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At least 1,675 records · Page 93Linked to original sources
PubMed1984

Bacteriophage Mu d1(Apr lac) generates vir-lac operon fusions in Shigella flexneri 2a.

Previous studies have demonstrated that expression of virulence in Shigella spp. is controlled by growth temperature. To study the regulation of virulence (vir) genes, we set out to develop a rapid, easily-assayed phenotype with which to measure expression of virulence. This report described a procedure for isolating vir-lac operon fusions in S. flexneri 2a by using the specialized transducing bacteriophage Mu d1(Apr lac) of Casadaban and Cohen (M. Casadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1976). Mu d1(Apr lac) lysogens were isolated and screened for loss of virulence and for temperature-dependent expression of the lactose genes on Mu d1(Apr lac). A recombinant plasmid carrying the Mu immunity gene was also introduced into lysogens of interest to stabilize the Mu d1(Apr lac) insertion and prevent possible thermal induction at 37 degrees C. The mutant which we isolated failed to penetrate tissue culture cells in the assay for virulence and produced almost 15-fold more beta-galactosidase when grown at 37 degrees C than when grown at 30 degrees C. The site of insertion of Mu d1(Apr lac) in this strain was shown to be in the 140-megadalton plasmid pSf2a140, which is known to be associated with virulence. P1L4-mediated transduction of the insertion into a virulent recipient demonstrated genetic linkage of Mu d1(Apr lac) with loss of virulence and temperature-dependent expression of beta-galactosidase. All of these features fulfill the phenotype expected for a Mu d1(Apr lac)-induced vir-lac operon fusion. This mutant provides us with a means of measuring expression of a gene function required for virulence by assaying for beta-galactosidase. The insertion will also serve as a starting point for mapping of genes on pSf2a140 which are necessary for expression of virulence.

Bacteriophage mu↗
PubMed1984

Isolation and analysis of two Escherichia coli K-12 ilv attenuator deletion mutants with high-level constitutive expression of an ilv-lac fusion operon.

A lysogenizing lambda phage, lambda dilv-lac11, was constructed to carry an ilvD-lac operon fusion. Expression from the phage of the ilvE and lacZ genes is controlled by an intact ilv control region also carried by this phage. Two spontaneous mutants of lambda dilv-lac11 that have high-level constitutive expression of the ilv-lac fusion operon were isolated by growth on a beta-chloroalanine selective medium. The mutants were shown by nucleotide sequence determination to contain large deletions (delta 2216, approximately 1.6 kilobases; delta 2219, approximately 1.9 kilobases), which in both cases remove the proposed ilv attenuator terminator. The rest of the ilv leader and promoter region DNA remains intact in these mutants. Deletion 2216 also removed part of the downstream ilvG gene, whereas delta 2219 extended through the entire ilvG gene into the ilvGE intercistronic region. A possible mechanism of deletion formation is discussed.

Bacteriophage lambda↗
PubMed1987

Chlamydial rRNA operons: gene organization and identification of putative tandem promoters.

We isolated and characterized the rRNA operons of murine Chlamydia trachomatis. By exhaustively screening a library of chlamydial DNA and by blot hybridization of genomic DNA, we showed that there are only two rRNA operons in C. trachomatis. S1 nuclease protection and primer extension analysis were used to map the 5' and 3' ends of the mature 16S and 23S transcripts in both rRNA cistrons and, additionally, to demonstrate the lack of intervening sequences in these genes. The 5' ends of the presumed primary rRNA transcript were located and found to originate at two tandem sites separated by 100 base pairs. The two tandem chlamydial rDNA transcripts were not differentially regulated. Their products were coordinately expressed and were detectable as early as 9 h postinfection. However, the upstream transcript was only 10% as abundant as the downstream transcript. The sequences surrounding the transcription initiation sites bore little homology with each other or with the classic Escherichia coli -10 and -35 promoter sequences. This finding suggests that chlamydial transcription signals may differ from those of previously studied procaryotes.

Base Sequence↗
PubMed1992

Integration host factor is required for positive regulation of the tdc operon of Escherichia coli.

A 14-bp segment in the promoter region of the tdcABC operon of Escherichia coli shows sequence identity with the consensus binding site for the E. coli integration host factor (IHF). In an himA (IHF-deficient) strain, expression of beta-galactosidase from a tdcB'-'lacZ protein fusion plasmid was about 10% of that seen with an isogenic himA+ strain. Threonine dehydratase activity from the chromosomal tdcB gene in the himA mutant was also about 10% of the wild-type enzyme level. Two different mutations introduced into the putative IHF-binding site in the fusion plasmid greatly reduced the plasmid-coded beta-galactosidase activity in cells containing IHF. In vitro gel retardation and DNase I footprinting analyses showed binding of purified IHF to the wild-type but not to the mutant promoter. IHF protected a 31-bp region between -118 and -88 encompassing the conserved IHF consensus sequence. These results suggest that efficient expression of the tdc operon in vivo requires a functional IHF and an IHF-binding site in the tdc promoter.

Amino Acid Sequence↗
PubMed1994

The dacF-spoIIA operon of Bacillus subtilis, encoding sigma F, is autoregulated.

The spoIIA operon of Bacillus subtilis encodes sigma F and two proteins that may regulate sigma factor activity. High level induction of the tricistronic spoIIA operon occurs early during spore formation. At later times, the locus is cotranscribed with the upstream gene dacF, which encodes a putative DD-carboxypeptidase. In this study, the regulation of dacF-spoIIA transcription has been analyzed. Expression of a dacF-lacZ transcriptional fusion during sporulation required sigma F but not the later-expressed sporulation-associated sigma factors. Induction of sigma F synthesis during vegetative growth caused expression of dacF-lacZ fusions. The dacF-spoIIA promoter sequence is similar to sequences of previously identified sigma F promoters. It is concluded that dacF-spoIIA is transcribed by E sigma F. We present evidence that dacF-spoIIA is also transcribed by E sigma G, as is the case for the three other promoters known to be transcribed by E sigma F.

Bacillus subtilis↗
PubMed1994

Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM series.

A series of Escherichia coli-mycobacteria shuttle plasmids for the isolation and study of gene regulatory sequences was constructed. These pJEM vectors contain an efficient transcription terminator and multiple cloning sites and allow either operon or gene fusions to lacZ. By constructing operon fusions with pJEM15, we assessed various previously characterized mycobacterial promoters in the fast-growing species Mycobacterium smegmatis and the slow-growing species M. bovis BCG. Our results suggest that M. smegmatis and M. bovis BCG RNA polymerases do not share the same specificity. To isolate new mycobacterial promoters, an M. tuberculosis DNA library was generated, using pJEM13, and screened in M. smegmatis. Several Lac+ clones were isolated, and the beta-galactosidase activity was measured.

Base Sequence↗
PubMed1999

The cyclic AMP receptor protein is dependent on GcvA for regulation of the gcv operon.

The Escherichia coli gcv operon is transcriptionally regulated by the GcvA, GcvR, Lrp, and PurR proteins. In this study, the cyclic AMP (cAMP) receptor protein (CRP) is shown to be involved in positive regulation of the gcv operon. A crp deletion reduced expression of a gcvT-lacZ fusion almost fourfold in glucose minimal (GM) medium. The phenotype was complemented by both the wild-type crp gene and four crp alleles that encode proteins with amino acid substitutions in known activating regions of CRP. A cyaA deletion also resulted in a fourfold decrease in gcvT-lacZ expression, and wild-type expression was restored by the addition of cAMP to the growth medium. A cyaA crp double deletion resulted in levels of gcvT-lacZ expression identical to those observed with either single mutation, showing that CRP and cAMP regulate through the same mechanism. Growth in GM medium plus cAMP or glycerol minimal medium did not result in a significant increase in gcvT-lacZ expression. Thus, the level of cAMP present in GM medium appears to be sufficient for regulation by CRP. DNase I footprint analysis showed that CRP binds and protects two sites centered at bp -313 (site 1) and bp -140 (site 2) relative to the transcription initiation site, but a mutational analysis demonstrated that only site 1 is required for CRP-mediated regulation of gcvT-lacZ expression. Expression of the gcvT-lacZ fusion in a crp gcvA double mutant suggested that CRP's role is dependent on the GcvA protein.

Bacterial Proteins↗
PubMed1995

The ntrBC genes of Azospirillum brasilense are part of a nifR3-like-ntrB-ntrC operon and are negatively regulated.

A cosmid able to complement the Nif- and nitrate-dependent growth phenotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2. A 6-kb DNA region was sequenced and showed two open reading frames (ORFs) identified as the ntrB and ntrC genes. An ORF1 located upstream from the ntrB gene and coding for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rhodobacter capsulatus and the ORF1 of Rhizobium leguminosarum, both located upstream from the ntrB gene in a complex operon. Two other unidentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypeptides were also observed. delta ORF1-ntrBC, ORF1, ntrB, and ntrC mutants obtained by recombination of suicide plasmids containing an insertion of a promoterless lacZ kanamycin cassette showed decreased nitrogenase activities and were unable to grow on nitrate as the sole N source. These phenotypes were restored by complementation with plasmids containing the ntrC gene. Analysis of lacZ transcriptional fusions suggested that the ORF1-ntrBC operon in Azospirillum brasilense is expressed from a promoter located upstream from the ORF1 and that it is negatively regulated by the ntrC gene product.

Amino Acid Sequence↗
PubMed2002

Widespread organisation of C. elegans genes into operons: fact or function?

A recent report by Blumenthal et al. provides convincing evidence that at least 15% of Caenorhabditis elegans genes are co-transcribed within over a thousand operons. Polycistronic transcription of gene clusters is very rare in eukaryotes. The widespread occurrence of operons in C. elegans thus raises some interesting questions about the origin and function of these multigenic transcriptional units.

Animals↗
PubMed2002

Effect of copy number and mRNA processing and stabilization on transcript and protein levels from an engineered dual-gene operon.

To study the effect of mRNA stability and DNA copy number on protein production from a dual-gene operon, a synthetic operon containing the reporter genes gfp and lacZ under the control of the araBAD promoter was placed in pMB1-based (approximately 100 copies/cell) and F plasmid-based (approximately 1 copy/cell) vectors. DNA cassettes encoding secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the two genes. Although the copy number of the pMB1-based vectors was approximately 100-fold greater than the copy number of the F plasmid-based vectors, transcript and protein levels from the pMB1-based vector were not 100-fold greater than from the F plasmid-based vectors. In identical plasmid backbones, different combinations of mRNA control elements were used to alter steady-state levels of transcripts. Control elements that amplified the stability of one coding region relative to another amplified the ratio of protein produced from those transcripts. The effects of mRNA stability control elements were greater at low inducer concentrations, where mRNA levels limit protein production, than at high inducer concentrations. Although we can alter mRNA and protein levels through copy number, induction level, and mRNA stability control elements, some aspect of gene expression remains dependent on inherent characteristics of the coding region.

Arabinose↗
PubMed2002

Modified model for the regulation of the tryptophan operon in Escherichia coli.

This article proposes a modification of the model developed by Sinha (1988) and Sen and Liu (1990) for the regulation dynamics of the tryptophan operon in E. coli based on a consistent overall balance of the agent repressing the mRNA transcription. The dynamics of the model are analyzed by means of continuation techniques and the influence of periodic fluctuations in the intracellular demand for tryptophan is addressed. The analysis provides deeper insight into the dynamics of this operon system and the results obtained may be a useful starting point for the optimization of tryptophan yield in bacterial cultures.

Bacterial Proteins↗
PubMed1985

Two operator sites separated by 599 base pairs are required for deoR repression of the deo operon of Escherichia coli.

DeoP1 and deoP2 promoter fragments from the deo operon of Escherichia coli have been transcriptionally fused to the galactokinase gene. From single-copy expression of these fusions it is shown that the deoR binding site of both deoP1 and deoP2 are necessary to achieve full repression of the deo operon by the deoR repressor. Repression of the promoters can be achieved either by supplying extra deoR repressor in trans or by introduction of an extra deoR binding site at a position between 224 and 997 bp upstream of the promoter. Furthermore, the deoP2 promoter is shown to be regulated in a cumulative way by both the deoR and the cytR repressors, while deoP1 is only regulated by the deoR repressor. DeoP2 is a strong promoter being 20 times stronger than araPBAD and four times stronger than deoP1.

Base Composition↗
PubMed1986

T7 RNA polymerase directed expression of the Escherichia coli rrnB operon.

Plasmids having Escherichia coli ribosomal DNA sequences under control of a promoter for T7 RNA polymerase have been constructed. Transcription of the rDNA sequences is dependent on T7 RNA polymerase because the tandem promoters for E. coli RNA polymerase, normally used to direct transcription of these sequences, have been removed. The entire 16S, 23S and 5S coding sequences from the rrnB operon can be efficiently transcribed by T7 RNA polymerase in vitro to yield full-length 30S precursor RNA. When such plasmids are placed into an E. coli strain containing a chromosomal copy of the gene for T7 RNA polymerase under control of the lac UV5 promoter, high-level synthesis of rRNAs from the plasmid can be induced by adding IPTG to exponentially growing cells. Subsequent addition of rifampicin to inhibit further initiation of transcription by E. coli RNA polymerase provides a simple method to study the fate of plasmid-coded rRNAs in the complete absence of host-coded rRNA synthesis. Gel electrophoretic analysis demonstrated that the rRNAs synthesized by T7 RNA polymerase in the presence of rifampicin are processed to their mature forms and assembled into ribosomal particles for at least 35 min after rifampicin addition. T7 RNA polymerase is also capable of efficient transcription of the entire rrnB operon in the reverse direction.

DNA Restriction Enzymes↗
PubMed1986

Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon.

The parB region of plasmid R1 encodes two genes, hok and sok, which are required for the plasmid-stabilizing activity exerted by parB. The hok gene encodes a potent cell-killing factor, and it is regulated by the sok gene product such that cells losing a parB-carrying plasmid during cell division are rapidly killed. Coinciding with death of the host cell, a characteristic change in morphology is observed. Here we show that the killing factor encoded by the hok gene is a membrane-associated polypeptide of 52 amino acids. A gene located in the Escherichia coli relB operon, designated relF, is shown to be homologous to the hok gene. The relF gene codes for a polypeptide of 51 amino acids, which is 40% homologous to the hok gene product. Induced overexpression of the hok and relF gene products results in the same phenomena: loss of cell membrane potential, arrest of respiration, death of the host cell and change in cell morphology. The parB region and the relB genes were cloned into unstably inherited oriC minichromosomes. Whereas the parB region also conferred a high degree of genetic stability to an oriC minichromosome, the relB operon (with relF) did not; therefore the latter does not appear to 'stabilize' its replicon (the chromosome). The function of the relF gene is not known.

Amino Acid Sequence↗
PubMed1987

Biological consequences of segmental alterations in mRNA stability: effects of deletion of the intercistronic hairpin loop region of the Rhodobacter capsulatus puf operon.

It has been proposed that intercistronic stem and loop structures located in the puf operon of the photosynthetic bacterium Rhodobacter capsulatus account for segmental differences in transcript stability and consequently, the differential expression of the B870 and reaction center (RC) proteins encoded by puf. We report here that deletion of these structures leads to a failure to detect as discrete fragments the B870-encoding 0.49 kb and 0.50 kb mRNA segments located upstream from the site of the hairpins. The absence of these stable transcript fragments is associated with altered stoichiometry of the B870 and RC pigment-protein complexes in the bacterial intracytoplasmic membrane and a decreased rate of cell growth under photosynthetic conditions. These results support the view that the hairpin loop structures of the puf intercistronic region function in vivo to impede exoribonucleolytic degradation of upstream mRNA and establish that segmental variations in mRNA stability have a biologically important role in regulating the expression of puf operon genes.

Base Sequence↗
PubMed1987

Cloning and light regulation of expression of the phycocyanin operon of the cyanobacterium Anabaena.

The biliprotein phycocyanin (PC) is a major constituent of the light-harvesting apparatus of cyanobacteria and red algae. A DNA fragment encoding the beta and alpha subunits of PC was isolated from a genomic library of the cyanobacterium Anabaena 7120 DNA. The single-copy PC genes are part of a larger operon which consists of five open reading frames (ORFs) encoding, in order, the beta and alpha subunits of PC, two linker polypeptides associated with PC in phycobilisome rods, and a fifth ORF, which may encode a linker polypeptide involved in attachment of the phycobilisome rod to the core of the structure. The operon yields three major transcripts, the first of which (1.4 kb) encodes only the PC subunits. A second (3.6 kb) encodes all five ORFs, and appears to arise from partial read-through of a terminator following the PC subunit genes. The third transcript (1.4 kb) encodes the last two ORFs. The relative levels of the three transcripts in vivo are modulated by light intensity, but they are not altered by the removal of fixed nitrogen from the growth medium. The site of light regulation appears to be the terminator following the PC genes, rather than a promoter.

Amino Acid Sequence↗
PubMed1990

The three operators of the lac operon cooperate in repression.

We tested the effect of systematic destruction of all three lac operators of the chromosomal lac operon of Escherichia coli on repression by Lac repressor. Absence of just one 'pseudo-operator' O2 or O3 decreases repression by wild-type tetrameric Lac repressor approximately 2- to 3-fold; absence of both 'pseudo-operators' decreases repression greater than 50-fold. O1 alone represses under these conditions only approximately 20-fold. Dimeric active Lac repressor (iadi) represses the wild-type lac operon to about the same low extent. This indicates that cooperative interaction between lac operators is due to DNA loop formation mediated by tetrameric Lac repressor. Under conditions where loop formation is impossible, occupation of O3 but not of O2 may lead to weak repression. This suggests that under these conditions CAP activation may be inhibited and that stopping transcription at O2 does not significantly contribute to repression.

Base Sequence↗