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At least 595 records · Page 33Linked to original sources
PubMed2001

Transient promoter formation: a new feedback mechanism for regulation of IS911 transposition.

IS911 transposition involves a free circular transposon intermediate where the terminal inverted repeat sequences are connected. Transposase synthesis is usually driven by a weak promoter, p(IRL), in the left end (IRL). Circle junction formation creates a strong promoter, p(junc), with a -35 sequence located in the right end and the -10 sequence in the left. p(junc) assembly would permit an increase in synthesis of transposase from the transposon circle, which would be expected to stimulate integration. Insertion results in p(junc) disassembly and a return to the low p(IRL)- driven transposase levels. We demonstrate that p(junc) plays an important role in regulating IS911 transposition. Inactivation of p(junc) strongly decreased IS911 transposition when transposase was produced in its natural configuration. This novel feedback mechanism permits transient and controlled activation of integration only in the presence of the correct (circular) intermediate. We have also investigated other members of the IS3 and other IS families. Several, but not all, IS3 family members possess p(junc) equivalents, underlining that the regulatory mechanisms adopted to fine-tune transposition may be different.

Bacterial Proteins↗
PubMed1987

Patterns of somatic mutations in immunoglobulin variable genes.

The mechanism responsible for somatic mutation in the variable genes of antibodies is unknown and may differ from previously described mechanisms that produce mutation in DNA. We have analyzed 421 somatic mutations from the rearranged immunoglobulin variable genes of mice to determine if the nucleotide substitutions differ from those generated during meiosis and if the presence of nearby direct and inverted repeated sequences could template mutations around the variable gene. The results reveal a difference in the pattern of substitutions obtained from somatic mutations vs. meiotic mutations. An increased frequency of T:A to C:G transitions and a decreased frequency of mutations involving a G in the somatic mutants compared to the meiotic mutants is indicated. This suggests that the mutational processes responsible for somatic mutations in antibody genes differs from that responsible for mutation during meiosis. An analysis of the local DNA sequences revealed many direct repeats and palindromic sequences that were capable of templating some of the known mutations. Although additional factors may be involved in targeting mutations to the variable gene, mistemplating by nearby repeats may provide a mechanism for the enhancement of somatic mutation.

Animals↗
PubMed1986

Altered DNA conformations in the gene regulatory region of torsionally-stressed SV40 DNA.

We used mung bean nuclease to probe the SV40 genome for DNA unwinding and unpairing. Cleavage occurred at a limited number of specific sites in supercoiled, but not relaxed DNA. The number and location of cleavage sites depended upon Mg2+ concentration. Without Mg2+, cutting occurred mainly in one early denaturation region located 3' to the t antigen gene and within the T antigen gene intron. With Mg2+, cleavage occurred at a number of alternative sites in the genome. Certain Mg2+ concentrations favored cleavage in the gene regulatory region. These cleavages were mapped at single nucleotide resolution and occurred in both transcriptional enhancers and upstream from the start of major late gene transcription. The cleavages occurred between 5 bp inverted repeat sequences, consistent with the recognition of unusually small cruciform structures.

Animals↗
PubMed1987

Optimization of gene expression in Streptomyces lividans by a transcription terminator.

The ability of an inverted repeat sequence (IRS) from the 3' end of the aph gene from Streptomyces fradiae to induce transcription termination in vivo has been examined. As a model system, a DNA fragment encoding the human interferon alpha 2 inserted in the Streptomyces plasmid pIJ702 was used. When the IRS was inserted downstream from this sequence and transcription assayed in Streptomyces lividans, highly efficient (approximately 90%) transcription termination was observed occurring immediately after the 3' terminus of the dyad. In contrast, gene constructions lacking the IRS transcribed longer mRNAs. Moreover, the IRS gave rise to increased amounts of the hIFN alpha 2 suggesting that the putative stem-loop structure stabilised the transcript.

Base Sequence↗
PubMed1987

Reaction conditions affect the specificity of bromoacetaldehyde as a probe for DNA cruciforms and B-Z junctions.

The reaction of bromoacetaldehyde (BAA) was investigated further with recombinant plasmids containing tracts of (CG)16, in pRW756, or (CA)32, in pRW777, which adopt left-handed Z-structures under the influence of negative supercoiling. The cruciform structures adopted by the inverted repeat sequences near the replication origins of the pBR322 vectors served as internal controls for the number of unpaired bases. The extent of reaction with the B-Z junctions and the cruciforms was dependent on the reaction and analysis conditions, the method of preparation of BAA, ionic conditions, and the amount of negative supercoiling. In contrast to the previous results of Kang and Wells, B-Z junctions in addition to cruciforms do react with BAA. However, more forcing conditions are required to detect this reaction since B-Z junctions appear to be less reactive than the single stranded loops of cruciforms. The site of reaction with DNA was readily mapped with high precision at the nucleotide level. Also, a simple method is described for determining the concentration of BAA as well as its intrinsic reactivity in a given ionic medium.

Acetaldehyde↗
PubMed1992

Upstream binding sequences of the XylR activator protein and integration host factor in the xylS gene promoter region of the Pseudomonas TOL plasmid.

The xylR and xylS genes, which encode the positive regulators of the TOL plasmid catabolic pathways, are adjacent genes on the TOL plasmid and are transcribed from divergent promoters. Transcription from the xylS gene promoter, Ps, is positively regulated by effector-activated XylR protein and requires the specific RNA polymerase sigma 54 subunit (RpoN). Deletions and point mutations in the Ps upstream region localized the site of XylR interaction to the region between -133 bp and -207 bp (with respect to the transcriptional start of the xylS messenger), which contains an inverted sequence repeat largely homologous to the motif recognised by XylR in the XylR-regulated 'upper' catabolic pathway promoter, Pu. Gel retardation experiments showed binding of IHF to the Ps promoter region. Corresponding sequences showing good homology to the IHF-binding consensus were identified close to the Ps Promoter (between -35 bp and -47 bp, Ps proximal site) and further upstream overlapping the XylR recognition sequence (Ps distal site). In the latter case IHF recognition motifs were found well conserved on both strands at nearly the same position (between -140 bp and -152 bp on the upper and between -141 bp and -153 bp on the lower strand). Expression from Ps, either under inducing or non-inducing conditions, was, however, only slightly influenced by the absence of IHF in an IHF-deficient mutant and thus activation of Ps, like that of other sigma 54-dependent promoters which are rich in Ts, does not absolutely require IHF protein.

Amino Acid Sequence↗
PubMed1995

A novel initiator regulates expression of the nontissue-specific helix-loop-helix gene ME1.

The mouse ME1 gene (HEB, REB and GE1, homologues in human, rat and chick, respectively) is a member of the nontissue-specific helix-loop-helix (HLH) gene family that includes E2A, E2-2 and Drosophila daughterless. We have examined the factors that control ME1 gene expression. ME1 is a single copy gene that spans > or = 150 kb of DNA and contains > 10 exons. Transcription was directed by an unusual initiator element that contained a 13 bp poly d(A) tract flanked by palindromic and inverted repeat sequences. Both RNase protection and primer extension analyses mapped the ME1 transcriptional start site to the center of the 13 bp poly d(A) tract. The ME1 initiator and its proximal sequences were required for promoter activity, supported basal levels of transcription, and contributed to cell type-specific gene expression. Other cis-elements utilized by the TATA-less ME1 promoter included a cluster of Sp1 response elements, E-boxes and a strong repressor. Collectively, our results suggest that the ME1 initiator and other cis-elements in the proximal promoter play an important role in regulating ME1 gene expression.

Animals↗
PubMed2006

Tobacco plastid ribosomal protein S18 is essential for cell survival.

Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Deltarps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.

Cell Survival↗
PubMed1979

Molecular characterization of a small Haemophilus influenzae plasmid specifying beta-lactamase and its relationship to R factors from Neisseria gonorrhoeae.

The ampicillin-resistant Haemophilus influenzae strain Ve445 which caused purulent meningitis and septicaemia in a newborn child in Germany contained a 4.4 megadalton (Mdal) plasmid (pVe445) and produced a TEM type beta-lactamase. The transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pVe445 DNA proved that the structural gene for the beta-lactamase resided on this plasmid genome. Molecular DNA-DNA hybridization studies and electron microscope DNA heteroduplex analysis indicated that pVe445 probably contained 38 to 41% of the ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. The TnA fragment present in pVe445 most likely does not contain both of the inverted repeat sequences of TnA. DNA-DNA polynucleotide sequence studies indicated that the 4.4 Mdal plasmid pVe445 was unrelated to the 30 to 38 Mdal H. influenzae R plasmids but was closely related to the 4.1 Mdal ampicillin resistance specifying H. influenzae plasmid RSF0885 isolated in the U.S.A. The H. influenzae plasmid pVe445 shared 91% of its base sequences with the beta-lactamase specifying Neisseria gonorrhoeae plasmid pMR0360 (4.4 Mdal) and had 85% of its base sequences in common with the beta-lactamase specifying N. gonorrhoeae plasmid pMR0200 (3.2 Mdal). All of the four 3.2 to 4.4 Mdal beta-lactamase specifying R plasmids of H. influenzae and N. gonorrhoeae investigated probably have a common evolutionary origin.

Ampicillin↗
PubMed1985

Molecular analysis of an antibiotic resistance plasmid, pAV5, and its derivative plasmids in Acinetobacter calcoaceticus.

The non-conjugative plasmid pAV5 specifies resistance to kanamycin/neomycin (KmR) and tetracycline (TcR). Physical evidence is presented to show that pAV5 gives rise to two plasmids, pAV51 (KmR) and pAV52 (TcR), which are formed by deletion of apparently non-overlapping segments of pAV5. Expression of TcR has been obtained in Escherichia coli and is associated with a 1.9 kb HindIII fragment found in pAV5 and in pAV52. Expression of KmR has been obtained in E. coli and is associated with a 1.3 kb PstI fragment found in pAV5 and pAV51. Evidence is presented that the KmR gene is flanked by inverted repeat sequences and is therefore tentatively identified as a transposon, designated Tn4411. The KmR gene specifies an aminoglycoside 3'-phosphotransferase-type I (APH(3')-I) enzyme.

Acinetobacter↗
PubMed1986

Analysis of inverted repeat DNA in the genome of Rhodomicrobium vannielii.

The DNA of Rhodomicrobium vannielii was analysed for the presence of inverted repeat sequences (IR DNA) by S1 nuclease digestion. Approximately 7% of chromosomal DNA was found to be IR DNA which comprised two size classes. The large IR DNA was heterogeneous and contained species in the size range 100-700 bp. The smaller size class contained species of 17 and 27 bp. Both size classes of IR DNA hybridized to many chromosomal restriction fragments, suggesting that these IR DNA sequences are dispersed throughout the genome. Hybridization studies also indicated sequence homology between the two classes of IR DNA and suggested that the 17 and 27 bp IR DNA sequences may exist in clusters.

Autoradiography↗
PubMed2000

Critical nucleotides in the interaction of CatR with the pheBA promoter: conservation of the CatR-mediated regulation mechanisms between the pheBA and catBCA operons.

The promoter of the plasmid-borne pheBA genes encoding enzymes for phenol degradation resembles the catBCA promoter and is activated by CatR, the regulator of the chromosomally encoded catechol-degradative catBCA genes in Pseudomonas putida. In this study, site-directed mutagenesis of the pheBA promoter region was performed. The interrupted inverted repeat sequence of the CatR recognition binding site (RBS) of the pheBA promoter is highly homologous to that of the catBCA promoter. However, the RBS was shown not to be the sole important feature for high-affinity binding of CatR to this site. Mutagenesis of the activation binding site (ABS) of CatR, which overlaps the -35 hexamer sequence TTGGAT of the promoter, revealed that the two G nucleotides in this sequence are important for promoter activity but not for CatR binding. All other substitutions made in the ABS negatively affected both the promoter activity and CatR binding. The spacer sequence of the pheBA and catBCA promoters between the -10 and -35 hexamers is 19 bp, which is longer than optimal. However, reducing the spacer region of the pheBA promoter was not sufficient for CatR-independent promoter activation. An internal binding site (IBS) for CatR is located downstream of the transcriptional start site of the catBCA genes and it negatively regulates the operon. A similar IBS was identified in the case of the pheBA operon and tested for its functionality. The results indicate a conservation of CatR-mediated regulation mechanisms between the pheBA promoter and the catBCA promoter. This universal mechanism of CatR-mediated transcriptional activation could be of great importance in enabling catechol-degrading bacteria to expand their substrate range via horizontal transfer of the phenol degradative genes.

Bacterial Proteins↗
PubMed2000

Control of periplasmic nitrate reductase gene expression (napEDABC) from Paracoccus pantotrophus in response to oxygen and carbon substrates.

The napEDABC operon of Paracoccus pantotrophus encodes a periplasmic nitrate reductase (NAP), together with electron-transfer components and proteins required for the synthesis of a fully functional enzyme. Previously, it had been shown that high NAP activity was observed when P. pantotrophus was grown aerobically on highly reduced carbon sources such as butyrate or caproate, but not when cultured on more oxidized substrates such as succinate or malate. The enzyme is not present to any extent when the organism is grown anaerobically under denitrifying conditions, regardless of the carbon source. Transcriptional analyses of the nap operon have now identified two initiation sites which were differentially regulated in response to the carbon source, with expression being maximal when cells were grown aerobically with butyrate. Analysis of a P. pantotrophus mutant (M6) deregulated for NAP activity identified a single C-->A transversion in a heptameric inverted-repeat sequence that partially overlapped the proximal promoter. Transcription analysis of this mutant revealed that expression of nap was completely derepressed under all growth conditions examined. Taken together, these findings indicate that nap transcription is negatively regulated during anaerobiosis, such that expression is restricted to aerobic growth, but only when the carbon source is highly reduced.

Anaerobiosis↗
PubMed2003

An inducible tellurite-resistance operon in Proteus mirabilis.

Tellurite resistance (Te(r)) is widespread in nature and it is shown here that the natural resistance of Proteus mirabilis to tellurite is due to a chromosomally located orthologue of plasmid-borne ter genes found in enteric bacteria. The P. mirabilis ter locus (terZABCDE) was identified in a screen of Tn5lacZ-generated mutants of which one contained an insertion in terC. The P. mirabilis terC mutant displayed increased susceptibility to tellurite (Te(s)) and complementation with terC carried on a multicopy plasmid restored high-level Te(r). Primer extension analysis revealed a single transcriptional start site upstream of terZ, but only with RNA harvested from bacteria grown in the presence of tellurite. Northern blotting and reverse transcriptase-PCR (RT-PCR) analyses confirmed that the ter operon was inducible by tellurite and to a lesser extent by oxidative stress inducers such as hydrogen peroxide and methyl viologen (paraquat). Direct and inverted repeat sequences were identified in the ter promoter region as well as motifs upstream of the -35 hexamer that resembled OxyR-binding sequences. Finally, the 390 bp intergenic promoter region located between orf3 and terZ showed no DNA sequence identity with any other published ter sequences, whereas terZABCDE genes exhibited 73-85 % DNA sequence identity. The ter operon was present in all clinical isolates of P. mirabilis and Proteus vulgaris tested and is inferred for Morganella and Providencia spp. based on screening for high level Te(r) and preliminary PCR analysis. Thus, a chromosomally located inducible tellurite resistance operon appears to be a common feature of the genus Proteus.

Bacterial Proteins↗
PubMed2003

Two MerR homologues that affect copper induction of the Bacillus subtilis copZA operon.

Copper ions induce expression of the Bacillus subtilis copZA operon encoding a metallochaperone, CopZ, and a CPx-type ATPase efflux protein, CopA. The copZA promoter region contains an inverted repeat sequence similar to that recognized by the mercury-sensing MerR protein. To investigate the possible involvement of MerR homologues in copZA regulation, null mutations were engineered affecting each of four putative MerR-type regulators: yyaN, yraB, yfmP and yhdQ. Two of these genes affected copper regulation. Mutation of yhdQ (hereafter renamed cueR) dramatically reduced copper induction of copZA, and purified CueR bound with high affinity to the copZA promoter region. These results suggest that CueR is a direct regulator of copZA transcription that mediates copper induction. Surprisingly, a yfmP mutation also reduced copper induction of copZA. Sequence analysis suggested that yfmP was cotranscribed with yfmO, encoding a putative multidrug efflux protein. The yfmPO operon is autoregulated: a yfmP mutation derepressed the yfmP promoter and purified YfmP bound the yfmP promoter region, but not the copZA promoter region. Since the yfmP mutant strain was predicted to express elevated levels of the YfmO efflux pump, it was hypothesized that copper efflux might be responsible for the reduced copZA induction. Consistent with this model, in a yfmP yfmO double mutant copper induction of copZA was normal. The results demonstrate the direct regulation of the B. subtilis copper efflux system by CueR, and indirect regulation by a putative multidrug efflux system.

Amino Acid Sequence↗
PubMed2004

Unique organization and regulation of the mrx fimbrial operon in Xenorhabdus nematophila.

Xenorhabdus nematophila, a Gram-negative bacterium belonging to the Proteus clade of the family Enterobacteriaceae, forms a mutualistic association with the soil nematode Steinernema carpocapsae. The nematode invades insects and releases Xenorhabdus into the haemolymph, where it participates in insect killing. To begin to understand the role of fimbriae in the unique life cycle of Xenorhabdus, the organization and expression of the mrx fimbrial operon was analysed. The mrx operon contained only five structural genes (mrxACDGH), making it one of the smallest chaperone-usher fimbrial operons studied to date. Unlike the mrp operon of Proteus mirabilis, a site-specific recombinase was not linked to the mrx operon. The intergenic region between the major fimbrial gene (mrxA) and the usher gene (mrxC) lacked a mrpB-like gene, but contained three tandem inverted repeat sequences located downstream of mrxA. A 940 nt mrxA-containing mRNA was the major transcript produced in cells growing on agar, while an mrx polycistronic mRNA was produced at low levels. A canonical sigma(70) promoter, identified upstream of mrxA, was not subject to promoter inversion. Fimbriae were not produced in an lrp-mutant strain, suggesting that the leucine-responsive regulatory protein, Lrp, plays a role in the regulation of the mrx operon. These findings show that the genetic organization and regulation of the mrx operon is in several respects distinct from other chaperone-usher fimbrial operons.

Amino Acid Sequence↗
PubMed1992

Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration.

The Escherichia coli ruvA and ruvB genes are involved in DNA repair and in the late step of homologous genetic recombination. We have demonstrated previously that the RuvA-RuvB protein complex in the presence of ATP promotes reabsorption of cruciform structures extruded from a supercoiled plasmid with an inverted repeat sequence. Because the cruciform structure is topologically analogous to the Holiday structure, we have proposed that the role of the RuvA and RuvB proteins in recombination is to promote a strand exchange reaction at the Holliday junction. Here, we studied the specific interaction of the RuvA-RuvB complex with the Holliday structure using synthetic analogs prepared by annealing four oligonucleotides. The affinities of the RuvA protein for synthetic Holliday junctions are much higher (> 20-fold) than for duplex DNA, and the affinities of the RuvA protein for the junctions are further enhanced (> 4-fold) by the interaction with the RuvB protein. The RuvA-RuvB protein complex in the presence of ATP promotes dissociation of the synthetic Holliday junction with homology in the central core into two halves by catalyzing branch migration to the DNA ends, but it does not affect the structure of the synthetic Holliday junction without the homology. The separation of the synthetic Holliday junction is a result of the activity of the RuvA-RuvB complex that promotes strand exchange and DNA unwinding. Furthermore, RuvA and RuvB promote the strand exchange reaction at the Holliday junctions made by RecA. These results provide further evidence that the RuvA-RuvB complex recognizes the Holliday junction and promotes branch migration in homologous recombination.

Bacterial Proteins↗
PubMed1980

Restriction endonuclease cleavage maps of the ampicillin transposons Tn2601 and Tn2602.

This paper reports the cleavage maps of ampicillin transposons Tn2601 and Tn2602, for restriction endonucleases BamHI, PvuII, AvaI, HincII, and HaeII. Both of the transposons are very similar to the well-known ampicillin transposon Tn3 in size, endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. A slight difference in the cleavage pattern among these three transposons was observed in the region around the BamHI site which was assumed to be a part of the repressor gene for transposition.

Ampicillin↗