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Biomedical subjects

R H Doi

Publications and source records attributed to R H Doi.

At least 109 records · Page 6Linked to original sources
PubMed1983

A survey of electropherotype relationships of bluetongue virus isolates from the western United States.

The genome profiles of 200 field isolates of bluetongue virus were obtained by extracting the double-stranded RNA genome from each isolate and separating the ten genome segments by polyacrylamide gel electrophoresis. These virus isolates, consisting of representatives of the four bluetongue virus serotypes existing in the U.S.A., were obtained during an epidemiological study commencing in 1979 in four western states of the United States. Analysis of the migration patterns of the ten double-stranded RNA genome segments revealed distinct variations of electrophoretic profiles of many isolates. These variations are discussed in relation to the year and geographical region in which the isolate was made, the species of animal from which the virus was obtained and the electropherotype relationships of the four bluetongue virus serotypes existing in the U.S.A.

Animals↗
PubMed1982

Peptide mapping of Bacillus subtilis RNA polymerase alpha factors and core-associated polypeptides.

An analysis of the peptide maps of the sigma factors and core-associated subunits of Bacillus subtilis RNA polymerase has revealed that all the sigma factors ad core-associated polypeptides are derived from separate genes and are not proteolytically modified products of the major 55,000-dalton sigma factor. A comparison of the peptide pattern of the major B. subtilis and Escherichia coli sigma factors revealed limited homology between them. Furthermore, antibody prepared against the 55,000-dalton B. subtilis sigma factor cross-reacted against the E. coli sigma factor, but not against any of the other B. subtilis sigma factors and core-associated polypeptides. These results unambiguously demonstrate the independently derived nature of the B. subtilis RNA polymerase core-associated subunits and the partial relationship between the major sigma factors of B. subtilis and E. coli.

Bacillus subtilis↗
PubMed1982

Translational block to expression of the Escherichia coli Tn9-derived chloramphenicol-resistance gene in Bacillus subtilis.

The Gram-negative product-encoding Tn9-derived chloramphenicol-resistance (Cmr) gene can be cloned but not phenotypically expressed in Bacillus subtilis. We show that, even when transcribed from B. subtilis promoters, the ribosomal binding site for the Cmr gene does not function well in B. subtilis. The Cmr gene product, chloramphenicol acetyltransferase (CmAcTase; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28), is detected in B. subtilis when the promoters, ribosomal binding sites, and initiation codons of B. subtilis genes are fused to the Cmr gene. These gene fusions lead to the in vivo production of mRNAs containing B. subtilis translation start signals followed in an open reading frame by the translation start site normally used by Escherichia coli to initiate translation of Cmr mRNA. Both fusion and native CmAcTase proteins are produced in E. coli, but only fusion CmAcTase is produced in B. subtilis. We conclude that the absence of native CmAcTase in B. subtilis is due to inability of the E. coli ribosomal binding site to function well in B. subtilis. Since fusion CmAcTase polypeptides are produced in E. coli, we conclude that these particular B. subtilis regulatory elements function heterologously in E. coli. The absence of a suitable binding site on the Cmr gene for B. subtilis ribosomes is consistent with reports that many E. coli genes are not expressed in B. subtilis and that E. coli mRNA functions poorly in B. subtilis in vitro translation systems. The functioning of B. subtilis regulatory sequences in E. coli is consistent with in vivo and in vitro data showing the expression of B. subtilis genes in E. coli. To confirm the hypothesis that the large CmAcTase proteins are NH2-terminal fusions of native CmAcTase we partially determined the sequence of one CmAcTase fusion protein.

Acetyltransferases↗
PubMed1981

Free sigma factor of Escherichia coli RNA polymerase can bind to DNA.

Free sigma factor from E. coli RNA polymerase holoenzyme was shown to associate with supercoiled pBR350 and pBRH4 plasmid DNA s by two methods. The banding pattern of sigma factor through a nondenaturing polyacrylamide slab gel was significantly altered in the presence of supercoiled DNA; sigma factor had little or no affinity to linear, double- or single-stranded DNA. At saturation, approximately one sigma factor was bound/200 base pairs of supercoiled pBR350 DNA. By the nitrocellulose filter trapping method, sigma factor was able to bind supercoiled pBRH4 DNA much more efficiently than linear double-stranded pBRH4 DNA. These results suggest that sigma factor plays a role in the binding of holoenzyme to DNA and may be involved in locally denaturing DNA in the promoter region as postulated by previous investigators.

DNA, Bacterial↗
PubMed1981

Free sigma subunit of Bacillus subtilis RNA polymerase binds to DNA.

The affinity of Bacillus subtilis RNA polymerase sigma and delta subunits to DNA was examined by a non-denaturing polyacrylamide slab gel electrophoresis method which made it possible to resolve DNA-bound and free subunits. The results revealed that sigma subunit, but not delta subunit had a relatively high affinity for double stranded DNA. The sigma subunit was bound maximally to super-coiled pGR1-3 plasmid DNA at a mass ratio of sigma/DNA of 0.7. With B. subtilis double stranded linear DNA one sigma subunit was bound per approximately 1,000 base pairs. The sigma-DNA complex was sufficiently stable for isolation by a molecular gel filtration column. The sigma subunit had much higher affinity for super-coiled than for linear pGR1-3 DNA or for linear double stranded or denatured DNA from B. subtilis, E. coli, and calf thymus. These results indicate that the free B. subtilis sigma subunit, in contrast to the E. coli sigma subunit, can bind by itself to DNA.

Bacillus subtilis↗
PubMed1980

Template-independent poly(A) x poly(U) synthesizing activity of different forms of Bacillus subtilis RNA polymerase.

Several, but not all, forms of bacillus subtilis RNA polymerase found in vegetative and sporulating cells can synthesize poly(A) x poly(U) in vitro. The vegetative delta-containing form of RNA polymerase (E delta) has little or no poly(A) x poly(U)-synthesizing activity, whereas RNA polymerase core (E) and sigma-containing core (E delta) both have significant activity. When purified vegetative delta factor was added to core, the core synthetic activity was reduced essentially to that of the vegetative enzyme E delta. When E sigma enzymes from vegetative and sporulating cells were compared for their salt sensitivity, it was found that the sporulation enzyme E sigma retained much more of its activity at 0.1 M KCl than the vegetative enzyme E sigma. Furthermore, when sporulation enzyme E delta 1 was compared with vegetative enzyme E sigma, it was found that the activity of the E sigma 1 form was much more resistant to high KCl concentrations than that of the vegetative E sigma form. These differences in enzyme activity, as affected by salt concentrations, suggest that the conformations of the sporulation E sigma and E delta 1 enzymes are different from that found in vegetative E sigma enzyme. These differences in conformation may be involved in selective gene expression during sporularion.

Bacillus subtilis↗
PubMed1980

Transcription-termination factor Rho from Bacills subtilis.

A protein has been isolated from Bacillus subtilis which has functions similar to that of transcription termination factor rho (rho) from Escherichia coli. The apparent molecular weight of the B. subtilis rho factor is about 80000-95000 as estimated by a non-denaturing polyacrylamide gel electrophoresis method. It contains two subunits with a molecular weight of 47000 as determined by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The rho factor shows poly(C)-dependent beta-gamma ATPase activity and depresses the activity of RNA synthesis from B. subtilis phage rho 29 DNA template with purified B. subtilis RNA polymerase holoenzyme. The specific activity of the poly(C)-dependent ATPase of the B. subtilis rho factor was significantly less than that of the E. coli rho factor. In the presence of rho factor fewer RNA transcripts were produced overall from the rho 29 template and smaller RNA transcripts with discrete sizes were made. These results suggest that the B. subtilis rho factor can catalyze transcription termination at specific sites on rho 29 phage DNA in vitro.

Adenosine Triphosphatases↗
PubMed1980

Purification and characterization of a kanamycin nucleotidyltransferase from plasmid pUB110-carrying cells of Bacillus subtilis.

The nucleotidyltransferase encoded by plasmid pUB110 was purified to greater than 95% purity with a 33% yield. The enzyme is a monomeric protein with a molecular weight of 34,000. The optimum pH for activity is 5, and the optimum MgCl2 concentration for activity is 18 mM. The enzyme, which is synthesized constitutively, is stable for several weeks at 4 degrees C. This enzyme would appear to be a good model gene product for the development of a pUB110 deoxyribonucleic acid-dependent in vitro protein-synthesizing system from Bacillus subtilis.

Bacillus subtilis↗
PubMed1980

Ultrastructural analysis of the effect of netropsin on sporulation of Bacillus subtilis.

An electron microscopic analysis of Bacillus subtilis cells revealed that netropsin blocked sporulation ultrastructurally at stages 0-I. These observations are consistent with previous results which indicated that cells were not committed to sporulation in the presence of the drug. Further, the addition of netropsin up to stage III of sporulation prevented the normal sharp increase in dihydrodipicolinate synthase activity which results in dipicolinic acid accumulation of stage IV. The addition of netropsin after stage III had much less effect on the synthesis of dihydrodipicolinate synthase and on sporulation. Thus, morphological events in sporulation are blocked early whereas a sporulation-associated enzyme may be affected at later stages. These data indicate than netropsin affects the expression of sporulation-associated genes in a differential manner.

Bacillus subtilis↗
PubMed1979

Sigma factor is not released during transcription in Bacillus subtilis.

The relationship between sigma (sigma) and delta (delta) factors of Bacillus subtilis RNA polymerase has been analyzed during initiation of RNA synthesis. When core enzyme (E) containing delta factor (E delta) binds to DNA, the delta factor is released with the formation of an E-DNA complex. The addition of sigma to the E-DNA complex results in the formation of a stable E sigma-DNA complex which can synthesize RNA upon addition of nucleoside triphosphates. Sigma factor, significantly, is not released from the core during RNA synthesis. These results suggest that delta and sigma factors can act sequentially during initiation of RNA synthesis with delta acting as a DNA recognition factor and sigma acting as an initiation factor. The results do not preclude the possibility that E sigma can initiate RNA synthesis correctly since E sigma alone can bind to DNA and initiate RNA synthesis.

Bacillus subtilis↗
PubMed1978

Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.

Amino Acids↗
PubMed1978

Delta factor can displace sigma factor from Bacillus subtilis RNA polymerase holoenzyme and regulate its initiation activity.

A protein with a molecular weight of 21,000 daltons is found associated with a fraction of Bacillus subtilis RNA polymerase core. This protein (delta) does not react with antibody made against sigma factor and has a peptide map which is significantly different from sigma factor. At ratios of 2:1 to 4:1 (delta:holoenzyme) the delta displaces sigma factor completely from the core and associates in a 1:1 ratio with core to form delta-core. Under the same incubation conditions sigma factor at a ratio of 10:1 (sigma factor:delta-core) does not displace delta from the delta-core. The delta-core has much less activity as compared to holoenzyme on various DNA templates. However, sigma factor does stimulate the activity of delta-core enzyme under conditions of RNA synthesis. These observations and the results of others suggest that delta-core enzyme binds initially to specific DNA sites followed by delta release from the core-DNA complex and that the sigma factor binds to the core-DNA complex to initiate RNA synthesis. Thus both delta and sigma factors are required in a sequential fashion for specific transcription to occur in B subtilis.

Bacillus subtilis↗