Gary Ramsay

Identification and characterization of the avian erythroblastosis virus erbB gene product as a membrane glycoprotein

Avian erythroblastosis virus causes erythroid leukemia and sarcomas in chickens. The viral oncogene responsible for these diseases, erb, is divided into two regions known as erbA and erbB, and recent evidence suggests that it is the erbB gene that is responsible for the transforming activity. From rats bearing avian erythroblastosis virus-induced sarcomas, we have obtained antisera which are specific for the erb gene products. Using such antisera, we have been able to characterize the erbB gene product as a 68,000 molecular weight protein. Pulse-chase and cell-free in vitro translation experiments show that the initial product is a 62,500 dalton protein which is initially modified to a 66,000 dalton protein, and then further modified to a 68,000 dalton form. These modifications could be shown to be associated with glycosylation and phosphorylation. Cell fractionation experiments revealed that the 66,000 and 68,000 dalton proteins were located in cell membrane fractions, and immunofluorescence results showed the erbB gene product to be expressed on the cell surface.

The product of the retroviral transforming gene v-myb is a truncated version of the protein encoded by the cellular oncogene c-myb

Avian myeloblastosis virus (AMV) is an oncogenic retrovirus that rapidly causes myeloblastic leukemia in chickens and transforms myeloid cells in culture. AMV carries an oncogene, v-myb, that is derived from a cellular gene, c-myb, found in the genomes of vertebrate species. We constructed a plasmid vector that allows expression of a portion of the coding region for v-myb in a procaryotic host. We then used the myb-encoded protein produced in bacteria to immunize rabbits. The antisera obtained permitted identification of the proteins encoded by both v-myb and chicken c-myb. The molecular weights of the products of v-myb and c-myb (45,000 and 75,000 respectively) indicate that the v-myb protein is an appreciably truncated version of the c-myb protein.

Human proto-oncogene N-myc encodes nuclear proteins that bind DNA.

N-myc is a gene whose amplification has been implicated in the genesis of several malignant human tumors. We have identified two proteins with molecular weights of 65,000 and 67,000 encoded by N-myc. The abundance of these proteins in tumor cells was consonant with the extent of amplification of N-myc. The two proteins apparently arose from the same mRNA, were phosphorylated, were exceptionally unstable, were located in the nucleus of cells, and bound to both single- and double-stranded DNA. These properties suggest that the products of N-myc and of the related proto-oncogene c-myc may have similar biochemical functions and that N-myc may be a regulatory gene. Our findings sustain the view that inordinate expression of N-myc may contribute to the genesis of several different human tumors.

Phosphorylation of specific sites in the gag-myc polyproteins encoded by MC29-type viruses correlates with their transforming ability.

The putative transforming proteins of the four acute leukaemia viruses belonging to the MC29 subgroup were shown to be phosphorylated in vivo. Comparison of the MC29 and CM11 encoded phosphoproteins revealed identical tryptic phosphopeptide maps, with both the gag and myc domains being phosphorylated. In contrast, the MH2 phosphoprotein was only phosphorylated on the gag domain. Analysis of partial transformation‐defective MC29 deletion mutants revealed that the deletions had removed the v‐myc specific phosphopeptides. Phosphoamino acid analysis showed that these deleted phosphopeptides were phosphorylated on threonine. Moreover, a back mutant that had regained transforming ability had regained these phosphopeptides. These studies correlate the phosphorylation of the gag‐myc protein with the transformation capability of the virus.

Altered pathogenicity of avian myelocytomatosis (MC29) viruses with mutations in the v-myc gene

Avian myelocytomatosis virus MC29 inoculated into the wing web of 1-day-old Brown Leghorn chickens causes a high incidence of tumors, predominantly endotheliomas that are apparently induced directly by the action of the viral myc gene. Mutants of MC29, in which portions of the v-myc gene have been deleted and which have reduced ability to transform macrophages in vitro, induce few tumors, among which lymphomas and osteopetrosis predominate. Analysis of lymphomas from birds infected with mutant MC29 suggested that they were a result of helper virus action rather than the mutant MC29.

Analysis of cells transformed by defective leukemia virus OK10: Production of noninfectious particles and synthesis of Pr76gag and an additional 200,000-dalton protein

Abstract Nonproducer cells transformed by the defective leukemia virus (DL V), OK10, have been analyzed. Unlike nonproducer cells transformed by the other avian defective leukemia viruses examined so far, the OK10-transformed cells were found to release noninfectious particles. Analysis of these particles indicated that they contained the viral gag gene proteins but lacked env and pol gene products. In agreement with these results analysis of [ 35 S]methio-nine-labeled cell extracts of these nonproducer clones by immune precipitation showed that of the three viral structural protein precursors Pr769 gag , gPr95 env , and Pr180 gag-pol only Pr76 gag could be detected. In addition, a 200,000 molecular weight protein (OK10-200K) was identified in the cell extracts which by using specific antisera, was shown to be related to the gag and pol gene products but not to the product of the env gene. Tryptic peptide analysis of the OK10-200K protein confirmed the immunological data in that the OK10-200K protein was shown to contain all but one of the Pr1809 gag-pol methionine tryptic peptides plus unique peptides which were specific for OK10 and not related to the env gene product. One of these OK10-specific peptides was also shown to comigrate with one of the putative mac gene product tryptic peptides of the MC29-110K protein. These data indicate a novel gene order for a DLV.

Cell transformation by avian defective leukaemia viruses

A comparative study of seven independently isolated defective leukaemia viruses has been carried out. Phenotypic analysis of the chicken bone marrow cells transformed in vitro allowed the separation of these seven viruses into three groups based on the differentiation phenotype of the transformed cell. Nucleic acid hybridization studies revealed that these seven viruses had acquired cellular sequences. Interestingly, these studies also showed that the viruses within the same biological grouping had acquired related sequences. This indicates that viruses that have acquired the same or similar cellular sequences have very similar oncogenic capabilities. Analysis of proteins expressed in cells transformed by these viruses demonstrated that the cellular sequences were usually inserted within the gene for the viral core proteins, gag. Therefore the cellular sequences are expressed as a gag-related fusion protein which has an amino-terminal region derived from the gag gene and a carboxy-terminal half derived from the cellular sequences. Two exceptions to this are discussed. The general conclusion from these studies is that defective leukaemia viruses transform cells by virtue of acquired host cellular sequences. The ability of these viruses to transform cells and the target cell specificity of the transformation depends on these cellular sequences.

TRANSFORMATION DEFECTIVE MUTANTS OF AEV AND MC29 AVIAN LEUKEMIA VIRUSES SYNTHESIZE SMALLER GAG-RELATED PROTEINS

ABSTRACT Avian erythroblastosis virus (AEV) and myelocytomatosis virus 29 (MC29) are replication defective oncoviruses capable of causing acute leukemias as well as other neoplasms in the chicken. Hematopoietic cells transformed by AEV have properties of erythroblasts, those transformed by MC29 resemble macrophages. The uninfected target cells of these viruses are similar to the corresponding transformed cells in their phenotype of differentiation. AEV and MC29 viruses are also capable of transforming fibroblasts in vitro. Clones of fibroblasts transformed by AEV and MC29 can be distinguished by the pattern of transformation parameters they express. The different biological properties of the two virus strains have been attributed to the specific action of two new oncogenes designated as erb for AEV and mac for MC29. Candidate transforming proteins of these viruses are p75 AEV and p110 MC29, consisting of a portion related to the gag -gene product and a non- gag related portion (different for each virus). A mutant of AEV was isolated which had lost the ability to transform erythroblasts but which retained the ability to transform fibroblasts. This mutant, td 359 AEV, was found to have a small deletion in the non- gag related portion of p75 AEV, suggesting that this molecule is responsible for erythroblast transformation. The p40 AEV protein synthesized in an in vitro translation system using RNA from td 359 AEV was unaltered in size. Three mutants of MC29 were found which synthesize altered gag -related proteins of approximately 100k, 95k and 90k daltons, respectively. Preliminary experiments indicate that these mutants have a reduced potential to transform macrophages while having retained their ability to transform fibroblasts.