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R Losick

Publications and source records attributed to R Losick.

At least 163 records · Page 9Linked to original sources
PubMed1980

Mapping a cloned gene under sporulation control by inserttion of a drug resistance marker into the Bacillus subtilis chromosome.

A segment of Bacillus subtilis deoxyribonucleic acid (DNA) previously cloned in Escherichia coli contains a gene (the 0.4-kilobase [kb] gene) whose transcription is activated at an early stage of spore development. To map the genetic location of the 0.4-kb gene, we constructed a hybrid plasmid that inserts a chloramphenicol resistance determinant into the B. subtilis chromosome by recombination at a site of homology between cloned B. subtilis DNA and the chromosome. This hybrid plasmid (p1949-2) was constructed from the E. coli plasmid pMB9, the B. sultilis chloramphenicol resistance plasmid pCM194 (whose replication function was inactivated), and B. subtilis DNA from the vicinity of the 0.4-kb gene. Transformation of B. subtilis cells to drug resistance by p1949-2 was dependent upon the B. subtilis RecE+ phenotype and resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the 0.4-kb gene region of the chromosome. Chloramphenicol resistance in cells transformed by p1949-2 was mapped to the purA-cysA region of the B. subtilis chromosome, a region. In addition, DNA adjacent to the 0.4-kb gene was shown to contain the wild-type allele of genetic marker (tms-26) from that region.

Bacillus subtilis↗
PubMed1977

Purification and comparative properties of the delta and sigma subunits of RNA polymerase from Bacillus subtilis.

Bacillus subtilis delta protein is a 21 500-Mr polypeptide that can be isolated in association with RNA polymerase holoenzyme from uninfected bacteria and with modified forms of RNA polymerase from cells infected with phage SP01 [Pero, J., Nelson, J. and Fox, T. (1975) Proc. Natl Acad. Sci. U.S.A. 72,1589]. Although no function has been assigned to delta protein in uninfected cells, this host polypeptide enhances the specificity of transcription by phage-modified forms of RNA polymerase that contain SP01-coded regulatory subunits. This report describes the purification of delta and sigma proteins from uninfected B. subtilis and examines the comparative effects of these polypeptides on transcription by core RNA polymerase. Purified sigma polypeptide was found to stimulate the transcription of phage DNA while having little effect on RNA synthesis with the synthetic DNA poly(dA-dT) as template. In contrast, purified delta protein markedly depressed the transcription of poly(dA-dT) while having little effect on enzyme activity with phage DNA as template. The inhibitory effect of delta protein on poly (dA-dT) transcription was strongly dependent on the presence of KC1 in the RNA synthesis reaction mixture.

Bacillus subtilis↗
PubMed1977

Cloned Bacillus subtilis DNA containing a gene that is activated early during sporulation.

An endonuclease restriction fragment of Bacillus subtilis DNA has been identified that contains a gene whose transcription is activated early during the process of spore formation. This 4.4 kilobase (kb) DNA was detected by hybridizing electrophoretically separated Eco R1 restriction fragments with a radioactively labeled RNA of 0.4 kb from sporulating cells. The 4.4 kb B. subtilis DNA was then cloned and amplified in E. coli by insertion into the plasmid vector pMB9. Using the cloned B. subtilis DNA as a hybridization probe, we were able to detect the 0.4 kb transcript in total RNA from pulse-labeled bacteria. In wild-type cells, the gene coding for the 0.4 kb RNA was turned on within the first 30 min of spore formation. Although transcribed normally in a mutant blocked at stage II of spore development, the gene for the 0.4 kb RNA was not turned on in six different mutants blocked at stage 0 of sporulation. We conclude that the cloned B. subtilis DNA contains a gene whose transcription is regulated by events occurring at the onset of spore development.

Bacillus subtilis↗
PubMed1976

Bacillus subtilis RNA polymerase and its modification in sporulating and phage-infected bacteria.

Bacillus subtilis RNA polymerase holoenzyme consists of the subunits beta', beta, sigma, alpha, delta, and omega. In sporulating bacteria and in bacteria infected with phages SP01 and SP82, this enzyme undergoes changes in subunit composition and transcriptional specificity that could play a regulatory role in gene transcription. Sporulating bacteria may contain a specific component that inhibits the activity of the sigma subunit of polymerase probably by interfering with the binding of sigma-polypeptide to core enzyme. The hypothetical inhibitor may be metabolically unstable, since its activity is rapidly depleted from sporulating cells in the presence of chloramphenicol. Inhibition of sigma-polypeptide activity may restrict the transcription of phage DNA an infected sporulating cells. Although lacking the sigma-subunit, RNA polymerase purified from sporulating cells contains sporulation-specific subunits of 85,000 and 27,000 daltons. In SP01-infected bacteria, the sigma-subunit is replaced by phage-induced subunits. Purified enzyme containing the protein product of SP01 regulatory gene 28 directs the transcription of phage middle genes in vitro, while enzyme containing phage-induced polypeptides V and VI preferentially copies late genes. Accurate transcription of middle and late genes in vitro requires the host delta-subunit of polymerase (or high ionic strength) but not sigma-subunit. Phage PBS2 induces an entirely new multisubunit RNA polymerase that specifically transcribes PBS2 DNA in vitro. This enzyme is synthesized de novo after infection and does not arise by modification of the B. subtilis holoenzyme.

Antigen-Antibody Reactions↗
PubMed1976

The program of protein synthesis during sporulation in Bacillus subtilis.

The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression. At various stages of growth and spore formation, cells of B. subtilis were pulse-labeled with (35)S-methionine. Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis. We report that sporulating cells restricted or "turned off" the synthesis of certain polypeptides characteristic of the vegetative phase of growth. In certain cases, this "turn off" was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation. Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium. The synthesis of these sporulation-specific proteins was "turned off" in a temporally defined sequence throughout the period of spore formation. Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared. Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.

Bacillus subtilis↗
PubMed1975

RNA polymerase from sporulating Bacillus subtilis. Purification and properties of a modified form of the enzyme containing two sporulation polypeptides.

A new form of DNA-dependent RNA polymerase termed enzyme III has been purified from sporulating cells of Bacillus subtilis. In addition to the subunits of core RNA polymerase (beta', beta, alpha, and omega), enzyme III contains sporulation-specific polypeptides of 85,000 (P85) and 27,000 (P27) daltons. P85 corresponds to an RNA polymerase-binding protein previously identified by precipitation of RNA polymerase from crude extracts of sporulating cells with antibody directed against core enzyme. Both P85 and P27 co-purified with RNA polymerase highly purified by gel filtration, DEAE-cellulose chromatography, phosphocellulose chromatography, and glycerol gradient centrifugation. Enzyme III bound more tightly to phosphocellulose and sedimented more rapidly during zone centrifugation than did RNA polymerase lacking the sporulation polypeptides. RNA polymerase containing P85 and P27 transcribed B. subtilis DNA about 4.5 times more actively than did core RNA polymerase, although both enzymes exhibited similar activities with poly(dA-dT) and phage phie DNA as templates. Enzyme III and core RNA polymerase also differed in their response to increasing concentrations of Mg2+ and KCl.

Bacillus subtilis↗
PubMed1975

Antibody directed against Bacillus subtilis rho factor purified by sodium dodecyl sulfate slab gel electrophoresis. Effect on transcription by RNA polymerase in crude extracts of vegetative and sporulating cells.

Antibody directed against rho factor from vegetative Bacillus subtilis was prepared by immunizing a rabbit with denaturated rho polypeptide isolated by electrophoresis of partially purified DNA-dependent RNA polymerase on a sodium dodecyl sulfate-polyacrylamide slab gel. Antiserum to rho reacted specifically with native rho polypeptide but not with core RNA polymerase as judged by complement fixation and by an immunodiffusion assay. Anti-rho antibody also inhibited the ability of rho to stimulate transcription of phage phie DNA but failed to inhibit transcription of poly(dA-dT) by core enzyme. Specific antibody was also raised against a mixture of the beta and beta' subunits of RNA polymerase purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The effect of the anti-rho gamma-globulin on the transcription of phage phie DNA by RNA polymerase in crude extracts of vegetative and sporulating cells was examined. Anti-rho antibody markedly inhibited the transcription of phage DNA by RNA polymerase partially purified from vegetative bacteria by ammonium sulfate fractionation but had little effect on transcription of the phage DNA template by enzyme from sporulating cells. Addition of purified rho to a vegetative extract that had been depleted of rho by treatment with the anti-rho antibody restored active transcription of phage DNA. However, addition of purified rho to an antibody-treated extract of sporulating cells had little effect on phie RNA synthesis. These findings suggest that sporulating cells contain a component that interferes with the activity of the rho subunit of RNA polymerase.

Antibodies, Bacterial↗
PubMed1975

Rifampin resistance mutation of Bacillus subtilis altering the electrophoretic mobility of the beta subunit of ribonucleic acid polymerase.

The rifampin-resistance mutation of LS3,an asporogenous mutant of Bacillus subtilis 3610, leads to altered mobility of the beta subunit of ribonucleic acid polymerase in sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. This finding argues that the rifampin-resistance mutation is located in the structural gene coding for the beta polypeptide.

Bacillus subtilis↗
PubMed1974

Chloramphenicol restores sigma factor activity to sporulating Bacillus subtilis.

The sigma subunit of RNA polymerase from sporulating Bacillus subtilis is markedly inhibited in its ability to direct active transcription of phage varphie DNA in vitro. Treatment of sporulating bacteria with chloramphenicol rapidly restores sigma activity, suggesting that sporulating cells contain an inhibitor of sigma that is physiologically unstable or that becomes unstable after drug treatment. The hypothetical inhibitor is depleted exponentially with an apparent half-life of 11 min at 37 degrees .

Bacillus subtilis↗
PubMed1974

An immunological assay for the sigma subunit of RNA polymerase in extracts of vegetative and sporulating Bacillus subtilis.

The activity of the sigma subunit of Bacillus subtilis RNA polymerase decreases markedly during the first hours of sporulation [T.G. Linn et al. (1973) Proc. Nat. Acad. Sci. USA 70, 1865-1869]. We have prepared antibody against RNA polymerase holoenzyme to determine the fate of sigma polypeptide during spore formation. This antiserum specifically and independently precipitates sigma and core polymerase from crude extracts of B. subtilis as judged by both sodium dodecyl sulfate and urea gel electrophoresis of the precipitates. We report that crude extracts of sporulating cells lacking sigma activity contain as much sigma polypeptide as extracts of vegetative cells. However, sigma polypeptide in extracts from sporulating cells is apparently only weakly associated with RNA polymerase, as indicated by the failure of sigma to co-purify efficiently with core enzyme during phase partitioning. The loss of sigma activity and the weak binding of sigma to core enzyme occurs normally in a mutant blocked at an intermediate stage of sporulation (SpoII-4Z) and in wild-type bacteria sporulating in 121B medium, Difco sporulation medium, or Sterlini-Mandelstam resuspension medium. In contrast, sigma in two mutants (SpoOa-5NA and SpoOb-6Z) blocked at an early stage of spore formation remains active and tightly associated with RNA polymerase during stationary phase.

Animals↗
PubMed1973

Isolation of a new RNA polymerase-binding protein from sporulating Bacillus subtilis.

RNA polymerase was precipitated from extracts of radioactively labeled vegetative and sporulating Bacillus subtilis with antiserum prepared against vegetative core polymerase. The precipitates were solubilized and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antiserum added to an extract of vegetative B. subtilis precipitated only the known subunits of core RNA polymerase, but antiserum added to an extract of sporulating cells precipitated a new polypeptide of 70,000 daltons in addition to the subunits of core enzyme. The 70,000-dalton polypeptide precipitated from an extract of a mixture of vegetative and sporulating B. subtilis, separately labeled with two different radioisotopes, contained only the radioisotope characteristic of the sporulating cells. The 70,000-dalton protein has been freed of core RNA polymerase and extensively purified by chromatography on phosphocellulose. Precipitation of the purified 70,000-dalton protein by the anti-polymerase serum requires the prior addition of vegetative or sporulation core RNA polymerase. The reaction is specific since the purified protein is not precipitated during antibody precipitation of either phage lambda repressor or bovine serum albumin. The RNA polymerase-binding protein appears during the third hour of sporulation and is apparently not synthesized by the sporulation-defective mutant rfr 10.

Animals↗