Analysis of viral DNA sequences in hamster cells transformed by herpes simplex virus type 2.

Herpes simplex virus type 2 (HSV-2) DNA was treated with four restriction endonucleases (EcoRI, HincIII, Bgl II, and Xba I) and eight fragments were purified and labeled with 32P in vitro. The kinetics of renaturation of each of the fragments was measured in the presence of DNA extracted from 333-8-9, a hamster cell line transformed by UV light-inactivated HSV-2 strain 333, and from a series of cloned derivatives and their tumor lines. All of the lines examined contained a partial set of viral sequences present at only a few copies per cell. Passage of the cell lines in tissue culture or in animals resulted in partial loss of viral DNA. Two blocks of sequences were present in most of the lines examined; those mapping at positions 21--33 of the HSV-2 genome were detected in seven of seven cell lines tested and those at positions 60--65 were detected in six of eight. Other sequences from the L component can also be present in the DNA of HSV-2-transformed hamster cells.     

Reduction of caveolin and caveolae in oncogenically transformed cells.

Caveolae are flask-shaped non-clathrin-coated invaginations of the plasma membrane. In addition to the demonstrated roles for caveolae in potocytosis and transcytosis, caveolae may regulate the transduction of signals from the plasma membrane. Transformation of NIH 3T3 cells by various oncogenes leads to reductions in cellular levels of caveolin, a principal component of the protein coat of caveolae. The reduction in caveolin correlates very well with the size of colonies formed by these transformed cells when grown in soft agar. Electron microscopy reveals that caveolae are morphologically absent from these transformed cell lines. These observations suggest that functional alterations in caveolae may play a critical role in oncogenic transformation, perhaps by disrupting contact inhibition in transformed cells.     

Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression.

The adenovirus type 12 (Ad12) DNA sequences integrated into the DNA of four lines of Ad12-transformed hamster cells are extensively methylated. Methylation in mammalian cell DNA is believed to occur predominantly at 5'-C-G-3' sequences. The majority, although not all, of the 5'-C-C-G-G-3' sequences present in integrated Ad12 DNA are methylated. Ad12 DNA isolated from purified virions, on the other hand, is not methylated to any significant extent. The segments of the integrated viral DNA comprising early genes, which are expressed as mRNA in two lines of Ad2-transformed hamster cells, are undermethylated in comparison to late viral segments, which are not expressed and are extensively methylated. In contrast, in two lines of Ad12-induced rat brain tumor cells, some of the late viral genes have been shown to be expressed as mRNA. The segment of the integrated Ad12 DNA that comprises these late genes, the EcoRI B fragment, is undermethylated in comparison to the extensive methylation of the same fragment in Ad12-transformed hamster cells. Thus, there appears to exist a striking inverse correlation between the levels of methylation of specific DNA segments and the extent to which these segments are expressed as mRNA. The functional significance of this correlation remains to be determined. It may provide a clue to understanding the regulation of gene expression in transformed cells and perhaps in eukaryotic cells in general.     

Mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral DNA sequences.

The viral DNA sequences in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV-1) virions, by full-length linear BPV-1 DNA, or by a defined transforming subgenomic DNA segment of BPV-1 were examined by reassociation kinetics and blot hybridization. In all cases, the transformed cells contained multiple copies of BPV-1 DNA, present exclusively as supercoiled or nicked circular extrachromosomal molecules or as a slowly migrating complex of circular viral DNA molecules. In the transformed cell lines established from cells transfected with full-length linear BPV-1 DNA, there was recircularization of the input DNA which in some cases resulted in the loss of the restriction site used in the linearization of the DNA. In the transformed cell lines established with the defined subgenomic segment there was circularization of the DNA accompanied by the acquisition of new sequences or duplication and rearrangement of the BPV-1 sequences. In contrast to other well-studied virus transformation systems, no integration of the BPV-1 genome into the host chromosome could be detected under conditions sensitive enough to detect 0.1-0.2 viral genome equivalent. It was concluded that maintenance of transformation may be mediated by nonintegrated viral DNA.     

Molecular cloning of murine endogenous viral sequences and expression of a newly constructed recombinant murine leukemia virus DNA in transfected mink cells.

In the process of molecularly cloning unintegrated proviral DNA from NIH-3T3 mouse cells infected with Rauscher murine leukemia virus, we observed the occurrence of clones with inserts smaller than the expected Rauscher murine leukemia virus fragments. The insert of one of these clones, lambda.Xe-1, was characterized in more detail. It had a size of 3.5 kilobases. The restriction map was similar but not identical to that of the envelope regions of Moloney and Rauscher murine leukemia viruses. After ligation to previously cloned Moloney murine leukemia viral sequences and transfection of the ligated DNA into mink lung cells a nondefective xenotropic murine leukemia virus, XH-19, was isolated. Restriction mapping of proviral DNA isolated from mink lungs cells chronically infected with XH-19 showed the presence of Moloney murine leukemia virus-derived sequences coupled to xenotropic viral sequences.     

Integration of avian sarcoma virus DNA sequences in transformed mammalian cells.

DNA from six avian sarcoma virus (ASV)-transformed mammalian cell lines was digested with the restriction endonucleases EcoRI, Xho I, or Sal I, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filter strips, and hybridized with specific ASV [32P]cDNA probes. DNA from all of the ASV-transformed cell lines yielded three common virus-specific DNA fragments (2.4, 1.8, and 1.3 X 10(6) daltons) upon cleavage with EcoRI. Xho I appeared to cleave at least once within the integrated provirus and yielded a common fragment of 3.3 X 10(6) daltons as well as a second virus-specific DNA fragment whose size varied from 4.0 to 5.0 X 10(6) daltons in the different transformed cell lines. Sal I did not cleave within the provirus and yielded a single major virus-specific fragment of about 11 X 10(6) daltons in all transformed lines examined. Using specific cDNA probes, we show that the 1.8 X 10(6)-dalton EcoRI fragment contains sequences homologous to the 3' end of the viral RNA as well as to the src region of the viral genome. These studies clearly demonstrate that the same region on the ASV genome is utilized for provirus integration in different ASV-transformed cell lines.     

Evolution of type C viral genes: preservation of ancestral murine type C viral sequences in pig cellular DNA.

Domestic pigs (Sus scrofa) and other members of the family Suidae have multiple copies of type C viral gene sequences in the cellular DNA of all their tissues. Partially homologous viral gene sequences are also found in cellular DNA of rodents, particularly Muridae. The results lead to the conclusion that type C viral genes were introduced into the Suidae lineage as a result of trans-species infection by an ancestral xenotropic murine virus. The rate of evolution of the virogene sequences in the pig appears to be much slower than that of genes that have remained in the rodent lineage; this may be a consequence of transfer from a shorter-lived animal (the rodent) to a longer-lived one (the pig). We estimate the time of gene transmission as 5-10 million years ago and conclude that the present-day porcine type C virogenes most closely approximate the viral genes as they were several million years ago in the rodent lineage.     

Amplification of integrated viral DNA sequences in polyoma virus-transformed cells.

Polyoma virus (Py) transformation of rat cells requires integration of viral genomes into the host DNA, which generally occurs in a partial or full head-to-tail tandem arrangement. The instability of this structure was previously demonstrated by the high rate of loss of integrated Py genomes in the presence of viral large tumor (T) antigen. We now show that integrated Py DNA sequences can also undergo amplification. We studied two rat cell lines transformed by the ts-a Py mutant, which codes for a thermolabile large T antigen. In a derivative of the ts-a H6A cell line, we have observed loss of full-length Py DNA molecules from the integrated tandem ("curing"), accompanied by the creation of new tandem repeats of two segments of viral DNA corresponding to 38% and 10% of the viral genome, each containing the origin of DNA replication. In the ts-a H3A cell line, which contains an integrated partial tandem of about 1.3 viral genomes with three distinct deletions, propagation at 33 degrees C resulted in the generation of full tandem repeats of a 94% Py DNA "unit" (including two 3% deletions), an 85% "unit" (including a 3% and the 12% deletion), or both. Amplification of integrated viral DNA was not observed in cells propagated at 39.5 degrees C, the nonpermissive temperature for large T antigen function. Amplification of integrated Py DNA sequences thus requires an active large T antigen and can generate a full tandem of integrated viral DNA molecules long after the initial integration event.     

Restriction enzyme analysis of mouse cellular type C viral DNA: emergence of new viral sequences in spontaneous AKR/J lymphomas.

The topography of endogenous type C viral sequences in mouse cellular DNA was investigated by EcoRI nuclease restriction and application of the Southern blotting technique. The DNAs from one outbred and five inbred strains were resolved into 20-35 fragments containing viral sequences, distributed in unique, though related, patterns for each mouse strain. Different normal tissues from the same animal were indistinguishable in their DNA patterns, suggesting that tissue differentiation is not associated with gross alteration in the topography of endogenous type C virus sequences. Tumor tissues from spontaneous lymphomas of AKR/J mice were similarly analyzed. In four out of seven individual tumors we detected the emergence of one or two new virus-containing DNA fragments. The mass of these fragment varied, indicating different insertion sites of the new viral sequences. The detection of these new viral sequences suggests that each tumor was composed of descendents of only one or a few cells.     

Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases.

The DNAs from five independent simian virus 40 (SV40) transformants of the BALB/c3T3 mouse cell line were digested with either the HpaII or the BamHI restriction endonuclease and the resulting fragments were fractionated by gel electrophoresis. The DNA fragments were denatured in situ in the gel and transferred to a membrane filter. Fragments containing viral DNA were detected by hybridization with high specific activity 32P-labeled SV40 complementary RNA (cRNA) synthesized in vitro. Each of the lines yielded a small number of fragments containing SV40 DNA and the fragments from each line were different. This observation shows that the structure of the integrated SV40 DNA and/or its location in the host DNA are different in each line.     

Molecular cloning and characterization of DNA sequences encoding rat and human atrial natriuretic factors.

A cDNA copy of the message encoding rat atrial natriuretic factor (ANF) has been cloned in Escherichia coli, and its nucleotide sequence was determined. ANF appears to be synthesized as a larger precursor, atrial pronatriodilatin. The cDNA has an open reading frame potentially encoding a protein of 152 amino acids, of which the first 24 amino acids strongly resemble a signal sequence. This is followed by a sequence with 80% homology to a second vasoactive protein, porcine cardiodilatin. The ANF peptide is contained in the COOH-terminal portion of the protein. The DNA sequence corresponding to human ANF is also presented and displays a high degree of homology to its rat counterpart. These data provide further evidence for the expression in cardiac atria of a multifactor system that may contribute to the regulation of blood pressure and extracellular fluid volume.     

Immunologic selection against simian virus-40 transformed cells: Concomitant loss of viral antigens and early viral gene sequences

A clonal line of highly oncogenic "spontaneously transformed" mouse cells (T AL/N clone 3) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned. The clone of SV40-transformed cells (subclone 1) expressed SV40-specific T (nuclear) and transplantation antigens but was 100 times less tumorigenic than the parent T AL/N clone 3 cells. When large numbers of subclone 1 cells (10(4)-10(5)) were injected into syngeneic AL/N mice, tumors were produced. From the tumors, cell lines were established in culture, all of which were consistently negative for T antigen. Tumor lines tested were found not to contain SV40-specific transplantation antigen and had again become highly tumorigenic. The original subclone 1 cells contained about one copy of SV40 DNA per diploid amount of cell DNA, as well as RNA complementary to the early region of the SV40 genome. The T antigen-negative cells from tumor line 124 contained approximately 0.5 copy of SV40 DNA per diploid equivalent and did not synthesize any detectable virus-specific RNA. Reassociation kinetic analysis with restriction enzyme fragments of viral DNA demonstrated that the cells from tumor line 124 (and also the clones of this line) had lost DNA sequences predominantly from the early region of the SV40 genome. The results indicate that a set of stably integrated SV40 DNA sequences can be present in a cell without the expression of viral antigens.     

Baboon endogenous virus genome: molecular cloning and structural characterization of nondefective viral genomes from DNA of a baboon cell strain.

Several heterogeneities in the baboon endogenous virus (BaEV) genomes that are present in the DNA of normal baboon tissues and the baboon cell strain BEF-3 have been described previously. To study these genomes, we cloned BaEV proviruses from BEF-3 cellular DNA into the lambda vector Charon 4A. Of the four full-length clones isolated, one was nondefective as determined by transfection. The sequence of a portion of this clone was found to code for amino acids 61-91 in the p30 region of the gag gene. This identification allowed us to align the restriction map with the BaEV genetic map. One heterogeneity, a BamHI site 2.4 kilobases (kb) from the proviral 5' end, was located close to the gag-pol junction; another, a BamHI site 1.4 kb from the 5' end of the genome, corresponded to the gag p30 coding sequence for amino acids 32-34; and a third, a Xho I site, was near the 3' end of the pol gene. To select the nondefective BaEV genomes from BEF-3 cells, we infected permissive cells with virus produced by BEF-3 cells and also transfected BEF-3 cellular DNA into permissive cells. The BaEV genomes in the permissive recipient cultures were then analyzed by restriction enzyme analysis. These nondefective genomes were found to be heterogeneous with respect to the gag-pol BamHI site and the Xho I site, but all were found to contain the BamHI site 1.4 kb from the 5' end of the genome.     

Instability of integrated viral DNA in mouse cells transformed by simian virus 40

The state and organization of simian virus 40 (SV40) DNA in tsA mutant-transformed mouse clones were examined early after agar selection in an attempt to elucidate the mechanisms that actively generate the diverse integration patterns found in transformed cells. Although recently selected as a cloned population from agar, A21 cells displayed extremely heterogeneous SV40 DNA patterns when analyzed by agarose gel electrophoresis and Southern blot hybridization. Reselection of clones in agar from A21 at 33 degrees C or 39.5 degrees C and DNA analysis by hybridization demonstrated (i) simplification of the number of integration sites in the new clones; (ii) new sites of integrated SV40 DNA in high molecular weight cell DNA fragments generated by digestion with restriction endonuclease Bgl II; (iii) relatedness between clones with respect to integrated viral sequence arrangement; and (iv) persistence of free viral DNA forms. The majority of free viral DNA appeared to be full length, nondefective SV40 DNA, although a subpopulation of defective viral molecules was also detected. No detectable free SV40 DNA could be observed in A21 clonal derivatives isolated by growth in agar at 39.5 degrees C, indicating that the persistence of free viral forms was regulated by the A gene. These results suggest that the heterogeneity in viral sequences in the A21 cells was generated within a cloned population from which new clones can be derived with different transformed phenotypes and integration patterns.     

Internal organization of long repetitive DNA sequences in sea urchin genomes.

In keeping with earlier reports, we have found that reassociated long repeat DNA from sea urchins is thermostable, indicating the absence of evolutionarily diverged families of repeated sequences. However, we found that when fragments of radiolabeled long repeat DNA were denatured and reassociated with intact long repeat driver DNA, then sheared to 350 basepairs and assayed for thermal stability, the level of mismatch found in the duplexes varied inversely with the length of the starting fragments. This effect was shown to be due directly to the physical size of the molecules involved in reassociation. These results are consistent with, and support a model for, long repeat DNA in which short units of repetition are arranged in precise arrays. The significance of this arrangement of sequence units within long repeat DNA is discussed.     

Inverted repeat nucleotide sequences in the genomes of Marek disease virus and the herpesvirus of the turkey.

The DNAs of two herpesvirus, the oncogenic Marek disease virus and the serologically related herpesvirus of the turkey, were studied by electron microscopy. On the basis of fold-back molecules observed in single-stranded DNA from both viruses, structures have been derived from the overall nucleotide sequence arrangement in their genomes. Although differing in molecular weight, the genomes of Marek disease virus and turkey herpesvirus are both constructed according to the same plan--two regions of unique nucleotide sequence, each enclosed by inverted repeat sequence. The genome structure of these viruses therefore closely resembles that of herpes simplex virus rather than the biologically more similar herpesvirus Epstein--Barr virus, H. saimiri, and H. ateles.