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J A Huberman

Publications and source records attributed to J A Huberman.

At least 55 records · Page 3Linked to original sources
PubMed1991

Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins.

The silent mating-type loci of Saccharomyces cerevisiae, HML and HMR, are flanked by transcriptional silencers that have ARS activity (i.e., they function as replication origins when in plasmids). To test whether these ARS elements are chromosomal origins, we mapped origins near HML (close to the left telomere of chromosome III). Our results indicate that the HML-associated ARS elements either do not function as chromosomal replication origins or do so at a frequency below our detection level, suggesting that replication from a silencer-associated origin in each S phase is not essential for the maintenance of transcriptional repression at HML. Our results also imply that the ability of a DNA fragment to function as an ARS element in a plasmid does not ensure its ability to function as an efficient chromosomal replication origin. Telomere proximity is not responsible for inactivating these ARS elements, because they are not detectably functional as chromosomal origins even in genetically modified strains in which they are far from the telomere.

Chromosome Mapping↗
PubMed1990

Ambiguities in results obtained with 2D gel replicon mapping techniques.

Recently, two 2-dimensional (2D) gel techniques, termed neutral/neutral and neutral/alkaline, have been developed and employed to map replication origins in eukaryotic plasmids and chromosomal DNA (1-11). The neutral/neutral technique, which requires less DNA for analysis, has been preferentially used in recent studies. We show here that the signal predicted for an origin is not detected using the neutral/neutral technique if the origin is located near the end of the analyzed restriction fragment. We also demonstrate that analysis of the same batch of DNA by the two different mapping techniques can generate apparently contradictory results: in some situations where neutral/alkaline 2D analysis indicates that a certain origin is always used, neutral/neutral 2D analysis suggests that the origin is not always used. Several possible explanations for this type of disagreement between the two techniques are discussed, and we conclude that it is important to use both techniques in combination in order to minimize possible misinterpretations.

DNA, Fungal↗
PubMed1988

Organization of replication of ribosomal DNA in Saccharomyces cerevisiae.

Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the nontranscribed spacer region, (ii) only a fraction of the potential origins are utilized in a single S phase, and (iii) the replication forks moving counter to the direction of transcription of the 37S precursor RNA stop at or near the termination site of transcription. Consequently, most ribosomal DNA is replicated unidirectionally by forks moving in the direction of transcription and most replicons are larger than the repeat unit. The significance of this finding for the replication of abundantly transcribed genes is discussed.

Chromosome Mapping↗
PubMed1988

Two-dimensional gel electrophoretic method for mapping DNA replicons.

We describe in detail a method which allows determination of the directions of replication fork movement through segments of DNA for which cloned probes are available. The method uses two-dimensional neutral-alkaline agarose gel electrophoresis followed by hybridization with short probe sequences. The nascent strands of replicating molecules form an arc separated from parental and nonreplicating strands. The closer a probe is to its replication origin or to the origin-proximal end of its restriction fragment, the shorter the nascent strands that are detected by the probe. The use of multiple probes allows determination of directions of replication fork movement, as well as locations of origins and termini. In this study, we used simian virus 40 as a model to demonstrate the feasibility of the method, and we discuss its applicability to other systems.

Animals↗
PubMed1987

The in vivo replication origin of the yeast 2 microns plasmid.

We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2 micron plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 +/- 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2 microns plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA.

DNA Replication↗
PubMed1986

Both DNA topoisomerases I and II relax 2 micron plasmid DNA in living yeast cells.

Measurements at various temperatures of the linking number of yeast 2 microns plasmid DNA in wild-type cells and in cells bearing mutations in the DNA topoisomerase I and II genes show that bulk 2 microns plasmid minichromosome are maintained in a relaxed state by the combined action of topoisomerases I and II. Bulk 2 microns minichromosomes are not under torsional stress in vivo and are not substrates for a putative gyrase-like topoisomerase.

DNA Topoisomerases, Type I↗
PubMed1986

Characterization of DNA sequences associated with residual nuclei of Saccharomyces cerevisiae.

We have used two different approaches to determine whether particular DNA sequences are specifically associated with high-salt-treated residual nuclei of Saccharomyces cerevisiae. First, libraries of yeast DNA in phage lambda were probed with nick-translated total nuclear or residual nuclear DNA from unsynchronized yeast cells. None of the plaques gave a significantly stronger or weaker signal with the residual nuclear probe than with the total nuclear probe. Second, DNA was purified from whole nuclei or residual nuclei which had been isolated from cells in G1, G1/S, early S, or nuclear division. This DNA was "dot-blotted" and then probed with specific yeast DNA sequences. Ribosomal DNA was 2- to 3-fold enriched in residual nuclei in late G1, G1/S, and early S, and 2 microns plasmid DNA sequences were 3- to 5-fold depleted during nuclear division and early G1. However, ARS1, TRP1, CEN6, and a telomere sequence were neither enriched nor depleted at any time during the cell cycle.

Base Sequence↗
PubMed1985

Method of mapping DNA replication origins.

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.

Chromatography↗
PubMed1984

Effects of 2',3'-dideoxynucleosides on mammalian cells and viruses.

Previous studies on the biological effects of the 2',3'-dideoxynucleosides (ddNs) have shown that while ddAdo is lethal to E. coli, ddThd has minimal effects on the growth of mammalian cell lines and that it inhibits retrovirus infection of some cell lines but not others. Previous studies have also shown that the 5'-triphosphate of ddThd, ddTTP, selectively inhibits cellular DNA polymerases beta and gamma and retroviral reverse transcriptases. Cellular DNA polymerase alpha is relatively resistant to ddTTP. We have extended these findings to show that the 5'-triphosphates of the other 3 ddNs (ddATP, ddCTP, and ddGTP) affect cellular DNA polymerases alpha, beta, and gamma in the same fashion as does ddTTP. We also show that all four ddNs in concentrations up to 100 microM have negligible effects on the growth of NIH Swiss 3T3 cells. These negligible effects may be due to inefficient intracellular phosphorylation of each nucleoside to the triphosphate. We have determined that, in several different cell lines, ddThd is phosphorylated only at a very slow rate to ddTTP, and in the one cell line tested (monkey CV-1 cells), ddAdo and ddGuo are also poorly phosphorylated. Both ddAdo and ddGuo, and probably ddThd, are converted by CV-1 cells to additional unknown compounds which may have biological activity. The four ddNs display effects of different magnitudes on certain virus infections. Although 30 microM ddThd inhibits herpes simplex I infection of CV-1 cells by 50%, 30 microM ddAdo has no effect. Infection of NIH Swiss 3T3 cells by 334C murine leukemia virus is inhibited 70-80% by ddAdo, ddCyd, and ddThd at 50 microM, but inhibition by 50 microM ddGuo is 100%.

Animals↗
PubMed1984

Isolation and initial characterization of residual nuclear structures from yeast.

Residual nuclear structures have previously been isolated from a wide range of eukaryotic organisms. When nuclei are isolated from Saccharomyces cerevisiae and then treated with 1.95 M NaCl and DNase I, sedimentable residual structures are obtained similar in several respects to structures isolated from organisms previously studied. These yeast residual nuclear structures retain less than 7% of nuclear DNA, less than 17% of nuclear RNA and less than 50% of nuclear proteins. Electron microscopy suggests that these structures are derived from the nuclear interior and are composed of a sparse fibrogranular network. Replicating DNA is preferentially bound to these yeast residual nuclear structures, just as it is to residual nuclear structures from other organisms.

Cell Nucleus↗
PubMed1983

In vitro DNA synthesis by an alpha-like DNA polymerase bound to replicating simian virus 40 chromosomes.

Simian virus 40 chromosomes carry out replicative DNA synthesis in vitro which is sensitive to aphidicolin and to N-ethylmaleimide, resistant to 2',3'-dideoxythymidine-5'-triphosphate, and proportional to the amount of chromosome-associated alpha-like polymerase. Thus, an alpha-like DNA polymerase (alpha polymerase or delta polymerase) is responsible for in vitro DNA synthesis.

Aphidicolin↗
PubMed1982

Both parental deoxyribonucleic acid strands at each replication fork of replicating simian virus 40 chromosomes are cut by a single-strand-specific endonuclease.

We have measured the relative accessibility to a single-strand-specific endonuclease of the single-stranded DNA on the leading and lagging sides of replication forks in replicating simian virus 40 (SV40) chromosomes. To do this we have digested replicating SV40 chromosomes with a single-strand-specific endonuclease (P1 nuclease) and then characterized the intermediate and final products of digestion by sucrose gradient sedimentation and agarose gel electrophoresis. P1 nuclease rapidly and specifically cleaves parental DNA strands at replication forks, yielding intermediate and final cleavage products which are consistent with an approximately equal rate of nuclease cleavage on both sides of the fork. Thus, single-stranded DNA is approximately as accessible to P1 nuclease on the leading side of the fork as on the lagging side; the simplest interpretation of this observation is that the stretch of single-stranded DNA on the leading side is as long as that on the lagging side.

Centrifugation, Density Gradient↗
PubMed1980

Two-dimensional analysis of proteins sedimenting with simian virus 40 chromosomes.

The nonhistone proteins sedimenting in low-salt glycerol gradients with simian virus 40 chromosomes were analyzed by two-dimensional gel electrophoresis, utilizing nonequilibrium pH gradients as the first dimension and sodium dodecyl sulfate-gel electrophoresis as the second dimension. By densitometric quantitation of the radiolabeled proteins present in each fraction of the gradients, it was possible to identify sedimenting with all or a fraction of the simian virus 40 chromosomes. VP-1 sedimented with simian virus 40 chromosomes; additional evidence for its binding to chromosomes was obtained by immunochemical techniques. Four proteins (Mr 25,000, pI 6.0; Mr 32,000, pI 7.2; Mr 35,000, pI 8.5; and Mr 80,000, pI 7.2) sedimented with specific subsets of chromosomes.

Centrifugation, Density Gradient↗