Molecular cloning of the intronless EJ ras oncogene using a murine retrovirus shuttle vector

We have inserted a genomic clone of the human EJ bladder oncogene into a murine retrovirus shuttle vector. Cotransfection of this shuttle vector DNA containing the activated ras oncogene with molecularly cloned Moloney murine leukemia virus into NIH/3T3 cells was able to rescue a replicating and transforming retrovirus. The viral DNA from infected cells was excised by fusion to mouse COP-5 cells and recloned into Escherichia coli as plasmid DNA. Analysis of the recloned plasmids by size, restriction enzyme mapping, and DNA sequence indicated that approximately 5% of the recloned plasmids contained the intronless EJ ras oncogene.     

Studies on the Nucleic Acid Sequences of Kirsten Sarcoma Virus: a Model for Formation of a Mammalian RNA-Containing Sarcoma Virus

The genetic information contained in the Kirsten and Moloney strains of mammalian RNA-containing sarcoma viruses has been analyzed by RNA . (3)H-DNA hybridization. Kirsten sarcoma virus has been found to possess two distinct sets of nucleic acid sequences. One set of sequences is contained in murine type C helper virus, and the other set is contained in rat type C helper virus. Moloney sarcoma virus contains sequences of murine type C helper virus but not of rat type C helper virus. The results indicate that Kirsten sarcoma virus arose through a process of recombination between Kirsten murine leukemia virus and nucleic acid sequences found in rat cells. A model is suggested for the formation of transforming type C viruses involving the transduction of oncogenic information.     

Molecular cloning, genomic analysis, and biological properties of rat leukemia virus and the onc sequences of Rasheed rat sarcoma virus.

Rasheed rat sarcoma virus (RaSV) has been shown to code for a protein of 29,000 Mr not present in replication-competent rat type C helper virus (RaLV)-infected cells. This protein is a fused gene product consisting of a portion of the RaLV p15 gag protein and the transformation-specific 21,000 Mr (p21) ras protein, which is also found in Harvey murine sarcoma virus. We now report the molecular cloning of both the SD-1 (Sprague-Dawley) strain of RaLV and the transforming ras sequences of RaSV. Heteroduplex analysis of these cloned DNAs demonstrated that the RaSV ras gene (v-Ra-ras) was inserted into the rat type C viral genome with a small deletion of RaLV genetic information in the 5' region of the gag gene and that the v-Ra-ras gene (0.72 kilobase pair) is homologous to and colinear with the p21 ras gene of Harvey murine sarcoma virus (v-Ha-ras). Restriction enzyme mapping confirmed the homology demonstrated by heteroduplex mapping, showing strong site conservation of restriction endonucleases known to cleave v-Ha-ras. Cloned v-Ra-ras DNA transformed NIH 3T3 cells, inducing the synthesis of the p29 RaSVgag-ras protein.     

Construction and isolation of a transmissible retrovirus containing the src gene of Harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type 1

We constructed lambda recombinants containing the Harvey murine sarcoma virus genome and the thymidine kinase (tk) gene of herpes simplex virus type 1 linked to each other. The tk gene was located in a position downstream from both the long terminal repeat and the src gene of Harvey murine sarcoma virus. The DNAs of the lambda recombinants were used to transfect NIH3T3 mouse fibroblasts in order to obtain Harvey murine sarcoma virus DNA-induced foci of transformed cells. The transformed foci were superinfected with a helper-independent retrovirus, and new individual retrovirus were isolated from the superinfected foci. The new viruses could induce focus formation on NIH3T3 cells and could convert NIH3T3(TK-) cells into TK+ cells by carrying the herpes simplex virus type 1 tk gene into the TK- cells. From virus-infected cells, we isolated nonproducer foci on NIH3T3 cells and TK+ transformants on NIH3T3(TK-) cells containing one such new viral genome coding for the dual properties. The new retroviral sequence in the nonproducer cells could be rescued into virus particles at high titers by superinfection with a helper-independent retrovirus. A hybridization analysis indicated that the recombinant virus contained both the Harvey murine sarcoma virus src sequence and the tk gene sequence in a single RNA species approximately 4.9 kilobases long. We concluded that retroviruses can be used as true vectors for genes other than genes that lead to oncogenesis.     

Transformation/neodifferentiation of human skin fibroblasts by acute oncornaviruses and dexamethasone

We demonstrated that the Kirsten murine sarcoma virus (KiMSV) and the Harvey murine sarcoma virus (HaMSV) converted human skin fibroblasts (HSF) into adipocytes. Adipocytic conversion of HSF by KiMSV and HaMSV was dependent on the presence of glucocorticosteroids. The Kirsten murine leukemia virus, the Harvey murine sarcoma [corrected] virus and the amphotropic helper virus (AP292) were ineffective by themselves. Balb murine sarcoma virus and Moloney murine sarcoma virus were, to a lesser degree, able to effect adipocytic conversion of HSF. In contrast, the feline sarcoma virus and the simian sarcoma virus did not cause this conversion. Together, the results suggest a role for certain oncogenes and glucocorticosteroids in the transformation/neodifferentiation of human cells.     

Production of chimeric protein coded by the fused viral H-ras and human N-ras genes in Escherichia coli

A new method for the production of a chimeric protein of two related genes has been developed. The nucleotide sequences of the region from the N terminus to the 86th amino acid (aa) residue of human N-ras and of the Harvey sarcoma virus (Ha-MuSV) H-ras are 80% homologous. We isolated the DNA fragment encoding the N-terminal portion up to the 70th aa residue from plasmid pH-1 which encodes the total genome of Ha-MuSV, and the DNA fragment encoding the C-terminal portion from the 40th aa to the C terminus from plasmid p6a1 which includes the human N-ras cDNA but lacks the N-terminal portion. After partial digestion of both fragments with phage lambda exonuclease, which creates 3'-protruding ends, a hybrid was formed between 73% homologous single-stranded DNA portions at the 3' ends of both fragments. The hybrid was recloned on pBR322 after repairing with Escherichia coli DNA polymerase I and DNA ligase. The chimeric v-H/N-ras gene composed of the N-terminal portion of v-H-ras gene and the remaining region of N-ras gene was inserted into an expression vector containing two tandem trp promoters and a terminator, and expressed in E. coli. The chimeric protein was found to accumulate to approx. 10% of total cellular proteins.     

Structure of the provirus within NIH 3T3 cells transfected with Harvey sarcoma virus DNA.

NIH 3T3 cells transformed with unintegrated Harvey sarcoma virus (HSV) linear DNA generally acquired a complete HSV provirus. Infection of these transformed cells with Moloney murine leukemia helper virus was followed by release of infectious particles. The HSV provirus within these transfected cells was convalently joined to nonviral DNA sequences and was termed "cell-linked" HSV DNA. The association of this cell-virus DNA sequence with the chromosomal DNA of a transfected cell was unclear. NIH 3T3 cells could also become transformed by transfection with this cell-linked HSV DNA. In this case, the recipient cells generally acquired a donor DNA fragment containing both the HSV provirus and its flanking nonviral sequences. After cells acquired either unintegrated or cell-linked HSV DNA, the newly established provirus and flanking cellular sequences underwent amplifications to between 5 and 100 copies per diploid cell. NIH 3T3 cells transfected with HSV DNA may acquire deleted proviral DNA lacking at least 1.3 kilobase pairs from the right end of full-length HSV 6-kilobase-pair DNA (corresponding to the 3'-proximal portion of wild-type HSV RNA). Cells bearing such deleted HSV genomes were transformed, indicating that the viral transformation gene lies in the middle or 5'-proximal portion of the HSV RNA genome. However, when these cells were infected with Moloney murine leukemia helper virus, only low levels of biologically active sarcoma virus particles were released. Therefore, the 3' end of full-length HSV RNA was required for efficient transmission of the viral genome.     

Construction and isolation of a transforming murine retrovirus containing the src gene of Rous sarcoma virus.

Recombinant murine retroviruses containing the src gene of the avian retrovirus Rous sarcoma virus were isolated. Such viruses were isolated from cells after transfection with DNAs in which the src gene was inserted into the genome of the amphotropic murine retrovirus 4070A. The isolated viruses had functional gag and pol genes, but they were all env defective since the src gene was inserted in the middle of the env gene coding region. Infectious transforming virus could be isolated only from cells transfected with DNA constructions in which the src gene was in the same polarity as that of a long terminal repeat of the amphotropic viral genome. These recombinant viruses encoded a pp60src protein with a molecular weight similar to that of the Schmidt-Ruppin strain of Rous sarcoma virus. In addition, the src protein(s) of these recombinant viruses was as active as protein kinases in the immune complex protein kinase assay. Intravenous injection of helper-independent Moloney and Friend murine leukemia virus pseudotypes of the src recombinant viruses into 6-week-old NIH Swiss mice resulted in the appearance of splenic foci within 2 weeks, splenomegaly and, later after infection (8 to 10 weeks), anemia. Infectious transforming virus could be recovered from the spleens of diseased animals. Such viruses encoded pp60src but not p21ras or mink cell focus-forming virus-related glycoproteins.     

Generation of a recombinant Moloney murine leukemia virus carrying the v-src gene of avian sarcoma virus: transformation in vitro and pathogenesis in vivo.

A Moloney murine leukemia virus (M-MuLV) recombinant carrying the v-src gene of avian sarcoma virus was generated by the introduction of a cloned portion of v-src from Schmidt-Ruppin A avian sarcoma virus into a molecular clone of M-MuLV provirus at the recombinant DNA level. The v-src sequences (lacking a portion of the 5' end of v-src) were inserted into the p30 region of the M-MulV gag gene so that M-MuLV gag and v-src were in the same reading frame. Transfection of this chimeric clone, pMLV(src), into NIH 3T3 cells which were constitutively producing M-MuLV gag and pol protein resulted in the formation of foci of transformed cells. Infectious and transforming virus could be recovered from the transformed cells. This virus was designated M-MuLV(src). M-MuLV(src)-transformed cells contained two novel proteins of 78 and 90 kilodaltons. The 78-kilodalton protein, p78gag-src, contained both gag and src determinants, exhibited kinase activity in an immune kinase assay, and is probably a fusion of Pr65gag and src. The 90-kilodalton protein, which is of the appropriate size to be the gPr80gag fused to src, contained gag determinants as well as a V8 protease cleavage fragment typical of the carboxy terminus of avian sarcoma virus pp60src. However, it could not be immunoprecipitated with an anti-v-src serum. M-MuLV(src)-transformed cells showed elevated levels of intracellular phosphotyrosine in proteins, although the elevation was intermediate compared with cells transformed with wild-type v-src. M-MuLV and amphotropic murine leukemia virus pseudotypes of M-MuLV(src) were inoculated into newborn NIH Swiss mice. Inoculated mice developed solid tumors at the site of inoculation after 3 to 6 weeks, with most animals dying by 14 weeks. Histopathological analysis indicated that the solid tumors were mesenchymally derived fibrosarcomas that were both invasive and metastatic.     

Oncogenic genes and human malignancy.

All vertebrates possess a series of genes which are homologs of the oncogenic genes of acute transforming retroviruses. Two lines of evidence suggest that these genes may play a role in the development of human malignancy: (1) DNA from a variety of human tumors transforms NIH 3T3 mouse fibroblasts and the transforming genes from a number of carcinomas, sarcomas, and hematological malignancies have been identified as members of a family of genes, the ras family, closely related to the oncogenic genes of the Harvey and Kirsten murine sarcoma viruses; and (2) correlations exist between the chromosomal localizations of certain oncogenes and the chromosomal breakpoints in specific translocations and deletions in certain human malignancies. In three separate hematological malignancies, alterations in more than one oncogenic gene may be involved in the neoplastic process.     

High level expression of p55, a thyroid hormone binding protein which is homologous to protein disulfide isomerase in a retroviral vector

To develop an efficient system for a high level expression of a human cellular thyroid hormone binding protein (p55) in eukaryotic cells, a full-length p55 cDNA was inserted into a Harvey murine sarcoma virus-derived vector (pHTBr) and transfected into mouse NIH 3T3 cells. The expressed p55 has a molecular weight of 55,000 and is recognized by the human specific anti-p55 monoclonal antibody. Similar to the endogenous p55, the expressed p55 is localized on endoplasmic reticulum and nuclear envelope. Moreover, p55 was specifically labeled by N-bromoacetyl-3,3',5-triiodo-L-thyronine. Thus, the expressed p55 is structurally indistinguishable from the endogeneous p55. pHTBr was packaged into a virus with the aide of an amphotropic virus. Infection by pHTBr-containing virus yielded a 2-11 fold higher expression than the endogeneous p55 in NIH3T3, rat GH3, human HepG2 cells and a mouse monoclonal antibody secreting hybridoma.     

Cell Line Derived from a Murine Sarcoma Virus (Moloney Pseudotype)-Induced Tumor: Cultural, Antigenic, and Virological Properties

A cell line derived from a murine sarcoma virus (Moloney pseudotype)-induced tumor has been established. It retains oncogenicity, releases both sarcoma and leukemia viruses, and has virus-induced cellular antigens.     

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen.

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into a retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of psi 2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10(6) cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.     

Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185

The neu oncogene, which is frequently activated in neuro- and glioblastomas of BDIX rats, was originally identified in the NIH 3T3 focus-forming assay. cDNA clones of the normal and transforming alleles of neu have been isolated. When these clones are inserted into the expression vector pSV2, they direct the synthesis of p185, the neu gene product. The transforming cDNA clone yields foci when transfected onto a NIH 3T3 monolayer, but the normal cDNA does not. The construction of in vitro recombinants between the normal and transforming cDNAs has allowed the determination of the mutation responsible for the activation of the neu proto-oncogene. A single point mutation changes a valine in the transmembrane domain of the predicted protein product insert to a glutamic acid. The DNAs from four independent cell lines containing activated neu oncogenes contain the identical mutation at this position.