Biomedical subjects

N Baran

Publications and source records attributed to N Baran.

29 records · Page 2Linked to original sources

PubMed1983

"Onion skin" replication of integrated polyoma virus DNA and flanking sequences in polyoma-transformed rat cells: termination within a specific cellular DNA segment.

Replication of integrated polyoma virus DNA and flanking cellular sequences was studied in an inducible line of polyoma-transformed rat cells, designated the LPT line, that contains a single viral integration site. Chromosomal DNAs were purified from LPT cells treated with the virus-inducing agent mitomycin C and from untreated cells and were digested with restriction enzymes. The digests were analyzed by the Southern blotting technique. The virus DNA and a recombinant plasmid containing flanking cell DNA were used as hybridization probes. The analysis showed that mitomycin C treatment caused a more than 10-fold amplification of restriction fragments extending up to about 2.0 kilobase pairs into the cellular DNA flanking one end of the viral insertions, defined as the left joint. Fragments extending beyond this region were not amplified. These results showed that (i) integrated polyoma virus DNA undergoes multiple rounds of replication in mitomycin C-treated LPT cells and (ii) the replication extends into the flanking sequences and is arrested within a 0.40-kilobase-pair cellular DNA segment located about 2.0 kilobase pairs beyond the left joint. This segment may include a terminator of a normal cellular replicon.

Cell Line↗

PubMed1983

Integration of polyoma virus DNA into chromosomal DNA in transformed rat cells causes deletion of flanking cell sequences.

In order to find out whether polyoma virus (Py) integration into chromosomes causes rearrangements in the cell DNA flanking the integration site, we have mapped the flanking sequences in the inducible LPT line of Py-transformed rat cells and the corresponding sequences in normal rat fibroblasts, and then compared the two maps. To carry out this study we have cloned a segment including Py DNA and flanking sequences in the bacteriophage vector lambda gt WES and subcloned the flanking cell DNA in a bacterial plasmid. We performed a Southern blot analysis of LPT and rat fibroblast DNA digested with various restriction enzymes and used the cloned flanking cell DNA and Py DNA as hybridization probes. Autoradiography of the LPT DNA blots revealed two sets of fragments. One set includes fragments containing both Py and cell DNA sequences; the second set consists of fragments which contain no virus DNA sequences, and are identical to the fragments observed in the corresponding normal rat DNA digests. These data indicate that LPT cells are heterozygous with respect to the Py inserts. The same data were used to map the flanking sequences in the two types of cells. A comparison between the two maps revealed that a 3.0 kb cell DNA segment, which is located next to the unoccupied integration site in the normal rat chromosomes, has been deleted from the LPT chromosome which carries Py DNA, but not from the LPT chromosome which does not carry the virus DNA. The implications for papovavirus integration are discussed.

Animals↗

PubMed1982

Integration site of polyoma virus DNA in the inducible LPT line of polyoma-transformed rat cells.

The structure of the polyoma virus (Py) integration site in the inducible LPT line of Py-transformed rat cells was determined by biochemical methods of gene mapping. LPT cell DNA was digested with various restriction enzymes. The digestion products were electrophoresed in agarose gels and transferred onto nitrocellulose sheets by Southern blotting. Fragments containing viral or cell DNA sequences, or both, were identified by hybridization with Py DNA or with a cloned flanking cell DNA probe. Cleavage of LPT DNA with enzymes that restrict the Py genome once generated linear Py DNA molecules and two fragments containing both cell and viral DNA sequences. Cleavage of LPT DNA with enzymes which do not restrict Py DNA generated series of fragments whose lengths were found to differ by increments of a whole Py genome; the smallest fragment in each series was found to be longer than the viral genome. These data indicate that LPT cultures contain Py insertions of various lengths integrated into the same chromosomal site in all the cells. The length heterogeneity of the viral insertions is due to the presence of 0, 1, 2, 3. . . Py genomes arranged in a direct tandem repeat within invariable sequences of viral DNA. Double-digestion experiments were also carried out with the above enzymes and with enzymes that cleave the Py genome at multiple sites. The data obtained in these experiments were used to construct a physical map of the integration site. This map showed that the early region of the virus remained intact even in the smallest insertion (which contains no whole duplicated genomes), whereas the late region was partially duplicated and split during integration. The smallest insertion is colinear with the Py physical map over a region including the entire Py genome and at least a part of the duplicated segment. This structure could give rise to nondefective circular viral DNA molecules by single homologous recombination events. Similar recombination events may occur at a higher frequency in the longer insertions, which include longer regions of homology, and may yield many more free viral genomes. The presence of these insertions in LPT cells could thus be one of the factors which account for the high inducibility of the LPT line.

Animals↗

PubMed1979

Electron microscopic mapping of RNA transcribed from the late region of polyoma virus DNA.

The polyoma virus (Py) RNA species transcribed from the L DNA strand of the "late" region of the Py genome in Py-infected mouse cells have been mapped by hybridization with specific fragments of Py DNA followed by electron microscopic visualization of the hybrids. Total cellular polyadenylated Py-specific RNA molecules having an S value in the range of 16S to 20S were purified by oligodeoxythymidylic acidcellulose column chromatography, preparative hybridization with Py DNA, and sucrose gradient centrifugation. Cytoplasmic Py-specific RNA was similarily purified, except that it was not fractionated by sucrose gradient centrifugation. Hybrids of these RNA molecules and Py DNA fragments were spread for electron microscopy by either the cytochrome c technique or the bacteriophage T4 gene 32 protein method. The polyadenylic acid at the 3'-end of the RNA in the hybrids was identified by labeling with simian virus 40 DNA circles to which polybromodeoxyuridylic acid tails had been covalently attached. These experiments revealed the presence of three L DNA strand transcripts in both RNA preparations. Two of these RNA molecules were found to be spliced from chains transcribed from two noncontiguous parts of the late region. The third molecule either is a continuous transcript of the entire late region or contains a splicing feature which is too small to be reliably observed by the electron microscope methods used. The 5'-ends of the three RNA species map within a region extending from 68 to 70 map units on the Py restriction endonuclease map. Each of the two spliced molecules contains a 5'-terminal leader sequence transcribed from a DNA segment with an estimated length of 60 to 110 nuvleotides. The 3'-ends of the leaders map at 66.7 +/- 1.0 and 66.4 +/- 0.50 map units. In these molecules the 5'-ends of the other part (the main body) map at 59.4 +/- 0.90 and 49.4 +/- 2.0 map units, respectively. The 3'-termini of all three RNA species map at 24 to 25 map units.

DNA, Viral↗

PubMed1977

Circular dimers of a lambda DNA in infected, nonlysogenic Escherichia coli.

Covalently closed circular dimers of phage lambda DNA have been found in Escherichia coli infected with lambda. These dimers can be formed by either the lambda Red or Int systems, by a nonrecombinational replicative mechanism requiring the activity of the lambda O and P genes or by joining of the cohesive ends. Dimers mediated by the E. coli Rec system have not been observed. Those formed by the Int system often result from recombination between different DNA molecules; however, the Red-mediated dimers may be a result of replicative extension of a single DNA molecule. Trimers have also been observed but studied only briefly.

Coliphages↗

PubMed1977