Nancy Quintrell

Transcription of mouse mammary tumor virus genes in tissues from high and low tumor incidence mouse strains.

Abstract Mouse mammary tumor virus-specific nucleotide sequences are present in the DNA of high and low tumor incidence mouse strains (Varmus et al., 1972). To examine regulation of these genes at the transcriptional level, we have measured and characterized the MMT-virus † -specific RNA present in tumors, lactating mammary glands, spleens and livers from several mouse strains. Single-stranded DNA complementary to MMT virus 70 S RNA was synthesized by virus-associated DNA polymerase and hybridized to large excesses of unlabeled cell RNA. This SS DNA is highly specific for MMT virus sequences and copied from at least 60% of the genome. Complete hybridization of MMT virus SS DNA to tissue RNA was observed, using optimum annealing conditions (0·6 m -NaCl, 68 °C) and a stringent assay for hybridization (digestion of unhybridized DNA with a single strand-specific nuclease). Under these conditions, we can achieve reproducible measurement of the amount of virus-specific RNA per cell. Hybrid formation was further documented by density gradient centrifugation, and the character of the hybrids was assessed by thermal denaturation studies. Our principal findings include: 1. (1) large but variable amounts of virus-specific RNA (30 to 1000 genome equivalents per cell) in virus-producing tumors and lactating mammary glands from strains GR, Rill, C3H/an and DBA/2 mice; 2. (2) low amounts of MMT virus RNA (1·7 genome equivalents per cell) in virusfree mammary tumors from strain BALB/c mice; 3. (3) large quantities of MMT virus RNA (1300 genome equivalents per cell) in Leydig cell tumors containing incomplete virus particles in the cytoplasm; 4. (4) strain-dependent variation in the amount of virus-specific RNA (1 to 200 genome equivalents per cell) in lactating mammary glands from low tumor incidence strains (129, C68, C57BL/6, I and BALB/c); 5. (5) low levels of MMT virus-specific RNA (0.1 to 1 genome equivalent per cell) in livers and spleens from high and low incidence strains; 6. (6) correlation between virus production and amount of MMT virus RNA in the progeny of backcrosses between strains GR and C57 BL/6 mice; 7. (7) accurate base pairing, as judged by thermal stability, in hybrids between MMT virus SS DNA and RNA from virus-producing tissues, and minor degrees (3 to 5%) of base mismatching in hybrids formed between MMT virus DNA and RNA from virus-negative tissues. The results indicate that transcriptional controls are important in regulation of MMT virus gene expression and that hormonal and genetic factors may influence transcriptional control.

The low molecular weight RNAs of Rous sarcoma virus

Abstract Chromatography of Rous sarcoma virus RNA on methylated albumin-kieselguhr has allowed the identification of four classes of intravirion RNA: a substantial amount of 4 S RNA, a discrete 7 S component, small quantities of 18 and 28 S RNA, and the 70 S RNA of the viral genome. The 4 S RNAs of the virus and its host cell have identical electrophoretic mobilities in 10% polyacrylamide gels and are methylated to the same extent. However, significant differences in nucleotide compositions are detectable. The virus apparently does not contain RNA corresponding to any of the other low molecular weight species of cellular RNA. The data are in accord with previous suggestions that RNA tumor viruses contain tRNA acquired from the host cell during assembly, but also indicate at least minor differences in composition between the cellular and viral 4 S RNA populations. The results of reconstruction experiments suggest that the viral 4 S RNA is not simply a contaminant derived from cellular debris.

Structure of viral DNA and RNA in mammalian cells infected with avian sarcoma virus.

Abstract The expression of retrovirus genes varies from one species of host to another and is probably regulated by cellular mechanisms. Thus, avian fibroblasts infected with avian sarcoma virus (ASV) produce virus and acquire a neoplastic phenotype, whereas ASV-infected mammalian (cells produce little or no virus and only rarely become neoplastic. We have explored the origins of these variations by analysing the viral DNA and RNA in several lines of mammalian cells infected by ASV. Our work was facilitated by recently developed techniques that permit the fractionation, detection and characterization of extremely small quantities of viral nucleic acids. Each line of ASV-infected mammalian cells contained either one or two copies of ASV provirus. Nevertheless, the amounts of stable viral RNA produced in these cells varied over a range of at least 100-fold. In permissive chicken cells, the four genes of ASV (gag, pol, env and src) appear to be expressed by means of three viral messenger RNAs (gag/pol, Mr 3.3 × 106; env, Mr 1.8 × 106; and src, Mr 1.1 × 106). The same viral mRNAs were found in ASV-transformed mammalian cells, although the relative amounts of the three species were different; the gag/pol mRNA predominated in permissive cells, the src, mRNA in mammalian cells. The previous description of these RNAs was revised by demonstrating that the src mRNA apparently contains a 5′ untranslated region composed of two distinct elements: a nucleotide sequence transposed from the 5′ end of the viral genome, and a nucleotide sequence contiguous to the 5′ boundary of src in the viral genome. We encountered several novel forms of viral mRNA: two distinctive src mRNAs in the same clone of cells; an env RNA appreciably larger than usual; and two mRNAs that appear to be encoded by the extreme right-hand end of the ASV provirus and adjacent cellular DNA. The first of these anomalies persisted when virus was rescued from the mammalian cells and then propagated in permissive cells, whereas the second and third anomalies were not apparent during the replication of rescued virus. Viral gene expression was attenuated in ASV-infected hamster cells that had reverted from a transformed to a normal phenotype: in particular, the amount of src mRNA was reduced by almost 100-fold. The revertant cells and their transformed siblings contained single identical proviruses located at apparently identical sites within the host genome. Thus, the change in viral gene expression that accompanies and perhaps causes reversion is not due to either translocation of the provirus or independent origins of the cell lines. We conclude that the enzymatic mechanisms for the genesis of ASV mRNAs are widely distributed among vertebrates: in particular, phylogenetically disparate host cells may utilize the same signals within the ASV genome for the splicing and polyadenylation of viral RNA: even anomalous signals can be interpreted identically across a broad phylogenetic range. However, the regulation of these mechanisms varies from one cell to another, and consistent differences may occur in the maturation of viral RNA within individual clones of transformed cells. In at least some instances, transcription may be initiated within the ASV provirus but continue uninterrupted into adjacent cellular DNA; thus, the mere insertion of viral DNA into the host genome might induce the expression of previously silent cellular genes. Attenuation of viral gene expression by the host cell can lead to reversion of the cell from a transformed to a normal phenotype. Our data indicate that the host modulates ASV gene expression principally by regulating the production of viral mRNAs. but the mechanism of this regulation remains to be elucidated.

Revertants of an ASV-transformed rat cell line have lost the complete provirus or sustained mutations insrc

Abstract We have isolated and characterized 12 revertants of a clonal line (B31) of avian sarcoma virus (ASV)-transformed rat-1 cells. The B31 cells contain a single normal ASV provirus, display the classical features of virally transformed cells, and revert to normal phenotype at low frequency. Revertants isolated after selective killing of transformed cells resemble uninfected rat-1 cells morphologically, fail to grow in suspension, and are at least 100-fold less tumorigenic than B31 cells. Two mechanisms of reversion have been identified in these cells. (i) Three of the revertant lines have lost the entire provirus, including both copies of the sequences repeated at the ends of the provirus; the manner in which the provirus is lost is not known. (ii) The other nine revertants retain a provirus of normal size and unaltered flanking cellular DNA: contain the same species of viral RNA at the same concentrations as in the parental line, B31; are susceptible to retransformation by wild-type ASV; and yield transformation-defective (td) virus after fusion with chicken cells. In one case, the rescued virus transforms chicken cells, but produces fusiform rather than normal foci and does not retransform rat cells morphologically. Hence these revertants arise as a consequence of nonconditional mutations (base substitutions or small deletions) in the viral transforming gene, src . In several cases, the revertant cells retransform spontaneously, or transforming virus appears in stocks of rescued td virus after passage through chicken cells, indicating back mutations to wild-type. Several of the rescued td viruses can also recombine to restore a wild-type phenotype. Analysis of the structure and enzymatic activity of products of src confirms that the revertant cells bear various mutations in src .

Homologies among the nucleotide sequences of the genomes of C-type viruses

Abstract DNA was synthesized with detergent-disrupted virions of several C-type viruses and used to measure the extent of homology among the genomes of these viruses by molecular hybridization. The DNA was reacted with viral RNA under conditions which permit saturation of most if not all complementary nucleotide sequences in the RNA. This technique provides a quantitative estimate of the extent of homology among viral RNAs and is superior to current procedures that measure the fraction of DNA hybridized to an excess of viral RNA. The genomes of RD-114 and Crandell virus are at least 85% related, whereas there is no detectable homology among the genomes of RD-114 virus, feline sarcoma-leukemia viruses, murine leukemia virus, avian sarcoma virus, and visna virus. The genome of feline sarcoma-leukemia viruses, like that of RD-114 virus, is at least partly homologous to DNA from normal cats, suggesting that normal cats harbor endogenous genes coding for components of at least two classes of C-type viruses.

SYNTHESIS OF VIRAL RNA IN CELLS INFECTED BY AVIAN SARCOMA VIRUSES

ABSTRACT. Cells infected with avian sarcoma virus synthesize viral RNA by transcribing an integrated DNA provirus. Transcription is catalyzed by an RNA polymerase of the host and may initiate outside of the provirus, generating a precursor which must be further processed in order to produce mature viral genome and messengers. Infected cells contain small amounts of RNA complementary to the viral genome, but the principal stable products of viral RNA synthesis are molecules with polarity identical to that of the viral genome. The production of viral messengers can be interrupted by the antibiotic cordycepin without major effect on primary transcription. Avian cells producing virus contain at least two separate classes of viral messenger RNA (mol. wts. ca. 3 & times; 10 6 and 1.3 & times; 10 6 ) which could permit independent expression of different viral genes. Mammalian cells transformed by avian sarcoma virus but not producing virus probably transcribe provirus into a large nuclear RNA (mol. wt. ca. 3 & times; 10 6 ), but only the smaller class of viral messenger (1.3 & times; 10 6 ) is detectable in cytoplasm and at least one viral gene (envelope glycoprotein) may not be represented in the messengers. Avian sarcoma virus-infected hamster cells which have reverted to a normal phenotype retain provirus and generate messenger for the viral transforming gene, but the amount of this message is substantially smaller than in the parental transformed cells; we can account for the revertant phenotype only by attributing it to a reduction in dose for the product of the viral transforming gene.

Mutants of rous sarcoma virus with extensive deletions of the viral genome

Abstract Deletion mutants of Rous sarcoma virus (RSV) have been isolated from a stock of Prague RSV which had been irradiated with ultraviolet light. Quail fibroblasts were infected with irradiated virus and transformed clones isolated by agar suspension culture. Three clones were obtained which did not release any virus particles. Analysis of DNA from these non-producer clones with restriction endonucleases and the Southern DNA transfer technique indicated that the clones carry defective proviruses with deletions of approximately 4 × 10 6 daltons of proviral DNA. The defective proviruses, which retain the viral transformation ( src ) gene, contain only 1.7–2.0 × 10 6 daltons of DNA. Multiple species of viral RNA containing the sequences of the src gene were detected in these clones; some of these RNAs may contain both viral and cellular sequences. The protein product of the src gene, p60 src ( Brugge and Erikson, 1977 ), was also synthesized in the nonproducer clones. However these clones did not contain the products of the group-specific antigen ( gag ), DNA polymerase ( pol ), or envelope glycoprotein ( env ) genes, nor did they contain the 35 and 28 S RNA species which are believed to represent the messengers for these viral gene-products. The properties of these mutants indicate that expression of the src gene is sufficient to induce transformation. These clones may represent useful tools for the study of the expression of this region of the genome.

Of birds and mice and men: Comments on the use of animal models and molecular hybridization in the search for human tumor viruses

Infection of either duck or mammalian cells with Rous sarcoma virus introduces a new set of genes into the chromosomal DNA of the host cell. This event is a prerequisite for both replication of the virus and neoplastic transformation of the cells. By contrast, virus‐induced mammary carcinoma in the mouse can be inherited disease without requirement for genetic information beyond that already present in the mouse genome. This communication illustrates certain molecular correlates of the preceding biological observations, and comments on the use of these correlates in designing and executing the search for RNA tumor viruses of man.