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Biomedical subjects

R H Doi

Publications and source records attributed to R H Doi.

At least 73 records · Page 4Linked to original sources
PubMed1989

Synthesis and refolding of human tissue-type plasminogen activator in Bacillus subtilis.

A 1.6 kb cDNA fragment encoding the mature part of the human tissue-type plasminogen activator (t-PA) was subcloned into a Bacillus subtilis dual plasmid expression system [Le Grice et al., Gene 55 (1987) 95-103]. Expression of the tPA gene in this vector was regulated by the inducible Escherichia coli lac elements, as well as a strong phage-T5-derived promoter and ribosome-binding site preceding the polylinker. The 5' end of the tPA gene corresponding to the N terminus of mature t-PA was fused in phase to the third codon present in the polylinker region of the expression vector, p602/22, to form p602-t-PA. B. subtilis containing p602-t-PA, when induced with isopropyl-beta-D-thiogalactopyranoside, produced large amounts of immunoreactive t-PA (approx. 20 micrograms/ml). As expected, t-PA was not secreted into the culture media, but was localized in intracellular inclusion bodies and was found to be enzymatically inactive. However, enzymatic activity could be regained following complete reduction followed by slow oxidation of the solubilized inclusion bodies. The recombinant t-PA (rt-PA) showed, after purification, a smaller molecular size than melanoma t-PA, probably due to lack of glycosylation in the Bacillus system. Like melanoma t-PA, rt-PA exhibited tremendous stimulation of plasminogen activation in the presence of fibrin. Our results illustrate that B. subtilis, when supplied with the proper transcriptional/translational regulatory elements, can be an effective system for expression of heterologous gene products.

Bacillus subtilis↗
PubMed1989

Bluetongue virus-17 fusion protein ns1 expressed in Escherichia coli by pUC vectors.

The cDNA coding for the major nonstructural protein, NS1, of bluetongue serotype 17 (BTV-17) was cloned previously. Using pUC plasmids, we have successfully expressed the NS1 protein in Escherichia coli as a LacZ-NS1 fusion protein. The recombinant NS1 protein reacted with rabbit anti-BTV-17 antiserum, and was thus immunologically indistinguishable from the native BTV-17 NS1 protein. This was the first bluetongue viral protein to be produced in a bacterial system.

Blotting, Western↗
PubMed1989

Bacillus subtilis subtilisin gene (aprE) is expressed from a sigma A (sigma 43) promoter in vitro and in vivo.

In vitro studies demonstrated that the Bacillus subtilis subtilisin gene (aprE) could be transcribed by RNA polymerase holoenzyme reconstituted from core and sigma A factor obtained from vegetative cells. Upstream deletions (from -45) reduced the amount of transcription from the promoter. A deletion downstream of the promoter that overlapped a putative downstream minor promoter did not affect transcription from the sigma A promoter, which indicated that the putative downstream promoter is not utilized in vivo. S1 nuclease mapping studies showed that there was a low level of transcription from the subtilisin promoter during the growth phase and that the site of transcription initiation was the same during log and stationary phases. We conclude from these findings that there is only one promoter for the subtilisin gene and that it can be transcribed by the sigma A form of RNA polymerase in vitro.

Bacillus subtilis↗
PubMed1989

Recombinant DNA probe for serotype-specific identification of bluetongue virus 17.

The double-stranded RNA genome from 117 field isolates of bluetongue virus (BTV) serotypes 10, 11, 13, and 17 was blotted onto nitrocellulose paper and hybridized with a radioactively labeled cloned copy of DNA genome segment 2 of BTV-17. Viral RNA from BTV prototype strains 2, 10, 11, 13, and 17 were used as controls. The probe hybridized only with the viral RNA from prototype BTV-17 virus and field isolates of BTV-17. There was no cross hybridization with field isolates of BTV serotypes 10, 11, and 13. A complementary DNA probe developed from genes coding for BTV serotype specificity was effectively used in a slot-blot hybridization system for efficiently characterizing the viral serotype.

Animals↗
PubMed1988

Expression and secretion of human atrial natriuretic alpha-factor in Bacillus subtilis using the subtilisin signal peptide.

Using the signal peptide of the Bacillus subtilis subtilisin gene (aprE) and a synthetic cDNA corresponding to the mature region of the human atrial natriuretic alpha-factor (hANF), we have constructed a secretion vector. B. subtilis cells, when transformed with this vector, secrete immunoreactive hANF peptides into the medium at about 500 micrograms/liter. The hANF is the first human gene product to be secreted from B. subtilis using this signal peptide. We have used promoters active during vegetative growth or sporulation and hosts deficient in several extracellular proteases but some proteolysis of the secretion products still occurs. In addition, both cell growth and sporulation are adversely affected by hANF production. Possible explanations for this observation are inefficient secretion of the atrial hormone or toxicity of the precursor or mature peptide.

Atrial Natriuretic Factor↗
PubMed1988

The cloning of full-length genome segments 2, 5, 6, and 8 of bluetongue virus (BTV) serotype 17 and studies of their genetic relatedness to United States BTV serotypes.

The double-stranded (ds) RNA genome segments 2, 5, 6, and 8, which encode the outer capsid proteins P2 and P5, and the two nonstructural proteins, NS1 and NS2, respectively, of bluetongue virus (BTV) serotype 17 have been cloned into pBR322. The length of the cloned genes indicates that the entire respective dsRNA genome has been cloned in each case. The four cloned genes were used as 32P-labeled probes to hybridize to PAGE-separated United States BTV serotypes 2, 10, 11, 13, and 17. Of these, the segment 6 clone was identified as being highly conserved and hybridized to all serotypes. Segment 8 was less conserved and segment 5 even less. The segment 2 clone was serotype specific. None of the probes hybridized with dsRNA from epizootic hemorrhagic disease virus (EHDV).

Animals↗
PubMed1988

Comparison of dot-blot and Northern blot hybridizations in the determination of genetic relatedness of United States bluetongue virus serotypes.

Dot-blot and Northern blot hybridization methods to determine the genetic relatedness of United States bluetongue virus serotypes 2, 10, 11, 13, and 17 were compared. Both plasmid and insert DNA probes from cloned BTV-17 dsRNA segments 2, 5, 6, and 8 were hybridized to dsRNA from the BTV serotypes and epizootic hemorrhagic disease virus (EHDV). Stringencies of hybridization were kept identical, and experiments differed only in the method in which dsRNA was applied to the membranes (dot-blot or Northern blot). The Northern blot hybridization method yielded more consistent results, and it was visually and unequivocally shown to which dsRNA segment a cDNA probe bound, since the dsRNA segments were separated by PAGE prior to blotting and hybridization. In contrast, the dot-blot hybridization method gave less consistent results. Identical results for plasmid and insert probes were obtained for Northern blot hybridizations but not for dot-blot hybridizations. At least two probes could be used simultaneously in Northern blot hybridizations.

Blotting, Northern↗
PubMed1988

Cloning and nucleotide sequence of senN, a novel 'Bacillus natto' (B. subtilis) gene that regulates expression of extracellular protein genes.

A new 'Bacillus natto' gene, senN, that regulates the expression of several extracellular proteins in B. subtilis has been cloned and sequenced. senN codes for a small, highly basic protein with an amino acid sequence different from the products coded by the regulatory genes sacQ, sacV, prtR and hpr. SenN stimulates gene expression at the transcriptional level. A putative homologous locus has been detected in the B. subtilis chromosome by Southern blotting.

Amino Acid Sequence↗
PubMed1988

rpoD operon promoter used by sigma H-RNA polymerase in Bacillus subtilis.

Three promoters direct transcription of the sigA (rpoD) operon in Bacillus subtilis. Promoters P1 and P2 are used during the exponential growth phase, whereas P3 is used only during the stationary phase. We examined the use of these promoters in promoter-probe plasmids and found that expression from P3 was prevented by a mutation in spoOH, which encodes the secondary RNA polymerase sigma factor sigma H. Moreover, we found that sigma H-containing RNA polymerase efficiently and accurately used the P3 promoter in vitro. Evidently, this operon, which is essential for exponential growth, is transcribed during the early phase of sporulation by this secondary form of RNA polymerase. Comparison of the nucleotide sequences of the P3 promoter and the spoVG promoter, which also is used by sigma H-RNA polymerase, revealed sequences at the -10 and -35 regions of these promoters that may signal recognition of promoters by sigma H-RNA polymerase.

Bacillus subtilis↗
PubMed1987

Promoter switching during development and the termination site of the sigma 43 operon of Bacillus subtilis.

Sequencing data indicated that the RNA polymerase sigma 43 operon of Bacillus subtilis consisted of three genes, P23 (function unknown), dnaE (DNA primase), and rpoD (sigma 43) (Wang and Doi 1986a). S1 nuclease mapping experiments with RNA from various stages of growth demonstrated the presence of two overlapping sigma 43 promoters that controlled the expression of the operon during growth and a sigma 37 promoter that regulated the expression of the operon during the sporulation phase. This promoter switching mechanism ensured that this important operon would be expressed during different nutritional states of the cell and also illustrated a function for the minor RNA polymerase sigma 37 holoenzyme in the expression of genes which are normally expressed during the logarithmic phase of growth. The location of the transcription termination signal confirmed that the sigma 43 operon consists of three genes.

Bacterial Proteins↗
PubMed1987

In vitro expression of a Tn9-derived chloramphenicol acetyltransferase gene fusion by using a Bacillus subtilis system.

A coupled in vitro protein-synthesizing system has been developed with components derived totally from Bacillus subtilis. The system synthesized specific gene products from various exogenous DNA templates, including B. subtilis phage phi 29, plasmid pUB110, and a heterologous B. subtilis-Escherichia coli gene fusion containing the transposon Tn9-derived chloramphenicol acetyltransferase (cat) gene. The gene fusion product was able to show CAT activity, bind specifically to a Sephacryl-chloramphenicol column, and react immunologically against anti-CAT antiserum. The fidelity of this in vitro system was demonstrated by the synthesis of gene products identical to that made in vivo. We suggest that this system may be used to study the regulation of gene expression in vitro.

Acetyltransferases↗
PubMed1987

Developmental expression of three proteins from the first gene of the RNA polymerase sigma 43 operon of Bacillus subtilis.

The first gene of the Bacillus subtilis RNA polymerase sigma 43 operon, P23, has a protein-coding capacity of 23,000 daltons. Sequence analysis revealed three potential translational initiation sites within the same reading frame, which could encode proteins of 23,000 (P23), 19,000 (P19), and 9,000 (P9) daltons, respectively. An internal promoter (P3), which is expressed only during the sporulation stage, is located between the second and the third translational start sites. By protein fusion to the Escherichia coli beta-galactosidase gene, we showed that all three translational initiation sites of the P23 gene are used in vivo in both E. coli and B. subtilis, and regulation for differential expression of the three proteins during the development of B. subtilis is coupled to the transcriptional promoter switching mechanism. The physiological function of these multiple gene products is unknown and is currently under investigation.

Bacillus subtilis↗
PubMed1986

Determination of the signal peptidase cleavage site in the preprosubtilisin of Bacillus subtilis.

Bacillus subtilis subtilisin is predicted to be synthesized as a preproenzyme according to the sequence analysis of its gene. We have synthesized the [35S] methionine-labeled preprosubtilisin in vitro and processed the precursor to prosubtilisin by the addition of membrane vesicles derived from vegetative cells of B. subtilis and Triton X-100. Radiosequencing of the prosubtilisin allowed the precise determination of the signal peptidase cleavage site. The preprosubtilisin was found to have a 29-amino-acid-long signal peptide with the signal peptidase cleavage sequence of AlaGln-AlaAla. Fusion of the signal peptide sequence to the mature TEM beta-lactamase structural gene allowed the production of an active and secreted form of beta-lactamase in vivo. An N-terminal sequence analysis of this product indicated that the observed in vivo signal peptidase cleavage site was exactly the same as that determined by in vitro analysis. During the development of the in vitro processing system, we demonstrated that the replacement of the subtilisin transcription regulatory sequence by a vegetative promoter allowed the vegetative expression and secretion of subtilisin. Thus, the late expression of the native subtilisin gene is mainly controlled at the transcription level and the secretion/processing systems are available for vegetative production of subtilisin.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence and organization of Bacillus subtilis RNA polymerase major sigma (sigma 43) operon.

The gene coding for Bacillus subtilis RNA polymerase major sigma 43, rpoD, was cloned together with its neighboring genes in a 7 kb EcoRI fragment. The complete nucleotide sequence of a 5 kb fragment including the entire rpoD gene revealed the presence of two other genes preceding rpoD in the order P23-dnaE-rpoD. The dnaE codes for DNA primase while the function of P23 remains unknown. The three genes reside in an operon that is similar in organization to the E. coli RNA polymerase major sigma 70 operon, which is composed of genes encoding small ribosome protein S21 (rpsU), DNA primase (dnaG), and RNA polymerase sigma 70 (rpoD). There is a relatively high degree of base and amino acid homology between the DNA primase and sigma genes. The most significant differences between the two operons are observed in the molecular size of the first genes (P23 and rpsU), the complete lack of amino acid homology between P23 and S21, the molecular weights of the two rpoD genes, the size of the intercistronic region between the first two genes, and the regulatory elements of the operon.

Amino Acid Sequence↗
PubMed1986

Use of the Bacillus subtilis subtilisin signal peptide for efficient secretion of TEM beta-lactamase during growth.

We report the development of an efficient Bacillus subtilis secretory system, with the secreted product stably maintained in the medium for 100 h. The system is based on characterization of the subtilisin signal peptidase cleavage site and promoters, catabolite repression of sporulation, presence of a vegetative secreting mechanism, and availability of a protease-deficient strain.

Bacillus subtilis↗