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PubMed1996

Genetic and sequence analysis of the cos region of the temperate Pseudomonas aeruginosa bacteriophage, D3.

The location and structure of the cos ends of bacteriophage D3, which infects Pseudomonas aeruginosa strain PAO, has been determined using a combination of deletion analysis, transposon mutagenesis, and sequencing directly off the phage DNA. Phage D3 was found to have 9-bp 3' cos ends, making it the first phage of a Gram-negative organism known to have 3' cos ends. A 700-bp region flanking the cos site was necessary for efficient transduction of D3 cosmid derivatives. This region was found to contain incomplete inverted repeat sequences flanking the cos site, along with adenine-rich repeats homologous to coliphage gama Ter binding sites. Possible IHF binding sites were also present.

Base Sequence↗
PubMed2003

Extremely complex pattern of microsatellite mutation in the germline of wheat exposed to the post-Chernobyl radioactive contamination.

The molecular structure of rare variants at 13 microsatellite loci found in a population of wheat plants grown for one generation in the heavily contaminated 30 km exclusion zone around the Chernobyl Nuclear Power Plant and in a control population was compared. Evidence for rare alterations (variants) was obtained for all 13 loci, including gain and loss of repeats, as well as the complete loss of microsatellite bands. The ratio between gains and losses among variants in the control group was similar to that in the exposed group. Sequencing of variants at six microsatellite loci found in the exposed population revealed extremely complex pattern of germline mutations, including complete deletions of loci, a bias towards mutations with gains and losses of multiple repeat units, and relatively frequent insertions of DNA of unknown origin. The occurrence of large deletions at two loci may be attributed to direct and inverted repeats sequences located just upstream and downstream of the array. The results of our study also suggest that the majority of mutations within the studied wheat microsatellite loci are represented by gains and losses of multiple repeat units, implying that a simple model of replication slippage cannot account for mutation events at these loci. Our data also support the conclusion that the spectra of spontaneous and radiation-induced mutation in wheat may be similar.

Base Sequence↗
PubMed2001

Complete genome sequence of the shrimp white spot bacilliform virus.

We report the first complete genome sequence of a marine invertebrate virus. White spot bacilliform virus (WSBV; or white spot syndrome virus) is a major shrimp pathogen with a high mortality rate and a wide host range. Its double-stranded circular DNA genome of 305,107 bp contains 181 open reading frames (ORFs). Nine homologous regions containing 47 repeated minifragments that include direct repeats, atypical inverted repeat sequences, and imperfect palindromes were identified. This is the largest animal virus that has been completely sequenced. Although WSBV is morphologically similar to insect baculovirus, the two viruses are not detectably related at the amino acid level. Rather, some WSBV genes are more homologous to eukaryotic genes than viral genes. In fact, sequence analysis indicates that WSBV differs from all known viruses, although a few genes display a weak homology to herpesvirus genes. Most of the ORFs encode proteins that bear no homology to any known proteins, either suggesting that WSBV represents a novel class of viruses or perhaps implying a significant evolutionary distance between marine and terrestrial viruses. The most unique feature of WSBV is the presence of an intact collagen gene, a gene encoding an extracellular matrix protein of animal cells that has never been found in any viruses. Determination of the genome of WSBV will facilitate a better understanding of the molecular mechanism underlying the pathogenesis of the WSBV virus and will also provide useful information concerning the evolution and divergence of marine and terrestrial animal viruses at the molecular level.

Amino Acid Sequence↗
PubMed1996

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome.

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.

Animals↗
PubMed1988

Nucleotide sequence, transcription and deduced function of a gene involved in polyketide antibiotic synthesis in Streptomyces coelicolor.

The BamHI fragment containing the actIII gene, from the actinorhodin (Act) biosynthetic gene cluster of Streptomyces coelicolor A3(2), was sequenced. The derived amino acid sequence for the actIII gene shows homology to known oxidoreductases and the actIII product is believed to be responsible for catalysing a beta-keto reductive step during assembly of the Act polyketide chain. High resolution transcript mapping identified the transcription start point at 33 nucleotides upstream of the putative translation start codon. The transcript ends in a large invertedly repeated sequence. In vivo promoter-probe studies suggest that efficient transcription of the actIII gene requires the product of the actII gene.

Alcohol Oxidoreductases↗
PubMed2003

New insertion sequence elements in the upstream region of cfiA in imipenem-resistant Bacteroides fragilis strains.

The 747-bp cfiA gene, which encodes a metallo-beta-lactamase, and the regions flanking cfiA in six imipenem-resistant and four imipenem-susceptible Bacteroides fragilis strains isolated in Japan were analyzed by PCR and DNA sequencing. The nucleotide sequences of the cfiA genes (designated cfiA(1) to cfiA(10)) of all 10 strains tested varied from that of the standard cfiA gene from B. fragilis TAL2480. However, putative proteins encoded by the cfiA variants contained conserved amino acid residues important for zinc binding and hairpin loop formation, suggesting that cfiA variants have the capability of producing metallo-beta-lactamases with full catalytic activities. PCR assay indicated that six metallo-beta-lactamase-producing, imipenem-resistant strains had an insertion mutation in the region immediately upstream of cfiA. Nucleotide sequencing of the PCR-amplified fragments along with the upstream region of cfiA revealed that there were five new kinds of insertion sequence (IS) elements (designated IS612, IS613, IS614, IS615, and IS616, with a size range of 1,594 to 1,691 bp), of which only IS616 was found to be almost identical to IS1188, one of the IS elements previously identified in the upstream region of cfiA. These elements had target site duplications of 4 or 5 bp in length, terminal inverted repeats (14, 15, or 17 bp in size), and a large open reading frame encoding a putative transposase which is required for the transcription of IS elements. Each element was inserted such that the transcriptional direction of the transposase was opposite to that of cfiA. A computer-aided homology search revealed that, based on the homology of their putative transposases, the sizes of their terminal inverted repeat sequences, and their target site duplications, IS612, IS613, IS614, and IS615 belong to the IS4 family, which includes IS942, previously found in some drug-resistant B. fragilis strains, but that IS616 belongs to the IS1380 family. All the IS elements appear to have putative promoter motif sequences (the -7 region's TAnnTTTG motif and the -33 region's TTG or TG) in their end regions, suggesting that the IS elements provide a promoter for the transcription of cfiA upon insertion. These data provide additional proof that various IS elements may exist to provide a promoter to express the cfiA gene.

Amino Acid Sequence↗
PubMed1993

Cloning of type XVII collagen. Complementary and genomic DNA sequences of mouse 180-kilodalton bullous pemphigoid antigen (BPAG2) predict an interrupted collagenous domain, a transmembrane segment, and unusual features in the 5'-end of the gene and the 3'-untranslated region of the mRNA.

Thus far, 16 distinct vertebrate collagens (types I-XVI) have been delineated. In this study, we have cloned a mouse collagenous protein, the 180-kDa bullous pemphigoid antigen (BPAG2). Isolation of over-lapping clones, together with 5' and 3' rapid amplification of cDNA ends, allowed delineation of the entire coding sequence. The 5' and 3' ends of the mRNA transcripts were confirmed by primer extension and anchored reverse transcription polymerase chain reaction analyses. The deduced polypeptide contained 1,433 amino acids, including a collagenous domain that consisted of 13 separate segments. Computer analysis of the deduced amino acid sequence demonstrated the presence of a membrane-associated segment. Examination of the 5' end of the BPAG2 gene revealed that the 295-base pair (bp) exon 1 contained two segments of (T)13AA and TT(A)11, whereas exon 2 was shown to contain the translation initiation codon. The 3' end of the mRNA transcript identified two 6-bp inverted repeat sequences that could form a stem for a 30-bp hairpin loop followed by a series of U residues. Comparison of mouse and human BPAG2 sequences demonstrated 86% homology and the unit of evolutionary period of 4.2 million years. In summary, we have cloned full-length mouse BPAG2 cDNA sequences that encode a collagenous polypeptide. We propose that this polypeptide be designated as the alpha 1-chain of type XVII collagen.

Amino Acid Sequence↗
PubMed1987

Genetic analysis of the interaction of the insertion sequence IS903 transposase with its terminal inverted repeats.

The insertion sequence IS903 has perfect, 18-base-pair terminal repeats that are the presumed binding sites of its transposase. We have isolated mutations throughout this inverted repeat and analyzed their effect on transposition. We show that every position in the inverted repeat (with the possible exception of position 4) is important for efficient transposition. Furthermore, various substitutions at a single position can have a wide range of effects. Analysis of these hierarchical effects suggests that transposase contacts the minor groove in the region from position 13 to position 16 but makes major groove (or more complex) interactions with the outer portion of the inverted repeat. Our data indicate that the transposase exhibits relaxed specificity for the "second" end of a transposed segment; the defect in transposition of virtually all mutant inverted repeats can be rescued by a wild-type end. However, this rescue exhibits a pronounced position effect; in most cases, it is efficient only when the wild-type end is close to the 3' end of the transposase gene. This confirms the cis-acting nature of the transposase protein and suggests the initial transposase-inverted repeat interaction is the rate-limiting step in transposition. From the behavior of transposons with one mutant and one wild-type end, we infer that the inverted repeat contains two functional domains--one for initial complex formation with transposase and the other for effective completion of transpositional recombination. To support this hypothesis we show that an end with a mutation in one domain can significantly rescue an end with a mutation in the other domain.

Binding Sites↗
PubMed1999

Molecular characterization of the PmrA regulon.

The two-component system PmrA/PmrB of Salmonella enterica controls expression of several loci including those mediating modifications in the lipopolysaccharide that result in polymyxin resistance. To gain insight in the regulation of polymyxin resistance, we mapped the transcription start sites of the PmrA-regulated genes pmrC, pmrG, pbgPE, and ugd and identified a conserved sequence in the promoter region of the first three genes. His-tagged PmrA protein could gel shift DNA fragments containing the promoters of the pmrC, pmrG, and pbgPE genes but not the udg promoter. DNase I footprinting analysis of the pmrC, pmrG, and pbgPE promoters indicate that phosphorylated as well as unphosphorylated PmrA bind to a 16-base pair imperfect inverted repeat sequence (5'-TTAAKTTCTTAAKGTT-3'), which is found 40, 80, and 38 nucleotides upstream from the transcription start sites of the pmrC, pmrG, and pbgPE genes, respectively. Our data suggest that a PmrA dimer activates transcription of the divergent pmrG and pbgPE promoters by binding to a single site in the pmrG-pbgPE intergenic region and that the ugd gene is regulated by the PmrA/PmrB system only indirectly.

Bacterial Proteins↗
PubMed2001

Molecular characterization of Pseudomonas putida KT2440 rpoH gene regulation.

The rpoH gene of Pseudomonas putida KT2440 encoding the heat-shock sigma factor sigma(32) was cloned and sequenced, and the translated gene product was predicted to be a protein of 32.5 kDa. The unambiguous role of the gene as a sigma factor was confirmed because the cloned P. putida gene complemented the growth defect, at 37 and 42 degrees C, of an Escherichia coli rpoH mutant strain. Primer extension analysis showed that in P. putida the rpoH gene is expressed from three promoters in cells growing at 30 degrees C. Two of them, P1 and P3, share homology with the sigma(70)-dependent promoters, while the third one, P2, shows a typical sigma(24)-consensus sequence. The pattern of transcription initiation of the rpoH gene did not change in response to different stresses, i.e. a sudden heat shock or the addition of aromatic compounds. However, the predicted secondary structure of the 5' region of the mRNA derived from the three different promoters suggests regulation at the level of translation efficiency and/or mRNA half-life. An inverted repeat sequence located 20 bp downstream of the rpoH stop codon was shown to function as a terminator in vivo in P. putida growing at temperatures from 18 to 42 degrees C.

Base Sequence↗
PubMed1997

Occurrence of deletions, associated with genetic instability in Streptomyces ambofaciens, is independent of the linearity of the chromosomal DNA.

The chromosomal structures of mutant strains of Streptomyces ambofaciens which have arisen from genetic instability were investigated by using pulsed-field gel electrophoresis and probing with sequences cloned from the unstable region which maps near the ends of the linear chromosomal DNA. The chromosomal structures of seven mutant strains harboring large deletions were classified into three types. (i) Deletions internal to one chromosomal arm were characterized in two of the mutant strains. In these strains, a linear chromosomal structure was retained, as were parts of the terminal inverted repeats sequences (TIRs) and the proteins bound to them. (ii) Four of the mutants presented a deletion including all sequences from the TIRs. A junction fragment homologous to sequences originating from internal region of both arms was characterized. Consequently, the chromosomal DNA of these strains was deduced to be circularized. Furthermore, chromosomal stability was assessed in the progeny of these circular DNA mutants. Additional deletion events were detected in 11 mutants among the 13 strains isolated, demonstrating that circular chromosomes do not correspond to a stabilization of the chromosome structure and that the occurrence of deletion could be independent of the presence of chromosomal ends. (iii) A mutant with DNA amplification was shown to have a linear chromosome with a deletion of all sequences between the amplified region and the end of the chromosome. The other chromosomal arm remained unaffected by deletion and associated with protein.

Bacterial Proteins↗
PubMed1983

The nucleotide sequence of myosin light chain (L-2A) mRNA from embryonic chicken cardiac muscle tissue.

The nucleotide sequence of a cDNA clone (pML10) for chicken cardiac myosin light chain is described. The cDNA insert contains 613 nucleotides representing the entire coding sequence, with the exception of nine NH2-terminal amino acids, and the full 3'-non-coding region of 146 nucleotides. The missing 5' terminus of the mRNA, not represented in the clone pML10, was obtained by extension of the cDNA using a 43 nucleotide long internal EcoR1 fragment as a primer. The non-coding region contains several direct and inverted repeated sequences and the polyadenylation signal sequence AATAAA. The coding portion exhibits non-random usage of synonymous codons with a strong bias for codons ending in G and C.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence of the promoter-distal region of the tra operon of plasmid R100, including traI (DNA helicase I) and traD genes.

The nucleotide sequence of the promoter-distal region of the tra operon of R100 was determined. There are five open reading frames in the region between traT and finO, and their protein products were identified. Nucleotide sequences of plasmid F corresponding to the junction regions among the open reading frames seen in R100 were also determined. Comparison of these nucleotide sequences revealed strong homology in the regions containing traD, traI and an open reading frame (named orfD). The TraD protein (83,899 Da) contains three hydrophobic regions, of which two are located near the amino-terminal region. This protein also contains a possible ATP-binding consensus sequence at the amino-terminal region and a characteristic repeated peptide sequence (Gln-Gln-Pro)10 at the carboxy-terminal region. The TraI protein (191,679 Da) contains the sequence motif conserved in an ATP-dependent DNA helicase superfamily in its carboxy-terminal region. The protein product of orfD, which is probably a new tra gene (named traX), contains 65% hydrophobic amino acids, especially rich in alanine and leucine. There exist non-homologous regions between R100 and F that could be represented as four I-D (insertion or deletion) loops in heteroduplex molecules. Assignment of each loop to the strand of R100 or F was , however, found to be the reverse from that previously assumed. The three I-D loops that were located between traT and traD, between traD and traI, and between traI and finO had no terminal inverted repeat sequences nor had they any homology with known insertion sequences, while the fourth was IS3, located within the finO gene of F. The sequences in the I-D loops, except IS3, may also code for proteins that are, however, likely to be nonessential for transfer of plasmids.

Amino Acid Sequence↗
PubMed2000

IS1414, an Escherichia coli insertion sequence with a heat-stable enterotoxin gene embedded in a transposase-like gene.

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E. coli (ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed that astA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the -1 reading frame in reference to the toxin gene. In vitro expression of the predicted M(r)- approximately 46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414. Preliminary experiments to show IS1414 translocation were unsuccessful. Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.

Alleles↗
PubMed1991

Specific DNA binding of the TraM protein to the oriT region of plasmid R100.

The product of the traM gene of plasmid R100 was purified as the TraM-collagen-beta-galactosidase fusion protein (TraM*) by using a beta-galactosidase-specific affinity column, and the TraM portion of TraM* (TraM') was separated by collagenolysis. Both the TraM* and TraM' proteins were found to bind specifically to a broad region preceding the traM gene. This region (designated sbm) was located within the nonconserved region in oriT among conjugative plasmids related to R100. The region seems to contain four core binding sites (designated sbmA, sbmB, sbmC, and sbmD), each consisting of a similar number of nucleotides and including a homologous 15-bp sequence. This result, together with the observation that the TraM* protein was located in the membrane fraction, indicates the possibility that the TraM protein has a function in anchoring the oriT region of R100 at the sbm sites to the membrane pore, through which the single-stranded DNA is transferred to the recipient. sbmC and sbmD, each of which contained a characteristic inverted repeat sequence, overlapped with the promoter region for the traM gene. This suggests that the expression of the traM gene may be regulated by its own product.

Amino Acid Sequence↗
PubMed1982

Expression of cloned herpesvirus genes. I. Detection of nuclear antigens from herpes simplex virus type 2 inverted repeat regions in transfected mouse cells.

Three different recombinant plasmids containing the entire 15-kilobase L and S inverted repeat sequence of herpes simplex virus type 2 DNA have been introduced into cultured Ltk- or BSC cells by both the calcium and DEAE-dextran transfection procedures. In each case, after 24 h approximately 1% of the cells gave strongly positive nuclear staining when assayed by immunofluorescence with hyperimmune antisera made against early and immediate-early infected-cell polypeptides. The nuclear fluorescence pattern and intensity mimicked that observed within 2 to 3 h after infection of Ltk- cells with either herpes simplex virus type 1 or type 2 wild-type virus. Herpes simplex virus type 1 (KOStsB2)-infected Ltk- cells under nonpermissive conditions did not express these antigens in the nucleus. Therefore, we conclude that either one or both of the 185,000- and 110,000-molecular-weight immediate early proteins, or some other as yet unknown gene product encoded entirely within the inverted repeats, can be transiently expressed in large amounts in transfected cells in the absence of other viral genes or accompanying virion components. Permanent mouse cell lines derived from transfection with these plasmids by using the thymidine kinase coselection procedure did not express sufficient nuclear antigen to be detectable by immunofluorescence.

Animals↗
PubMed1997

Cloning, characterization and functional analysis of groESL operon from thermophilic cyanobacterium Synechococcus vulcanus.

Genes encoding 10914 Da and 58267 Da polypeptides homologous to groES and groEL of Escherichia coli were cloned and sequenced from a thermophilic cyanobacterium, Synechococcus vulcanus. The deduced amino acid sequence of the GroEL protein was much more homologous to GroELs of other cyanobacteria which accompany GroES than another GroEL homolog of S. vulcanus (GroEL2) reported previously (M. Furuki, N. Tanaka, T. Hiyama, and H. Nakamoto, Biochim. Biophys. Acta 1294 (1996) 106-110). We designate the gene as groEL1 to distinguish it from the non-operon forming groEL2 gene. A 9-base pair inverted repeat sequence (TTAGCACTC-N9-GAGTGCTAA) was located upstream of the promoter region of groEL1, which was absent in groEL2. Southern blot analysis indicated that only one groESL1 operon was present in the genomic DNA of S. vulcanus. The amount of the bicistronic, 2.3 kb transcript of groESL1 operon increased 30-fold within 30 min upon heat shock. The increase was completely inhibited by chloramphenicol, suggesting the involvement of heat-induced production of a polypeptide. Introduction of the cloned groEL1 gene into a groEL defective mutant of E. coli resulted in the complementation of heat sensitivity, which contrasted with the previous result with groEL2.

Amino Acid Sequence↗
PubMed1991

Mechanisms of insertional mutagenesis in human genes causing genetic disease.

Examples of the insertion of less than 10 bp of DNA sequence into human gene-coding regions causing genetic disease were collated in order to study the underlying causative mechanisms. The nature of these insertions was found to be consistent with several mechanisms of mutagenesis including: (1) slipped mispairing mediated by direct repeats or runs of identical bases and (2) the templated misincorporation of bases by secondary-structure intermediates whose formation is facilitated by palindromic (inverted repeat) sequences, quasi-palindromic sequences or symmetric elements. Both the size and position of insertions were found to be non-random and highly dependent upon the surrounding DNA sequence. Inferred mechanisms of insertional mutagenesis thus appear to be very similar to those involved in the causation of gene deletions.

Base Sequence↗