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PubMed2002

Transposition of cyanobacterium insertion element ISY100 in Escherichia coli.

The genome of the cyanobacterium Synechocystis sp. strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY100 in 22 copies is the most abundant. A typical ISY100 member is 947 bp long and has imperfect terminal inverted repeat sequences. It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS630, indicating that ISY100 belongs to the IS630 family. To determine whether ISY100 has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY100 with the chloramphenicol resistance gene, substituted for the transposase gene of ISY100, at another site and introduced the plasmid into an Escherichia coli strain already harboring a target plasmid. Mini-ISY100 transposed to the target plasmid in the presence of IPTG at a very high frequency. Mini-ISY100 was inserted into the TA sequence and duplicated it upon transposition, as do IS630 family elements. Moreover, the mini-ISY100-carrying plasmid produced linear molecules of mini-ISY100 with the exact 3' ends of ISY100 and 5' ends lacking two nucleotides of the ISY100 sequence. No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc1/mariner family elements, Tc1 and Tc3, which transpose to the TA sequence, have. These findings suggest that ISY100 transposes to a new site through the formation of linear molecules, such as Tc1 and Tc3, by excision. Some Tc1/mariner family elements leave a footprint with an extra sequence at the site of excision. No footprints, however, were detected in the case of ISY100, suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system. ISY100 transposes in E. coli, indicating that it transposes without any host factor other than the transposase encoded by itself. Therefore, it may be able to transpose in other biological systems.

Amino Acid Sequence↗

PubMed1982

2-micrometers DNA-like plasmids in the osmophilic haploid yeast Saccharomyces rouxii.

DNA plasmids were detected in two independent strains of Saccharomyces rouxii among 100 yeast strains other than Saccharomyces cerevisiae tested. The plasmids, pSR1 and pSR2, had almost the same mass (approximately 4 X 10(6) daltons) as 2-micrometers DNA of S. cerevisiae. pSR1 and pSR2 gave identical restriction maps with restriction endonucleases BamHI, EcoRI, HincII, HindIII, and XhoI, and both lacked restriction sites for PstI, SalI, and SmaI. These maps, however, differed significantly from that of S. cerevisiae 2-micrometers DNA. Restriction analysis also revealed two isomeric forms of each plasmid and suggested the presence of a pair of inverted repeat sequences in the molecules where intramolecular recombination took place. DNA-DNA hybridization between the pSR1 and pSR2 DNAs indicated significant homology between their base sequences, whereas no homology was detected between pSR1 and pJDB219, a chimeric plasmid constructed from a whole molecule of 2-micrometers DNA, plasmid pMB9, and a 1.2-kilobase DNA fragment of S. cerevisiae bearing the LEU2 gene. A chimeric plasmid constructed with pSR1 and YIp1, the larger EcoRI-SalI fragment of pBR322 ligated with a 6.1-kilobase DNA fragment of S. cerevisiae bearing the HIS3 gene, could replicate autonomously in an S. cerevisiae host and produced isomers, presumably by intramolecular recombination at the inverted repeats.

Base Sequence↗

PubMed1987

Modulation of Bacillus subtilis alpha-amylase promoter activity by the presence of a palindromic sequence in front of the gene.

Upstream of the promoter of the Bacillus subtilis alpha-amylase gene (amyE) derived from an alpha-amylase hyper-producing strain, there is an inverted repeat sequence (palindromic sequence), which has a free energy of 21.2 kcal/mol due to the formation of stable stem-loop structure. The role of the palindromic sequence for the expression of amyE was studied using a plasmid encoding the amyE'-'bla (E. coli beta-lactamase) fused gene in an alpha-amylase-deficient B. subtilis mutant as the host. By the presence of the palindromic sequence, the transcription activity of the amyE promoter was enhanced approximately 6 fold by starch (3%) in the medium and was less repressed by glucose.

Bacillus subtilis↗

PubMed1993

Identification of a new transcriptional unit that yields a gene product within the unique sequences of the short component of the herpes simplex virus 1 genome.

The herpes simplex virus genome 1 consists of two unique stretches, long (UL) and short (U(S)), each flanked by inverted repeat sequences. The U(S) sequence has been previously reported to contain 12 open reading frames designated U(S)1 through U(S)12. This report demonstrates the existence of a 13th open reading frame within the U(S) sequence, designated U(S)8.5. The U(S)8.5 sequence is located between, and overlaps in part with, the domains of the U(S)8 and U(S)9 genes. Its transcription is initiated within the coding sequence of U(S)8, and its transcript decays earlier than that of U(S)8. On the basis of the size of its RNA (1.2 kb) and map position, it is likely that the U(S)8.5 transcript is 3' coterminal with the U(S)8 and U(S)9 mRNAs at the single polyadenylation signal which serves these genes. The nucleotide sequence of the U(S)8.5 open reading frame predicts that its product is a 151-amino-acid basic, hydrophilic polypeptide. To determine whether the U(S)8.5 encodes a protein, a sequence encoding 23 amino acids that contains an epitope reacting with a known monoclonal antibody to human cytomegalovirus protein was inserted in frame after the predicted fifth codon of the U(S)8.5 gene. The recombinant virus carrying this epitope induced the synthesis of a protein reactive with the monoclonal antibody in immunoblots. The tagged protein localized in nucleoli of cells infected with the recombinant virus.

Amino Acid Sequence↗

PubMed1982

Molecular cloning of the white locus region of Drosophila melanogaster using a large transposable element.

We report the molecular cloning of a chromosome segment including the white locus of Drosophila melanogaster. This region was isolated using a deficiency extending from the previously cloned heat-shock puff sequences at 87A7 to a large transposable element containing the loci white and roughest.FB-NOF, a 7.5 kb element with partial homology to a family of inverted repeat sequences (Potter et al., 1980), is found very near the deficiency breakpoint, and is followed by DNA originating from the white locus region. Sequences totalling 60 kb surrounding this initial entry point were obtained by the cloning of successively overlapping fragments from a wild-type strain. Several rearrangement breakpoints have been mapped relative to the cloned DNA; these define the limits of the white locus and further differentiate the "white proximal region", thought to function in gene regulation, from the remainder of the locus. Insertion of the dispersed repetitive element copia into the white locus is observed in strains carrying the white-apricot allele. Analysis of several white-apricot revertants suggests that copia insertion is responsible for the apricot eye color phenotype.

Journal Article↗

PubMed1990

Identification and characterization of a defective SSV1 genome integrated into a tRNA gene in the archaebacterium Sulfolobus sp. B12.

Within the chromosome of the archaebacterium Sulfolobus sp. B12, a 7.4 kb region was identified which displayed extensive sequence similarities to the 15.5 kb genetic element SSV1 carried by the same strain both as a circular form and as a site-specifically integrated copy. DNA sequence analysis indicated that this 7.4 kb region (designated SSV1intB) represented an SSV1-like element distinguishable from the full-length integrated copy (designated SSV1intA) by extensive deletions and point mutations. The physical organization of DNA sequences of SSV1intB indicated that this element was integrated at the same attP site as previously identified for SSV1intA. A comparison of the DNA sequences at the left attachment sites of SSV1intA and SSV1intB revealed that they both represented very similar putative arginine tRNA genes followed by a 10 bp inverted repeat sequence. S1 nuclease mapping experiments indicated that these tRNA genes are transcribed.

Archaea↗

PubMed1997

Pyroglutamic acid and iron regulate the expression of the pcp gene in Pseudomonas fluorescens MFO.

Pyrrolidone carboxyl peptidase (Pcp) is an aminopeptidase (EC 3.4.11.8) able to specifically remove the L-pyroglutamyl residue from the amino-terminus of polypeptides. Since nothing was known concerning the regulation and function of Pcps, a mutant of a milk-isolated strain lacking Pcp activity (Pseudomonas fluorescens MB1), was constructed by homologous recombination using a transcriptional fusion between pcp and a reporter gene (uidA). The wild-type and mutant strains were grown in synthetic media and in milk to investigate the environmental effects on pcp transcription. The expression of pcp and of the transcriptional fusion pcp::uidA was not sensitive to environmental conditions like temperature, osmolarity or nitrogen and phosphate starvation but was induced by the product of the enzymatic activity, pyroglutamic acid (pGlu). The expression of the native gene and the fusion in inducing conditions was also controlled by the iron concentration. The identification in the pcp promoter sequence of putative ferric uptake regulator (Fur) binding sites suggests a transcriptional regulation in a Fur-dependent fashion. Two other putative regulatory stretches, corresponding to inverted repeated sequences with perfect and imperfect symmetry, were also identified. pGlu and iron are therefore at least two of the transcriptional effectors of pcp expression.

Amino Acid Sequence↗

PubMed1985

Complete nucleotide sequence of the E. coli N-acetylneuraminate lyase.

The nucleotide sequence of the cloned DNA, 1,243 bp in length coding for N-acetylneuraminate lyase (N-acetylneuraminate pyruvate lyase; NPL) of Escherichia coli has been determined. Nucleotide sequence and amino acid analysis have assigned the open reading frame for NPL, starting with the ATG near its 5'terminus. The molecular weight calculated from the predicted amino acid sequence was 32,640 daltons, being in good agreement with that of a NPL subunit estimated by the SDS-PAGE method and amino acid composition. Several signal sequences conserved in the promoter regions of E. coli were found in the npl gene. They were the Shine-Dalgarno sequence, the Pribnow box and the sequence coserved in the "-35 region" and they were separated to each other with preferable spacing for an efficient transcription. Downstream from the termination codon, the inverted repeat sequence was present, followed by 4 successive T's.

Amino Acid Sequence↗

PubMed1991

Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point.

From examination of published DNA sequences of genes found inserted at a specific site in integrons, all genes are shown to be associated, at their 3' ends, with a short imperfect inverted repeat sequence, a 59-base element or relative of this element. The similarity of the arrangement of gene inserts in the integron and in the Tn7 transposon family is described. A refined consensus for the 59-base element is reported. Members of this family are highly diverged and the relationship of a group of longer elements to the 59-base elements is demonstrated. The ability of 59-base elements of different length and sequence to act as sites for recombination catalysed by the integron-encoded DNA integrase is demonstrated, confirming that elements of this family have a common function. The ability of elements located between gene pairs to act as recombination sites has also been demonstrated. The recombination cross-over point has been localized to the GTT triplet which is conserved in the core sites, GTTRRRY, found at the 3' end of 59-base elements. Recombination at the core site found in inverse orientation at the 5' end of the 59-base elements was not detected, and the sequences responsible for orientation of the recombination event appear to reside within the 59-base element. A model for site-specific insertion of genes into integrons and Tn7-like transposons is proposed. Circular units consisting of a gene associated with a 59-base element are inserted into an ancestral element which contains neither a gene nor a 59-base element.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1993

Identification and characterization of IS1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family.

Insertion sequence (IS) elements are mobile genetic elements found in prokaryotes. We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements. By subcloning a single strain of M. pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event. The nucleotide sequences of the element, designated IS1138, and the target site into which it inserted were determined. IS1138 consists of 1288 bp with 18 bp perfect terminal inverted repeats. Sequence analysis of the target site before and after insertion of IS1138 identified a 3 bp duplication of target DNA flanking the element. The predicted amino acids encoded by the major open reading frame of IS1138 share significant similarity with the transposases of the IS3 family. Southern hybridization analysis indicates that repetitive sequences similar to IS1138 are present in most, if not all, strains of M. pulmonis, but IS1138-like sequences were not detected in other mycoplasmal species.

Amino Acid Sequence↗

PubMed2005

Characterization of a carbapenemase-producing clinical isolate of Bacteroides fragilis in Scandinavia: genetic analysis of a unique insertion sequence.

In 2003 a Bacteroides fragilis blood culture isolate (K2-28) was recovered from a 61-y-old male with severe general atherosclerosis during treatment with meropenem. K2-28 was shown to possess a functional metallo-beta-lactamase with a reduction in imipenem MIC from 256 to 3 mg/l in the presence of EDTA using the MBL E-test strip. PCR results were for positive for the cfiA gene. Analysis of the cfiA from K2-28 revealed it was 100% identical to previously described cfiA-1 genes. Analysis of the upstream region of cfiA revealed a novel insertion sequence (IS) element, being most similar (94% identity) to IS612 recently described from Japan designating the element within the IS4 family. The element possessed a perfect terminal inverted repeat sequence at the distal ends of the IS element and provided a putative promoter for transcription of the cfiA gene. The distance between the hybrid promoter and the cfiA start codon was 158 base pairs and inserted into a different DNA sequence upstream of cfiA to that previously reported. The -10 promoter region was most similar to that of IS613 (100%) and the -35 promoter region to IS612 (100%), demonstrating the plasticity of these genetic regions.

Bacterial Proteins↗

PubMed1981

Kinetic Complexity, Homogeneity, and Copy Number of Chloroplast DNA from the Marine Alga Olisthodiscus luteus.

The kinetic complexity of chloroplast DNA isolated from the chromophytic alga Olisthodiscus luteus has been determined. Using optical reassociation techniques, it was shown that the plastid DNA of this alga reacted as a single component with a second order rate constant of 4.1 molar(-1) and second(-1) (Cot((1/2)) 0.24 molar second) under conditions equivalent to 180 millimolar Na(+) and 60 degrees C. Given the 92 x 10(5) dalton complexity calculated for this chloroplast genome, an Olisthodiscus cell contains 650 plastome copies. Although this complement remains constant throughout the growth cycle of the organism, the ploidy level of an individual chloroplast shows significant plasticity and is dependent upon the number of chloroplasts present per cell. Experiments with the DNA fluorochrome Hoechst dye 33258 (bisbenzimide) demonstrate that plastids isolated from all phases of cell growth each possess a ring-shaped nucleoid containing detectable DNA. Olisthodiscus chloroplast DNA showed no sequence mismatch when thermal denaturation profiles of reassociated chloroplast DNA were examined, thus all plastome copies are essentially identical. Finally, reassociation studies demonstrated that no foldback (short inverted repeat) sequences were present in the plastid genome although significant hairpin loop structures were observed in control nuclear DNA samples.

Journal Article↗

PubMed1995

Molecular analysis of OmpR binding sequences involved in the regulation of ompF in Escherichia coli.

OmpR, the transcriptional regulatory protein of ompF, had not been previously shown to specifically bind to the -70 to -60-bp region of ompF. We show that the -102 to -76-bp sequence of ompF has a high affinity binding site for OmpR and produced a single OmpR/ompF complex (complex b). Extension of this DNA fragment to include an inverted repeat sequence located between the -71 and -64-bp region resulted in the formation of a second, slower migrating complex (complex a). A -102 to -58-bp fragment containing a substitution of the -70 CG bp was able to form complex b, but not complex a. A mutant OmpR protein derived from a strain that can not repress ompF was unable to form complex a, while complex b was formed normally. Deletion of the -70 CG bp resulted in incomplete repression of OmpF. These results suggest that OmpR binds to the -71 to -64-bp region and that this sequence plays a role in the regulation of ompF in Escherichia coli.

Bacterial Outer Membrane Proteins↗

PubMed1996

Characterization of IS1272, an insertion sequence-like element from Staphylococcus haemolyticus.

We have previously shown (G. L. Archer, D. M. Niemeyer, J. A. Thanassi, and M. J. Pucci, Antimicrob. Agents Chemother. 38:447-454, 1994) that some methicillin-resistant staphylococcal isolates contain a partial deletion of the genes (mecR1 and mecI) that regulate the transcription of the methicillin resistance structural gene (mecA). When a fragment of DNA inserted at the point of the mecR1 deletion was used as a probe, hybridization with multiple bands was detected for Staphylococcus haemolyticus genomic DNA. In the present study, DNA sequencing of four unique clones recovered from a lambda library of S. haemolyticus revealed identical 1,934-bp elements. Each element, designated IS1272, contained 16-bp terminal inverted repeats (sequence identity, 15 of 16 bp) and two open reading frames of 819 and 687 bp; there were no flanking target site duplications. Database searches yielded amino acid homology with proteins predicted to be encoded by open reading frames from a putative insertion sequence element from Enterococcus hirae. DNA probes from each end and the middle of IS1272 were hybridized with restriction endonuclease-digested genomic DNA from clinical S. haemolyticus, Staphylococcus epidermidis, and Staphylococcus aureus isolates. Each of the 20 or more copies of the element found in S. haemolyticus isolates was intact, and copies were found in most chromosomal SmaI fragments. S. aureus and S. epidermidis isolates contained mostly incomplete fragments of the element, and there were many more hybridizing fragments in methicillin-resistant than in methicillin-susceptible isolates. IS1272, which appears to be primarily resident in S. haemolyticus, has disseminated to multiple staphylococcal species and is prevalent in multiresistant isolates.

Amino Acid Sequence↗

PubMed1994

The UAS of the yeast GAPDH promoter consists of multiple general functional elements including RAP1 and GRF2 binding sites.

The upstream activating sequence (UAS) of TDH3, one of three genes encoding glyceraldehyde phosphate dehydrogenase in Saccharomyces cerevisiae, was characterized by using a series of external and internal deletion mutants of the TDH3 upstream region. The levels of activation by these deletions of transcription mediated through either the segment of TDH3 promoter or the segment of ADH1 (alcohol dehydrogenase 1 gene) promoter were quantitatively examined and the region between -583 and -447 was found to be required for full transcriptional activation with either promoter segment. It has been demonstrated that the protein binding site involved in the formation of two DNA-protein complexes is identical with the consensus RAP1 binding sequence by methylation interference assay. Surprisingly, the UAS fragment composed of the 22-mer sequence containing exclusively a RAP1 binding sequence showed full activation, suggesting that the RAP1-dependent transcriptional activation is a primary positive control in the TDH3 gene expression. In addition, a pair of inverted repeat sequences homologous to the binding sequence for GRF2, another yeast trans-acting factor, and directly repeated sequences containing a CATCC motif were also found upstream and downstream, respectively, of the RAP1 binding site. Deletion analysis suggested that these elements could also function as regulatory elements for transcription.

Base Sequence↗

PubMed1997

Cloning of a gene encoding nisin resistance from Lactococcus lactis subsp. lactis M189 which is transcribed from an extended 210 promoter.

A 56-kb plasmid was identified in Lactococcus lactis subsp. lactis (L. lactis) M189 which encodes resistance to nisin (Nis(R)) following mobilization of the plasmid into L. lactis LM0230. The Nis(R) determinant was localized on a 1.6-kb HindIII fragment by DNA restriction fragment deletion and subcloning. An open reading frame (ORF) of 957 bases was identified by sequence analysis and its transcription start site was mapped by primer extension. The ORF is flanked by two regions which exhibit complete homology to parts of the inverted repeat sequences of IS981 and ISS1T. The promoter for transcription was found to consist of an extended -10 site (TgTGtTATAAT) that lacks a -35 site. Function of the extended -10 promoter was demonstrated by its ability to express the promoterless cat gene from Staphylococcus aureus. Base substitution analysis revealed that the TgTGt extension is essential for promoter efficiency in L. lactis.

Journal Article↗

PubMed1991

Synthetic gene design to investigate the role of cis-acting DNA structural elements in regulation of gene expression in vivo.

To delineate the DNA structural elements responsible for transcriptional control in vivo, we have developed a novel approach taking advantage of the degeneracy of the genetic code. Using synthetic oligonucleotides as structural cassettes we have been able to replace, within a gene, segments of DNA coding for the same amino acid sequence but capable of adopting unusual DNA structures and monitor the effect of such structural elements on gene expression in vivo. We find that the presence of an inverted repeat sequence, with a potential to adopt cruciform structure, within the beta-galactosidase gene down regulates its expression in vivo.

Amino Acid Sequence↗

PubMed1996

The same molecular mechanism at the maternal meiosis I produces mono- and dicentric 8p duplications.

We studied 16 cases of 8p duplications, with a karyotype 46,XX or XY,dup(8p), associated with mental retardation, facial dysmorphisms, and brain defects. We demonstrate that these 8p rearrangements can be either dicentric (6 cases) with the second centromere at the tip of the short arm or monocentric (10 cases). The distal 8p23 region, from D8S349 to the telomere, including the defensin 1 locus, is deleted in all the cases. The region spanning from D8S252 to D8S265, at the proximal 8p23 region, is present in single copy, and the remaining part of the abnormal 8 short arm is duplicated in the dicentric cases and partially duplicated in the monocentric ones. The distal edge of the duplication always spans up to D8S552 (8p23.1), while its proximal edge includes the centromere in the dicentric cases and varies from case to case in the monocentric ones. The analysis of DNA polymorphisms indicates that the rearrangement is consistently of maternal origin. In the deleted region, only paternal alleles were present in the patient. In the duplicated region, besides one paternal allele, some loci showed two different maternal alleles, while others, which were duplicated by FISH analysis, showed only one maternal allele. We hypothesize that, at maternal meiosis I, there was abnormal pairing of chromosomes 8 followed by anomalous crossover at the regions delimited by D8S552 and D8S35 and by D8S252 and D8S349, which presumably contain inverted repeated sequences. The resulting dicentric chromosome, 8qter-8p23.1(D8S552)::8p23.1-(D8S35)-8q ter, due to the presence of two centromeres, breaks at anaphase I, generating an inverted duplicated 8p, dicentric if the breakage occurs at the centromere or monocentric if it occurs between centromeres.

Abnormalities, Multiple↗