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PubMedDate not supplied

[An evaluation of the identification technics with IS6110 and Mt308 used as molecular markers for Mycobacterium tuberculosis strains isolated in Romania].

During the past years, new diagnostic technologies based on DNA methods including PCR, were developed in the aid of tuberculosis control. Among several DNA sequences, an insertion sequence IS6110, found to be specific to mycobacteria belonging to M. tuberculosis complex were used. However, recent studies showed the absence of this IS6110 in 3-4% of M. tuberculosis strains isolated from patients of Vietnamese origin. In order to evaluate the frequency of IS6110 among a series of strains isolated in Romania, we have performed the PCR technique for amplification of 2 DNA specific regions of M. tuberculosis complex. The sequences of primers were as shown before (Guesdon et al., 1990). For this study, 219 strains have been isolated from tuberculous patients, in "Marius Nasta" Institute of Bucharest, Romania. We have shown that genomic DNA from 3.6% of 219 strains evaluated in our study do not contain the IS6110 sequence and could determine some identification problems when this molecular sequence is targeted.

DNA, Bacterial↗

PubMed1994

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia.

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after gamma-ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 bp insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 bp terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5' and 3' ends of dTnp1 together with a perfect palindrome located after the 5' inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.

Amino Acid Sequence↗

PubMed1988

Cloning and transposon insertion mutagenesis of virulence genes of the 100-kilobase plasmid of Salmonella typhimurium.

We have cloned regions of the 100-kilobase (kb) plasmid, pStSR100, of Salmonella typhimurium SR-11 that confer virulence to plasmid-cured S. typhimurium. Cells carrying recombinant plasmids that conferred virulence were selected by inoculating mice orally with recombinant libraries in virulence plasmid-cured S. typhimurium and harvesting isolates that infected spleens. Three plasmids, pYA401, pYA402, and pYA403, constructed with the cosmid vector pCVD305 conferred wild-type levels of virulence to plasmid-cured S. typhimurium and had a common 14-kb DNA insert sequence. Another recombinant plasmid, pYA422, constructed with the vector pACYC184, conferred to plasmid-cured S. typhimurium a wild-type 50% lethal dose (LD50) level, but mice died more slowly than when infected with wild-type S. typhimurium. Furthermore, pYA422 conferred the ability to cause a higher, but not a wild-type, level of splenic infection on plasmid-cured S. typhimurium. pYA422 had a 3.2-kb insert sequence which mapped to the center of the 14-kb common sequence of the cosmid clones. Transposon Tn5 insertion mutations in pYA403 inhibited virulence to various degrees, and when transduced into the native virulence plasmid of S. typhimurium, these Tn5 insertions decreased virulence to degrees similar to those observed when the Tn5 insertions were present in pYA403. vir-22::Tn5 in pStSR100 greatly lowered infection of spleens relative to unmutagenized virulence plasmid, while vir-26::Tn5 and vir-27::Tn5 lowered splenic infection to lesser degrees. At least three proteins were encoded by pYA403 containing 23 kb of insert sequence and subclone pYA420, containing the 14-kb common insert sequence present in all of the cosmid clones. One of these proteins, with an apparent molecular weight of 28,000, was also encoded by pYA422. The Tn5 insertion that most attenuated virulence, vir-22::Tn5, inhibited synthesis of the 28,000-molecular-weight protein. The vir-22::Tn5 insertion was complemented by recombinant plasmids encoding only the 28,000-molecular-weight protein, suggesting a role of this protein in virulence. However, recombinant plasmids, exemplified by pYA422, that encoded only the 28,000-molecular-weight protein did not confer full virulence.

Animals↗

PubMed1995

Sequence and distribution of IS1312: evidence for horizontal DNA transfer from Rhizobium meliloti to Agrobacterium tumefaciens.

Two novel insertion sequences, IS1312 and IS1313, were found in pTiBo542, the Ti plasmid of Agrobacterium tumefaciens strains Bo542 and A281. Nucleotide sequencing and Southern hybridization revealed that IS1312 and IS1313 are homologous to Rhizobium meliloti ISRm1 and ISRm2, respectively. IS1312, ISRm1, and another Agrobacterium insertion sequence, IS426, belong to the same IS3 family of insertion sequences; however, IS1312 is more closely related to the Rhizobium ISRm1 than it is to the Agrobacterium IS426. The distribution patterns of these insertion elements and their sequence similarities suggest that IS1312 and IS1313 were horizontally transferred from R. meliloti to A. tumefaciens.

Agrobacterium tumefaciens↗

PubMed1989

Nascent secretory chain binding and translocation are distinct processes: differentiation by chemical alkylation.

We have investigated the effects of chemical alkylation of microsomal membranes on nascent chain binding and translocation. Assays were conducted using either full-length or truncated preprolactin transcripts in combination with a reconstituted membrane system consisting of proteolyzed rough microsomes and the cytoplasmic domain of the signal recognition particle receptor. Treatment of rough microsomes with N-ethylmaleimide was observed to inhibit preprolactin processing at a site other than the signal recognition particle or the signal recognition particle receptor. As formation of a translocation competent junction between the ribosome/nascent chain complex and the membrane has recently been demonstrated to require GTP (Connolly, T., and R. Gilmore. J. Cell Biol. 1986. 103:2253-2261), the effects of membrane alkylation on this parameter were assessed. N-ethylmaleimide treatment did not inhibit nascent chain targeting or GTP-dependent signal sequence insertion. Translocation of the targeted and inserted nascent chain was, however, blocked. These data indicate (a) that the process of nascent chain translocation is distinct from targeting and signal sequence insertion, and (b) translocation of the peptide chain across the membrane is mediated by an N-ethylmaleimide-sensitive membrane protein component(s). To further substantiate the observation that nascent chain targeting and signal sequence insertion can be distinguished from translocation, the temperature dependencies of the two phenomena were compared. Signal sequence insertion occurred at low temperatures (4 degrees C) and was maximal between 10 and 15 degrees C. Translocation was only observed at higher temperatures and was maximal between 25 and 30 degrees C.

Alkylation↗

PubMed1990

Nucleotide-sequence of insertion element IS15 delta IV from plasmid pBP11.

The nucleotide sequence of an insertion element in R-factor R1767 derivative pBP11 was determined. It is almost overall identical with IS15 delta, IS26 and IS46. Like IS46 it flanks one end of the sul-bla determinant and is involved in amplification of the resistance cassette. The significance for this process of a palindrome comprising part of IS15 delta IV is discussed.

Base Sequence↗

PubMed2005

Characterization of IS1501 mutants of Leptospira interrogans serovar pomona.

Leptospira interrogans is a diverse species in which individual serovars have distinctive restriction fragment length polymorphisms that are useful in strain identification. Many of these polymorphisms can be detected using hybridization probes derived from insertion sequences; an observation that suggests these IS elements are active and can transpose in L. interrogans. Two spontaneous mutants of L. interrogans serovar Pomona strain RZ11 were isolated by immune selection and characterized. Changes in the size and antigenicity of LPS from these mutants were detected. Genetic analysis showed that both mutants have additional copies of an IS3-like element, designated IS1501, that are not present in the parental strain. One mutant, GT211, has a single additional copy of IS1501, whereas the other mutant, GT210 has three additional copies of IS1501 relative to strain RZ11. IS1501 transposition generated 3-bp direct repeats from target sequences flanking the insertion site. RT-PCR analysis of transcripts at altered loci showed IS1501 transcripts extended into adjacent sequences. These data are the first to show spontaneous transposition of an endogenous Leptospira insertion sequence, and suggest that IS1501 may be capable of gene activation.

Antigens, Bacterial↗

PubMed1999

Spontaneous tandem amplification and deletion of the shiga toxin operon in Shigella dysenteriae 1.

Only one species of Shigella, Shigella dysenteriae 1, has been demonstrated to produce Shiga toxin (Stx). Stx is closely related to the toxins produced by Shiga toxin-producing Escherichia coli (STEC). In STEC, these toxins are often encoded on lambdoid bacteriophages and are major virulence factors for these organisms. Although the bacteriophage-encoded stx genes of STEC are highly mobile, the stx genes in S. dysenteriae 1 have been believed to be chromosomally encoded and not transmissible. We have located the toxin genes of S. dysenteriae 1 to a region homologous to minute 30 of the E. coli chromosome, within a 22.4 kbp putative composite transposon bracketed by IS600 insertion sequences. This region is present in all the S. dysenteriae 1 strains examined. Tandem amplification occurs via the flanking insertion sequences, leading to increased toxin production. The global regulatory gene, fnr, is located within the stx region, allowing deletions of the toxin genes to be created by anaerobic growth on chlorate-containing medium. Deletions occur by recombination between the flanking IS600 elements. Lambdoid bacteriophage genes are found both upstream and within the region, and we demonstrate the lysogeny of Shigella species with STEC bacteriophages. These observations suggest that S. dysenteriae 1 originally carried a Stx-encoding lambdoid prophage, which became defective due to loss of bacteriophage sequences after IS element insertions and rearrangements. These insertion sequences have subsequently allowed the amplification and deletion of the stx region.

Bacterial Toxins↗

PubMed1995

Iron transport genes of the pJM1-mediated iron uptake system of Vibrio anguillarum are included in a transposonlike structure.

The pJM1 genes encoding the proteins involved in iron transport in the anguibactin iron uptake system were found to be flanked by insertion sequences in a composite transposonlike structure. These Vibrio anguillarum insertion sequences, ISV-A1 and ISV-A2, are related to IS903, IS102, and the ISVs found in Vibrio parahaemoliticus, Vibrio mimicus, and non-O1 Vibrio cholerae flanking various tdh (thermostable direct hemolysin) genes. The inverted repeats at the ends of ISV-A1 and ISV-A2 have no more than three mismatches when compared to the inverted repeats of the other ISVs or IS903 and IS102. ISV-A1 and ISV-A2 are flanked by 9-bp direct repeats, which is the number of bases that are duplicated upon IS903 or IS102 transposition. The similarities found between the V. anguillarum ISVs and the other ISVs as well as IS903 and IS102 suggest that they derive from a common ancestral insertion sequence. At the end of ISV-A1 there is a -35 sequence region followed by a -10 sequence found in the pJM1 sequence immediately outside the ISV. This promoter region is followed by an open reading frame with the potential to encode a polypeptide of 26,985 Da whose function is still unknown. The functionality of this promoter has been demonstrated and expression analysis showed that the promoter is regulated by the iron concentration of the media.

Amino Acid Sequence↗

PubMed1998

IS195, an insertion sequence-like element associated with protease genes in Porphyromonas gingivalis.

Porphyromonas gingivalis is recognized as an important etiologic agent in adult and early-onset periodontal disease. Proteases produced by this organism contribute to its virulence in mice. Protease-encoding genes have been shown to contain multiple copies of repeated nucleotide sequences. These conserved sequences have also been found in hemagglutinin genes. In the process of studying the genetic loci containing the conserved repeated sequences, we have characterized a prtP gene homolog from P. gingivalis W83 encoding a cysteine protease with Lys-X specificity. However, this prtP gene was interrupted by an insertion sequence-like element which we designated IS195. Furthermore, IS195 and another element, IS1126, were present downstream of prtP gene homologs (kgp) found in P. gingivalis H66 and 381. IS195, a 1,068-bp insertion sequence-like element, contained 11-bp inverted repeats at its termini and was bordered by 9-bp direct repeats presumed to be a transposition-mediated target site duplication. Its central region contained one large open reading frame encoding a predicted 300-amino-acid protein which appeared to be a transposase. We isolated two naturally occurring variants of P. gingivalis W83, one carrying IS195 within the coding region of the prtP gene and another containing an intact prtP gene. Biochemical characterization revealed a lack of trypsin-like Lys-X specific proteolytic activity in the P. gingivalis W83 variant carrying the disrupted prtP gene. Studies using a mouse model revealed a reduction of virulence resulting from insertion of IS195 into the coding region of the prtP gene. An allelic-exchange mutant defective in the prtP gene also was constructed and tested in vivo. It displayed intermediate virulence compared to that of the wild-type and prtP::IS195 mutant strains. We conclude that the Lys-X cysteine protease contributes to virulence in soft tissue infections.

Amino Acid Sequence↗

PubMed2004

Construction of an Enterococcus faecalis Tn917-mediated-gene-disruption library offers insight into Tn917 insertion patterns.

Sequencing the insertion sites of 8,865 Tn917 insertions in Enterococcus faecalis strain OG1RF identified a hot spot in the replication terminus region corresponding to 6% of the genome where 65% of the transposons had inserted. In E. faecalis, Tn917 preferentially inserted at a 29-bp consensus sequence centered on TATAA, a 5-bp sequence that is duplicated during insertion. The regional insertion site preference at the chromosome terminus was not observed in another low-G+C gram-positive bacterium, Listeria monocytogenes, although the consensus insertion sequence was the same. The 8,865 Tn917 insertion sites sequenced in E. faecalis corresponded to only approximately 610 different open reading frames, far fewer than the predicted number of 2,400, assuming random insertion. There was no significant preference in orientation of the Tn917 insertions with either transcription or replication. Even though OG1RF has a smaller genome than strain V583 (2.8 Mb versus 3.2 Mb), the only E. faecalis strain whose sequence is in the public domain, over 10% of the Tn917 insertions appear to be in a OG1RF-specific sequence, suggesting that there are significant genomic differences among E. faecalis strains.

Bacterial Proteins↗

PubMed1996

Heminested inverse PCR for IS6110 fingerprinting of Mycobacterium tuberculosis strains.

A heminested inverse PCR (HIP) for the amplification of sequences flanking the Mycobacterium tuberculosis insertion sequence IS6110 has been developed. The method depends upon primers that anneal to IS6110 at sites between its 5' end and the closest BsrFI site. The accuracy of HIP was demonstrated by the amplification of sequences within plasmid constructs carrying one or two copies of the insertion sequence IS986 in different orientations. The identities of the amplicons produced from strains carrying a single copy of IS6110 were verified by nucleotide sequencing. Analyses of 204 M. tuberculosis strains including those involved in outbreaks showed that IS6110 HIP is highly discriminatory and reproducible. HIP fingerprinting of these 204 strains generated 136 distinct types, and its discriminatory power was equivalent to that of standard restriction fragment length polymorphism analysis. The method is therefore of value for the rapid fingerprinting of M. tuberculosis strains for epidemiological purposes.

Base Sequence↗

PubMed1988

Leukemogenicity of Moloney murine leukemia viruses carrying polyoma enhancer sequences in the long terminal repeat is dependent on the nature of the inserted polyoma sequences.

The leukomogenicity of Moloney murine leukemia virus (M-MuLV) variants with chimeric long terminal repeats (LTRs) containing sequences from polyomavirus was studied. We previously showed that insertion of the B enhancer element from the PyF101 variant into the M-MuLV LTR between the M-MuLV enhancers and promoter abolished leukemogenicity. PyF101 differs from wild-type polyoma in that it can productively infect undifferentiated F9 embryonal carcinoma cells; this is due to alterations in the B enhancer element. Two additional chimeric M-MuLVs were generated that contained the B enhancers from wild-type polyoma and also from a second host range variant (PyF441), which differs from wild-type polyoma by only a single base change. In contrast to Mo+PyF101 M-MuLV, both Mo+Pywt and Mo+-PyF441 M-MuLV induced T-lymphoid leukemia in neonatal NIH Swiss mice with the same time course as wild-type M-MuLV. Thus the lack of leukemogenicity of Mo+PyF101 M-MuLV was related to the exact nature of the PyF101 B enhancers. While both Mo+Pywt and Mo+PyF441 M-MuLVs induced leukemia, they showed differences when the resulting tumors were examined. First, approximately one-third of the tumors induced by Mo+Pywt M-MuLV contained proviruses which lacked polyoma sequences, while all of the tumors induced by Mo+PyF441 M-MuLV contained proviruses with the chimeric LTR. Second, a majority of tumors induced by Mo+Pywt M-MuLV (and also wild-type, M-MuLV) showed proviral integrations near one or more of the cellular c-myc, pim-1, or pvt-1 loci. In contrast, tumors induced by Mo+PyF441 M-MuLV showed infrequent integrations at these loci.

Animals↗

PubMed1985

Mapping of DNase I-hypersensitive sites in the 5' and 3' long terminal repeats of integrated moloney murine leukemia virus proviral DNA.

The chromatin state of integrated Moloney murine leukemia virus (M-MuLV) proviral DNA was investigated. Nuclei from M-MuLV-infected mouse NIH 3T3 cells were digested with limited amounts of DNase I, and hypersensitive (HS) sites were mapped by the indirect end labeling technique. Particular emphasis was placed on the 5' long terminal repeat (LTR), since viral transcription initiates there. M-MuLV proviral DNA showed two strong DNase I-HS sites in the 5' LTR, one coincident with the transcription initiation (cap) site and the other with the transcriptional enhancers. Two weaker DNase I-HS sites were also detected in internal proviral DNA. The 3' LTR also showed a strong HS site in the region of the enhancers, but an HS site at the cap site of the 3' LTR was not detected. Thus, the chromatin configurations of the 5' and 3' LTRs of integrated M-MuLV proviruses appear to be different. The chromatin configuration of M-MuLV proviruses which contain LTR insertions of polyomavirus enhancer sequences was also studied. The 5' LTR of M-MuLV proviruses containing polyoma enhancer sequences substituted for the M-MuLV enhancers showed two strong HS sites, one in the polyoma sequences and one at the cap site. The 5' LTR of M-MuLV proviruses containing polyoma enhancer sequences inserted into the wild-type M-MuLV LTR between the cap site and the M-MuLV enhancers showed three HS sites. Two HS sites corresponded to those of the wild-type M-MuLV LTR, whereas the third mapped to the inserted polyoma sequences. The HS site associated with the inserted polyoma sequences was considerably stronger than the M-MuLV-associated HS sites.

Animals↗

PubMed1993

Site-specific photocross-linking reveals that Sec61p and TRAM contact different regions of a membrane-inserted signal sequence.

A chemically charged amber suppressor tRNA was used to introduce the photoactivatable amino acid (Tmd)Phe at a selected position within the signal sequence of the secretory protein preprolactin. This allowed the interactions of the NH2-terminal, the central, and the COOH-terminal regions of the signal sequence to be investigated during insertion into the membrane of the endoplasmic reticulum (ER). We found that different regions of the nascent chains were photocross-linked to different ER proteins. The TRAM protein (translocating chain-associating membrane protein) contacts the NH2-terminal region of the signal sequence while the mammalian Sec61p contacts the hydrophobic core of the signal sequence and regions COOH-terminal of this. These results suggest that the ER translocation complex is composed of heterologous protein subunits which contact distinct regions of nascent polypeptides during their membrane insertion.

Acylation↗

PubMed2002

Sequence variability of the 5' UTR in isolates of hepatitis C virus in China.

BACKGROUND: Hepatitis C virus infection is a great issue in China; however, there is very little information on genotyping investigations based on sequence variability in the 5'untranslated (5'UTR) reported. The present study was to define the sequence variability based on the sequence divergences of the 5'UTR of the virus. METHODS: Sequences of 91 isolates from patients with chronic hepatitis C from Yunnan, southwest China, were sequenced and genotypes were defined according to the sequence divergences of the 5'UTR of the virus. RESULTS: Eighty-six isolates were classified into 3 clades (previously termed groups or major types) by the methods proposed by Chan et al in 1992 and phylogenetic analysis based on nucleotide sequence divergences within the 5'UTR. Fifty-six percent of the isolates were classified into clade 3, 35% into clade 1, and 34.9% into clade 2. New genotypes 1f, 2h, 3h and 3i were defined. In addition, 3 novel sequences were discovered, respectively with an 18-nt sequence deletion (corresponding to nucleotide position -173 to -156), a 28-nt sequence insertion, and a 40-nt sequence insertion, between -56 and -55. Of these isolates, 56% possessed a "G" at position -66 in place of the "T" that is present in all previously reported sequences. CONCLUSIONS: These HCV variants, evolved or remained in this area, may be of great significance in diagnosis and treatment of hepatitis C patients.

5' Untranslated Regions↗

PubMed1996

IS6501-anchored PCR for the detection and identification of Brucella species and strains.

A new strategy was developed to analyse the polymorphism of the genome of Brucella spp. All species of the Brucella genus contain several copies (between 10 and 40) of an insertion sequence, IS6501 (known also as IS711). The position of copies of this insertion sequence appears to differ in each species and this can be used to discriminate between them. A new polymerase chain reaction test, called IS-anchored-PCR (IS-an-PCR) was developed. It was based on a combination of a primer bound on the sequence of IS6501 with a second primer chosen arbitrarily. The patterns obtained reflect the position of the insertion sequence in the genome. This method can be used to identify Brucella and to discriminate between different species, strains within a species and between the vaccine strain B19 and the corresponding 'wild-type' B. abortus A1.

Base Sequence↗

PubMed1983

Promoter mutations that allow nifA-independent expression of the nitrogen fixation nifHDKY operon.

The nifHDKY operon of Klebsiella pneumoniae encodes for structural polypeptides of nitrogenase and requires the nifA gene product for transcription. Mutations that allow transcription of the nifHDKY operon in absence of the nifA gene product were characterized in plasmids containing the regulatory region of nifHDKY and nifH fused in phase to lacZ. beta-Galactosidase activity served as a measure for nifH expression. Most mutations were located in the nif regulatory region and included insertion sequence 2 (IS2) insertions, a sequence duplication, and a base substitution. In Escherichia coli, beta-galactosidase activity expressed from the mutant plasmids in the absence of nifA was 6-30% of the nifA-activated, parental level. Expression from most mutant plasmids was further increased by nifA. In K. pneumoniae, IS2-containing plasmids expressed low levels of beta-galactosidase and responded poorly, if at all, to activation by nifA, whereas expression from other mutant types was similar to that observed in E. coli. Nucleotide sequence analysis of two mutants indicated that sequences within 41 base pairs upstream to the nifH coding sequence were involved in nif-specific regulation. The results suggest that an inverted repeat element in this region, which could theoretically form a cruciform structure in the DNA, is involved in the transcriptional control of the nifHDKY operon.

Bacterial Proteins↗