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PubMed1988

Sequence analysis of the gtfC gene from Streptococcus mutans GS-5.

The nucleotide sequence of the gtfC gene, which codes for glucosyltransferase synthesizing both water-soluble and water-insoluble glucans, and its flanking regions from Streptococcus mutans GS-5, was determined. Although the gtfC gene (4218 bp) is preceded by a Shine-Dalgarno (SD) sequence, a promoter-like sequence for this gene could not be identified. The gtfC gene product composed of 1375 amino acid residues (approx. 153 kDa) is generally hydrophilic with three small hydrophobic domains. Two direct repeating units were found near the C terminus of the peptide. The gtfC gene has extensive homology with the previously sequenced gtfB gene. The homologous regions correspond to the signal sequence, an internal region, and the direct repeating units of the peptide. An open reading frame preceded by an SD sequence and followed by an inverted repeat sequence was found immediately downstream from the gtfC gene. The combined sequences of the gtfB and gtfC genes as well as flanking regions suggest that the two gtf genes and the small downstream coding region could be coordinately expressed within an operon. The possible evolution of the gtfC gene in S. mutans GS-5 is also discussed.

Amino Acid Sequence↗
PubMed1992

A Coxiella burnetti repeated DNA element resembling a bacterial insertion sequence.

A DNA fragment located on the 3' side of the Coxiella burnetii htpAB operon was determined by Southern blotting to exist in approximately 19 copies in the Nine Mile I genome. The DNA sequences of this htpAB-associated repetitive element and two other independent copies were analyzed to determine the size and nature of the element. The three copies of the element were 1,450, 1,452, and 1,458 bp long, with less than 2% divergence among the three sequences. Several features characteristic of bacterial insertion sequences were discovered. These included a single significant open reading frame that would encode a 367-amino-acid polypeptide which was predicted to be highly basic, to have a DNA-binding helix-turn-helix motif, to have a leucine zipper motif, and to have homology to polypeptides found in several other bacterial insertion sequences. Identical 7-bp inverted repeats were found at the ends of all three copies of the element. However, duplications generated by many bacterial mobile elements in the recipient DNA during insertion events did not flank the inverted repeats of any of the three C. burnetii elements examined. A second pair of inverted repeats that flanked the open reading frame was also found in all three copies of the element. Most of the divergence among the three copies of the element occurred in the region between the two inverted repeat sequences in the 3' end of the element. Despite the sequence changes, all three copies of the element have retained significant dyad symmetry in this region.

Amino Acid Sequence↗
PubMed1994

Requirement for double-strand breaks but not for specific DNA sequences in herpes simplex virus type 1 genome isomerization events.

Herpes simplex virus type 1 (HSV-1) genome isomerization occurs as a result of DNA replication-mediated homologous recombination between several sets of inverted repeat sequences present in the viral DNA. The frequency with which this recombination occurs has been demonstrated to be dependent upon DNA homology length rather than specific sequences. However, the smallest of the viral inverted repeats, the alpha sequence, has been shown to function as a recombinational hot spot, leading to speculation that this sequence may represent a specific element through which genome isomerization is mediated. To investigate this apparent paradox, a quantitative transient recombination assay system was developed and used to examine the recombinogenic properties of a panel of alpha sequence mutants. This analysis revealed that the presence of both the pac1 and pac2 elements was both necessary and sufficient for the induction of high-frequency recombination events by the alpha sequence. However, it was the double-strand break promoted by pac1 and pac2 during cleavage and packaging at the alpha sequence, and not the DNA sequences of the elements themselves, which appeared to be critical for recombination. This was illustrated (i) by the inability of the same pac1 and pac2 sequences to mediate inversion events in cells infected with an HSV-1 mutant which was competent for DNA replication-dependent recombination but defective for the cleavage and packaging process and (ii) by the ability of double-strand breaks generated in non-HSV-1 DNA by an in vivo-expressed restriction endonuclease to significantly stimulate the initiation of recombination events in virus-infected cells. Thus, the alpha sequence appears to act as a hot spot for homologous recombination simply because it happens to coincide with the site of the double-strand break which is generated during the cleavage and packaging process, not because it contains discrete sequences which are required for this activity. However, it was found that this enhanced recombinogenicity disappeared when the element was flanked by regions of extensive sequence homology, particularly that of the large inverted repeats which flank the alpha sequence at its natural site in the HSV-1 genome. These findings are consistent with a model for HSV-1 genome isomerization in which recombination is initiated primarily by multiple random double-strand breaks which arise during DNA replication across the inverted repeats of the genome, rather than by a single specific break which occurs at the alpha sequence during the cleavage and packaging process.

Animals↗
PubMed1993

Conformational studies of hairpin sequences from the ColE1 cruciform.

Inverted repeat sequences derived from the ColE1 cruciform were investigated by nuclear magnetic resonance (NMR) and UV spectroscopy. It was shown that 15 different sequences exist as stable hairpin structures over a range of buffer conditions and DNA concentrations. Experiments with six oligomers (1-6) containing the native stem sequence and five base loops, found that the two hairpins with the wild-type loops (1-2) served as upper and lower bounds for the thermodynamic stability of all the other sequences. NMR experiments, including rotational correlation time measurements and NOESY spectra, were then performed on 1, the most stable hairpin sequence to begin to uncover a structural basis of its stability.

Bacteriocin Plasmids↗
PubMed1992

Delimitation of cohesive ends site (cos) of Streptomyces temperate bacteriophage R4.

The cohesive ends site (cos) of actinophage R4 was delimitated by assaying the in vivo packaging activity of cosmid derivatives in Streptomyces lividans. A region of 66 bp from -30 to +36 from the center of cohesive ends was required for the basal level of packaging activity. Two additional regions outside the basal sequences from -39 to -31 and from +37 to +97 were necessary for the high level of activity, defined as the accessory sequences. Direct- or inverted-repeat sequences were found within the delimitated region, which might be involved in the recognition by the terminase of actinophage R4.

Bacteriophages↗
PubMed2000

PBF-2 is a novel single-stranded DNA binding factor implicated in PR-10a gene activation in potato.

Elicitor-induced activation of the potato pathogenesis-related gene PR-10a requires a 30-bp promoter sequence termed the ERE (elicitor response element) that is bound by the nuclear factor PBF-2 (PR-10a binding factor 2). In this study, PBF-2 has been purified to near homogeneity from elicited tubers through a combination of anion-exchange and DNA affinity chromatography. Evidence demonstrates that inactive PBF-2 is stored in the nuclei of fresh tubers and becomes available for binding to the ERE upon elicitation. A protein with an apparent molecular mass of 24 kD (p24) is a DNA binding component of PBF-2. A cDNA encoding p24 has been cloned and encodes a novel protein with a potential transcriptional activation domain that could also act as a single-stranded DNA binding domain. Both PBF-2 and the cDNA-encoded protein bind with high affinity to the single-stranded form of the ERE in a sequence-specific manner. The inverted repeat sequence of the ERE, TGACAnnnnTGTCA, is critical for binding of this factor in vitro and for PR-10a expression in vivo, supporting the role of PBF-2 as a transcriptional regulator.

Amino Acid Sequence↗
PubMed1992

Physical mapping of repetitive extragenic palindromic sequences in Escherichia coli and phylogenetic distribution among Escherichia coli strains and other enteric bacteria.

Repetitive extragenic palindromic (REP) sequences are highly conserved inverted repeat sequences originally discovered in Escherichia coli and Salmonella typhimurium. We have physically mapped these sequences in the E. coli genome by using Southern hybridization of an ordered phage bank of E. coli (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) with generic REP probes derived from the REP consensus sequence. The set of REP probe-hybridizing clones was correlated with a set of clones expected to contain REP sequences on the basis of computer searches. We also show that a generic REP probe can be used in Southern hybridization to analyze genomic DNA digested with restriction enzymes to determine genetic relatedness among natural isolates of E. coli. A search for these sequences in other members of the family Enterobacteriaceae shows a consistent correlation between both the number of occurrences and the hybridization strength and genealogical relationship.

Base Sequence↗
PubMed1993

Characterization and partial nucleotide sequence of the DNA fingerprinting probe Ca3 of Candida albicans.

The moderately repetitive Ca3 fragment of Candida albicans has been used as an effective DNA fingerprinting probe in epidemiological studies. EcoRI digestion of Ca3 DNA results in seven fragments of 4.2 kb (A), 2.98 kb (B), 2.85 kb (C), 0.77 kb (D1), 0.77 kb (D2), 0.38 kb (E), and 0.30 kb (F). Five of these EcoRI fragments have been mapped in the 5'-3' order C B D1 A D2. The intact Ca3 probe and the three largest EcoRI fragments, A, B, and C, were individually used to probe Southern blots of EcoRI-digested DNA of a set of test strains, transverse alternating field electrophoresis-separated chromosomes of strain 3153A, and Northern (RNA) blots of test strain 3153A. Fragments, A, B, and C each generate a different Southern blot hybridization pattern with EcoRI-digested whole-cell DNA; Ca3 sequences are present in at least five of seven separable chromosomes and a minichromosome of strain 3153A; fragments A, B, and C are distributed differently on chromosomes; and fragments A, B, and C do not cross-hybridize. Ca3 hybridizes to three major transcripts of 2.8, 2.3, and 1.5 kb. Fragment A hybridizes intensely to the 1.5-kb transcript, while fragments B and C both hybridize intensely to the 2.8- and 2.3-kb transcripts. The B fragment, which contains 2,980 bp and contributes to the major portion of the Ca3 pattern, was sequenced. Both direct and inverted repeat sequence motifs were identified. These results provide us with initial insights into the evolution of the Ca3 pattern and the nature of the probe.

Base Sequence↗
PubMed1998

Extraordinary features in the Chlamydomonas reinhardtii chloroplast genome: (1). rps2 as part of a large open reading frame; (2). A C. reinhardtii specific repeat sequence.

We have determined the DNA sequence of the 3574-bp chloroplast DNA fragment of Chlamydomonas reinhardtii formed by the overlap of BamHI fragment 3 and EcoRI fragment 5. This sequence encodes most of rps18 and orf570, an unidentified open reading frame that contains a 150 amino acid domain with high homology to the N-terminal part of 30 S ribosomal protein S2 of other chloroplast, cyanobacterial and bacterial genomes. Between these two sequences lies a highly repetitive sequence element of 500 bp, that is composed of multiple direct and inverted repeat sequences that occur in rearranged, but highly conserved form in at least 36 locations in the C. reinhardtii chloroplast genome. Among the conserved repeat sequences in the C. reinhardtii chloroplast genome we identified the borders of the inverted repeats near atpB and rps4. This might indicate that the conserved sequence elements are remainders of gene rearrangements in the chloroplast genome that occurred by relocations of the inverted repeats.

Amino Acid Sequence↗
PubMed1989

Mutational analysis of IS10's outside end.

We present the genetic analysis of a large number of mutations in the outside end of insertion sequence IS10. (i) The terminal inverted repeat sequence is probably the primary site of transposase binding. Mutations in this region fall into phenotypic classes which correspond to their map locations, suggesting that this region may consist of several distinct functional segments. Similarities between the organization of IS10's inverted repeat and those of other transposable elements are discussed. (ii) Base pairs 23-42 include a consensus binding sequence for one of the IS10 transposition host factors, IHF. The phenotypes of mutations in this region suggest that IHF is the major host factor for outside-end transposition activity in vivo and that base pairs throughout this region are important for the IHF interaction. (iii) Mutations in bp 43-61 do not affect outside-end transposition activity but do affect, in expected ways, previously identified determinants involved in expression and regulation of transposase. (iv) Some mutations in bp 23-42 also affect transposase expression; the possibility that IHF negatively regulates transcription initiation is discussed.

Base Composition↗
PubMed1975

Sequence organization of the human genome.

The organization of three sequence classes--single copy, repetitive, and inverted repeated sequences--within the human genome has been studied by renaturation techniques, hydroxylapatite binding methods, and DNA hyperchromism. Repetitive sequence classes are distributed throughout 80% or more of the genome. Slightly more than half of the genome consists of short single copy sequences, with a length of about 2 kb interpersed with repetitive sequences. The average length of the repetitive sequences is also small and approximates the length of these sequences found in other organisms. The sequence organization of the human genome therefore resembles the sequence organization found in Xenopus and sea urchin. The inverted repeats are essentially randomly positioned with respect to both sequence class and sequence arrangement, so that all three sequence classes are found to be mutally interspersed in a portion of the genome.

Base Sequence↗
PubMed1994

Complete nucleotide sequence of a circular plasmid from the Lyme disease spirochete, Borrelia burgdorferi.

We have determined the complete nucleotide sequence of a small circular plasmid from the spirochete Borrelia burgdorferi Ip21, the agent of Lyme disease. The plasmid (cp8.3/Ip21) is 8,303 bp long, has a 76.6% A+T content, and is unstable upon passage of cells in vitro. An analysis of the sequence revealed the presence of two nearly perfect copies of a 184-bp inverted repeat sequence separated by 2,675 bp containing three closely spaced, but nonoverlapping, open reading frames (ORFs). Each inverted repeat ends in sequences that may function as signals for the initiation of transcription and translation of flanking plasmid sequences. A unique oligonucleotide probe based on the repeated sequence showed that the DNA between the repeats is present predominantly in a single orientation. Additional copies of the repeat were not detected elsewhere in the Ip21 genome. An analysis for potential ORFs indicates that the plasmid has nine highly probable protein-coding ORFs and one that is less probable; together, they occupy almost 71% of the nucleotide sequence. Analysis of the deduced amino acid sequences of the ORFs revealed one (ORF-9) with features in common with Borrelia lipoproteins and another (ORF-2) having limited homology with a replication protein, RepC, from a gram-positive plasmid that replicates by a rolling circle (RC) mechanism. Known collectively as RC plasmids, such plasmids require a double-stranded origin at which the Rep protein nicks the DNA to generate a single-stranded replication intermediate. cp8.3/Ip21 has three copies of the heptameric motif characteristically found at a nick site of most RC plasmids. These observations suggest that cp8.3/Ip21 may replicate by an RC mechanism.

Amino Acid Sequence↗
PubMed1996

Transcriptional organization and regulation of the dnaK and groE operons of Chlamydia trachomatis.

The transcriptional organization and regulation of the dnaK and groE heat shock operons of Chlamydia trachomatis were studied and found to resemble those of the cognate operons of Bacillus subtilis and Clostridium acetobutylicum. The gene order is conserved (hrcA-grpE-dnaK), but no dnaJ homolog could be identified in this region. The dnaK operon was transcribed as a low-abundance polycistronic mRNA whose levels did not increase upon exposure to heat shock. In contrast, a more abundant 2.3-kb mRNA encoding only the dnaK sequence was detectable, and its steady-state level increased upon heat shock. The transcription initiation sites of the dnaK and groE operons were found to be preceded by sequences that resemble an Escherichia coli sigma70 consensus promoter. Upstream of each putative promoter is an inverted repeat sequence which resembles a similar element (CIRCE [controlling inverted repeat of chaperone expression]) found upstream of the dnaK and groE operons in at least 27 eubacterial species. In vitro transcription studies utilizing partially purified C. trachomatis RNA polymerase demonstrated that the regions containing the putative promoter elements of the dnaK and groE operons are functional, although heat shock-regulated expression could not be demonstrated.

Amino Acid Sequence↗
PubMed1996

Evaluation of tuberculosis transmission in a community by 1 year of systematic typing of Mycobacterium tuberculosis clinical isolates.

Interhuman transmission of Mycobacterium tuberculosis was investigated by using molecular typing, including restriction fragment length polymorphism with probes IS6110, DR (direct repeat) and PGRS (polymorphic GC-rich sequence) and a PCR method using the inverted repeat sequences of IS6110 as primers. From 105 patients hospitalized for tuberculosis during a 1-year survey in three hospitals in Paris, France, 111 isolates were collected and analyzed. Eighty-eight patients were infected with genetically different isolates, demonstrating the clonal heterogeneity of M. tuberculosis in these patients originating from various geographical areas. Fourteen patients were infected by strains clustered with identical fingerprints. An epidemiological relatedness was demonstrated for isolates from only seven of these patients. Thus, the typing of isolates from all tuberculous patients in hospitals during 1 year allows the detection of transmission in the general community. This would improve the case findings, thereby further improving the detection of outbreaks.

Adult↗