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R Rothstein

Publications and source records attributed to R Rothstein.

72 records · Page 4Linked to original sources
PubMed1989

A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase.

A hyper-recombination mutation was isolated that causes an increase in recombination between short repeated delta sequences surrounding the SUP4-omicron gene in S. cerevisiae. The wild-type copy of this gene was cloned by complementation of one of its pleiotropic phenotypes, slow growth. DNA sequence of the clone revealed a 656 amino acid open reading frame capable of encoding a protein homologous to the bacterial type I topoisomerase. No homology was detected with previously identified eukaryotic topoisomerases. Construction of double mutants with either of the two known yeast topoisomerase genes revealed synergistic effects on growth suggesting overlapping functions. Expression of bacterial topoisomerase I in yeast can fully complement the slow growth defect of a null mutation. We have named this locus TOP3 and suggest that it defines a novel eukaryotic topoisomerase gene.

Amino Acid Sequence↗
PubMed1989

Elevated recombination rates in transcriptionally active DNA.

We have examined the effect of RNA polymerase II-dependent transcription on recombination between directly repeated sequences of the GAL10 gene in S. cerevisiae. Direct repeat recombination leading either to plasmid loss or conversion was examined in isogenic strains containing null mutations in the positive activator, GAL4, or the repressor, GAL80. A 15-fold increase in the rate of plasmid loss is observed in cells constitutively expressing the construct compared with cells that are not. Conversion events that retain the integrated plasmid are not stimulated by expression of the repeats. Northern analysis of strains containing plasmid inserts with various promoter mutations suggests that the stimulation in recombination is mediated by events initiating within the integrated plasmid sequences.

Crossing Over, Genetic↗
PubMed1989

The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptionally regulated gene.

We have previously shown direct-repeat recombination events leading to loss of a plasmid integrated at the GAL10 locus in Saccharomyces cerevisiae are stimulated by transcription of the region. We have examined the role of two recombination- and repair-defective mutations, rad1 and rad52, on direct repeat recombination in transcriptionally active and inactive sequences. We show that the RAD52 gene is required for transcription-stimulated recombination events leading to loss of the integrated plasmid. Similarly, Gal+ events between the duplicated repeats that retain the integrated plasmid DNA (Gal+ Ura+ replacement events) are reduced 20-fold in the rad52 mutant in sequences that are constitutively expressed. In contrast, in sequences that are not expressed, the rad52 mutation reduces plasmid loss events by only twofold and Gal+ Ura+ replacements by fourfold. We also observe an increase in disome-associated plasmid loss events in the rad52 mutant, indicative of chromosome gain. This event is not affected by expression of the region. Plasmid loss events in rad1 mutant strains are reduced only twofold in transcriptionally active sequences and are not affected in sequences that are repressed. However, the rad1 and rad52 double mutant shows a decrease in plasmid loss events greater than the sum of the decreases in the rates of this event displayed by either single mutant in both constitutive and repressed DNA, indicating a synergistic interaction between these two genes. The synergism is limited to recombination since the rad1 rad52 double mutant is no more sensitive when compared with either single mutant in its ability to survive radiation damage. Finally, the recombination pathway that remains in the double mutant is positively affected by transcription of the region.

DNA Repair↗
PubMed1988

Mitotic sectored colonies: evidence of heteroduplex DNA formation during direct repeat recombination.

In yeast meiosis, ascosporal colonies are sometimes sectored for a marker--i.e., half the colony has one allele and half has the other. This is interpreted as replicative resolution of heteroduplex DNA (hDNA) formed as a recombination intermediate. We have looked for similar evidence of hDNA formation during mitotic recombination between two repeated sequences on the same chromosome. The two repeats, an ochre suppressor and a wild-type tRNA gene, are separated by plasmid DNA and the URA3 marker. Recombination between the repeats excises the URA3 gene and one copy of the repeat, leaving either the wild-type tRNA or the suppressor on the chromosome. A red/white color assay is used to distinguish between the two. We find that some colonies that have lost the URA3 gene are sectored for the suppressor. This suggests that hDNA is formed across the anticodon during the recombination event and then resolved by replication. The disruption of either of two genes involved in recombination and repair, RAD1 and RAD52, does not significantly alter the frequency of sectored colony formation during plasmid excision.

Canavanine↗
PubMed1988

SAM2 encodes the second methionine S-adenosyl transferase in Saccharomyces cerevisiae: physiology and regulation of both enzymes.

In Saccharomyces cerevisiae the SAM1 and SAM2 genes encode two distinct forms of S-adenosylmethionine (AdoMet) synthetase. In a previous study we cloned and sequenced the SAM1 gene (D. Thomas and Y. Surdin-Kerjan, J. Biol. Chem. 262:16704-16709, 1987). In this work, the SAM2 gene was isolated by functional complementation of a yeast double-mutant strain, and its identity was ascertained by gene disruption. It has been sequenced and compared with the SAM1 gene. The degree of homology found between the two genes shows that SAM1 and SAM2 are duplicated genes. Using strains disrupted in one or the other SAM gene, we have studied the regulation of their expression by measuring the steady-state level of mRNA after growth under different conditions. The results show that the expression of the two SAM genes is regulated differently, SAM2 being induced by the presence of excess methionine in the growth medium and SAM1 being repressed under the same conditions. The level of mRNA in the parental strain shows that it is not the sum of the levels found in the two disrupted strains. This raises the question of how the two AdoMet synthetases in S. cerevisiae interact to control AdoMet synthesis.

Amino Acid Sequence↗
PubMed1987

Concerted deletions and inversions are caused by mitotic recombination between delta sequences in Saccharomyces cerevisiae.

Deletions of a tyrosine tRNA suppressor gene, SUP4-o, are mediated by recombination between short repeated delta sequences in Saccharomyces cerevisiae. The arrangement of the five solo delta sequences that surround the SUP4 locus was established by DNA sequence analysis. Seven deletion classes were identified by genomic blotting. DNA sequence analysis also showed that the delta sequences within a 6.5-kilobase region of the SUP4 locus were the endpoints of these events. In three of these classes, an adjacent interval surrounded by delta sequences was inverted in concert with the deletion. The frequency of all deletion classes decreased in strains that contained mutations in the recombination and repair gene RAD52. We present two gene conversion mechanisms by which these rearrangements could have been generated. These models may also explain deletions between repeated sequences in other systems.

Base Sequence↗
PubMed1984

Intestinal obstruction due to bezoars.

A patient with acute intestinal obstruction due to unrecognized gastric phytobezoars is presented. There was no history of prior gastric or other abdominal surgery. At laparotomy several fragments of rubbery material were discovered obstructing the ileocecal valve. Exploration of the upper gastrointestinal tract revealed two partially fragmented bezoars in the stomach which were removed through a gastrotomy. After surgery the patient recounted the ingestion of persimmons a few weeks before the onset of her symptoms. A review of the literature reveals bezoars to be an infrequent, although not an altogether rare, cause of small bowel obstruction in patients without antecedent gastric surgery. A careful dietary history should accompany all patients presenting with acute intestinal obstruction.

Bezoars↗
PubMed1983

Pulmonary embolism.

Pulmonary embolism is one of the major diagnostic challenges facing the emergency physician, since the failure to rapidly diagnose and treat this condition can result in early mortality. Presently, as many as 100,000 preventable deaths a year may be caused by pulmonary embolism. A high index of suspicion and a well thought out approach to the diagnosis and treatment of this elusive condition should help emergency physicians to improve upon these statistics.

Blood Gas Analysis↗
PubMed1981

Modification of the bacteriophage vector M13mp2: introduction of new restriction sites for cloning.

The construction of two new derivatives of the bacteriophage cloning vector M13mp2 is described. One derivative, mWJ22, contains a new HindIII site while the other, mWJ43, contains a new BamHI site. These new sites were both introduced at the EcoRI site at amino acid five of the 145 amino acid-long fragment of Escherichia coli beta-galactosidase within the phage. The new restriction sites do not disrupt the blue color detection system of M13mp2; therefore insertion of cloned fragments results in colorless plaques on indicator plates for the new derivatives.

Base Sequence↗
PubMed1979

Deletions of a tyrosine tRNA gene in S. cerevisiae.

Genetic fine structure analysis of a tyrosine tRNA in yeast revealed that complete deletions of the gene occurred at an unusually high frequency. Among 56 spontaneous mutations at the SUP4 locus, 16 were classified as deletions as judged by their failure to recombine with any other mutations known to map within the gene. Physical analysis of each deletion confirmed the genetic result. The deletions fall into two size classes: ten are 2100 bp deletions and six are 2800 bp deletions. These results imply that the physical structure of the region surrounding the SUP4 locus, which is known to contain short repeated segments, has a direct role in promoting deletions.

Chromosome Deletion↗
PubMedDate not supplied

Epidemiological use of rectal proliferation measures.

Measures of rectal mucosal proliferation have been developed and used in research clinical settings, but their utility for larger-scale epidemiological studies remains uncertain. We assessed the suitability of bromodeoxyuridine (BrdUrd) and proliferating cell nuclear antigen (PCNA)-labeling indices (LIs) in the setting of a multicenter clinical trial of adenoma recurrence. Subjects at participating practices were asked to permit biopsy of normal rectal mucosa during a colonoscopy scheduled for other reasons. PCNA and BrdUrd labeling was performed, and corresponding LIs were computed. In general, subjects were willing to undergo biopsy during their scheduled procedures; less than 10% refused. Specimen preparation for PCNA was acceptable; the mean number of scorable crypts (+/- SE) was 12.99 +/- 0.37. Preparation for BrdUrd labeling was less successful, with a higher proportion of unscorable specimens and a lower mean number of scorable crypts. Among the 54 specimens with both LIs computed, the LI for PCNA was modestly higher than that for BrdUrd LI (4.1 +/- 0.2 and 3.7 +/- 0.2 respectively; P = 0.03). The rank order correlation between the two indices was 0.38). There was variation across centers in the PCNA LIs but few differences according to number of crypts scored. Measurement of rectal mucosal proliferation is feasible among endoscopy patients in large studies if PCNA is used; BrdUrd seems more difficult. The relationship between these two labels requires further study.

Adenoma↗