proteolytic cleavage. The deletion mutant dl313 which lacks DNA coding for the last 70 C-terminal amino acids of E1A products also produced multiple species which suggested that post-translational modifications involved in their generation do not take place in this region of the proteins. The N-terminal serum was effective in detecting neither the truncated 1.1-kb mRNA product predicted for the mutant hr1 nor the product of the small 0.6-kb E1A mRNA, suggesting that these species are either very short-lived in infected cells or exist in a conformation in which the amino terminus is inaccessable to the antibody.

The regulation of myogenin gene expression during the embryonic development of the mouse.

The myogenic program can be activated in cultured cells by each of four basic-helix-loop-helix (bHLH) transcription factors, the expression of which is strictly controlled, both temporally and spatially, during embryonic development. To begin to understand the mechanisms by which these regulators are regulated themselves, we have used transgenic animals to define the minimal sequences required for the complete recapitulation of the temporal and spatial expression pattern of the myogenin gene during embryogenesis. We show that this can be achieved with only 133 bp of 5'-flanking DNA and identify two essential motifs, which are consensus binding sites for the bHLH proteins and for the proteins of the RSRF family. We show further that these sequences, when juxtaposed to a heterologous promoter, are capable of imposing the myogenin expression pattern. We conclude that the proper regulation of myogenin requires a bHLH protein, most probably Myf-5, the only myogenic bHLH factor known to be present in the embryo at the time that myogenin is activated, and an RSRF-like binding activity. Furthermore, the expression pattern of a mutant myogenin promoter lacking the RSRF site reveals the existence of at least two populations of cells within the myotomes and of novel rostrocaudal gradients of expression.

Detection of cellular proteins associated with human adenovirus type 5 early region 1A polypeptides.

Antisera prepared against synthetic peptides corresponding to the amino and carboxy termini of human adenovirus type 5 (Ad5) early region 1A (E1A) proteins were used to identify polypeptides that are associated with these viral species in lytically infected KB cells. Proteins were sought which coprecipitated with E1A polypeptides using both sera and which were not recognized in extracts from mock-infected cells by either serum. Four such species were identified with apparent molecular weights of 68K, 65K, and a doublet at about 105K. A fifth species migrating with a molecular weight in excess of 250K was also identified consistently with E1A-C1 but not E1A-N1 serum. Addition of an excess of the appropriate synthetic peptide to the immunoprecipitation mixtures prevented the precipitation of all of these species. Mixing experiments demonstrated that all species were cellular proteins expressed in normal uninfected KB cells and in addition showed that an association with E1A proteins could take place in vitro. Studies carried out with the mutants pm975 and hr1 indicated that while the 105K doublet and the greater than 250K species were found with the products of both the 1.1- and 0.9-kb E1A mRNAs, 65K and 68K appeared to be primarily associated with those of the 1.1-kb mRNA. Finally, the 105K doublet and greater than 250K were shown to be phosphoproteins. These data indicated that Ad5 E1A proteins may function in a complex with cellular polypeptides which includes species of 105K, 68K, 65K, and possibly a large protein of greater than 250K.

Co-purification of protein kinase activity with the 58 000 Dalton polypeptide coded for by the early 1B region of human adenovirus type 5.

Extracts from human adenovirus type 5 (Ad5)-transformed cells were fractionated by ammonium sulphate precipitation, DEAE-Sephacel chromatography and glycerol gradient centrifugation. In all cases, protein kinase activity co-purified with the 58 000 mol. wt. polypeptide (58K) coded for by the early 1B region of Ad5. Kinase activity was also precipitated by an antiserum raised against a synthetic peptide corresponding to the predicted carboxy terminus of 58K. These data suggest that protein kinase activity is associated with 58K, either intrinsically or in an enzyme bound to 58K.

External cell surface protein phosphorylation in normal and Rous sarcoma virus transformed chick embryo fibroblasts.

Normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts growing on plastic dishes were incubated with ATP (gamma 32P) in situ to detect external cell surface protein kinase activity. Under the conditions employed, 32P was incorporated exclusively into proteins, specifically those at the external cell surface, as radioactivity was removed by trypsin treatment of labeled whole cells. In addition, exogenous histones were phosphorylated when added to the reaction mixture. Cyclic nucleotides had virtually no effect on 32P incorporation, suggesting that little or no cyclic nucleotide-dependent protein kinase activity was present at the external cell surface. Cell surface protein kinase activity was higher in transformed than in normal cells, and, using a temperature-sensitive RSV src mutant, this difference was shown to be transformation-specific. Several differences were observed in the cell surface proteins phosphorylated in normal and transformed cells and at least two of these were transformation-specific. These data suggest that changes in external cell surface protein phosphorylation are associated with RSV transformation and thus could play a role in the formation of the transformed cell phenotype.

Characterization of a novel insertional mouse mutation, kkt: A closely linked modifier of Pax1.

We describe a novel transgene insertional mouse mutant with skeletal abnormalities characterized by a kinked tail and severe curvature of the spine. The disrupted locus is designated kkt for "kyphoscoliosis kinked tail." Malformed vertebrae including bilateral ossification centers and premature fusion of the vertebral body to the pedicles are observed along the vertebral column, and the lower thoracic and lumbar vertebrae are the most affected. Some of the homozygous kkt neonates displayed two backward-pointing transverse processes in the sixth lumbar vertebra (L6) that resembled the first sacral vertebra, and some displayed one forward- and one backward-pointing transverse process in L6. The fourth and fifth sternebrae were also fused, and the acromion process of the scapula was missing in kkt mice. The skeletal abnormalities are similar to those observed in the mouse mutant undulated (un). The transgene is integrated at the distal end of chromosome 2 close to the Pax1 gene, as revealed by FISH analysis. However, mutation of the Pax1 gene is responsible for the un phenotype, but the Pax1 gene in the kkt mice is not rearranged or deleted. Pax1 is expressed normally in kkt embryos and in the thymus of mature animals, and there is no mutation in its coding sequence. Thus, the skeletal abnormalities observed in the kkt mutant are not due to a lack of functional Pax1. Mouse genomic sequences flanking the transgene and PAC clones spanning the wild-type kkt locus have been isolated, and reverse Northern analysis showed that the PACs contain transcribed sequence. Compound heterozygotes between un and kkt (un(+/-)/kkt(+/-)) display skeletal abnormalities similar to those of un or kkt homozygotes, but they have multiple lumbar vertebrae with a split vertebral body that is more severe than in homozygous un or kkt neonates. Furthermore, the sternebrae are not fused and no backward-pointing transverse processes are detected in L6. It is therefore apparent that these two mutations do not fully complement each other, and we propose that a gene in the kkt locus possesses a unique role that functions in concert with Pax1 during skeletal development.

Studies on the phosphorylation of the 58000 dalton early region 1B protein of human adenovirus type 5.

The 58000 dalton (58K) protein coded for by early region 1B of human adenovirus type 5 (Ad5) was found to be phosphorylated. At least three major tryptic phosphopeptides were identified and the average number of phosphates per 58K molecule was estimated to be between two and three. Thus, it was possible that each phosphopeptide contained just one phosphate group. The ratio of phosphoserine to phosphothreonine was about 2 to 1 on average and essentially no phosphotyrosine was detected. No evidence was found to suggest that cAMP-dependent protein kinase was involved in the phosphorylation of 58K. Previous studies have shown that 58K was phosphorylated when immunoprecipitates containing Ad5 early region 1 proteins were incubated in vitro with [gamma-32P]ATP. Analysis of the phosphopeptides of 58K labelled under these conditions indicated a large number of phosphorylation sites which differed from those found in vivo. Thus, the action of kinases in the in vitro phosphorylation of 58K in immunoprecipitates did not mimic the enzymic activity responsible for 58K phosphorylation in vivo.

Acylation of the 176R (19-kilodalton) early region 1B protein of human adenovirus type 5.

Antipeptide sera were prepared in rabbits against synthetic peptides corresponding to the predicted amino and carboxy termini of the early region 1B 176R (19-kilodalton [kDa]) protein of human adenovirus type 5. Both antisera specifically immunoprecipitated the 19- and 18.5-kDa forms of the 176R protein observed previously with antitumor sera. These data suggested that both species are full-length molecules of 176 residues. To identify posttranslational modifications that could explain the formation of these multiple species and possibly their known association with membranes, studies were carried out to determine whether they are glycosylated or acylated. Neither the 19- nor the 18.5-kDa species appeared to be a glycoprotein, however, they were labeled with [3H]palmitate and [3H]myristate, indicating that both species are acylated. Thus, whereas acylation does not appear to be the cause of the multiple species, it could play a role in the membrane association of these viral proteins. The acylation of 176R was found to be unusual. The fatty acid linkage was resistant to treatment with hydroxylamine or methanol-KOH, suggesting that acylation was through an amide bond. In addition, both palmitate and myristate were present in 176R, suggesting either a lack of specificity in the acylation reaction or the existence of more than one acylation site.

Binding of cellular polypeptides to human adenovirus type 5 E1A proteins produced in Escherichia coli.

Cellular proteins of 300, 107, 105, 68 and 65 kDa have previously been shown to associate specifically with the early region 1A (E1A) proteins of human adenovirus type 5. In the present study we report that, to varying degrees, these proteins also were capable of binding to E1A products produced in Escherichia coli from plasmids carrying cDNAs corresponding to the 1.1- and 0.9-kb E1A mRNAs. When these purified E1A proteins were mixed in solution with extracts from mock-infected human cells, the 68- and 65-kDa species bound very efficiently to the 1.1-kb mRNA product and somewhat less so with that of the 0.9-kb mRNA. The 107-, 105-, and 300-kDa species bound poorly, if at all, to both E1A products. Using the E1A 1.1-kb mRNA product which had been covalently attached to Sepharose beads, the 68-, 65-, and 300-kDa species bound efficiently, and binding of protein which migrated in SDS gels in the region of the 107- and 105-kDa species was also observed. In addition to these proteins, several other cellular polypeptides of 30, 33, 75, 95, 150, 180, and greater than 300 kDa were shown to bind to E1A-Sepharose and thus may also be E1A-binding proteins. The present data confirm the specificity of the previously identified cellular proteins for E1A products and show that binding of the 300-, 65-, and 68-kDa species does not require the presence of any other viral polypeptide. In contrast, the inefficient binding of the 107- and 105-kDa species to Escherichia coli-expressed E1A protein may suggest that these interactions require either eukaryotic-specific post-translational modifications of the E1A protein, or the presence of additional Ad5 gene products.

Progressive cardiac fibrosis and myocyte injury in v-fps transgenic mice. A model for primary disorders of connective tissue in the heart?

Transgenic mice that express v-fps protein-tyrosine kinase have severe cardiac or neurologic abnormalities and a high incidence of lymphoid or mesenchymal tumors. The cardiac lesions of v-fps transgenic mice were examined at less than 1, 2, 3, 6, 14, 26, and 43 weeks of age (total N = 19) and compared with nontransgenic littermate controls (N = 34). Three of eight transgenic animals 1 to 4 days old showed moderate proliferation of connective tissue elements most evident along the septal endocardium of the right ventricle. Variable cardiac hypertrophy was seen grossly in 2- to 14-week-old transgenic animals, and marked biventricular dilatation was present in those 6 to 43 weeks of age. Sections of all transgenic mice 3 weeks or older revealed characteristic lesions that consisted of cellular fibrosis in subendocardial, subepicardial, and perivascular sites, with associated proliferation of pericytes and fat cells. Atrophy, degeneration, and loss of entrapped myocytes were noted in transgenic animals as early as 2 weeks of age, but frank coagulative necrosis or myocytolysis was absent at all ages studied. Nonetheless, pleomorphic nuclei were found in occasional myocytes late in disease. Inflammatory cells were rare, as confirmed immunohistochemically (Thy-1 [pan-T], L3T4 [CD4], Lyt-2 [CD8], interleukin-2 receptor [activated lymphocyte], Mac-1 [macrophage], and B220 [pan-B]). Monoclonal immunoreactivity to the v-fps transgene product was positive predominantly in nonmyocyte cardiac constituents. Collectively, the data suggest a primary proliferative abnormality of connective tissue elements in the heart, accompanied by secondary myocyte damage.

A novel nuclear receptor corepressor complex, N-CoR, contains components of the mammalian SWI/SNF complex and the corepressor KAP-1.

Transcriptional silencing by many transcription factors is mediated by the nuclear receptor corepressor (N-CoR). The mechanism by which N-CoR represses basal transcription involves the direct or indirect recruitment of histone deacetylases (HDACs). We have isolated two multiprotein N-CoR complexes, designated N-CoR-1 and N-CoR-2, which possess histone deacetylase activity that is mediated by distinct HDACs. Based on Western blotting using antibodies against known subunits, the only HDAC found in the N-CoR-1 complex was HDAC3. In contrast, N-CoR-2 contained predominantly HDAC1 and HDAC2 as well as several other subunits that are found in the Sin3A.HDAC complex. Using mass spectrometry and Western blotting, we have identified several novel components of the N-CoR-1 complex including the SWI/SNF-related proteins BRG1, BAF 170, BAF 155, BAF 47/INI1, and the corepressor KAP-1 that is involved in silencing heterochromatin. Indirect immunofluorescence has revealed that both KAP-1 and N-CoR colocalize throughout the nucleus. These results suggest that N-CoR is found in distinct multiprotein complexes, which are involved in multiple pathways of transcriptional repression.

Identification of human adenovirus early region 1 products by using antisera against synthetic peptides corresponding to the predicted carboxy termini.

Synthetic peptides were prepared which corresponded to the carboxy termini of the human adenovirus type 5 early region 1B (E1B) 58,000-molecular-weight (58K) protein (Tyr-Ser-Asp-Glu-Asp-Thr-Asp) and of the E1A gene products (Tyr-Gly-Lys-Arg-Pro-Arg-Pro). Antisera raised against these peptides precipitated polypeptides from adenovirus type 5-infected KB cells; serum raised against the 58K carboxy terminus was active against the E1B 58K phosphoprotein, whereas serum raised against the E1A peptide immunoprecipitated four major and at least two minor polypeptides. These latter proteins migrated with apparent molecular weights of 52K, 50K, 48.5K, 45K, 37.5K, and 35K, and all were phosphoproteins. By using tryptic phosphopeptide analysis, the four major species (52K, 50K, 48.5K, and 45K) were found to be related, as would be expected if all were products of the E1A region. The ability of the antipeptide sera to precipitate these viral proteins thus confirmed that the previously proposed sequence of E1 DNA and mRNA and the reading frame of the mRNA are correct. Immunofluorescent-antibody staining with the antipeptide sera indicated that the 58K E1B protein was localized both in the nucleus and in the cytoplasm, especially in the perinuclear region. The E1A-specific serum also stained both discrete patches in the nucleus and diffuse areas of the cytoplasm. These data suggest that both the 58K protein and the E1A proteins may function in or around the nucleus. These highly specific antipeptide sera should allow for a more complete identification and characterization of these important viral proteins.

Establishment and characterization of hamster cell lines transformed by restriction endonuclease fragments of adenovirus 5.

We have established a library of hamster cells transformed by adenovirus 5 DNA fragments comprising all (XhoI-C, 0 to 16 map units) or only a part (HindIII-G, 0 to 7.8 map units) of early region 1 (E1: 0 to 11.2 map units). These lines have been analyzed in terms of content of viral DNA, expression of E1 antigens, and capacity to induce tumors in hamsters. All cells tested were found to express up to eight proteins encoded within E1A (0 to 4.5 map units) with apparent molecular weights between 52,000 (52K) and 25K. Both G and C fragment-transformed lines expressed a 19K antigen encoded within E1B (4.5 to 11.2 map units), whereas an E1B 58K protein was detected in C fragment-transformed, but not G-fragment-transformed, lines. No clear distinction could be drawn between cells transformed by HindIII-G and by XhoI-C in terms of morphology or tumorigenicity, suggesting that the E1B 58K antigen plays no major role in the maintenance of oncogenic transformation, although possible involvement of truncated forms of 58K cannot be ruled out. Sera were collected from tumor-bearing animals and examined for ability to immunoprecipitate proteins from infected cells. The relative avidity of sera for different proteins was characteristic of the cell line used for tumor induction, and the specificity generally reflected the array of viral proteins expressed by the corresponding transformed cells. However, one notable observation was that even though all transformed lines examined expressed antigens encoded by both the 1.1- and 0.9-kilobase mRNAs transcribed from E1A, tumor sera made against these lines only precipitated products of the 1.1-kilobase message. Thus, two families of E1A proteins, highly related in terms of primary amino acid sequence, appear to be immunologically quite distinct.