Deletion analysis of a complex promoter for a developmentally regulated gene from Bacillus subtilis

Banner, Carl; Moran, Charles P.; Losick, Richard

doi:10.1016/s0022-2836(83)80023-0

Cited by 117 publications

(75 citation statements)

References 23 publications

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“…As indicated above, only the first 17 amino acids of the predicted 26-amino-acid peptide generated from the SPS2 insert of pAP290APst are from the same frame as the SPS2 gene product itself. Since the presence of pAP762 and pAP290 inhibited sporulation but the presence 1 FIG. 4.…”

Section: Resultsmentioning

confidence: 95%

“…Such studies have contributed to the delimitation of control sequences and have demonstrated that common factors are involved in the expression of distinct genes (1,3,4,22,27,28,32). We tested whether the 5' end of a sporulation-specific gene of Saccharomyces cerevisiae, when present at high copy number, titrates a transcriptional regulatory molecule required for the expression of a subset of sporulation-specific genes.…”

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confidence: 99%

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Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae.

Percival‐Smith¹,

Segall²

1987

Mol. Cell. Biol.

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We found that the introduction into a yeast cell of a high-copy-number plasmid containing the 5' end of the SPS2 gene, a sporulation-specific gene of Saccharomyces cerevisiae, led to a reduction in the efficiency of spore formation. The plasmid pAP290, which contains the sequence from -138 to + 152 of the SPS2 gene, caused a fivefold reduction in spore formation; the presence of the plasmid had no effect on transcription of the chromosomal SPS2 gene. A plasmid containing only the sequence upstream of the TATA box of the SPS2 gene (-350 to -68) was unable to inhibit the completion of sporulation, whereas the downstream sequence, from -70 to +404, although unable by itself to inhibit sporulation, could do so when provided with an upstream fragment containing the CYCI upstream activation sequence. Deletion of 22 base pairs from pAP290, which introduced a frameshift after codon 17 of the SPS2 gene and reduced the open reading frame to 26 amino acids, generated a plasmid (pAP290APst) which could no longer inhibit sporulation. The SPS2 inserts of pAP290 and pAP290APst were found to direct equivalent levels of sporulation-specific transcription. We conclude from these results that the presence of both the SPS2 promoter (or a substitute promoter) and the initial coding sequence of the SPS2 gene is required in the high-copy-number plasmid to generate the asporogenous phenotype. We speculate that the accumulation of a protein containing the amino-terminal portion of the SPS2 gene product, synthesized from the transcripts of the truncated plasmid-borne copies of the SPS2 gene, prevents ascus formation.

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Section: Resultsmentioning

confidence: 95%

mentioning

confidence: 99%

Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae.

Percival‐Smith¹,

Segall²

1987

Mol. Cell. Biol.

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“…The importance of the UAS in the activation of spoVG transcription has been clearly demonstrated (3,31), but the mechanism by which it functions has yet to be determined. In an effort to understand this mechanism, the spoVG promoter-regulatory region was subjected to mutational analysis.…”

Section: Resultsmentioning

confidence: 99%

“…Since spoVG promoter mutations that disrupt ECH-DNA interaction eliminate spoVG transcriptional activity (6,32), it is likely that EuH is primarily responsible for the utilization of the spoVG promoter. An A+T-rich upstream activation sequence (UAS), positioned 4 bp away from the spoVG -35 promoter element, is necessary for optimal transcription from the spoVG promoter (3) as well as for the interaction of the AbrB protein with the spoVG promoter in vitro (9,26).…”

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confidence: 99%

Analysis of the upstream activating sequence and site of carbon and nitrogen source repression in the promoter of an early-induced sporulation gene of Bacillus subtilis

Frisby

Zuber

1991

J Bacteriol

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The transcription from the spoVG promoter of Bacillus subtilis is induced at the start of the stationary phase of growth and is dependent on the expression of the spoOA, spoOB, and spoOH genes. It is repressed in cells grown in the presence of excess glucose and glutamine and is under the negative control of the abrB gene. The spoOA and spoOB gene products function to suppress the negative control exerted by abrB. Transcription initiation requires the form of RNA polymerase holoenzyme that contains the spoOH gene product, cH. Optimal transcription also requires an upstream A-T-rich region termed the upstream activating sequence (UAS). The mechanism of UAS function was examined through mutational analysis of the spoVG promoter region. Deletion of the UAS or positioning the UAS one half turn or one full turn of the DNA helix upstream of its location in wild-type spoVG resulted in a severe reduction in promoter activity. Deletion of most of the UAS abolished the abrB-dependent repression of spoVG transcription. Higher activity was observed when the UAS was inserted 10 bp (one turn of the helix) upstream than when the sequence was repositioned either 5 or 13 bp upstream. Sequences upstream of the UAS were found not to be involved with the position-dependent function of the UAS. Positioning the UAS 42 or 116 bp upstream eliminated the stimulatory effect of the sequence on spoVG transcription. These data indicate that the UAS functions effectively when it is in close proximity to the -35 region. In vitro transcription analysis indicated that the deletion and insertion mutations affecting the UAS impair RNA polymerase-spoVG promoter interaction. Deletion of the UAS showed that the negative effect of exogenous glucose and glutamine is not dependent on the UAS but is exerted at a site within or near the -35 and -10 regions.Endospore formation in Bacillus subtilis is a developmental process triggered by nutritional deprivation. It can be divided into seven well-defined stages, beginning with the initiation of sporulation (stage 0) and ending with mother cell lysis (stage VII) and liberation of the mature spore (24). Initiation of sporulation requires the products of at least seven spoO genes (spoOA, spoOB, spoOE, spoOF, spoOH, spoOJ, and spoOK) to begin the cascade of events leading to the expression of numerous sporulation-associated genes (13,24). A mutation in any one of the spoO genes blocks the sporulation process before any morphological changes within the cell are observed. The spoVG gene has served as a system for examining the activation of gene transcription at the onset of sporulation because it is under spoO control and is induced as cells enter the stationary phase of growth in sporulation medium (23,33). Furthermore, transcription of spoVG is repressed when cells are grown in a medium containing a high concentration of glucose and glutamine, preferred carbon and nitrogen sources which inhibit sporulation when present in excess (24,27).Transcription of spoVG is dependent upon the products of spoOA, spoOB, a...

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“…Although the use of plasmid vectors has been successful in some instances (Segall & Losick, 1977;Bonamy & Szulmajster, 1982) serious limitations have been encountered with many of the standard plasmid vectors. The increase in gene copy number which results when chromosomal genes are cloned into multicopy plasmids may result in deleterious effects on the host cell (Kawamura et al, 1981;Banner et al, 1983). Difficulties have also been encountered when attempting to clone fragments of DNA larger than about 2.5 kbp in certain plasmid vectors (Gryczan & Dubnau, 1982).…”

Section: Introductionmentioning

confidence: 99%

Construction of Improved Bacteriophage 105 Vectors for Cloning by Transfection in Bacillus subtilis

Jones

Errington²

1987

Microbiology

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483A series of improved phage vectors have been constructed, based on Bacillus subtilis bacteriophage 4105, which can be used to clone genes in B. subtilis by direct transfection of protoplasts. The new vectors, designated 4105523, 4105524, 4105527 and 4105528, show frequencies of plaque formation that are equal to those of wild-type 4105. This represents at least a 1 0-fold improvement over 4 105J9, the vector used in previous cloning experiments. Two of the new vectors 4105527 and $105528 incorporate a mutation, cts-52, that renders the prophage temperature inducible. This has made it possible to devise a rapid small-scale procedure for screening progeny phage for the presence of inserted DNA. The usefulness of the new vectors is illustrated in the accompanying paper by cloning more than 20 B. subtilis sporulation genes.

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Deletion analysis of a complex promoter for a developmentally regulated gene from Bacillus subtilis

Cited by 117 publications

References 23 publications

Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae.

Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae.

Analysis of the upstream activating sequence and site of carbon and nitrogen source repression in the promoter of an early-induced sporulation gene of Bacillus subtilis

Construction of Improved Bacteriophage 105 Vectors for Cloning by Transfection in Bacillus subtilis

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