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M Botchan

Publications and source records attributed to M Botchan.

At least 37 records · Page 2Linked to original sources
PubMed1986

Induction of specific transcription by RNA polymerase III in transformed cells.

RNA polymerase III (pol III) transcripts of the highly repeated mouse B2 gene family are increased in many oncogenically transformed murine cell lines. In cells transformed by simian virus 40, the small, cytoplasmic B2 RNAs are present at 20-fold-higher levels than in normal cells (M. R. D. Scott, K. Westphal, and P. W. J. Rigby, Cell 34:557-567, 1983; K. Singh, M. Carey, S. Saragosti, and M. Botchan, Nature [London] 314:553-556). We found that transcripts of the highly repeated B1 gene family are also increased 20-fold upon simian virus 40 transformation and showed that these RNAs result from pol III transcription. In contrast, transcripts from less highly repeated pol III templates such as the 5S, 7SL, 7SK, 4.5SI, tRNAMet, and tRNAPro genes are unaffected. The expression of the B2 RNAs in isolated nuclei shows that the augmentation is due mainly to an increased rate of transcription by pol III. There is thus specific transformation-inducible pol III transcription. We developed an in vitro transcription assay which utilizes genomic DNA as a template to study the transcription of all members of a repetitive gene family in their native context. This assay reproduces the low cytoplasmic levels of B1 compared with B2 RNAs suggesting that this ratio is dictated by intrinsic signals in the DNA.

Animals↗
PubMed1985

Association of crossover points with topoisomerase I cleavage sites: a model for nonhomologous recombination.

Nonhomologous DNA recombination is frequently observed in somatic cells upon the introduction of DNA into cells or in chromosomal events involving sequences already stably carried by the genome. In this report, the DNA sequences at the crossover points for excision of SV40 from chromosomes were shown to be associated with eukaryotic topoisomerase I cleavage sites in vitro. The precise location of the cleavage sites relative to the crossover points has suggested a general model for nonhomologous recombination mediated by topoisomerase I.

Animals↗
PubMed1985

Isolation of cellular genes differentially expressed in mouse NIH 3T3 cells and a simian virus 40-transformed derivative: growth-specific expression of VL30 genes.

We constructed and screened a cDNA library made from simian virus 40 (SV40)-transformed NIH 3T3 cells, and we isolated cDNAs representing genes that are differentially expressed between the parental cell and its SV40-transformed derivative. We found only a small number of cDNAs representing such genes. Two isolated cDNA clones represented RNAs expressed at elevated levels in the transformed cell line in a manner relatively independent of growth conditions. The expression of two other cDNAs was growth specific because transformed cells and nonconfluent parental cells contained higher levels of the homologous RNAs than did confluent, contact-inhibited parental cells. Another cDNA was well expressed in confluent parental and confluent transformed cells, but not in nonconfluent cells. The expression of some of these cDNAs varied strikingly in different mouse cell lines. Thus the genotype or histories of different cell lines can also affect the expression of certain genes. Interestingly, the only cDNA isolated that was expressed exclusively in the transformed cell was from an SV40 message. We focused on a growth-specific cDNA which we show is derived from a mouse endogenous retrovirus-like family called VL30. We sequenced the 3' long terminal repeat (LTR) of this transcriptionally active VL30 gene. This LTR has good homology with other VL30 LTR sequences, but differences occur, particularly upstream of the VL30 promoter. We found that VL30 gene expression varied in different mouse cell lines such that C3H cell lines had very low levels of VL30 transcripts relative to NIH 3T3 cell lines. However, Southern analysis showed that both cell lines had about the same number of VL30 genes homologous to our probe and that the position of the majority of these genes was conserved. We discuss possible explanations for this difference in VL30 expression.

Animals↗
PubMed1984

DNA sequence studies of simian virus 40 chromosomal excision and integration in rat cells.

Cell fusion between simian CV1 cells and the simian virus 40-transformed rat cell line 14B, which contains a single copy of integrated simian virus 40 DNA, results in chromosomal excision of viral DNA. A heterogeneous population of circular molecules containing both viral and cellular DNA is detected in the replicating pool. We present the DNA sequences across six novel junctions created by these excision events and use this information to define the parental genomic sequences involved in this form of "illegitimate" recombination. The data were analyzed to discover whether any common structural feature(s) could be detected at these sites. In each case a redundancy of either two or three base-pairs was found at the precise points of cross-over in both parental DNA molecules. The cross-over points were further distinguished by the presence of at least one copy of the sequence 5'Pyr-T-T3' in either of the homologous sequences that define the cross-over points. Additional stretches of homology are found extending from the homologous cross-over points. To explore the possibility that the selection of the cross-over sites is determined by the free energy of base-pairing, we have used the program of Zuker & Stiegler (1981) to form model heteroduplexes between single-stranded parental DNA molecules. In some cases model heteroduplexes were formed that paired the cross-over points, although these structures were of dubious thermodynamic stability. We therefore conclude that, while the redundancies at the cross-over points must play some role in these processes, other factors aside from simple base-pairing across replicating structures must also be involved. In order to expand our analysis of the recombination events that accompany transformation of rat cells by simian virus 40, we determined the DNA sequences across one of the sites on the rat genome that served as the target for the integration event that engendered the 14B line. Our analysis of this DNA showed that: (1) viral and chromosomal DNA share three base-pairs of homology at the site of cross-over; (2) the cross-over site in the rat genome is adjacent to the trinucleotide 5'Pyr-T-T3'; and (3) the homology shared by the virus and chromosome does not resemble the homology reported at another integration locus, but is similar in that it is flanked on one side by alternating purine and pyrimidine nucleotides.

Animals↗
PubMed1984

Transformation mediated by the SV40 T antigens: separation of the overlapping SV40 early genes with a retroviral vector.

A murine retroviral vector has been used to separate physically the overlapping genes encoded by SV40. This minimal retroviral vector contains LTRs and other cis-acting signals required for infectious RNA virus propagation. We placed the SV40 early region within this DNA and after transfection of cells producing helper Moloney murine leukemia virus, SV40 retroviruses (MV40) could be rescued. Cytoplasmic spliced large T and small t transcripts, as well as unspliced transcripts, are packaged into virions with equal efficiency. Pure SV40 large T retroviruses can be cloned from these heterogeneous virus stocks by secondary transformation of rodent cells. The large T retrovirus stocks morphologically transform primary or established mouse and rat lines with high efficiency. There is little difference in transformation either by agar assay or focus formation between retroviruses carrying both SV40 genes or large T alone. We present quantitative data that demonstrate that abortive transformation of rodent cells by SV40, transient expression of the transformed phenotype after infection, is not manifested by MV40. Thus abortive transformation is not the result of a weakly dominant transforming gene, but rather of the normally inefficient mode of integration and early gene expression of SV40 upon infection of rodent cells.

Antigens, Viral, Tumor↗
PubMed1984

Simian virus 40 T antigen is required for viral excision from chromosomes.

We describe experiments that show that simian virus 40 (SV40) T antigen is required for viral excision from host chromosomes at some point prior to or during the homologous recombination events that create circular wild-type virus. Two recombinant SV40-pBR322 plasmids were constructed such that homologous recombination across similar-sized but different duplications of SV40 would reconstitute wild-type viral DNA. One plasmid (pSVED) was constructed such that the duplication separates the viral early T-antigen promoter from the coding sequences; the other recombinant (pSVLD) contains a duplication of the late viral sequences and thus maintains a complete T-antigen gene. These plasmids were individually established in Rat 2 cells via cotransformation with the herpes virus Tk gene. Both classes of cell lines contained integrated tandem arrays of the plasmids and yielded equivalent levels of infectious virus after cell fusions with COS-7 cells; however, only the T+ lines yielded virus after cell fusion with CV-1 cells. These results are consistent with the notion that viral excision is initiated by T-antigen-mediated in situ replication of viral DNA as proposed in the "onion skin" model. In contrast, both plasmids yielded infectious virus when transiently introduced via transfection into CV-1 cells. This latter finding is discussed in terms of the possible induction of cellular repair and recombination pathways evoked by the introduction of damaged DNA into the nucleus.

Animals↗
PubMed1983

Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat.

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.

Animals↗
PubMed1983

Bovine papilloma virus contains an activator of gene expression at the distal end of the early transcription unit.

Bovine papilloma virus (BPV) contains a cis-acting DNA element which can enhance transcription of distal promoters. Utilizing both direct and indirect transient transfection assays, we showed that a 59-base-pair DNA sequence from the BPV genome could activate the simian virus 40 promoter from distances exceeding 2.5 kilobases and in an orientation-independent manner. In contrast to the promoter 5'-proximal localization of other known viral activators, this element was located immediately 3' to the early polyadenylation signal in the BPV genome. Deletion of these sequences from the BPV genome inactivated the transforming ability of BPV recombinant plasmids. Orientation-independent reinsertion of this 59-base-pair sequence, or alternatively of activator DNA sequences from simian virus 40 or polyoma virus, restored the transforming activity of the BPV recombinant plasmids. Furthermore, the stable transformation frequency of the herpes simplex virus type 1 thymidine kinase gene was enhanced when linked to restriction fragments of BPV DNA which included the defined activator element. This enhancement was orientation independent with respect to the thymidine kinase promoter. The enhancement also appeared to be unrelated to the establishment of the recombinant plasmids as episomes, since in transformed cells these sequences are found linked to high-molecular-weight DNA. We propose that the enhancement of stable transformation frequencies and the activation of transcription units are in this case alternate manifestations of the same biochemical events.

Base Sequence↗
PubMed1980

Integration of viral DNA sequences in cells transformed by adenovirus 2 or SV40.

We have cloned and propagated in prokaryotic vectors the viral DNA sequences that are integrated in a variety of cells transformed by adenovirus 2 or SV40. Analysis of the clones reveals that the viral DNA sequences sometimes are arranged in a simple fashion, collinear with the viral genome; in other cell lines there are complex arrangements of viral sequences in which tracts of the viral genome are inverted with respect to each other. In several cases the nucleotide sequences at the joints between cell and viral sequences have been determined: usually there is a sharp transition between cellular and viral DNAs. The viral sequences are integrated at different locations within the genomes of different cell lines; likewise there is no specific site on the viral genomes at which integration occurs. Sometimes the viral sequences are integrated within repetitive cellular DNA, and sometimes within unique sequences. In some cases there is evidence that the viral sequences along with the flanking cell DNA have been amplified after integration. The sequences that flank the viral insertion in the line of SV40-transformed rat cells known as 14B have been used as probes to isolate, from untransformed rat cells, clones that carry the region of the chromosome in which integration occurred. Analysis of the structure of these clones by restriction endonculease digestion and heteroduplex formation shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration.

Adenoviruses, Human↗
PubMed1980

Characteristics of an SV40-plasmid recombinant and its movement into and out of the genome of a murine cell.

A bacterial plasmid carrying the early region of SV40 (pOT) has been stably established in high molecular weight (hmw) DNA of mouse L cells by selection for the herpes virus thymidine kinase (tk) gene. DNA blotting has demonstrated that most cell lines contain multiple discrete copies of pOT, generally with an intact SV40 early region. No free copies of pOT have been detected. Both pOT and tk sequences may be amplified up to 20-200 copies of the SV40 early region. In contrast to the uniform staining pattern normally observed in SV40-transformed lines, indirect immunofluorescence using antiserum to the SV40 T antigen has demonstrated that the expression of the early region is heterogeneous in these cell lines. This fraction expressing T is characteristic of a given cell line, and varies from 0 to 99% positive. Several pOT cell lines have been fused to simian cells, and replicating low molecular weight DNAs were isolated from the heterokaryons. Transformation of E. coli with this DNA demonstrates that pOT can be rescued from hmw DNA in L cells and reestablished as a plasmid in E. coli. Excision is generally precise when pOT is introduced to the murine cells as supercoiled molecule, and imprecise when pOT is introduced in linear form.

Animals↗
PubMed1979

Retransformation of a simian virus 40 revertant cell line, which is resistant to viral and DNA infections, by microinjection of viral DNA.

We have isolated morphological transformants of cultured cells as dense foci on a monolayer of normal cells appproximately 4 weeks after microinjection of purified simian virus 40 DNA (200 to 400 molecules per cell) directly into the nucleus. Both Rat 1 (an established contact-inhibited rat embryo fibroblast line) and F1' 1--4 (a 5-fluorodeoxyuridine-selected flat revertant from the simian virus 40-transformed 14B cell line) were transformed with an efficiency of 5 to 10% of the cells injected. F1' 1--4 is not susceptible to retransformation by either viral or DNA infection (by calcium phosphate-facilitated cellular uptake), and as a result it had previously been thought to possess a host mutation preventing expression of the simian virus 40 genome.

Animals↗
PubMed1978

Isolation and characterization of T antigen-negative revertants from a line of transformed rat cells containing one copy of the SV40 genome.

Negative selection with FUdR produced revertants from the transformed rat line 14B, which contains one insertion of the SV40 viral genome (Botchan, Topp and Sambrook, 1976). 14B contains nuclear T antigen, grows to a high density, grows in low serum and is anchorage-independent. The revertants fall into three classes with regard to viral DNA sequences: the SV40 DNA is retained; the SV40 DNA is retained but has undergone a deletion; and the SV40 DNA is lost, generating a cured cell. This heterogeneity is not a result of long-term passage. The revertants arise with a frequency of one in 8.4 X 10(5) cells after as few as 12 passages. All three classes of revertants are T antigen-negative, density-sensitive, more serum sensitive than 14B and anchorage-dependent. These data argue for a direct role of the functioning viral genome in the maintenance of the transformed state, and that with 14B, the phenotypes of transformation are not virus gene dosage-dependent.

Antigens, Viral↗
PubMed1976

The arrangement of simian virus 40 sequences in the DNA of transformed cells.

High molecular weight DNA, isolated from eleven cloned lines of rat cells independently transformed by SV40, was cleaved with various restriction endonucleases. The DNA was fractionated by electrophoresis through agarose gels, denatured in situ, transferred directly to sheets of nitrocellulose as described by Southern (1975), and hybridized to SV40 DNA labeled in vitro to high specific activity. The location of viral sequences among the fragments of transformed cell DNA was determined by autoradiography. The DNAs of seven of the cell lines contained viral sequences in fragments of many different sizes. The remaining four cell lines each contain a single insertion of viral DNA at a different chromosomal location. The junctions between viral and cellular sequences map at different places on the viral genome.

Base Sequence↗