Biomedical subjects

D J Henner

Publications and source records attributed to D J Henner.

At least 37 records · Page 2Linked to original sources

PubMed1987

Transformation of Aspergillus nidulans with the hygromycin-resistance gene, hph.

Aspergillus nidulans strain G191 was transformed to hygromycin resistance using plasmid pDH25, which contains the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the A. nidulans trpC gene. Southern hybridizations of transformants revealed multiple, integrated copies of the vector. A pleiotropic effect conferring increased hygromycin B sensitivity was found to be associated with the A. nidulans pyrG89 allele. Plasmid pDH25 features a ClaI site immediately preceding the hph start codon thus permitting convenient replacement of the trpC sequences with other eukaryotic promoters.

Anti-Bacterial Agents↗

PubMed1987

Characterization and mapping of the Bacillus subtilis prtR gene.

A gene from Bacillus natto encoding a 60-amino-acid peptide has been previously described that, when cloned on a high-copy plasmid in B. subtilis, enhances production of alkaline protease, neutral protease, and levansucrase. An identical gene was isolated from B. subtilis and caused a similar phenotype when placed on a high-copy plasmid. Genetic mapping localized this gene near metB, distant from other pleiotropic genes causing similar effects. Deletion of this gene from the B. subtilis chromosome had no obvious phenotypic effect.

Amino Acid Sequence↗

PubMed1987

Construction and properties of an intracellular serine protease mutant of Bacillus subtilis.

An intracellular serine protease (ISP-1) mutant of Bacillus subtilis was created by introducing a frameshift into the coding region of the cloned gene. Intracellular protease activity in the mutant was very low, yet sporulation in both nutrient broth and minimal medium was normal. The rate of bulk protein turnover in the mutant was slightly slower than that in the wild-type strain. These results suggest that the gene for ISP-1 is not essential and that ISP-1 is not the major enzyme involved in protein turnover during sporulation.

Bacillus subtilis↗

PubMed1986

Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis.

A single-copy integration vector was used for the in vitro construction of translational fusions to the lacZ gene of Escherichia coli. Insertion of a single copy of the lacZ fusion into the B. subtilis chromosome leads to an easily detected Amy- phenotype. A trpE-lacZ fusion was constructed in which the trp promoter directs hybrid beta-galactosidase (beta Gal) synthesis. The level of beta Gal in a wild-type strain carrying the trpE-lacZ fusion in the chromosome is regulated by exogenous tryptophan, while a 5-methyltryptophan-resistant mutant constitutively synthesizes betaGal. A trpF-lacZ fusion was constructed and used to determine the effect of a frameshift mutation in the trpE gene on expression of the trpF-lacZ fusion. The frameshift mutation in trpE led to a three-fold reduction in the levels of the trpF-lacZ fusion. The levels of the betaGal activity of these integrated lacZ fusions appear to provide a quantitative measure of the expression of B. subtilis genes under single-copy conditions.

Amino Acid Sequence↗

PubMed1986

The organization and nucleotide sequence of the Bacillus subtilis hisH, tyrA and aroE genes.

The nucleotide sequence of approximately 3 kb of Bacillus subtilis DNA distal to the trp operon was determined. Three open reading frames were found and these were shown to encode the hisH, tyrA and aroE genes. Integrative plasmids were constructed to interrupt transcription through this region. These data suggest that these three genes can be transcribed from both the trp promoter preceding the trp operon and from a promoter within the trpA structural gene.

Amino Acid Sequence↗

PubMed1986

Identification of the pleiotropic sacQ gene of Bacillus subtilis.

The sacQ gene of Bacillus subtilis, a pleiotropic gene affecting the expression of a number of secreted gene products, has been identified as a small 46-amino-acid polypeptide. The increased expression of this polypeptide in strains carrying the sacQ36 allele, or in strains carrying the sacQ gene on a high copy plasmid, appears to be responsible for the phenotype of higher levels of proteases seen in these strains. A deletion of the sacQ gene had no apparent phenotype, indicating that it is not an essential gene.

Alleles↗

PubMed1986

Novel form of transcription attenuation regulates expression the Bacillus subtilis tryptophan operon.

Transcription of the trp operon of Bacillus subtilis is regulated in response to the availability of tryptophan. The first structural gene of the operon is preceded by a 204-base-pair transcribed leader region that contains a segment with the features of a procaryotic termination site. Transcription of the leader region was analyzed in vivo and in vitro to determine whether this putative termination site was used to regulate operon expression. When RNA was isolated from wild-type cells grown in the presence of excess tryptophan, transcripts of the operon ended at the putative termination site. In contrast, RNA isolated from cells grown in the absence of tryptophan or from a mutant strain which is constitutive for trp operon expression contained trp transcripts that extended beyond the termination site into the structural genes. To assess termination quantitatively in vivo, a trpE-lacZ fusion was constructed in which the trp promoter and leader region controls hybrid beta-galactosidase formation. The effects on hybrid beta-galactosidase levels of point mutations and deletions introduced into this leader region were determined. The results obtained establish that transcription of the trp operon structural genes is regulated in the leader region. This regulation appears to be mediated by the formation of alternative secondary structures of the leader transcript. In vitro transcription studies with wild-type and mutant templates provided additional evidence that the identified alternative RNA secondary structures regulate transcription termination. We hypothesize that binding of a tryptophan-activated regulatory protein to a specific segment of the nascent leader transcript prevents formation of one of the alternative secondary structures, thereby directing RNA polymerase to terminate transcription.

Amino Acid Sequence↗

PubMed1986

Regulatory elements common to the Bacillus pumilus and Bacillus subtilis trp operons.

The trp operon regulatory region of Bacillus pumilus was cloned and sequenced. The cloned B. pumilus trp promoter-leader region functioned in Bacillus subtilis to express the adjacent leukocyte interferon A gene on a multicopy transcriptional fusion plasmid, pBpIFI. In strains carrying this plasmid, anthranilate synthetase levels were elevated, possible due to titration of a B. subtilis trp regulatory factor by multiple copies of the transcript of the plasmid-borne B. pumilus trp leader region. The B. pumilus trp promoter was recognized efficiently in vitro by B. subtilis sigma 43 RNA polymerase. Approximately 12% of the transcripts produced in vitro terminated in the leader region immediately following synthesis of a transcript structure resembling rho-independent terminators of enteric bacteria. An analogous terminator exists in the B. subtilis trp leader transcript. Nucleotide sequence comparison of the B. pumilus and B. subtilis trp leader regions revealed conservation of these and other sequences that could form transcript secondary structures postulated to regulate transcription termination in B. subtilis (H. Shimotsu, M.I. Kuroda, C. Yanofsky, and D.J. Henner, J. Bacteriol. 166:461-471, 1986). We propose that two elements implicated in B. subtilis trp operon regulation are conserved in the related organism B. pumilus: alternative transcription antiterminator and terminator structures in the leader transcript, and a trans-acting factor present in limiting amounts that is required for transcription termination in the leader region.

Anthranilate Synthase↗

PubMed1986

Modulation of Bacillus subtilis levansucrase gene expression by sucrose and regulation of the steady-state mRNA level by sacU and sacQ genes.

In Bacillus subtilis, the extracellular enzyme levansurcrase is synthesized in the presence of sucrose. A termination structure between the transcription start site and the structural gene was the apparent site for regulation by sucrose of transcription into the structural gene. Sequence analysis of the sacB leader region from two strains constitutive for levansucrase synthesis showed a single base change in the stem of this termination structure. This single base change also led to the constitutive synthesis of a sacB'-'lacZ fusion, whereas the wild-type sacB'-'lacZ fusion was induced by the addition of sucrose. S1 nuclease mapping of sacB transcripts with probes labeled either within the termination structure or 3' to the termination structure showed that sucrose did not increase the number of transcripts extending into the termination structure; however, sucrose did increase the number of transcripts extending past the termination structure. Two pleiotropic mutations which affect the expression of levansucrase, sacQ36 hyperproducing [sacQ36(Hy)] and sacU32(Hy), were separately introduced into the strain carrying the sacB'-'lacZ fusion. These mutations each increased the expression levels of the sacB'-'lacZ fusion. S1 mapping showed increased levels of transcript initiating at the sacB promoter in strains with the sacQ36(Hy) and sacU32(Hy) mutations. This increased transcription appeared to be independent of the sucrose-regulated transcription termination, suggesting the existence of at least two different mechanisms for the regulation of sacB expression.

Bacillus subtilis↗

PubMed1985

Nucleotide sequence of the Bacillus subtilis tryptophan operon.

In Bacillus subtilis, tryptophan biosynthesis is one of the most thoroughly characterized biosynthetic pathways. Recombinant DNA methodology has permitted a rapid characterization of the tryptophan (trp) gene cluster at the molecular level. In this report the nucleotide sequence of the six structural genes together with the intercistronic regions and flanking regulatory regions are presented.

Bacillus subtilis↗

PubMed1984

Nucleotide sequence of the Bacillus subtilis trpE and trpD genes.

Several overlapping portions of the tryptophan (trp) operon of Bacillus subtilis have been cloned into plasmid pBR322. The nucleotide sequence of the region comprising the trpE and trpD genes and a portion of the trpC gene has been determined. When the deduced amino acid sequences of these genes are compared with their counterparts in Escherichia coli, several regions of striking homology are seen. The probable initiation codons for the trpE, D and C genes are each preceded by a recognizable Shine-Dalgarno sequence. The coding sequences for the trpE and trpD genes and for the trpD and trpC genes overlap slightly, leaving no intercistronic regions between the genes.

Amino Acid Sequence↗

PubMed1984

Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis.

The Escherichia coli lac operator has been placed on the 3' side of the promoter for the penicillinase gene of Bacillus licheniformis, creating a hybrid promoter controllable by the E. coli lac repressor. The E. coli lac repressor gene has been placed under the control of a promoter and ribosome-binding site that allows expression in Bacillus subtilis. When the penicillinase gene that contains the lac operator is expressed in B. subtilis on a plasmid that also produces the lac repressor, the expression of the penicillinase gene can be modulated by isopropyl beta-D-thiogalactoside (IPTG), an inducer of the lac operon in E. coli. A similar system was constructed from a promoter of the B. subtilis phage SPO-1 and the leukocyte interferon A gene, which allowed the controlled expression of interferon in B. subtilis. These two examples show that a functional control system can be introduced into B. subtilis from E. coli.

Bacillus↗

PubMed1984

Characterization of the Bacillus subtilis tryptophan promoter region.

The nucleotide sequence of the control region of the trp operon of Bacillus subtilis has been determined. The region was shown to contain the trp promoter by deletion analysis and by determination of the transcription start site. The trp promoter shows similarity to the consensus sequence for Escherichia coli and B. subtilis promoters. The presence of the trp control region on a high-copy-number plasmid confers resistance to the tryptophan analogue 5-methyltryptophan. It appears that an approximately 120-base-pair region comprising not only the trp promoter but also adjacent direct repeat sequences is necessary to confer 5-methyltryptophan resistance. We postulate that this region is involved in tryptophan regulation and confers 5-methyltryptophan resistance by titration of a trp regulatory protein. Removal of either the trp promoter or the adjacent direct repeat sequences abolished the 5-methyltryptophan-resistance phenotype. Placement of unrelated promoters adjacent to the direct repeat sequences restored 5-methyltryptophan resistance. This suggests that promoter activity is necessary for the regulatory function.

Bacillus subtilis↗

PubMed1984

Bacillus subtilis requires a "stringent" Shine-Dalgarno region for gene expression.

A series of plasmids was constructed differing only in the sequence of the Shine-Dalgarno region preceding the leukocyte interferon-A gene. This series of plasmids was used to test the efficiency of interferon expression in both Bacillus subtilis and Escherichia coli. In B. subtilis, interferon expression was much more sensitive to changes in the sequence of the Shine-Dalgarno region than in E. coli and it appeared to require more homology to the 3' end of 16S ribosomal RNA.

Bacillus subtilis↗

PubMed1984

Cloning and sequencing of the xylose isomerase and xylulose kinase genes of Escherichia coli.

A 4.2-kilobase-pair fragment of the Escherichia coli chromosome which contains the genes for xylose isomerase and xylulose kinase was cloned into plasmid pBR322. The hybrid plasmid (designated pECX14) complements strains deficient in either or both of the two enzymes. Deletion derivatives of pECX14 were used to localize the two genes on the cloned fragment. The entire nucleotide sequence of the cloned fragment was determined. Open reading frames which, if translated, would encode proteins of molecular weights 54,000 and 52,000 were found. These were identified as the isomerase and kinase structural genes, respectively.

Aldose-Ketose Isomerases↗

PubMed1984

Cloning of the neutral protease gene of Bacillus subtilis and the use of the cloned gene to create an in vitro-derived deletion mutation.

The neutral protease gene of Bacillus subtilis has been cloned, and its nucleotide sequence has been determined. The cloned gene was used to create an in vitro-derived deletion mutation, which was used to replace the wild-type copy of the gene. This deletion, in combination with a deletion of the alkaline protease gene, completely abolished protease production. The loss of the proteases had no detectable effect on growth, morphology, or sporulation.

Amino Acid Sequence↗

PubMed1983

Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis.

The subtilisin gene from B. amyloliquefaciens has been cloned and expressed under its own promoter on a high copy plasmid, pBS42, in Bacillus subtilis I-168 (Marburg strain). Greater than 95 percent of the expressed protease activity is secreted, and the activity is sensitive to inhibition by phenylmethylsulfonyl fluoride as expected for subtilisin. Bacillus subtilis transformants carrying the Bacillus amyloliquefaciens subtilisin gene in pBS42 (called pS4) secreted large amounts of a protein not seen in control pBS42 transformants. This protein migrated in SDS gels near the position of authentic subtilisin. The complete nucleotide sequence of the cloned gene has been determined using dideoxy sequencing methods. Ba131 exonuclease digestion studies at the 5' end of the gene have defined a 31 base pair stretch necessary for efficient expression of subtilisin. In addition to this putative promoter region, sequences have been assigned for ribosome binding, translation initiation, a signal peptide, the mature enzyme, and translation and transcription termination. A most interesting feature of the gene is a sequence of unknown function coding for roughly 75 amino acids between the signal sequence and the mature enzyme. It is proposed that this region serve as a pro-peptide as is commonly found in eukaryotic secreted proteases.

Amino Acid Sequence↗

PubMed1983

Construction of a vector for cloning promoters in Bacillus subtilis.

A versatile vector for cloning DNA fragments containing promoter activity in Bacillus subtilis was derived from plasmids pBR322, pUB110 and pC194. Selection is based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The plasmid contains a second selectable marker, neomycin resistance, and contains functional origins of replication for both B. subtilis and Escherichia coli.

Bacillus subtilis↗