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C Basilico

Publications and source records attributed to C Basilico.

At least 109 records · Page 6Linked to original sources
PubMed1984

Deletion of the origin of replication impairs the ability of polyomavirus DNA to transform cells and to form tandem insertions.

We examined the transforming properties of polyomavirus DNA molecules which can produce a functional large T-antigen but which are cis defective for viral DNA replication. The inability of these molecules to replicate results from the deletion of sequences comprising the viral replication origin. We found that even in the presence of a functional large T-antigen, transformation of rat cells by these viral DNAs was greatly reduced when compared with replication-competent parental DNA, and cells transformed by origin-minus mutants generally contained the integrated viral DNA in a nontandem arrangement. Therefore, polyomavirus large T-antigen promotes the establishment of transformation and tandem integration by interacting with the viral origin of DNA replication. This indicates that viral DNA synthesis is directly involved in these processes.

Animals↗
PubMed1984

New rat cell line that is highly susceptible to transformation by several oncogenes.

We describe here a new cell line, EL2, which spontaneously arose from primary rat embryo fibroblasts and has the distinctive property of being highly susceptible to a number of different transforming genes. The high susceptibility is expressed not only in high transformation frequencies but, most importantly, in an unusually high rate of growth of EL2 transformants under selective conditions, i.e., in soft agar or as foci. The biological characteristics of EL2 cells greatly accelerate the isolation of transformants from known oncogenes and could be useful to detect new transforming genes.

Animals↗
PubMed1983

Changes in the levels of viral and cellular gene-transcripts in the cell cycle of SV40 transformed mouse cells.

We have analyzed the regulation of transcription of integrated SV40 DNA and of five cellular genes during the cell cycle of two lines of SV40 transformed mouse 3T3 cells. These cells (ts SV3T3) are temperature sensitive for the expression of the transformed phenotype and at the nonpermissive temperature (39 degrees C) become arrested in G1 at low serum concentrations. SV40 specific RNAs are not detected either in the nuclear or in the cytoplasmic poly(A+)RNA of quiescent cells, suggesting control at the level of transcription. After serum stimulation, however, viral transcription increases and reaches its maximum during S-phase. The expression of a group of selected housekeeping genes has received parallel analysis to determine whether other cellular genes, beside the integrated SV40, are shut off in G1 arrested cells or are expressed in restricted periods of the cell cycle. We have found that, while the mRNAs for collagen, adenosinphosphoribosiltransferase (APRT) and the mouse major histocompatibility complex (H2) are present throughout the cell cycle, the genes coding for the multifunctional protein CAD and dehydrofolate reductase are cell-cycle regulated.

Adenine Phosphoribosyltransferase↗
PubMed1982

Changes in the topography of early region transcription during polyoma virus lytic infection.

We have studied the pattern of transcription of the early region of polyoma virus DNA after the onset of the late phase of lytic infection of mouse cells. Following initiation of viral DNA synthesis, the early/late switch is accompanied not only by efficient production of late mRNAs but also by the appearance of previously unidentified early-strand RNAs which have certain structural features in common with the classical early mRNAs. Stable poly(A)+RNAs have been identified by blot analysis and S1 nuclease mapping that are not detected early during infection or in polyoma virus-transformed cells. One group consists of transcripts whose 5' ends map 150-200 nucleotides upstream from the major early 5' ends (at positions 148 and 153 on the polyoma virus genome) but whose splicing pattern and poly(A) addition sites are indistinguishable from those of mRNAs produced early in infection. The 5' exons of these early region transcripts contain an open translational reading frame that extends from nucleotide positions 5,255 to 124 and is capable of encoding a basic protein of 53 amino acids. Transcription of these RNAs does not appear to be negatively regulated by large tumor antigen. A transcript of 1,800 nucleotides appears to map predominantly between 93 and 26 map units and does not contain sequences present in the early mRNA 5' exons. These data suggest that, after the onset of polyoma DNA replication, the activation of new early-strand promoters leads to the expression of previously untranscribed viral DNA sequences.

Antigens, Viral↗
PubMed1982

Requirements for excision and amplification of integrated viral DNA molecules in polyoma virus-transformed cells.

The integration of polyoma virus DNA into the genome of transformed rat cells generally takes place in a tandem head-to-tail arrangement. A functional viral large tumor antigen (T-Ag) renders this structure unstable, as manifested by free DNA production and excision or amplification of the integrated viral DNA. All of these phenomena involve the mobilization of precise genomic "units," suggesting that they result from intramolecular homologous recombination events occurring in the repeated viral DNA sequences within the integrated structures. We studied polyoma ts-a-transformed rat cell lines, which produced large T-Ag but contained less than a single copy of integrated viral DNA. In all of these lines, reversion to a normal phenotype (indicative of excision) was extremely low and independent of the presence of a functional large T-Ag. The revertants were either phenotypic or had undergone variable rearrangements of the integrated sequences that seemed to involve flanking host DNA. In two of these cell lines (ts-a 4A and ts-a 3B), we could not detect any evidence of amplification even after 2 months of propagation under conditions permissive for large T-Ag. An amplification event was detected in a small subpopulation of the ts-a R5-1 line after 2 months of growth at 33 degrees C. This involved a DNA fragment of 5.1 kilobases, consisting of the left portion of the viral insertion and about 2.5 kilobases of adjacent host DNA sequences. None of these lines spontaneously produced free viral DNA, but after fusion with 3T3 mouse fibroblasts, R5-1 and 4A produced a low level of heterogeneous free DNA molecules, which contained both viral and flanking host DNA. In contrast, the ts-a 9 cell line, whose viral insertion consists of a partial tandem of approximately 1.2 viral genomes, underwent a high rate of excision or amplification when propagated at temperatures permissive for large T-Ag function. These results indicate that the high rate of excision and amplification of integrated viral genomes observed in polyoma-transformed rat cells requires the presence of regions of homology (i.e., repeats) in the integrated viral sequences. Therefore, these events occur via homologous intramolecular recombination, which is promoted directly or indirectly by the large viral T-Ag.

Animals↗
PubMed1982

Induction of sister chromatid exchange by polyoma large viral tumor antigen in transformed rat fibroblasts.

The frequency of sister chromatid exchange (SCE) was determined in rat fibroblasts transformed by wild-type polyoma virus or by a mutant temperature sensitive for viral large tumor antigen function (ts-a). Elevated SCE frequencies were observed in two wild-type transformed cell lines growing at 37 degrees and in four ts-a-transformed lines upon growth at the permissive temperature for large viral tumor antigen (33 degrees). The increase in SCE frequency in ts-a-transformed cells at 33 degrees was reversed by growth at 39 degrees (nonpermissive for T-antigen function). An increase in SCE at 33 degrees was not observed in untransformed cells or in a ts-a-transformed cell line which makes a defective large viral tumor antigen. These results suggest that large viral tumor antigen can induce SCEs. Since large viral tumor antigen is also responsible for amplification of integrated viral DNA sequences (4), we tried to correlate this phenomenon with the increased SCE frequency. However, increasing SCE artificially by growing cells in the presence of 12-O-tetradecanoylphorbol-13-acetate did not result in amplification of integrated viral DNA in the absence of large viral tumor antigen function. Thus, there is no simple causal relationship between increased SCE and amplification.

Animals↗
PubMed1981

Viral gene expression in polyoma virus-transformed rat cells and their cured revertants.

We have studied transcription of integrated viral DNA sequences in a variety of ts-a polyoma virus-transformed rat cells and cured revertants (which had undergone excision of variables amounts of integrated viral DNA) to characterize the structure of viral mRNA's produced in these lines under conditions in which integrated DNA is stable. Our results indicate that cells containing intact early region sequences, either in single-copy or tandem insertions, produce mRNA's indistinguishable from those observed early in lytic infections; sequences complementary to the polyoma late region were not transcribed from integrated viral DNA. Cured revertants no longer encoded full-length early mRNA's , but produced viral transcripts whose 3' ends mapped at an alternative early region polyadenylic acid attachment site at 99 map units or extended in to flanking host sequences. The phenotype of these revertant cells correlated with the abundance of these transcripts, suggesting that the transforming function(s) of polyoma virus controls the cellular phenotype in a dose-dependent manner. Unexpected results were obtained from studies of cells containing tandem repeats of defective viral DNA in which the polyadenylic acid attachment signal at 25.8 map units and surrounding sequences were deleted. In these cases, polyadenylated mRNA's were observed that contained sequences complementary to the early strand of the polyoma late region. These mRNA's (some larger than 8 kilobases) originated at the viral early promoter, extended into the late region, and continued into the early region of the contiguous repeat in the tandem. The multimeric mRNA's produced contained defective early regions in tandem with late region sequences. S1 analysis indicated that whereas the 5' early region sequences of readthrough transcripts were spliced in the usual manner, internal early region repeats were either unspliced or used only one of the small early region splices. When deletions in the viral readthrough transcripts were observed. This suggests that sequences nearby the AAUAAA sequence at 26 map units may control transcription termination of the polyoma early region.

Animals↗
PubMed1981

Cell transformation mediated by chromosomal deoxyribonucleic acid of polyoma virus-transformed cells.

To study the mechanism of deoxyribonucleic acid (DNA)-mediated gene transfer, normal rat cells were transfected with total cellular DNA extracted from polyoma virus-transformed cells. This resulted in the appearance of the transformed phenotype in 1 X 10(-6) to 3 X 10(-6) of the transfected cells. Transformation was invariably associated with the acquisition of integrated viral DNA sequences characteristic of the donor DNA. This was caused not by the integration of free DNA molecules, but by the transfer of large DNA fragments (10 to 20 kilobases) containing linked cellular and viral sequences. Although Southern blot analysis showed that integration did not appear to occur in a homologous region of the recipient chromosome, the frequency of transformation was rather high when compared with that of purified polyoma DNA, perhaps due to "position" effects or to the high efficiency of recombination of large DNA fragments.

Animals↗
PubMed1980

Decreased initiation of DNA synthesis in a temperature-sensitive mutant of hamster cells.

We have analyzed ongoing DNA replication in ts BN-2, a dna- mutant of BHK-21 cells (Nishimoto et al. '78). At the non-permissive temperature of 39.5 degrees C, inhibition of 3H-thymidine into acid-precipitable material begins 1 to 2 h after the cells are released from a block at the start of the S-phase. The fraction of nuclei incorporating 3H-thymidine is similar to that of wild-type cells through the synchronized S-phase of 8 h. Alkaline sucrose gradient analysis shows that pulse-labeled DNA from mutant cells is incorporated into high molecular weight material after 3 h at either the permissive or non-permissive temperature. DNA fiber autoradiograms reveal that, at 39.5 degrees C, the rate of replication fork movement is about 30% increased in the mutant as compared to wild-type cells. In the mutant cells, however, the interval between adjacent initiation sites is increased and the relative frequency of initiation events is decreased at the restrictive temperature. The results indicate that there is a block to ongoing replication in ts BN-2 at the level of initiation of synthesis on individual replication units; elongation of nascent chains is not inhibited.

Animals↗
PubMed1980

A temperature-sensitive mutation affecting S-phase progression can lead to accumulation of cells with a G2 DNA content.

Cultures of ts BN75, a temperature-sensitive mutant of BHK 21 cells, show a gradual biphasic drop in [3H]thymidine incorporation together with an accumulation of cells having a G2 DNA content when incubated at 39.5 degrees. However, when higher (41 degrees - 42 degrees) nonpermissive temperatures were used, the major block was in S-phase DNA synthesis. The cultures of ts BN75 shifted to 42 degrees at the start of the S phase, cell-cycle progress was arrested in the middle of S, while under these conditions wild-type BHK cells underwent at least one cycle of DNA synthesis. When ts BN75 cells growth-arrested at high temperature with a G2 DNA content were shifted to the permissive temperature (33.5 degrees C), the restart of DNA synthesis preceded the appearance of mitotic cells. These data suggest that the ts defect of ts BN75 cells might affect primarily the S phase of the cycle rather than the G2 phase.

Animals↗
PubMed1980

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.

Antigens, Neoplasm↗
PubMed1980

Relationship between integrated and nonintegrated viral DNA in rat cells transformed by polyoma virus.

Fischer rat fibroblasts transformed by polyoma virus contain, in addition to viral sequences integrated into the host genome, nonintegrated viral DNA molecules, whose presence is under the control of the viral A gene. To understand the mechanism of production of the "free" viral DNA, we have characterized the DNA species produced by several rat lines transformed by wild-type virus or by ts-a polyoma virus and compared them with the integrated viral sequences. Every cell line tested yielded a characteristic number of discrete species of viral DNA. The presence of defectives was a very common occurrence, and these molecules generally carried deletions mapping in the viral "late" region. The production of multiple species of free viral DNA was not due to heterogeneity of the transformed rat cell population, and its pattern did not change upon fusion with permissive mouse cells. Analysis of the integrated viral DNA sequences in the same cell lines showed, in most cases, a full head-to-tail tandem arrangement of normal-size and defective molecules. The free DNA produced by these lines faithfully reflected the integrated species. This was true also in the case of a cell line which contained a viral insertion corresponding to approximately 1.3 polyoma genomes, with each of the repeated portions of the viral DNA molecule carrying a different-size deletion. These results support the hypothesis that the free DNA derives from the integrated form through a mechanism of homologous recombination leading to excision and limited replication.

Animals↗
PubMed1979

Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function.

Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the "curing" of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for "curing" by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo "curing" with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable.

Animals↗