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M Zannis-Hadjopoulos

Publications and source records attributed to M Zannis-Hadjopoulos.

62 records · Page 4Linked to original sources
PubMed1985

Properties of some monkey DNA sequences obtained by a procedure that enriches for DNA replication origins.

Twelve clones of monkey DNA obtained by a procedure that enriches 10(3)- to 10(4)-fold for nascent sequences activated early in S phase (G. Kaufmann, M. Zannis-Hadjopoulos, and R. G. Martin, Mol. Cell. Biol. 5:721-727, 1985) have been examined. Only 2 of the 12 ors sequences (origin-enriched sequences) are unique (ors1 and ors8). Three contain the highly reiterated Alu family (ors3, ors9, and ors11). One contains the highly reiterated alpha-satellite family (ors12), but none contain the Kpn family. Those remaining contain middle repetitive sequences. Two examples of the same middle repetitive sequence were found (ors2 and ors6). Three of the middle repetitive sequences (the ors2-ors6 pair, ors5, and ors10) are moderately dispersed; one (ors4) is highly dispersed. The last, ors7, has been mapped to the bona fide replication origin of the D loop of mitochondrial DNA. Of the nine ors sequences tested, half possess snapback (intrachain reannealing) properties.

Animals↗
PubMed1984

Mammalian DNA enriched for replication origins is enriched for snap-back sequences.

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.

Animals↗
PubMed1983

Mapping of the 3'-end positions of simian virus 40 nascent strands.

Using the instability of replication loops as the basis for the isolation of replication origins, we have undertaken an analysis of the 3' ends of the extruded nascent strands of replicating simian virus 40 (SV40) DNA. DNA fragments containing the SV40 origin of replication were obtained by digesting highly purified replicative intermediates of SV40 with BamHI and then heating at 55 degrees C for 16h. The origin-containing fragments extruded under these conditions were purified and cloned into pBR322. We used restriction mapping to analyze 640 clones of the 674 that contained SV40 sequences. A large majority of the clones were found to contain rearrangements in the sequences of either pBR322 or SV40 and were disregarded. Those clones that contained legitimate SV40 and pBR322 sequences were presumed to have been derived from the extruded SV40 nascent strands and were further analyzed. A combination of restriction enzymes was used that allowed us to define the 3' ends with an accuracy of +/- 20 base-pairs. The results of restriction analysis were confirmed by nucleotide sequence analysis of selected clones. The results show that the replication forks move with a high degree of symmetry, with respect to the initiation site of DNA replication, and are consistent with the existence of pause sites for the extension of replication forks. From the clones analyzed, it appears that the center of the replication bubble is to the early side of the BglI site.

Base Sequence↗
PubMed1983

Flat revertants of temperature-insensitive transformants induced by simian virus 40 tsA mutants lose their ability to express T-antigen.

Temperature-insensitive transformants that contained simian virus 40 sequences at only one or a few sites in the rat chromosome and that were induced by a temperature-sensitive A gene mutant of simian virus 40 were used to select flat revertants (revertants that had lost the transformed phenotype). The isolation was performed at the nonpermissive temperature so as not to select against temperature-sensitive transformants. Nonetheless, all of the revertants examined had lost their ability to express the T-antigen at both temperatures, and all contained rearrangements of the integrated simian virus 40 sequences. These results are most compatible with the hypothesis that the T-antigen of simian virus 40 is required for the maintenance of the transformed state even in temperature-insensitive cell lines.

Animals↗
PubMed1983

Relationship of simian virus 40 tumor antigens to virus-induced mutagenesis.

We analyzed the mutation frequency to 8-azaguanine (8AZ) resistance in rat FR3T3 cells acutely infected with simian virus 40 wild type and tsA and early deletion mutants and in a series of temperature-sensitive (N) and temperature-insensitive (A) transformants derived from Chinese hamster lung (CHL) cells. Upon acute infection, the frequency of mutation to 8AZ resistance was raised at most by two- to eightfold over the spontaneous frequency, and it was independent of the presence of a functional 90,000-molecular-weight T antigen or 20,000-molecular-weight t antigen or both. Similarly, in the stable transformants of CHL cells, no correlation was found between functional T antigens and mutation to 8AZ resistance. It therefore seems unlikely that simian virus 40-induced transformation results from any mutagenic activity of this virus.

Animals↗
PubMed1981

The remarkable instability of replication loops provides a general method for the isolation of origins of DNA replication.

Closed superhelical replicative intermediates of SV40 DNA were stable during incubation at elevated temperatures for prolonged times. However, when the replicative intermediates were nicked or made linear by digestion either outside or inside of the replication loop, the nascent DNA strands were spontaneously extruded at a measurable rate even at 37 degrees C. The extruded DNA was double-stranded. The rate-limiting step in the extrusion was not the rate of branch migration. The instability of replication loops suggested a method for the isolation of replication origins. The method has been tested by a reconstruction experiment in which a small amount of linear SV40 replicative intermediates was mixed with a vast excess of cellular DNA. The mixture was heated overnight at 55 degrees C, and the spontaneously extruded nascent strands were isolated and inserted into pBR322. Plasmids containing SV40 DNA of less than genomic size, symmetrically distributed about the SV40 replication origin, were isolated. This result suggests that the method is generally applicable to the isolation of replication origins.

DNA Replication↗
PubMed1980

Inhibition of DNA chain elongation in a purine-auxotrophic mutant of Chinese hamster.

DNA synthesis has been examined in a purine-auxotrophic mutant cell line of Chinese hamster (V79 pur 1) under conditions of purine deprivation. At 6 h after the removal of purines from the growth medium, there is a decrease in semiconservative DNA replication. Alkaline velocity centrifugation of the DNA synthesized during a 1-min pulse under conditions of purine deprivation shows that approximately 50% of the newly replicated DNA is the size of Okazaki pieces. These are not incorporated into bulk DNA during a 1-h chase. If the purine supply is restored to the growth medium, these short DNA pieces are jointed to full-sized DNA within 1 h. DNA fiber autoradiolgraphy reveals a retardation in the rate of DNA replication fork movement but no apparent inhibition of initiation of synthesis on replication units within clusters actively engaged in replication. Our results indicate that purine deprivation specifically inhibits elongation of nascent dna chains.

Animals↗