Integrable alpha-amylase plasmid for generating random transcriptional fusions in Bacillus subtilis

O’Kane, Cahir J.; Stephens, M. A.; McConnell, David J.

doi:10.1128/jb.168.2.973-981.1986

Cited by 38 publications

(23 citation statements)

References 53 publications

(39 reference statements)

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“…Insertion of the plasmids within the dnaK gene was verified by Southern blotting. a-Amylase was assayed as described previously (45).…”

Section: Methodsmentioning

confidence: 99%

“…Then the cultures were divided into two halves at time zero and grown further at 30 and 48°C, respectively. At the indicated times, samples were taken and a-amylase was assayed as described previously (45). Strain JDB2 grown at 30°C (-) and 48°C (0); strain JDB1 grown at 30°C (U) and 48°C (O).…”

Section: T L S F E D a A F G K E T T I E I P R E E T C Cgaaacatgtaaagmentioning

confidence: 99%

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Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis

et al. 1992

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By using an internal part of the dnaK gene from BaciUlus megaterium as a probe, a 5.2-kb Hindlll fragment of chromosomal DNA of BaciUlus subtilis was cloned. Downstream sequences were isolated by in vivo chromosome walking. Sequencing of 5,085 bp revealed four open reading frames in the order orf9-grpEdnaK-dnaJ. orJ39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases. Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins. Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species. Primer extension studies revealed three potential transcription start sites, two preceding orJ39 (Si and S2) and a third one in front of grpE (S3). S2 and S3 were activated at a high temperature. Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb. RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease. A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in a-amylase activity after heat induction. A 9-bp inverted repeat was detected in front of the coding region of orJ39. This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria. The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed. The dnaK locus was mapped at about 2230 on the B. subtilis genetic map.The heat shock response is a homeostatic mechanism that enables cells to survive a variety of environmental stresses. It is characterized by the increased synthesis of a group of evolutionarily conserved proteins, heat shock proteins (HSPs), and is a universal feature of both prokaryotic and eukaryotic cells (35). When Eschertichia coli cells are shifted to a high temperature, the synthesis of a set of about 20 HSPs transiently increases and then declines rapidly to steady-state levels characteristic of the new ambient temperature (44).The highly conserved HSPs perform similar functions in all organisms. One of these functions is the well-established regulation of protein-protein interactions by the chaperonins (69). One of the most abundant HSPs, HSP70, is highly conserved in evolution. It is found in such diverse organisms as E. coli, Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens (14, 35).The dnaK gene of E. coli was originally discovered because mutations in it blocked bacteriophage lambda DNA replication at all temperatures (22,23). Subsequently, the dnaK gene product was shown to be essential for E. coli viability at high and low temperatures (22,30,46,51,52), and genetic evid...

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“…Insertion of the plasmids within the dnaK gene was verified by Southern blotting. a-Amylase was assayed as described previously (45).…”

Section: Methodsmentioning

confidence: 99%

Section: T L S F E D a A F G K E T T I E I P R E E T C Cgaaacatgtaaagmentioning

confidence: 99%

Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis

et al. 1992

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“…The repressor gene xre (24,41) (6,41) in a process which is dependent on the function of recA (27). We suggest that after elimination of Xre, transcription proceeds from two overlapping putative rightward promoters into ORF10.…”

Section: Resultsmentioning

confidence: 99%

“…PBSX is therefore not propagated by these particles (25,26). PBSX appears to be a particulate bacteriocin which has evolved from a bacteriophage.Induction of PBSX is controlled by the repressor gene xre (6,27,40,41). The amino acid sequence of Xre predicts that it is a helix-turn-helix (HLH) protein resembling other DNAbinding proteins such as lambda cI and Cro (14, 41).…”

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Genetic control of bacterial suicide: regulation of the induction of PBSX in Bacillus subtilis

et al. 1994

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PBSX is a phage-like bacteriocin (phibacin) of Bacilus subtilis 168. Bacteria carrying the PBSX genome are induced by DNA-damaging agents to lyse and produce PBSX particles. The particles cannot propagate the PBSX genome. The particles produced by this suicidal response kill strains nonlysogenic for PBSX. A 5.2-kb region which controls the induction of PBSX has been sequenced. The genes identified include the previously identified repressor gene xre and a positive control factor gene, pcf. Pcf is similar to known sigma factors and acts at the late promoter P which has been located distal to pcf. The first two genes expressed from the late promoter show homology to genes encoding the subunits of phage terminases.The defective bacteriophage PBSX of Bacillus subtilis 168 is a phage-like bacteriocin, or phibacin, with curious biological properties. PBSX lysogens exposed to DNA-damaging agents produce PBSX phage-like particles which kill other nonlysogenic Bacillus strains (25) by binding to and disrupting the cell wall. Each PBSX particle has a small head and a relatively long tail. Phage heads appear to contain randomly selected 13-kb segments of host DNA, which are not injected into susceptible cells. PBSX is therefore not propagated by these particles (25,26). PBSX appears to be a particulate bacteriocin which has evolved from a bacteriophage.Induction of PBSX is controlled by the repressor gene xre (6,27,40,41). The amino acid sequence of Xre predicts that it is a helix-turn-helix (HLH) protein resembling other DNAbinding proteins such as lambda cI and Cro (14, 41). However, little is known about the mechanism of action of Xre, and apart from the suggestion that xre and a neighboring gene, now called open reading frame 10 (ORF10), are regulated by Xre itself, nothing is known about how the late genes are regulated (41).All known structural and lytic protein genes have been shown to be clustered within a large operon of at least 19 kb in length, which appears to be expressed from a single late promoter region called PL (Fig. la) (40). A number of mutations in regulatory genes, including xin (noninducible for PBSX) and xhi (heat inducible for PBSX), are located proximal to mutations affecting phage head and tail proteins such as xhd, xtl, and xki (6, 38). A regulatory mutation, xhi-1479, renders PBSX thermoinducible (6). A 1.2-kb EcoRI fragment from a PBSX wild-type strain was found to complement the xhi-1479 mutation, and sequence analysis identified the xre gene as the site of the mutation (41). xhi-1479 is thus analogous to the cIts857 mutation of bacteriophage lambda (36 Media. B. subtilis and Escherichia coli were grown in LuriaBertani (LB) broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) or LB agar. For selection, media contained chloramphenicol (at 3 pug/ml for low-copy-number vectors and 5 jig/ml for higher-copy-number vectors) and kanamycin (5 pug/ml) for B. subtilis, or chloramphenicol (15 jig/ml) and ampicillin (100 pug/ml) for E. coli as appropriate. a-Amylase activity was detected by adding starch (...

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Enhancer Trapping in Plants

Chudalayandi

2010

Methods in Molecular Biology

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Advances in sequencing technology have led to the availability of complete genome sequences of many different plant species. In order to make sense of this deluge of information, functional genomics efforts have been intensified on many fronts. With improvements in plant transformation technologies, T-DNA and/or transposon-based gene and enhancer-tagged populations in various crop species are being developed to augment functional annotation of genes and also to help clone important genes. State-of-the-art cloning and sequencing technologies, which would help identify T-DNA or transposon junction sequences in large genomes, have also been initiated. This chapter gives a brief history of enhancer trapping and then proceeds to describe gene and enhancer tagging in plants. The significance of reporter gene fusion populations in plant genomics, especially in important cereal crops, is discussed.

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Integrable alpha-amylase plasmid for generating random transcriptional fusions in Bacillus subtilis

Cited by 38 publications

References 53 publications

Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis

Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis

Genetic control of bacterial suicide: regulation of the induction of PBSX in Bacillus subtilis

Enhancer Trapping in Plants

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