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M R Botchan

Publications and source records attributed to M R Botchan.

50 records · Page 3Linked to original sources
PubMed1986

Transient replication of bovine papilloma virus type 1 plasmids: cis and trans requirements.

A transient assay has been used to study bovine papilloma virus type 1 (BPV-1) replication. We show that BPV-1 early replication occurs faster than cellular DNA synthesis. Initial replication events are dependent on a gene product(s), encoded by the BPV-1 E1 open reading frame. Mutational analysis of the viral upstream regulatory region shows the requirement of two domains in cis for replication. Domain one, located outside of the viral 69% transforming fragment, is an enhancer-like activity and can be replaced by other known viral enhancers. Domain two lies within sequences previously defined as plasmid maintenance sequence 1. The apparent requirement for a proximal enhancer function for replication may explain why certain BPV-1 constructions, when linked to bacterial plasmid sequences, can be maintained extrachromosomally while others cannot.

Bovine papillomavirus 1↗
PubMed1986

A bovine papillomavirus type 1-encoded modulator function is dispensable for transient viral replication but is required for establishment of the stable plasmid state.

A bovine papillomavirus (BPV) type 1-encoded function (M) which is a negative regulator of viral plasmid replication has been described elsewhere (Berg et al. Cell, in press; Roberts and Weintraub, Cell, in press). We report here that expression of M, which is a repressor of transient BPV replication and is not required as a positive factor in these assays, is required for the establishment of the viral genome as a stable nuclear plasmid. This function is encoded in part by the 5' portion of the BPV E1 open reading frame, whereas the 3' part of this open reading frame encodes a positive replication function (R). The R function is required for early replication events. We used transient replication assays to define the phenotypes of mutants in both the R and M genes and complementation tests to show that R and M define two separate genes. We showed that R- and M- mutants could also complement each other in stable assays. In cotransfection experiments, M- mutants had a lethal effect on the growth of G418-resistant colonies, and in addition their morphological transformation efficiencies were reduced. The rare colonies which did appear contained the mutant DNA integrated into the cellular genome. R- mutants transformed with wild-type efficiency, and the mutant DNA was also found integrated. When cotransfected, R- and M- mutants could each be established as unrearranged plasmids.

Animals↗
PubMed1986

trans Activation of the simian virus 40 enhancer.

We describe experiments which demonstrated that the simian virus 40 (SV40) enhancer affects certain transcriptional units differently. We also found that a specific enhancer-transcriptional unit interaction can be regulated by trans-acting factors. Using transient assays, we examined the effects of the SV40 enhancer on herpesvirus thymidine kinase (tk) RNA levels when transcription was initiated either by the herpesvirus tk promoter or by an SV40 early promoter-tk fusion. We were unable to detect any effect of the enhancer on transcription from the tk promoter in CV-1 or HeLa cells. However, we found that the addition of T-antigen in trans allowed the enhancer to stimulate expression from the tk promoter. This induction by T-antigen did not require T-antigen-binding sites in cis and appeared to be an indirect effect. In contrast, tk expression from the SV40 early promoter fusion was greatly stimulated by the enhancer in CV-1 cells. Furthermore, in 293 cells the SV40 enhancer had only a marginal effect on the SV40 promoter-tk fusion, whereas it strongly stimulated tk expression from the tk promoter. Our results raise the possibility that the enhancer function may not show cell specificity per se; rather, the interaction between the enhancer and a specific gene may be responsible for cell specificity. We discuss these observations in terms of the SV40 early gene-to-late gene switch that occurs during SV40 lytic growth.

Animals↗
PubMed1985

Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors.

The establishment of bovine papillomavirus type 1 in somatic mammalian cells is mediated by extrachromosomal replication and stable maintenance of the viral genome as a multicopy nuclear plasmid. Previous studies indicated the requirement of viral gene expression for bovine papillomavirus type 1 replication and plasmid maintenance (M. Lusky and M. R. Botchan, Cell 36:391-401, 1984; Turek et al., Proc. Natl. Acad. Sci. U.S.A. 79:7914-7918, 1982). To define the viral genes which are necessary for this process, we constructed a series of specific mutations within the viral genome and assayed the resulting mutants for their ability to replicate extrachromosomally in mouse C127 cells. We report here that the bovine papillomavirus type 1 trans-acting replication factors were encoded by at least two distinct viral genes since the mutants fell into two complementation groups, rep and cop. Mutants (rep-) affecting the E1 open reading frame (ORF) failed to replicate bovine papillomavirus type 1 DNA extrachromosomally and would integrate into chromosomal DNA. We suggest that this gene product is one of the factors required to specifically preclude the integration event. Mutants (cop-) affecting the E7 ORF were maintained in the extrachromosomal state; however, the copy number of the mutant genomes was reduced 100-fold compared with that of wild-type DNA. Analysis of single-cell subclones showed that each cell contained the mutant genomes at a copy number of one to two, indicating that the cop- phenotype did not reflect a simple segregation defect. We propose that the gene defined by mutations in the E7 ORF played a crucial role in stably maintaining the copy number of the viral plasmid at high levels. Genomes with mutations in the cop and rep complementation groups, when cotransfected, rescued the wild-type phenotype, extrachromosomal replication with a high, stable copy number for both types of plasmids. Therefore, the gene products acted in trans, and the mutations were recessive to the wild-type functions. One specific rep- mutant showed a 30-fold-increased transformation efficiency when compared with that of the wild-type genome. In addition, morphological transformation mediated by the cop- mutants appeared to be unstable. These results imply that either or both of the replication functions played some role in regulating the expression of the viral transforming functions.

Animals↗
PubMed1985

Amplification of a bovine papillomavirus-simian virus 40 chimera.

A chimeric plasmid, pBOP, containing bovine papillomavirus (BPV) and the origin of replication from simian virus 40 (SV40) was constructed. The plasmid was established in mouse cells, where it was maintained stably as an autonomous BPV replicon. Lines carrying pBOP were fused to cells of COS-7, a simian line producing SV40 T antigen. Replication dependent on the SV40 origin and having the kinetics and approximate amplitude of an SV40 infection ensued. SV40 replication is therefore dominant over BPV replication, and the SV40 origin can conveniently be used to amplify lower-copy-number plasmids in mammalian cells.

Animals↗
PubMed1985

DNA-mediated gene transfer efficiency is enhanced by ionizing and ultraviolet irradiation of rodent cells in vitro. I. Kinetics of enhancement.

The enhancement effects of ionizing and ultraviolet radiation on the efficiency of DNA-mediated gene transfer were studied. The established cell line, Rat-2, consists of cells that are density-dependent contact-inhibited and produce flat monolayers in vitro. When these cells are infected with SV40 virus, a small fraction of cells becomes morphologically "transformed" due to the stable expression of the viral A-gene. Rat-2 cells are competent for DNA-mediated gene transfer, deficient in thymidine kinase activity (TK-), and will die in HAT selective media. Confluent Rat-2 cells were transfected with purified SV40 viral DNA (via calcium phosphate precipitation), irradiated with either X rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were nonlinear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X rays or 330 MeV/amu argon particles at the Berkeley BEVALAC showed a higher frequency of HAT+ colonies/survivor than unirradiated transfected cells. In both cases the enhancement contained a linear and a higher order component in dose, but the argon ions were at least twice more efficient than X rays in producing enhancement per unit dose. Rat-2 cells transfected with pOT-TK5, X-irradiated, and assayed for either TK transformation or A-gene transformation showed the same dose dependence for radiation enhancement. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK transformation by both X rays and ultraviolet radiation. SV40 A-gene products are not necessary for the radiation enhancement of the efficiency of gene transfer. This in vitro system will be used to study the lesions produced by ionizing radiation on mammalian cell DNA that may act as substrates for integration of exogenously introduced plasmid DNA.

Animals↗
PubMed1984

An enhancer sequence from bovine papilloma virus DNA consists of two essential regions.

A comprehensive structural analysis of an enhancer sequence from bovine papilloma virus DNA is presented based on the construction and functional analysis of 20 mutant derivatives. The results, obtained in CV-1 tissue culture cells, show that this enhancer is a small genetic element--only 40 bp in length--that contains two essential regions. The two regions exhibit homology to each other and to DNA fragments from other viral genomes that also act as enhancers in the assay used. However, there is a certain latitude in the sequences that have enhancer activity in CV-1 cells, even within the critical regions. The results are discussed with respect to the model that enhancers are binding sites for tissue specific transcription factors. The formation of Z-DNA might be involved in the enhancement process. However, single base pair transitions in an 8 base pair stretch of alternating purines and pyrimidines within the BPV enhancer which conserve this pattern destroy enhancer function.

Animals↗
PubMed1984

Characterization of the bovine papilloma virus plasmid maintenance sequences.

Bovine Papilloma Virus (BPV-1) establishes itself as a multicopy nuclear plasmid in somatic mammalian cells in culture. We report here that two discontinuous regions within the viral genome can independently support extrachromosomal replication of the Tn5 neomycinr gene in cells that provide viral factors in trans. The viral plasmid maintenance sequences (PMS) act in cis and will integrate along with the marker gene in cell lines that do not provide BPV-1 gene products. PMS-1 is localized within a 521 bp region upstream of the BPV-1 early transcription unit; PMS-2 has been localized to a 140 bp region within the putative reading frame for the E1 protein of the viral genome. Recombinant plasmids carrying either of the PMS elements are unrearranged and stably maintained at a constant copy number supernumerary to the resident BPV-1 genomes even in the absence of selective pressure. Specific deletion mutants within the viral genome show that BPV-1 gene products required for morphological transformation are dispensable for plasmid maintenance. In mouse cells cotransformed with such deletion derivatives and an unlinked marker gene (neomycinr or Tk), the marker genes integrate into the host genome while the BPV molecules are nonselectively carried as nuclear plasmids. This result implies that the BPV-1 genome must have signals that specifically preclude integration in the presence of transacting factors.

Animals↗
PubMed1983

High mutation frequency in DNA transfected into mammalian cells.

The lacI gene of Escherichia coli was used to score mutation in mammalian cells of simian virus 40-based recombinant DNA vectors that provide for replication and selection in both bacterial and mammalian cells. Plasmid DNA was introduced into COS7 simian cells by DEAE-dextran transfection, allowed to replicate in the mammalian cells, and then returned to E. coli for analysis. Mutants in lacI were observed at frequencies of one to several percent, compared with a spontaneous mutation rate in E. coli of less than 10(-5). The lesions include a large number of base substitutions, in addition to deletions, duplications, and more complex rearrangements, including insertion into the plasmid of sequences originating in the host genome. We discuss possible sources of the high mutation frequency and its implications for experiments involving DNA transfer.

Animals↗
PubMed1982

Fragment spanning the SV40 replication origin is the only DNA sequence required in cis for viral excision.

A 311-base pair fragment containing the SV40 origin of replication was linked to the chicken thymidine kinase gene on a recombinant plasmid. This molecule was transfected into human 143 thymidine kinase-deficient (TK-) cells, and colonies positive for thymidine kinase were selected. When cell lines derived from these colonies were fused to permissive simian cells that produce SV40 T antigen, the recombinant plasmid excised itself from the human cellular genome and replicated with a high copy number per cell. These results show that this segment of the viral genome is the only sequence required in cis to mediate SV40 excision and replication upon fusion to permissive cells. In addition, we have shown that excised plasmids apparently identical to the input DNA can be efficiently rescued in Escherichia coli. SV40 excision and replication may therefore be useful for the recovery of cloned genes from eukaryotic cells.

Animals↗
PubMed1982

Isolation and characterization of human DNA fragments with nucleotide sequence homologies with the simian virus 40 regulatory region.

A recombinant library of human DNA sequences was screened with a segment of simian virus 40 (SV40) DNA that spans the viral origin of replication. One hundred and fifty phage were isolated that hybridized to this probe. Restriction enzyme and hybridization analyses indicated that these sequences were partially homologous to one another. Direct DNA sequencing of two such SV40-hybridizing segments indicated that this was not a highly conserved family of sequences, but rather a set of DNA fragments that contained repetitive regions of high guanine plus cytosine content. These sequences were not members of the previously described Alu family of repeats and hybridized to SV40 DNA more strongly than do Alu family members. Computer analyses showed that the human DNA segments contained multiple homologies with sequences throughout the SV40 origin region, although sequences on the late side of the viral origin contained the strongest cross-hybridizing sequences. Because of the number and complexity of the matches detected, we could not determine unambiguously which of the many possible heteroduplexes between these DNAs was thermodynamically most favored. No hybridization of these human DNA sequences to any other segment of the SV40 genome was detected. In contrast, the human DNA segments isolated cross-hybridized with many sequences within the human genome. We tested for the presence of several functional domains on two of these human DNA fragments. One SV40-hybridizing fragment, SVCR29, contained a sequence which enhanced the efficiency of thymidine kinase transformation in human cells by approximately 20-fold. This effect was seen in an orientation-independent manner when the sequence was present at the 3' end of the chicken thymidine kinase gene. We propose that this segment of DNA contains a sequence analogous to the 72-base-pair repeats of SV40. The existence of such an "activator" element in cellular DNA raises the possibility that families of these sequences may exist in the mammalian genome.

Base Composition↗
PubMed1978

Another chromosomal assignment for a simian virus 40 integration site in human cells.

Somatic cell hybrids derived from fusion of GM637, a human cell line transformed by simian virus 40, and mouse B82 cells were examined for simian virus 40 T antigen, V antigen, and viral DNA. All hybrid cell lines that contained viral DNA were T-antigen positive. Cells that did not have viral DNA were T-antigen negative. We determined that there is a single viral insertion in these hybrid cells. Correlation of T-antigen expression and viral DNA with the partial complements of the human genome retained in the hybrids shwed that the inserted viral genome is in human chromosome 8. The integrated viral DNA is stable; free viral DNA found in GM637 does not insert at other potential sites in the human genome.

Antigens, Viral↗