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PubMed2004

Presence of insertion sequences (IS elements) in group B streptococci of bovine origin.

BACKGROUND & OBJECTIVES: Streptococcus agalactiae (group B streptococci, GBS) is one of the leading causative agents of human and animal infections. Recently it was demonstrated that integration of different IS elements could inactivate some of the GBS virulence properties. The presence of IS elements in human isolates has been studied while the bovine isolates were not investigated till now. The objective of the study was to perform IS analysis of a large number of bovine GBS and to use the IS elements for classification and molecular epidemiology of GBS strains. METHODS: A total of 101 GBS isolates obtained from the dairy cows were tested. These were analyzed by PCR and multiplex PCR. Southern hybridization was accomplished with the Enzo(TM) DNA Labeling and Detection Kit. The computer techniques were used for selection of the specific primers and for analysis of the sizes of PCR products. RESULTS: GBS isolates collected at three different dairy farms were studied for the presence of IS elements. Multiplex PCR was used for the fast screening. It was found that IS861 presented in 29 GBS isolates (28.7%), IS1548 in 9 (8.9%), ISSa4 in 48 (47.5%) and IS1381 in 26 isolates (25.7%). A total of 28 bovine GBS isolates (27.7%) did not possess any of the IS elements, 36 (35.6%) possessed, 35 (34.7%) possessed two and 2 (1.9%) possessed three different IS elements. The GBS with four different IS elements were not found. Taken together, 10 different variants of GBS strains were discovered. Two out of 10 variants being specific for 51 isolates (50.5%) were predominant in bovine GBS. The results of the study demonstrated that the presence of IS elements significantly varied in bovine GBS. INTERPRETATION & CONCLUSION: The present data demonstrated that variants of IS elements present in GBS genome could be used as effective criteria for molecular epidemiology. In future this approach could probably be used as an additional tool for the epidemiological control and prevention of other bacterial infections.

Animals↗
PubMed1989

[Transposition of insertional sequence IS21 in E. coli cells activated by an exogenous promotor].

Transpositional activity of the IS21-element was found to be activated during its cloning in the pUC18 vector plasmid. The activated IS-element restores ability to transposition of the deficient IS21-element present in the same cell. Simultaneously with the activated transposition the increase in frequencies of precise exclusion of IS-element is registered. The activation of IS-element is concluded to be directed by exogenous promoter.

DNA Transposable Elements↗
PubMed1994

TAP (transporter associated with antigen processing)-independent presentation of endogenously synthesized peptides is enhanced by endoplasmic reticulum insertion sequences located at the amino- but not carboxyl-terminus of the peptide.

Under most circumstances, cell surface MHC class I molecules display peptides derived from a cytosolic pool of proteins. The efficient presentation of such peptides requires the functioning of two MHC gene products [TAP1 and TAP2 (transporter-associated with Ag processing 1 and 2)] that form a complex that facilitates transmembrane movement of peptides from the cytosol to the endoplasmic reticulum, the site of peptide association with class I molecules. It has been previously shown that peptides can be presented in a TAP-independent manner in association with HLA A2.1 or H-2 Kd if they are expressed COOH-terminal to an endoplasmic reticulum insertion/signal sequence derived from the adenovirus E3/19K glycoprotein (Anderson et al., 1991. J. Exp. Med. 174: 489; Eisenlohr et al., 1992. Cell 71: 963). We show that: 1) the E3/19K signal sequence greatly enhances the presentation of each of four additional peptides tested in association with H-2 Kb or Kk, 2) the E3/19K signal sequence can be substituted by a signal sequence derived from beta-IFN, and 3) the E3/19K signal sequence does not function when located at the COOH terminus of antigenic peptides. These findings indicate that first, many peptides require TAP for efficient presentation to T cells, second, expression of peptides COOH-terminal to signal sequences is a generally applicable method of bypassing the TAP-dependence of peptide presentation and third, the leader sequence does not act to bypass TAP simply by increasing the hydrophobic nature of peptides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

PCR-based fingerprinting of Mycobacterium bovis isolates.

We investigated a number of PCR-based strategies for sub-typing isolates of Mycobacterium bovis. A panel of 15 PCR primers, designed from random sequences, insertion sequences and repetitive elements, were analyzed for their ability to allow differentiation between eight M. bovis isolates. PCR products were analyzed using agarose gel electrophoresis and polyacrylamide gel electrophoresis. Random primer based PCR, conducted with a variety of primers did not differentiate between isolates of M. bovis. Successful differentiation between isolates was achieved by the amplification of fragments located between DNA repetitive elements and insertion sequences of the M. bovis genome. PCR primers designed from the major polymorphic tandem repeat (MPTR) region and from insertion sequences IS6110 and IS986 allowed differentiation between isolates of M. bovis. This study is presented as a first step towards the development of PCR-based methods to differentiate between isolates.

Animals↗
PubMed1992

Selective detection of Mycoplasma fermentans by polymerase chain reaction and by using a nucleotide sequence within the insertion sequence-like element.

A new assay using the polymerase chain reaction to amplify a 206-nucleotide specific gene sequence within the insertion sequence-like element of Mycoplasma fermentans has been developed. The unique insertion sequence-like element exists in multiple copies in the M. fermentans genome. The assay selectively amplifies DNA from all strains of M. fermentans tested. In contrast, DNA from other species of human and nonhuman mycoplasmas, common tissue culture-contaminating mycoplasmas, and bacteria, as well as human, monkey, and mouse tissues do not produce the amplified DNA products specific for M. fermentans.

Animals↗
PubMed1990

Insertion of sequences containing the coat protein subgenomic RNA promoter and leader in front of the tobacco mosaic virus 30K ORF delays its expression and causes defective cell-to-cell movement.

The regulation of the internal open reading frame (ORF) of tobacco mosaic virus (TMV) that encodes the 30K movement protein was examined by constructing mutants in vitro with the putative coat protein subgenomic RNA promoter and leader sequences inserted upstream of the 30K ORF. A mutant with a 49-nucleotide fragment of the promoter region inserted replicated only transiently before being overtaken by a progeny wild-type virus with the insert deleted. A mutant with a 253-nucleotide promoter region fragment inserted replicated stably, and the inserted promoter was active in its new location. The production of 30K protein was not enhanced by this promoter/leader insertion to a level similar to that of coat protein. However, the accumulation of 30K protein was delayed, suggesting that different promoters/leader sequences determine the time of expression of the genes. This mutant was deficient in movement. A similar mutant, but with increased production of 30K protein, overcame the movement deficiency, suggesting that 30K protein is needed during the early stages of infection for efficient cell-to-cell movement of the virus.

Base Sequence↗
PubMed2004

Screening of isolates from faeces for carbapenem-resistant Bacteroides strains; existence of strains with novel types of resistance mechanisms.

Isolates from faecal samples (n = 224) from the UK and Hungary were screened for carbapenem-resistant Bacteroides strains and were consecutively investigated for the resistance mechanisms through detection of cfiA genes, the presence or lack of insertion sequence insertions in their upstream regions and the production of carbapenemase activities. In this way, a significant number of strains (n = 7, 3.1%) were recovered. They included 2 Bacteroides fragilis strains (one in each country) which harboured cfiA genes, but which were not activated by insertion sequence elements; this is reminiscent of some novel clinical B. fragilis strains. The cfiA-negative strains exhibited lower levels of carbapenem resistance and varying levels of carbapenemase activity, suggesting that other resistance mechanisms may also exist.

Bacterial Proteins↗
PubMed1983

Sequence analysis of 28S ribosomal DNA from the amphibian Xenopus laevis.

We have determined the complete nucleotide sequence of Xenopus laevis 28S rDNA (4110 bp). In order to locate evolutionarily conserved regions within rDNA, we compared the Xenopus 28S sequence to homologous rDNA sequences from yeast, Physarum, and E. coli. Numerous regions of sequence homology are dispersed throughout the entire length of rDNA from all four organisms. These conserved regions have a higher A + T base composition than the remainder of the rDNA. The Xenopus 28S rDNA has nine major areas of sequence inserted when compared to E. coli 23S rDNA. The total base composition of these inserts in Xenopus is 83% G + C, and is generally responsible for the high (66%) G + C content of Xenopus 28S rDNA as a whole. Although the length of the inserted sequences varies, the inserts are found in the same relative positions in yeast 26S, Physarum 26S, and Xenopus 28S rDNAs. In one insert there are 25 bases completely conserved between the various eukaryotes, suggesting that this area is important for eukaryotic ribosomes. The other inserts differ in sequence between species and may or may not play a functional role.

Animals↗
PubMed1994

Immunoreactivity of chimeric proteins carrying the HIV-1 epitope IGPGRAF. Correlation between predicted conformation and antigenicity.

Sera from HIV-1 infected individuals were examined for their reactivity to the principal neutralizing domain, IGPGRAF sequence, of the V3-loop of HIV-1. Four hybrid proteins carrying this sequence inserted in four different outer loops of a protein that makes up the capsid of an insect virus were used as antigen in a Western blot assay for this survey. All the four antigens showed different activity: sera that recognise all antigens to sera that reacted with only one of them. Competition experiments indicated that the antibodies recognised these proteins with different affinity. Molecular modelling of the hybrid proteins predicted that the inserted sequence adopted different conformations in each position. Comparison of predicted most stable conformations for IGPGRAF indicated that there is a close relationship between conformational similarity to a V3-loop reference structure and the degree of reactivity with sera.

Amino Acid Sequence↗
PubMed2006

Genetic association of blaSHV-5 with transposable elements IS26 and IS5 in Klebsiella pneumoniae from Taiwan.

A cloned 5,248-bp EcoRI fragment from the Klebsiella pneumoniae transferable plasmid pKP53 (> 70 kb) containing bla(SHV-5) was sequenced. Insertion sequences IS26 and IS5 were found downstream from bla(SHV-5). The DNA sequences of the genetic environment surrounding bla(SHV-5) were homologous to plasmid p1658/97 from Escherichia coli, containing a truncated recF gene and a truncated deoR gene upstream and downstream from bla(SHV-5), respectively. RecF may be involved in bla(SHV-5) translocation to the plasmid by RecF-dependent recombination. This novel genetic environment may be associated with the successful proliferation and/or expression of SHV-5 in K. pneumoniae strains from Taiwan.

Base Sequence↗
PubMed2000

Analysis of DYS19 and DYS287 polymorphisms in the Han population and three other ethnic groups of northeast China.

The allelic distribution of the Y-chromosome specific microsatellite DYS19 in the Han population and in the Daur, Oroqen and Ewenki ethnic groups (Northeast China) was analyzed by PCR and denatured polyacrylamide gel electrophoresis. The allelic distribution in the Han population group is as follows: A = 2.90%, B = 26.09%, C = 26.09%, D = 29.98%, E = 15.94%. This allelic distribution differs statistically significant from that observed in the three other ethnic groups (p < 0.05). Furthermore the polymorphism of the Y-chromosome specific Alu insert sequence DYS287 was tested in these four groups. However, no Alu sequence insert was found.

Alleles↗
PubMed1991

Identification and DNA sequence of a new Bacteroides fragilis insertion sequence-like element.

A new Bacteroides fragilis insertion sequence (IS)-like element has been identified, cloned, and sequenced. The element is 1598 base pairs in length. It is flanked by a 15-base pair imperfect inverted repeat and contains a large open reading frame which could encode a 430 amino acid protein. There is an 8-base pair duplication of genomic DNA sequences at the site of integration. One copy of the IS-like element is integrated within the 5' upstream sequence of the metallo-beta-lactamase gene ccrA, cloned from B. fragilis TAL3636. The IS-like element is integrated 19 bp upstream of the predicted initiation codon and, therefore, probably provides the transcriptional start signals for the CcrA gene.

Amino Acid Sequence↗
PubMed1998

Novel sequence organization and insertion specificity of IS605 and IS606: chimaeric transposable elements of Helicobacter pylori.

IS605, an insertion sequence (IS) that is unusual in containing homologs of genes for the single putative transposases of two other unrelated IS elements (IS200 and IS1341), was found in nearly one-third of a set of 238 independent isolates of the gastric pathogen Helicobacter pylori. Hybridization and PCR tests indicated that any strain carrying one of these ORFs also carried the other, which implies that both ORFs are in the same unit of transposition. The IS605 ends and target sites for insertion were identified by sequencing eight preexisting insertions in strain NCTC11638, corresponding empty sites in other strains, and new transpositions in E. coli of an IS605 derivative marked with a selectable chloramphenicol-resistance gene. These tests showed that IS605 is also unusual in: (1) having unique, not inverted repeat, ends; (2) not duplicating (or deleting) target sequences during transposition; and (3) inserting with its left (IS200-homolog) end next to 5'-TTTAA or 5'-TTTAAC. IS605 was implicated in at least two genome rearrangements in strain NCTC11638. A second member of the IS605 family, called IS606 (25% amino acid identity to IS605 in inferred proteins) was found in one-third of 38 H. pylori strains tested, many of which did not carry IS605. The features of these two chimaeric IS elements are discussed in terms of possible transposition mechanisms, IS element evolution, and effects of IS elements on genome organization and evolution in the microbes that they inhabit.

Base Sequence↗
PubMed1999

Mycoplasma orale has a sequence similar to the insertion-like sequence of M. fermentans.

Polymerase chain reaction (PCR) using the Mycoplasma fermentans insertion sequence-like element (ISLE) RW primer set amplifies DNA from Mycoplasma orale when more than 1 ng is present in the reaction tube. In this study, amplified products from 11 different clinical isolates and the ATCC prototype of M. orale were sequenced and compared to 206 bp amplicons from eight isolates and the ATCC strain of M. fermentans. The nucleotide sequences of the amplified M. orale products had high sequence homology (88-92%) to those from M. fermentans, but differed at several key positions. The M. orale products contained a DraI restriction enzyme site not found in any of the M. fermentans amplified products. Consistent with this finding, the PCR products from M. orale were digested by DraI while the PCR products from M. fermentans were resistant to DraI digestion. The results suggest that M. orale may carry a similar IS-like element that complicates but does not negate using the ISLE PCR assay designed to detect M. fermentans. It appears possible for the RW primers to amplify M. orale if the mycoplasmas are present at higher concentrations. The amplified products can be differentiated from those from M. fermentans by a rapid DraI restriction endonuclease digestion or by Southern blot analysis using the RW006 internal probe under highly stringent conditions.

Base Sequence↗
PubMed1999

Characterization of IS1541-like elements in Yersinia enterocolitica and Yersinia pseudotuberculosis.

We characterized Yersinia enterocolitica and Yersinia pseudotuberculosis insertion sequences related to insertion sequence 1541, recently identified in Yersinia pestis. For each of the two species, two insertion sequence copies were cloned and sequenced. Genetic elements from Y. pseudotuberculosis were almost identical to insertion sequence 1541, whereas these from Y. enterocolitica were less related. Phylogenetic analysis of the putative transposases encoded by insertion sequences from the three pathogenic members of the genus Yersinia showed that they clustered with those encoded by Escherichia coli and Salmonella enterica elements belonging to the insertion sequence 200/insertion sequence 605 group. Insertion sequences originating from Y. pestis and Y. pseudotuberculosis constitute a monophyletic lineage distinct from that of Y. enterocolitica.

Base Sequence↗
PubMed2003

Low free energy cost of very long loop insertions in proteins.

Long insertions into a loop of a folded host protein are expected to have destabilizing effects because of the entropic cost associated with loop closure unless the inserted sequence adopts a folded structure with amino- and carboxy-termini in close proximity. A loop entropy reduction screen based on this concept was used in an attempt to retrieve folded sequences from random sequence libraries. A library of long random sequences was inserted into a loop of the SH2 domain, displayed on the surface of M13 phage, and the inserted sequences that did not disrupt SH2 function were retrieved by panning using beads coated with a phosphotyrosine containing SH2 peptide ligand. Two sequences of a library of 2 x 10(8) sequences were isolated after multiple rounds of panning, and were found to have recovery levels similar to the wild-type SH2 domain and to be relatively intolerant to further mutation in PCR mutagenesis experiments. Surprisingly, although these inserted sequences exhibited little nonrandom structure, they do not significantly destabilize the host SH2 domain. Additional insertion variants recovered at lower levels in the panning experiments were also found to have a minimal effect on the stability and peptide-binding function of the SH2 domain. The additional level of selection present in the panning experiments is likely to involve in vivo folding and assembly, as there was a rough correlation between recovery levels in the phage-panning experiments and protein solubility. The finding that loop insertions of 60-80 amino acids have minimal effects on SH2 domain stability suggests that the free energy cost of inserting long loops may be considerably less than polymer theory estimates based on the entropic cost of loop closure, and, hence, that loop insertion may have provided an evolutionary route to multidomain protein structures.

Amino Acid Sequence↗