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Biomedical subjects

S R Kushner

Publications and source records attributed to S R Kushner.

90 records · Page 5Linked to original sources
PubMed1977

Analysis of genetic recombination between two partially deleted lactose operons of Escherichia coli K-12.

Genetic recombination between a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1) in Escherichia coli K-12 has been examined. Since the deletions were nonoverlapping, rare lactose-fermenting (Lac+) recombinants occurred and were detected qualitatively on lactose tetrazolium agar indicator plates as white papillae growing on the surface of red colonies or quantitively on lactose minimal agar plates. Formation of Lac+ recombinants required the recA, recB, and recC gene products. Indirect suppression of recB21 by sbcB15 led to an increase in the frequency of Lac+ recombinants over wild-type levels. recF143 did not appreciably alter the number of Lac+ progeny, whereas recL152 and sbcB15 strains yielded increased numbers of Lac+ recombinants. The nature and formation of Lac+ recombinants was also examined. Respreading analysis indicated that formation of recombinants occurred primarily as the cells entered early stationary phase on the surface of the minimal agar plates and that over 90% of the recombinants contained a phi80dIIlac+ prophage. Time-of-entry experiments suggested that the region of deoxyribonucleic acid between the two operons was not inverted as a result of the recombinational event.

Conjugation, Genetic↗
PubMed1976

A proposal for a uniform nomenclature for the genetics of bacterial protein synthesis.

A new genetic nomenclature for the macromolecules involved in bacterial protein synthesis is proposed and explained. Genes for ribosomal proteins are designated rsp, rpl and rpm while genes for ribosomal RNAs are rrs and rrl. Protein synthesis factors and ribosome assembly and modification activities are also consistantly named.

Bacterial Proteins↗
PubMed1976

Amplification in Escherichia coli of enzymes involved in genetic recombination: construction of hybrid ColE1 plasmids carrying the structural gene for exonuclease I.

Endo-R-HindIII restriction endonuclease fragments obtained from F30 and pMB9 plasmid DNAs were ligated in vitro and used to transform a recB21 recC22 sbcB15 strain of E. coli K-12. The inability of this strain to stably maintain pMB9 alone permitted the isolation of transformants that carried hybrid plasmids containing the sbcB+ allele. These transformants became sensitive to ultraviolet light and recombination defieient and showed a 25-fold increase in the level of exonuclease I activity. The stability of the sbcB hybrid plasmids and their effects on exonuclease I activity have also been determined in wild-type and recA1 genetic backgrounds. The presence of the plasmids results in a 7-fold increase in the level of exonuclease I in a wild-type strain and 15-fold increase in a recA1 strain. The increased activity in the recA1 mutant appears to be a result of increased plasmid stability in this genetic background.

Base Sequence↗
PubMed1975

Analysis of temperature-sensitive recB and recC mutations.

The in vivo pleiotropic effects associated with the temperature-sensitive recB270 and recC271 mutations have been correlated with the in vitro behavior of the recBC nucleases coded for by these alleles. The ATP-dependent breakdown of double-stranded DNA is essential for cell viability, radiation repair, and genetic recombination. Temperature sensitivity can be suppressed in vitro and in vivo.

Adenosine Triphosphate↗
PubMed1974

Isolation of exonuclease VIII: the enzyme associated with sbcA indirect suppressor.

recB and/or recC deficiency in Escherichia coli K-12 is indirectly suppressed by the presence of sbcA(-) mutations. sbcA(-) strains contain an increased level of an ATP-independent nuclease. Genetic and enzymatic tests indicate that this activity is not exonuclease III, exonuclease V (recB-recC nuclease), DNA polymerase I, or lambda exonuclease. This new enzyme (exonuclease VIII) has been purified 750-fold and shows a striking preference for double-stranded DNA over heat-denatured DNA. It does not act endonucleolytically on closed circular, single-stranded DNA as exonuclease V does. It also lacks a 3'-phosphatase function. Analysis on sodium dodecyl sulfate-polyacrylamide gels indicates that exonuclease VIII is not present in unsuppressed (sbcA(+)) strains. It is thought that sbcA determines some type of control function; the structural gene for exonuclease VIII is denoted by recE.

Chromatography, DEAE-Cellulose↗
PubMed1974

In vivo studies of temperature-sensitive recB and recC mutants.

Some in vivo properties of Escherichia coli K-12 strains carrying recB270 (formerly recBts1) and recC271 (formerly recCts1) mutations have been determined. Single recB270 and recC271 mutants appear normal at 30 C with regard to ultraviolet and mitomycin C sensitivity, recombination proficiency, and viability. At 43 C these strains become sensitive to ultraviolet and mitomycin C, while showing only a slight decrease in recombination proficiency. The viable titers of the single mutants are somewhat reduced at 43 C. Double mutant strains carrying polA1 and recB270 or recC271 are inviable at 43 C. The double mutant strain (recB270 recC271) is sensitive to both UV and mitomycin C at 30 C, but shows only slightly reduced recombination proficiency. At 43 C the strain resembles absolute recB and recC mutants in all respects. In addition, the double mutant strain exhibits a temperature-induced drop in viable titer. The triple mutant polA1 recB270 recC271 is viable at 30 C. Two hypotheses are advanced to explain these results.

Cell Survival↗
PubMed1974

Differential thermolability of exonuclease and endonuclease activities of the recBC nuclease isolated from thermosensitive recB and recC mutants.

The recBC nuclease (also called exonuclease V) has been partially purified from Escherichia coli K-12 strains carrying the thermosensitive recB270, recC271, and recB270 recC271 mutations. Of the multiple activities associated with the enzyme, only the adenosine 5'-triphosphate-dependent exonucleolytic hydrolysis of duplex deoxyribonucleic acid (DNA) is abnormally thermolabile. The exo- and endonucleolytic degradation of single-stranded DNA is no more thermosensitive than that catalyzed by the wild-type enzyme. These results suggest that the defects in genetic recombination, DNA repair, and the maintenance of cell viability observed in recBC mutants in vivo result primarily from the specific loss of adenosine 5'-triphosphate-dependent exonuclease active on duplex DNA.

Adenosine Triphosphate↗
PubMed1972

Indirect suppression of recB and recC mutations by exonuclease I deficiency.

recB(-)and recC(-) strains of Escherichia coli K12 are recombination deficient and sensitive to ultraviolet light and the drug, mitomycin C. We have reported that sbcB mutations indirectly suppress all three phenotypes and result in the loss of exonuclease I. In this publication we report the occurrence of other mutations that lead to loss of exonuclease I and indirectly suppress mitomycin and UV sensitivity but not recombination deficiency. These mutations (called xonA) are cotransducible with his and closely linked with sbcB. Both sbcB and xonA mutant strains appear to have identical residual amounts of nuclease activity on single-stranded DNA. It is hypothesized that exonuclease I possesses a second activity other than its known ability to degrade single-stranded DNA from a 3'-OH terminus. Accordingly, sbcB mutations would alter the second activity while xonA mutations would not.

Biodegradation, Environmental↗
PubMed1972

Genetic analysis of mutations indirectly suppressing recB and recC mutations.

Mutations in sbcB inactivate exonuclease I and suppress the UV-sensitive, mitomycin-sensitive, recombination-deficient phenotypes associated with recB and recC mutations. Mapping experiments have located sbcB about 0.4 minutes from the his operon at 38.0 on the standard map of E. coli. This places sbcB between supD and his. A four-point cross shows that sbcB lies between P2 attH and his. P2 eduction deleting the his operon beginning with P2 attH also deletes sbcB and produces the expected exonuclease I deficiency and suppression of recB(-). The occurrence of chemical-mutagen-induced and spontaneous mutations indirectly suppressing recB(-) and recC(-) is examined. Three lines of strains produce only sbcA mutations while only sbcB mutations occur in a fourth line. Explanations for this behavior are proposed in light of the ability of the first three lines to express sbcB mutations which they inherit by transduction.

Chromosome Mapping↗
PubMed1971

Genetic recombination in Escherichia coli: the role of exonuclease I.

The indirect suppression of recB(-) and recB(-)recC(-) mutations by the sbcB(-) allele is caused by the loss of a nuclease active on denatured DNA. Results from enzyme purifications and studies with a specific antiserum demonstrate that the activity present in sbcB(+) strains, and lost in sbcB(-) strains, is exonuclease I. It is likely that sbcB is the structural gene for exonuclease I. The loss of exonuclease I activity restores the recombination proficiency of Escherichia coli cells that has been lost by mutations in the recB and/or recC genes. This indicates that in the absence of the recB-recC-determined enzyme, exonuclease I prevents recombination. Hypothetical pathways illustrating this conclusion are presented.

Alleles↗
PubMed1969

Enzymatic repair of DNA, 1. Purification of two enzymes involved in the excision of thymine dimers from ultraviolet-irradiated DNA.

Two nucleases that catalyze the excision of photoproducts from UV-irradiated DNA have been extensively purified from M. luteus (M. lysodeikticus). The first enzyme, an endonuclease, has been purified 5000-fold and is free of conflicting nuclease activities. It introduces single-strand breaks into irradiated DNA but does not act on native or single-stranded DNA. The purified enzyme is activated but not dependent on Mg(++) and has an approximate molecular weight of 15,000. Photoproduct excision is absolutely dependent on the second enzyme, a magnesium requiring exonuclease. This enzyme, which has been purified 1000-fold, is devoid of conflicting nucleases. It hydrolyzes irradiated and unirradiated denatured DNA at the same rate, but has no activity on RNA. It only acts on double-stranded DNA which has been both irradiated and pretreated with the endonuclease. The combined action of the endo- and exonuclease results in the quantitative removal of photoproduct regions from UV-irradiated DNA. Approximately five nucleotides are released for every single-strand incision.

DNA↗
PubMedDate not supplied

Cloning the quinic acid (aq) gene cluster from Neurospora crassa: identification of recombinant plasmids containing both qa-2+ and qa-3+.

A 22.2-kb insert of Neurospora crassa DNA containing at least two of the genes from the inducible catabolic quinic acid pathway has been cloned into the cosmid vehicle pHC79 resulting in a recombinant plasmid, pMSK308. The qa-2+ locus (which encodes catabolic dehydroquinase) is functionally expressed in both Escherichia coli and qa-2 mutants of N. crassa transformed with pMSK308 plasmid DNA. Expression of the qa-3 gene (which encodes quinate dehydrogenase) is only detected upon reintroduction into N. crassa. Results were also obtained which suggested that the qa-4 gene, which maps between qa-2 and qa-3, may also be present on both pMSK308 and the previously described plasmid pVK88. Certain anomalies in the types of N. crassa transformants obtained with pMSK308 plasmid DNA were noted.

Alcohol Oxidoreductases↗
PubMedDate not supplied

The cloning and analysis of the aroD gene of E. coli K-12.

A 5.6-kb PstI fragment containing the structural gene (aroD) for 5-dehydroquinate hydrolyase (DHQase) of Escherichia coli K-12 has been cloned into recombinant plasmid pJKK12. The bacterial fragment contains two Bg/II, one HpaII, one SalI and one XhoI site, but no EcoRI, HindIII or BamHI sites. the DHQase activity extracted from strains harboring pJKK12 had properties identical to those of the enzyme isolated from wild-type E. coli. The native protein appears to be a dimer composed of two 31 500 dalton subunits. aroD6 strains transformed with pJKK12 had an 11-fold and 34-fold increase in activity compared to untransformed wild-type controls grown on L broth and minimal medium, respectively. No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains of E. coli. At least four constitutively expressed genes are encoded on the fragment.

Amino Acids↗