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R H Doi

Publications and source records attributed to R H Doi.

At least 91 records · Page 5Linked to original sources
PubMed1986

Sequence relationships of United States prototype and wild-type bluetongue virus RNA genomes investigated by northern blot hybridization analysis.

The 10 double-stranded RNA (dsRNA) genome segments of various isolates of bluetongue virus (BTV) were separated on a polyacrylamide gel, denatured in NaOH, and blotted onto 2-aminophenylthioether paper. Blotted dsRNA segments were detected, using radioactive probes, a cloned copy of DNA 70% fragment of genome segment 7 of BTV-17, whole genome BTV-17 copy DNA, or whole genome BTV-17 dsRNA. These probes detected sequence diversities in different isolates of BTV and these diversities are discussed in relation to the serotype and the electrophoretic migration patterns of the isolates.

Base Sequence↗
PubMed1985

A strong sequence homology exists between the major RNA polymerase sigma factors of Bacillus subtilis and Escherichia coli.

The Bacillus subtilis rpoD gene has been sequenced and the primary structure of its product deduced. The molecular weight calculated for the sigma 43 is 42,828. The DNA and protein sequences of sigma 43 exhibit extensive homology to the Escherichia coli rpoD gene and its sigma 70 product, especially in the C-terminal two-thirds of the sequence. Other proteins exhibiting partial homology with sigma 43 include the E. coli nusA protein, the E. coli htpR (heat-shock regulatory gene) product sigma 32, and specific DNA-binding proteins. No amino acid homology was found between sigma 43 and B. subtilis phage SP01 sigma gp28, phage T7 RNA polymerase, or E. coli DNA primase. The gene exhibits a strong ribosomal binding site and a typical rho-independent transcription terminator sequence. A typical transcription terminator sequence was not observed upstream from the sigma 43 gene. The sigma 43 gene may be part of an operon, resembling the situation found in the E. coli sigma operon.

Amino Acid Sequence↗
PubMed1985

Bacillus subtilis dnaE encodes a protein homologous to DNA primase of Escherichia coli.

Bacillus subtilis dnaE encodes a protein essential for DNA replication and is tightly linked to rpoD, the gene for the major sigma factor of RNA polymerase. We have now determined the 1809-base pair sequence of the dnaE coding region, which precedes rpoD and is transcribed in the same counterclockwise direction on the chromosome. From the DNA sequence, we found that the dnaE protein comprised 603 amino acids with a calculated molecular mass of 68,428 daltons. This protein had significant and extensive regions of homology with Escherichia coli DNA primase, the polymerase that synthesizes short RNA primers during discontinuous DNA replication. Features of the coding and flanking regions that may modulate dnaE expression include a relatively weak ribosomal binding site (delta G' = -13.8 kcal), the use of uncommon codons in the reading frame, and no obvious promoter sequence for either dnaE or rpoD. Together, these results suggest that dnaE codes for B. subtilis DNA primase and, in light of the similarities to the organization of the E. coli sigma operon, that expression of dnaE may be coregulated with rpoD in B. subtilis.

Amino Acid Sequence↗
PubMed1985

Genetic mapping of rpoD implicates the major sigma factor of Bacillus subtilis RNA polymerase in sporulation initiation.

We have mapped the chromosomal locus of rpoD, which encodes the major sigma factor of Bacillus subtilis RNA polymerase. The rpoD locus lay between aroD and lys, tightly linked to dnaE and inseparable from crsA. Marker order in this region was acf-aroD-dnaE-rpoD(crsA)-spoOG-lys. By transformation using cloned donor DNA from the rpoD region, we identified the gene immediately upstream of rpoD as dnaE, which coded for a 62,000 dalton protein essential for DNA replication. Both dnaE and rpoD were transcribed in the same direction, counterclockwise on the chromosome. The gene functions and organization in the rpoD region are thus similar to those of the E. coli sigma operon. We also used transformation to identify crsA47 as a mutation within the sigma coding region itself. The crsA alteration of sigma renders the sporulation process insensitive to glucose catabolite repression, and also restores sporulation ability to strains carrying early-blocked spoOE, spoOF, and spoOK mutations. Thus the major sigma factor and these spoO gene products directly or indirectly affect the same cellular function.

Bacillus subtilis↗
PubMed1985

Detecting bluetongue virus RNA in cell culture by dot hybridization with a cloned genetic probe.

A 70% copy of genome segment 7 of bluetongue virus (BTV)-17 has been cloned into the plasmid pBR-322. This cloned BTV segment when used as a radioactive probe will hybridize to BTV double-stranded RNA extracted from cell cultures and dotted onto nitrocellulose paper. This dot hybridization technique is therefore suitable for detecting and identifying BTV in cell culture. The specificity of cloned probes is discussed in relation to detecting gene sequences specific for either the bluetongue serogroup or different serotypes of BTV.

Animals↗
PubMed1985

Mutations that affect the translation efficiency of Tn9-derived cat gene in Bacillus subtilis.

We have isolated two spontaneous mutations that increase the expression of the Tn9-derived cat gene in Bacillus subtilis. These mutations, which appear to affect initiation of translation of chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) consist of a tandem duplication and triplication of a 55-base-pair sequence located at the 5' end of cat. Included in the repeated sequence are the Shine-Dalgarno site, initiation codon, and a region of dyad symmetry located within the structural portion of the cat gene. A striking feature of the mutated initiation sites is their potential to form stem-loop structures at the 5' end of the cat messenger RNA. Within the single-stranded loops of these structures are the ribosome binding site and initiation codon for the cat gene. It appears that the Gram-negative cat translation initiation site has mutated to permit efficient utilization in B. subtilis without directly affecting Shine-Dalgarno sequence homology. This report suggests that secondary structure in the vicinity of the Shine-Dalgarno site can exert a strong positive influence on the initiation of translation in B. subtilis.

Acetyltransferases↗
PubMed1985

Catabolite-resistant sporulation (crsA) mutations in the Bacillus subtilis RNA polymerase sigma 43 gene (rpoD) can suppress and be suppressed by mutations in spo0 genes.

The catabolite-resistant sporulation (crsA) mutation is able to overcome the repressive effect of glucose on sporulation in Bacillus subtilis. Three chromosomal crsA mutations, crsA1, crsA4, and crsA47, were transferred by the "gene conversion" process to B. subtilis plasmid pRPD11, which consists of the entire wild-type rpoD coding sequence, encoding the major sigma 43 factor of B. subtilis RNA polymerase, and pUB110. By DNA sequence analysis we showed that all three crsA mutations were identical two-base changes, CCT (proline) to TTT (phenylalanine), within the rpoD coding sequence. Furthermore, the crsA47 mutation restored spo0J and spo0K sporulation to wild-type levels and partially improved the sporulation efficiencies of spo0B, spo0D, and spo0F. Extragenic suppressors (scr) of crsA47 included mutations in spo0A, spo0D, spo0F, and spo0K plus other mutations that have not been specifically identified. Thus sigma 43 appears to be involved in catabolite repression by glucose, to interact either directly or indirectly with spo0 gene products, and to play an important role in the initiation of spore development in B. subtilis.

Bacillus subtilis↗
PubMed1985

Translational coupling in Bacillus subtilis of a heterologous Bacillus subtilis-Escherichia coli gene fusion.

Translational coupling was demonstrated in a gene fusion in which the promoter and the N-terminal region of the Bacillus subtilis subtilisin (aprA) gene were fused to a promoterless Tn9-derived chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene. Expression of this gene fusion results in the production of a native-sized CAT product, whereas the Tn9-derived CAT gene is usually not translated from its own ribosome binding site in B. subtilis (D. S. Goldfarb, R. L. Rodriguez, and R. H. Doi, Proc. Natl. Acad. Sci. USA 79:5886-5890, 1982). A 178-base-pair deletion, which removed part of the signal peptide and the propeptide of the aprA gene and created a translational stop codon 230 base pairs upstream of the CAT gene ribosome binding site, reduced expression of the CAT gene. A BamHI 10-mer linker insertion into this deletion site, which restored the reading frame and simultaneously removed the translation stop codon, restored CAT gene expression. The data indicate that expression of the CAT gene was dependent on translation of the truncated aprA gene into the ribosome binding site of the CAT gene.

Acetyltransferases↗
PubMed1985

Molecular cloning and hybridization studies on bluetongue virus serotype 17.

The dsRNA of bluetongue virus (BTV) serotype 17 has been reverse transcribed and dsDNA copies of the viral RNA have been cloned into the plasmid vector pBR-322. Segments ranging from 3 kilobases to less than 500 bases have been cloned and at present 1 of the clones has been identified by hybridization to the genome segment of its origin (genome segment 7). Identification of further clones is proceeding. The techniques for northern blotting BTV dsRNA onto 2-aminophenylthioether (APT) paper and the detection of the transferred RNA by 32P labelled dsRNA or cDNA probes have been standardized. Cross-hybridization studies can be used to detect genetic relationships of different serotypes and isolates of BTV.

Bluetongue virus↗
PubMed1984

Utilization of a Bacillus subtilis sigma 37 promoter by Escherichia coli RNA polymerase in vivo.

The promoter region of Bacillus subtilis subtilisin E was found to be composed of two overlapping promoters with their transcription starting sites separated from each other by 15 base pairs (Wong, S.-L., Price, C. W., Goldfarb, D. S., and Doi, R. H. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1184-1188). At least one of the promoters is transcribed by a minor form of B. subtilis RNA polymerase with a sigma factor of 37,000 daltons. In vitro transcription analyses and in vivo studies with promoter probe plasmids pKO-1 and pCED-6 demonstrated that Escherichia coli RNA polymerase was able to initiate transcription from the subtilisin promoter cluster. S1 nuclease-mapping studies with both in vivo and in vitro transcribed RNA from E. coli and B. subtilis illustrate that E. coli can initiate transcription from both promoters with the same transcription start points as B. subtilis. The promoter strength of this promoter cluster in E. coli, as expressed in terms of galactokinase units, was 64 units and represents weak promoter activity in the E. coli system. These data indicate that either the single E. coli RNA polymerase is able to recognize the minor sigma 37 promoter or E. coli contains a hitherto unrecognized minor RNA polymerase holoenzyme which is capable of recognizing a B. subtilis sigma 37 promoter. On the other hand the B. subtilis RNA polymerase holoenzymes have been quite promoter-specific in our experiments to date.

Bacillus subtilis↗
PubMed1984

Overlapping promoters transcribed by bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases.

A 471-base pair HindIII DNA fragment of Bacillus subtilis contains two overlapping promoters which are recognized in vitro by sigma 55- and sigma 37-containing RNA polymerase holoenzymes from B. subtilis. In vitro transcript analyses and S1 nuclease mapping experiments with in vivo RNA indicate that both enzymes initiate transcription from the same putative +1 site. Physiological studies with the promoter-containing DNA fragment inserted into the expression probe plasmid pCED6 and quantitative S1 nuclease mapping experiments with RNA isolated from various stages of growth indicate that expression from these overlapping promoters is greater during the early stationary phase than during growth. We propose that the cryptic gene controlled by these promoters is transcribed by the sigma 55 enzyme during growth and by the sigma 37 enzyme during early stationary phase.

Bacillus subtilis↗
PubMed1984

The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo.

A cloned Bacillus subtilis gene (sprE) expressed only during the stationary growth phase is shown to encode the subtilisin E protease, an enzyme associated with sporulation. We have determined the DNA sequence of the sprE promoter region and the promoter-proximal half of the structural gene. The sprE gene codes for a putative 29-residue signal peptide and a 77-residue leader peptide preceding the mature subtilisin sequence. By plasmid integration and phage PBS1 transduction, we have mapped the sprE locus between glyB and metD on the B. subtilis chromosome, a region also containing the hyperprotease-producing hpr gene. In vitro the sprE gene is transcribed by the minor form of RNA polymerase containing a 37,000-dalton sigma factor (sigma 37). We show by S1 nuclease mapping that sprE transcription initiates at dual start sites both in vitro and in vivo and that the promoter for the downstream site has a characteristic sigma 37 recognition sequence. We propose that the physiological role of the sigma 37 RNA polymerase is to transcribe a class of genes that are catabolite repressed, that encode extracellular enzymes, or that are expressed only during the stationary phase of growth.

Amino Acid Sequence↗
PubMed1984

Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases.

A mutant strain of Bacillus subtilis carrying lesions in the structural genes for extracellular neutral (nprE) and serine (aprA) proteases was constructed by the gene conversion technique. This mutant had less than 4% of the extracellular protease activity of the wild type and sporulated normally, indicating that neither of these sporulation-associated proteases is essential for development.

Bacillus subtilis↗
PubMed1983

A temporally regulated promoter from Bacillus subtilis is transcribed only by an RNA polymerase with a 37,000 dalton sigma factor.

A 1,250 base pair Bacillus subtilis chromosomal HindIII restriction fragment (S fragment) has been cloned into the B. subtilis expression-probe plasmid pGR71. The S fragment induces the expression of the pGR71 chloramphenicol resistance gene shortly after the initiation of sporulation. The transcriptional promoter responsible for the expression of this temporally regulated genetic element has been identified and mapped in vitro. This promoter is recognized exclusively by the minor B. subtilis RNA polymerase that contains the 37,000 dalton sigma factor.

Acetyltransferases↗
PubMed1983

Rapid methods for comparing the double-stranded RNA genome profiles of bluetongue virus.

Various double-stranded RNA extraction procedures, gel electrophoresis systems, and methods to detect the RNA bands in the gel were investigated to find the most rapid methods to obtain the genome profiles of bluetongue virus in small volumes (1-25 ml) of infected cell culture fluids. Rapid double-stranded RNA extraction procedures coupled with staining the acrylamide gel slabs with ethidium bromide or silver nitrate resulted in well-defined genome profiles from bluetongue virus infected cell cultures in 6-48 h. Radioactive labelling of viral RNA with 32P was time consuming, cumbersome and expensive. These techniques detect less than 0.5 micrograms of double-stranded RNA which can be obtained from one 1-ml well of a 24-well cluster plate of bluetongue virus infected cell monolayers. The methods were therefore suitable for rapid comparisons of the electropherotypes of multiple virus isolates.

Bluetongue virus↗
PubMed1983

Isolation and physical mapping of the gene encoding the major sigma factor of Bacillus subtilis RNA polymerase.

At least four sigma factors separately bind the Bacillus subtilis RNA polymerase core (beta beta' alpha 2), each conferring a different promoter specificity on the holoenzyme in vitro. Using the Broome-Gilbert immunological screening, we isolated recombinant lambda phages that carry rpoD, the gene for the most abundant sigma factor, sigma 55. These phages encode a 55,000-dalton protein whose size, immunological properties, and peptide map identify it as sigma 55. All the phages have in common two adjacent 3.5-kilobase EcoRI fragments from the B. subtilis chromosome; most carry additional genomic DNA. Deletion analysis localized rpoD to a 1.6-kilobase region, suggested the direction of its transcription, and found two additional genes near rpoD, which code for proteins of 62,000 and 17,000 daltons.

Bacillus subtilis↗