Biomedical subjects

S R Kushner

Publications and source records attributed to S R Kushner.

At least 73 records · Page 4Linked to original sources

PubMed1983

Amplification and purification of exonuclease I from Escherichia coli K12.

Employing the recombinant runaway replication plasmid pDPK13 [sbcB+], an exonuclease I-overproducing derivative of Escherichia coli K12 has been constructed. The strain SK4258 has exonuclease I activity 140-400-fold higher than wild type control levels. A new purification procedure has been developed such that the protein can be purified to near homogeneity and is free of endonuclease and RNase activities. The specific activity of the purified enzyme is 10-fold higher than reported previously (Ray, R.K., Reuben, R., Molineux, I., and Gefter, M. (1974) J. Biol. Chem. 249, 5379-5381). Native exonuclease I is a single polypeptide having Mr = 55,000 with a Stokes radius of 3.12 nm.

DNA↗

PubMed1983

Amplification of ribonuclease II (rnb) activity in Escherichia coli K-12.

A 7.1 kb HindIII-XhoI fragment of E. coli DNA which contains the structural gene for ribonuclease II (rnb) has been cloned in the recombinant plasmid pDK24. At least two constitutively expressed genes are encoded on the fragment as shown by maxicell analysis. On denaturing polyacrylamide gels RNase II appears as a single 72,000 dalton species. The approximate site of transcription initiation of the rnb gene has been mapped. Although derivatives of E. coli harboring pDK24 contained 10-fold more RNase II activity that wild type strains without the plasmid, the degradation rate of mRNA was similar in all strains tested. Strains deficient in both RNase II and polynucleotide phosphorylase appear inviable.

Cloning, Molecular↗

PubMed1983

Cloning and physical analysis of the pyrF gene (coding for orotidine-5'-phosphate decarboxylase) from Escherichia coli K-12.

The structural gene (pyrF) for orotidine-5'-phosphate decarboxylase (OMPase, EC 4.1.1.23) of Escherichia coli K-12 has been cloned as part of two PvuII fragments (1.2 and 0.9 kb) to form the recombinant plasmid pDK26. Extracts of E. coli [pDK26] had 80-fold higher levels of OMPase activity than wild-type strains without the plasmid. Maxicell analysis showed that pDK26 encoded two proteins of Mr 27 000 [pyrF(OMPase)] and 15 000 (Z) in addition to the ampicillin-resistance determinant. The approximate initiation site and direction of transcription of the pyrF gene have been determined. Extracts of strains that were deficient in polynucleotide phosphorylase (PNPase) had higher levels of OMPase activity than isogenic PNPase+ strains when one or two copies of the pyrF gene were present per cell either in the chromosome or on a low copy number plasmid. However, no significant difference in OMPase activity was seen in PNPase- strains that contained the pyrF gene cloned in a multicopy plasmid. Southern hybridization experiments showed that the yeast gene for OMPase (URA3) and the E. coli pyrF gene had less than 70% DNA sequence homology.

Base Sequence↗

PubMed1983

Physical and genetic characterization of the cloned sbcB (exonuclease I) region of the Escherichia coli genome.

A 17-kilobase (kb) HindIII fragment containing the structural gene for exonuclease I (sbcB) from Escherichia coli K-12 was physically and genetically characterized. The monomeric molecular weight of exonuclease I was 53,700, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 35S-labeled E. coli mini- and maxicells. The gene was in close proximity to two unidentified proteins with molecular weights of 15,200 and 13,100. No other polypeptides appeared to be constitutively synthesized from the 17-kb fragment. Genetically, no portion of the histidine operon or the shikimic acid transport gene (shiA) was detected on the fragment. Although the entire 17-kb fragment in the vector pMB9 was too unstable to be useful, a 7.6-kb BamHI-EcoRI fragment inserted into a variety of vectors was stable. A detailed restriction map of the fragment is presented. Several derivatives in the runaway-replication vectors pMB06 and pMOB45 yielded 20- to 52-fold increases in exonuclease I activity after a switch in growth temperature to 40 degrees C. Of six exonuclease I mutants examined by DNA-DNA hybridization, one (xonA6) appeared to have arisen from a 1.2-kb insertion into the structural gene for exonuclease I.

Cloning, Molecular↗

PubMed1983

Purification and characterization of orotidine-5'-phosphate decarboxylase from Escherichia coli K-12.

Using blue Sepharose affinity chromatography, we purified orotidine-5'-phosphate decarboxylase over 600-fold, to near homogeneity, from strains of Escherichia coli harboring the cloned pyrF gene on the multicopy plasmid pDK26. The purified enzyme has a subunit molecular weight of 27,000 but appears to be catalytically active as a dimer. In contrast to yeast enzymes, orotidine-5'-phosphate decarboxylase from E. coli is unstable at pH 6.0. The specific activity and Km values were 220 U/mg and 6 microM, respectively.

Carboxy-Lyases↗

PubMed1982

DNA repair in Escherichia coli: identification of the uvrD gene product.

A 2.9-kilobase (kb) Pvu II DNA fragment that contains the uvrD gene of Escherichia coli K-12 has been cloned in both low-copy and multiple-copy plasmid vehicles. The low-copy uvrD plasmid (pVMK49) complements a variety of uvrD, uvrE, and recL mutations. In contrast, the same strains carrying the 2.9-kb fragment in a multiple-copy plasmid (pVMK45) remain sensitive to ultraviolet light (UV). Additionally, pVMK45 transformants of wild-type E. coli are sensitive to UV and methyl methanesulfonate and appear to be recombination deficient. The cloned uvrD gene does not complement the dominant uvrD3 allele. The 2.9-kb Pvu II insert in these plasmids encodes a single 76,000-dalton protein, which, on the basis of insertional inactivation experiments with the Tn1000 transposon, must be the uvrD gene product. These data confirm earlier genetic analysis which suggested that recL, uvrE, and uvrD were all allelic. The direction of transcription of the uvrD gene has also been determined.

Base Composition↗

PubMed1981

Identification and characterization of recombinant plasmids carrying the complete qa gene cluster from Neurospora crassa including the qa-1+ regulatory gene.

The early reactions in the catabolism of quinic acid in Neurospora crassa are controlled by at least four genes which are clustered on linkage group VII. Three of the loci (qa-2, qa-4, and qa-3) encode enzymes that convert quinic acid to protocatechuic acid. The fourth gene (qa-1) encodes a positive regulatory protein which, in the presence of quinic acid, leads to the de novo synthesis of the other proteins in the qa cluster. This communication describes a series of recombinant plasmids that span 36.5 kilobases of linkage group VII and contain the coding sequences for qa-2, qa-4, qa-3, and the qa-1 regulatory protein. The plasmids were obtained by partial digestion of wild-type N. crassa DNA with EcoRI and ligation into the cosmid cloning vehicle pHC79. Two independently derived plasmids (pMSK331 and pMSK335), each containing 36.5-kilobase inserts, were shown by transformation back into N. crassa to contain the entire qa gene cluster. A preliminary physical organization of the gene cluster is presented. An improved procedure for the transformation of N. crassa with plasmid DNA is also described.

DNA Restriction Enzymes↗

PubMed1981

Genetic organization and transcriptional regulation in the qa gene cluster of Neurospora crassa.

A transcription map of the qa gene cluster of Neurospora crassa has been constructed by using cloned DNA fragments as hybridization probes. The mRNAs encoded in the previously identified qa-2 (3-dehydroquinate hydro-lyase, EC 4.2.1.10), qa-4 (dehydroshikimate dehydratase), qa-3 (quinate:NAD+ 3-oxidoreductase, EC 1.1.1.24), and qa-1 (regulatory protein) genes have been characterized. In addition, mRNAs encoded in two new genes in this cluster (qa-x, qa-y) have been identified. Regulation of this system occurs at the level of transcription and is under the combined control of quinic acid and the qa-1 protein. The qa cluster represents a group of adjacent coding sequences which occupy approximately 18 kilobases in linkage group VII. The expression of the qa-1 gene appears to be constitutive but also autoregulated. mRNAs encoded in genes flanking the qa gene cluster have also been identified.

Gene Expression Regulation↗

PubMed1980

Expression of the HIS3 gene of Saccharomyces cerevisiae in polynucleotide phosphorylase-deficient strains of Escherichia coli K-12.

The PstI and BamHI fragments, containing the HIS3 (imidazoleglycerol phosphate dehydratase) gene of yeast obtained from pYehis2, and the ColE1-derived plasmid pBR322 were ligated in vitro and used to transform hisB463 strains of Escherichia coli K-12. Expression of the cloned HIS3 gene from yeast was markedly enhanced (3--5-fold) in polynucleotide phosphorylase (pnp)-deficient strains of E. coli. The levels of both HIS3 and plasmid-encoded mRNAs were increased in pnp- strains carrying the chimeric plasmids, whereas there was little difference in the levels of pBR322-specific mRNAs in pnp+ and pnp- strains. This increase in HIS3 mRNA appeared to be related to specific stabilization of the eukaryotic message due to its unique structural features, since the half-life of the HIS3 mRNA increased from 1.5 to 18.7 min, whereas no increase in the half-lives of pBR322 vehicle mRNAs was observed. A physical map of the plasmid pYehis2 was constructed using restriction endonuclease and molecular cloning techniques.

Chromosome Mapping↗

PubMed1980

Isolation of plasmids carrying either the uvrC or uvrC uvrA and ssb genes of Escherichia coli K-12.

A 3.4 kb PstI fragment containing the uvrC gene of Escherichia coli K-12 has been cloned into pBR322. Plasmids carrying this PstI fragment, in either orientation (pGY3233, pGY4211) relative to the cloning vehicle, complement uvrC mutants. A second plasmid (pGY3243) with a 21 kb HindIII fragment is shown to complement mutations in uvrA and ssb (single-strand binding protein). A composite plasmid (pGY4610) containing pBR322 and PstI fragments derived from pGY3233 (3.4 kb) and pGY3243 (11.05 kb) complements the uvrC, uvrA and ssb mutations.

Cloning, Molecular↗

PubMed1979

Constitutive expression in Escherichia coli of the Neurospora crassa structural gene encoding the inducible enzyme catabolic dehydroquinase.

In Neurospora crassa the qa-2 gene, which encodes catabolic dehydroquinase, is under positive control exerted by the inducer quinic acid and an activator protein encoded in the closely linked qa-1 gene. In order to determine if this regulatory mechanism is maintained when the qa-2 gene is cloned on a recombinant plasmid and expressed in Escherichia coli, molecular cloning experiments have been performed using DNA isolated from a qa-1+ (inducible), a qa-1C (constitutive) and two qa-1 (non-inducible) strains of N. crassa. The results demonstrate that the level of expression of the qa-2 gene in E. coli is completely independent of the mutational state of the qa-1 gene. Moreover, the level of expression of the cloned qa-2 gene was unaffected by either an intracellularly produced inducer of catabolic dehydroquinase or by the general procaryotic positive effector, the CAP factor. The weight of evidence thus supports the conclusion that transcription of the N. crassa qa-2 gene in E. coli does not require the qa-1 activator protein and thus is not controlled by the same mecahnism which functions in N. crassa.

Centrifugation, Density Gradient↗

PubMed1979

Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA.

An efficient transformation system has been developed for Neurospora crassa that uses spheroplasts and pVK88 plasmid DNA. pVK88 is a recombinant Escherichia coli plasmid carrying the N. crassa qa-2(+) gene which encodes catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) and is part of the qa gene cluster. The recipient strain carries a stable qa-2(-) mutation and an arom-9(-) mutation, thus lacking both catabolic and biosynthetic dehydroquinase activities. Transformants were selected as colonies able to grow in the absence of an aromatic amino acid supplement. These colonies were qa-2(+) and had normal levels of catabolic dehydroquinase. DNA.DNA hybridization evidence with appropriate labeled probes indicates clearly that in some instances transformation involves the integration of bacterial plasmid sequences together with the qa-2(+) gene into the N. crassa genome. On the basis of genetic, enzyme assay, and DNA hybridization data, at least three types of transformation events can be distinguished: (i) replacement of the qa-2(-) gene by the qa-2(+) gene without any effect on the expression of the other genes in the qa cluster, (ii) linked insertion of a normal qa-2(+) gene accompanied by inactivation of the adjacent qa-4(+) gene, and (iii) insertion of a normal qa-2(+) gene at an unlinked site in the N. crassa genome. This newly integrated qa-2(+) genetic material is inherited in a typical Mendelian fashion. A low level of transformation has also been obtained by using linear total N. crassa DNA. Two such qa-2(+) transformants are unlinked to the qa-2(-) gene of the recipient.

DNA Restriction Enzymes↗

PubMed1979

Increased expression of a eukaryotic gene in Escherichia coli through stabilization of its messenger RNA.

The expression of a cloned eukaryotic gene [catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) (qa-2+) from Neurospora crassa] is dramatically increased (as much as 100-fold) in Escherichia coli strains deficient in polynucleotide phosphorylase (pnp) (polynucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) and RNase I (rna). The increased expression is controlled primarily by the absence of polynucleotide phosphorylase and appears to be specific for the eukaryotic gene. No increase in the specific activity of either chromosomal or plasmid-borne prokaryotic genes has been observed. In polynucleotide phosphorylase-deficient strains of E. coli the half-life of plasmid (pVK88, ampr qa-2+)-encoded mRNAs increases from 1.0 to 2.8 min. This increase must be due primarily to stabilization of the aq-2 mRNA because no increase in the half-lives of pBR322 vehicle mRNAs was observed in polynucleotide phosphorylase-deficient strains. These results suggest that there are inherent structural differences between prokaryotic and eukaryotic mRNAs.

DNA, Recombinant↗

PubMed1978

Transcription and translation in E. coli of hybrid plasmids containing the catabolic dehydroquinase gene from Neurospora crassa.

Two hybrid plasmids which carry the gene for Neurospora crassa catabolic dehydroquinase (C-DHQase) and complement an aroD6 (dehydroquinase-deficient) auxotroph of Escherichia coli have been analyzed. One of these contains a 2.9 kilobase (kb) fragment cloned in the HindIII site of plasmid pBR322 (pVK57) and the other contains a 6.8 kb fragment cloned in the PstI site (pVK88). Restriction enzyme mapping of these plasmids has demonstrated that the 2.9 kb fragment is totally contained within the 6.8 kb fragment. When the polarity of either the HindIII fragment or PstI fragment was reversed with respect to pBR322 no effect was observed on either the ability of the hybrid to complement an aroD- auxotroph or on the level of C-DHQase activity. In vivo transcription of plasmid pVK88 in both orientations was analyzed by RNA-DNA hybridization and by the techniques developed by Southern (1975). Approx. 40% of the plasmid-directed transcription occurred from the cloned PstI fragment and 60--70% of these N. crassa transcripts were encoded by the 2.9 kb HindIII fragment. The Southern technique allowed a further localization of the region of most extensive transcription to a 1.8 kb HindIII-EcoRI fragment. Biochemical analysis revealed that the C-DHQase protein produced by strains harboring pVK57 and pVK88 in either orientation was identical to the N. crassa enzyme. Furthermore, when these plasmids were segregated into minicells and labeled with 14C amino acids, the C-DHQase protein was synthesized at a level comparable to other plasmid-encoded proteins. Taken together, these experiments demonstrate that transcription is efficiently initiated in E. coli from a site on the cloned N. crassa DNA and that the resulting C-DHQase mRNA is efficiently and accurately translated.

DNA Restriction Enzymes↗

PubMed1978

Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products.

DNA Repair↗

PubMed1977

Transcription of ribosomal protein genes carried on F' plasmids of Escherichia coli.

The transcriptional activity of the DNA sequences coding for certain ribosomal proteins has been measured in Escherichia coli. Two partial diploid strains were isolated from mating of a recA, argD, aroE recipient and an Hfr with an origin at 69.5 min. One contained an F' element carrying the genes from 69.5 to 72.7 min including the rpsL locus (72.4) and the second was diploid for the genes from 69.5 to 71.5 min but did not include any mapped ribosomal protein loci. The molecular weights of the plasmids were estimated to be 140 and 110 x 10(6) daltons, respectively. The extent of in vivo transcription of the chromosomal genes on the plasmid and the ribosomal protein mRNA fraction of the total cellular RNA weer calculated from DNA-RNA liquid hybridization experiments using both DNA and RNA excess procedures. The results indicated a high degree of transcriptional activity concentrated in the ribosomal protein sequences with 83% of the F' chromosomal sequences transcribed into mRNA products representing about 0.12% of the total cellular RNA.

Conjugation, Genetic↗

PubMed1977

Conditionally lethal ribosomal protein mutants: characterization of a locus required for modification of 50S subunit proteins.

Mutagenized P1 bacteriophage were used to transduce a marker (aroE) adjacent to the cluster or ribosomal protein genes located at 72 min on the Escherichia coli chromosome. Linked temperature-sensitive transductants were isolated and characterized. A mutant unable to grow at 44 degrees was found to be defective in protein synthesis both in vivo and in vitro. At the restrictive temperature mutant cells lost all polyribosomes. Analysis of the ribosomal proteins revealed alterations in at least four 50S subunit proteins. The mutation (called rimE1, ribosomal protein modification) mapped between rpsE and aroE. It is suggested that the rimE locus is the structural gene for an activity that modifies a selected number of ribosomal proteins.

Coliphages↗

PubMed1977

Expression in Escherichia coli K-12 of the structural gene for catabolic dehydroquinase of Neurospora crassa.

The inducible quinic acid catabolic pathway of Neurospora crassa is controlled by four genes, the qa cluster which includes structural genes qa-2, qa-3, qa-4 for three enzymes and a regulatory gene, qa-1. In this paper we report the molecular cloning of at least the qa-2 gene which encodes the catabolic dehydroquinase (5-dehydroquinate hydro-lyase, EC 4.2.1.10). Endo.R.HindIII restriction endonuclease fragments of N. crassa DNA from a qa-1(c) (constitutive) mutant and of Escherichia coli plasmid pBR322 DNA were ligated in vitro and used to transform an aroD6 (5-dehydroquinate hydrolyase deficient) strain of E. coli K12. The recombinant plasmid (pVK55) isolated from one AroD(+) transformant (SK1518) contained, in addition to pBR322, two N. crassa HindIII fragments with molecular weights of 2.3 x 10(6) and 1.9 x 10(6). Derivatives of SK1518 cured of plasmid DNA were phenotypically Amp(s) and AroD(-). These cured strains, retransformed with pVK55, were phenotypically Amp(R) and AroD(+). Strains transformed with pVK55 possessed 5-dehydroquinate hydrolyase activity but no activity was present in any AroD(-) strain. The enzyme extracted from strains containing the recombinant plasmid was identical to N. crassa catabolic dehydroquinase by the criteria of heat stability, ammonium sulfate fractionation, immunological crossreactivity, molecular weight, and purification characteristics. This identity demonstrates that the N. crassa qa-2(+) gene is carried by the recombinant plasmid and is apparently transcribed and translated with complete fidelity. Furthermore, subunit assembly of the N. crassa polypeptides also occurs in E. coli, because the catabolic dehydroquinase is a multimer composed of approximately 20 identical subunits.

DNA Restriction Enzymes↗