PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “insertion sequence”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 487 records · Page 27Linked to original sources

A novel insertion sequence in the cryptic plasmid of Neisseria gonorrhoeae may alter the B protein at the translational level.

A variant of the cryptic plasmid of Neisseria gonorrhoeae, 4.4 kb in size, was isolated and characterized at the molecular level. This variant harbored a 156-bp insertion which was located between coordinates 3134 and 3135 within the putative cppB gene using the 4.2-kb cryptic plasmid, pJD1, as a reference. The insertion contained a novel EcoRI site and several elements of symmetry (both direct and inverted repeats). Stop codons present in the insertion interrupted the coding capacity of the cppB gene. Although the insertion was within one of two previously characterized 44-bp repeats purportedly involved in site-specific recombination, it was distinct from a 54-bp segment deleted in some cryptic plasmids. The presence of the insertion suggests a mechanism of modulating the expression of the cppB gene at the translational level through DNA rearrangement.

Amino Acid Sequence↗

Identification by subtractive hybridization of a novel insertion sequence specific for virulent strains of Porphyromonas gingivalis.

Subtractive hybridization was employed to isolate specific genes from virulent Porphyromonas gingivalis strains that are possibly related to abscess formation. The genomic DNA from the virulent strain P. gingivalis W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only Bacillus stearothermophilus IS5377, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS1598, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS1598 was detected in P. gingivalis W83, W50, and FDC 381 by Southern hybridization. All three P. gingivalis strains have been shown to possess abscess-forming ability in animal models. However, IS1598 was not detected in avirulent strains of P. gingivalis, including ATCC 33277. The IS1598 may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS1598 isolated in this study is a novel insertion element which might be a specific marker for virulent P. gingivalis strains.

Amino Acid Sequence↗

Replicative and conservative transpositional recombination of insertion sequences.

We have presented the results of experiments with IS903- and IS10- derived transposons that have led us to the following conclusions: The predominant mechanism of transpositional recombination of these IS elements is a donor-suicide process that results intermolecularly in a simple IS insertion. This process presumably involves little or no replication of the IS. Intramolecular transposition by this process normally results in nonviable products. However, in the particular situation where the transpositional target lies within the transposon, viable products are obtained; these are deletions and deletion-inversions. Deletions between an IS and a target lying outside the element (the conventional "adjacent deletion") occur by a fully replicative process analogous to the formation of cointegrate molecules in intermolecular transposition. The ability of an IS to promote adjacent deletions correlates closely with its ability to fuse replicons into a cointegrate. Before transposition can occur, a complex of the transposase and both IS ends is probably formed. Requirement for such a pretranspositional complex is suggested by the effect on transpositional frequency of changing the distance between the ends. Our results do not support any of the asymmetrical models for transposition. They are, however, compatible with a modified version of the symmetric model proposed by Shapiro (1979). It is interesting to note the similarity between the structures generated by intramolecular simple transposition of an inverse transposon and the circular structures apparently formed by retroviral and copia autointegrative transposition. Shoemaker et al. (1981a,b) and Flavell and Ish-Horowicz (1983) have characterized circular molecules from retrovirally infected cells and Drosophila tissue-culture cells, respectively. The structures of some of the circular molecules resemble deletions and deletion-inversions (Fig. 3B). To our knowledge, a circular species containing two long terminal repeats (LTRs) and an adjacent deletion, which we predict could only occur by a fully replicative process given the similarity in geometry of an LTR to an IS, have not been found. It would appear, then, that the molecule containing two LTRs acts as an inverse transposon, integrating into itself. Shoemaker et al. (1981b) and Flavell and Ish-Horowicz (1983) have also suggested that these products arise from molecules containing two LTRs. We suggest that the two inside LTR ends interact in a conservative, intramolecular, simple transpositionlike event.

DNA Replication↗

Repetitive nucleotide sequence insertions into a novel calmodulin-related gene and its processed pseudogene.

A gene containing a transposon-like human repeat element, called THE 1, has been isolated and characterized. The gene, termed T+, encodes a polypeptide resembling known calcium-binding proteins. The THE 1 element is present in the 3'-untranslated region of its message. The cDNA clone corresponding to the gene's mRNA product led to the identification of this gene. A processed RNA pseudogene related to the authentic gene has also been isolated. In addition to intron processing, this pseudogene differs from the gene in that it contains an interspersed Alu repeat instead of a THE 1 element in the 3'-untranslated region. Thus, we compare a site containing a THE 1 element to an ancestrally related transposon-less target site. The comparison suggests a retroviral-related mechanism of THE 1 insertion. This system is unusual in that the parent gene is associated with three distinct retrotransposition events: the parent gene was converted to a processed RNA pseudogene, an Alu repeat inserted into the pseudogene, and a THE 1 element inserted into the parent gene.

Amino Acid Sequence↗

Promotion of specific in vitro transcription by excised "TATA" box sequences inserted in a foreign nucleotide environment.

We have cloned into plasmid pBR322 a DNA fragment extending from position -32 to position -12 of the adenovirus type 2 major late promoter region (position +1 referring to the cap site). In vitro transcription experiments show that this 21 base pair sequence, which contains the Goldberg-Hogness or "TATA" box, is both necessary and sufficient for specific initiation of transcription by RNA polymerase B (or II). Furthermore, we show that similar sequences, randomly occurring in the bacterial plasmid pBR322, are also recognized by the RNA polymerase B transcription machinery and able to promote specific in vitro transcription. Finally, we discuss the possible importance of the nucleotide sequence of the start region in the actual efficiency of initiation of in vitro transcription.

Adenoviridae↗

Sensitivity of PCR targeting the IS2404 insertion sequence of Mycobacterium ulcerans in an Assay using punch biopsy specimens for diagnosis of Buruli ulcer.

Punch biopsy specimens from Mycobacterium ulcerans disease lesions were used to compare the sensitivities and specificities of direct smear, culture, PCR, and histopathology in making a diagnosis of M. ulcerans disease in a field setting. PCR for the insertion element IS2404 was modified to include uracil-N-glycosylase and deoxyuridine triphosphate instead of deoxythymidine triphosphate to reduce the risk of cross contamination. The "gold standard" for confirmation of clinically diagnosed Buruli ulcer was a definite histological diagnosis, a positive culture for M. ulcerans, or a smear positive for acid-fast bacilli (AFB), together with a possible histological diagnosis. For 70 clinically diagnosed cases of M. ulcerans disease, the modified PCR was 98% sensitive and gave a rapid result. The sensitivities of microscopy, culture, and histology were 42%, 49%, and 82%, respectively. The use of a 4-mm punch biopsy specimen was preferred to a 6-mm punch biopsy specimen since the wound was less likely to bleed and to need stitching. Given adequate technical expertise and the use of controls, the PCR was viable in a teaching hospital setting in Ghana; and in routine practice, we would recommend the use of Ziehl-Neelsen staining of biopsy specimens to detect AFB, followed by PCR, in AFB-negative cases only, in order to minimize costs. Histology and culture remain important as quality control tests, particularly in studies of treatment efficacy.

Adolescent↗

Cloning and structural analysis of the anthocyanin pigmentation locus Rt of Petunia hybrida: characterization of insertion sequences in two mutant alleles.

Anthocyanin biosynthesis in flowers of Petunia hybrida is controlled by the regulatory genes an1, an2 and an11. Seven classes of cDNA clones homologous to transcripts that are down-regulated in an1-, an2- and an11- mutants were isolated via differential cDNA cloning. Genetic mapping, antisense RNA experiments and analyses of mutant alleles demonstrated that one class of clones originated from the Rt locus. The rt gene has no introns and encodes a protein with homology to mammalian glucuronosyl transferases and flavonoid 3-O-glucosyltransferase (UF3GT) encoded by the bz1 gene from Zea mays. As the Rt locus controls the rhamnosylation of reddish anthocyanin-3-O-glucosides which is the first in a series of modifications that finally yield magenta or blue/purple coloured anthocyanins, this suggests that rt encodes an anthocyanin rhamnosyl transferase. Molecular analysis of two mutant rt alleles showed that their expression is blocked by different DNA insertion elements. Mutability of the rt-vu15 allele results from the presence of a 284 bp transposable element (dTph1) in the rt promoter region, causing a block in transcription. The protein coding region of the rt-r27 allele contains a 442 bp insertion (dTph3) resulting in premature polyadenylation of rt transcripts. Although dTph3 cannot transpose, it has sequence characteristics of transposable elements, suggesting that it is a defective member of a new family of transposable elements.

Alleles↗

The effect of two chemical mutagens ENU and MMS on MR-mediated reversion of an insertion-sequence mutation in Drosophila melanogaster.

The effects of two mutagens ENU and MMS characterized by different alkylation patterns have been studied on the reversion of an MR-induced singed mutation to wild-type. Reversion of this unstable singed mutation under the influence of MR is assumed to represent the removal or transposition of an insertion element. Since MR acts primarily in spermatogonia, the mutagens were fed to 1st instar larvae. Recessive lethal tests were carried out simultaneously to calibrate for the mutagenic effectiveness of the chemicals. For both powerful mutagens, it was observed that the frequency of reversion remained far below of what would have been expected on the basis of the mutagenic effectiveness, as registered in the lethal tests. Thus 1 mM ENU, 5 mM and 10 mM MMS did not affect the reversion frequency at all, and with 3 mM ENU only a doubling of the reversion frequency was observed, despite a 5-fold increase in the lethal frequency. The threshold at 1 mM EMU and the low effectiveness of 3 mM on the reversion process are taken as an indication that ENU affected the transposition process in an indirect manner, rather than the excision events themselves. The data obtained with Drosophila are consistent with the microbial observations in that mutation involving removal or transposition of an insertion element is not affected by mutagenic treatments. This finding may have consequences for the evaluation of induced genetic damage on the basis of the spontaneous load of genetic detriment in man. An incidental observation was that non-MR Cy larvae exhibited greater sensitivity to the induction of recessive lethals by MMS than MR-individuals.

Animals↗

Evidence for inserted sequences in the head region of nonmuscle myosin specific to the nervous system. Cloning of the cDNA encoding the myosin heavy chain-B isoform of vertebrate nonmuscle myosin.

The complete amino acid sequence of a vertebrate nonmuscle myosin heavy chain-B isoform (MHC-B, 1976 amino acids, 229 kDa) has been deduced by using cDNA clones from chicken brain libraries. The chicken nonmuscle MHC-B shows overall similarity in primary structure to other MHCs in the areas contributing to the ATP-binding site and actin-binding site. Similar to other nonsarcomeric MHC IIs, there is a short uncoiled tail sequence at the carboxyl terminus of the molecule. It is in the uncoiled tail sequence that the greatest number of differences in amino acids sequence between MHC-A and B were found, which allowed generation of isoform-specific antibodies. These antibodies were used to determine the relative content of MHC-A and MHC-B in various tissues. During the cloning of the cDNA encoding chicken brain MHC-B, we found a 63-nucleotide insertion encoding 21 amino acids located in the head region of the MHC near to the actin-binding site and a 30 nucleotide insertion encoding 10 amino acids near to the ATP-binding site. Analysis using S-1 nuclease showed that both inserts are expressed in a tissue-dependent manner; mRNA containing the inserts is present in tissues of the nervous system, but is absent from other non-muscle cells, which contain the noninserted isoform of MHC-B. Similar inserts were found in corresponding positions in human cerebellar mRNA. Antibodies raised against a peptide synthesized based on the 21 amino acid insert found in chickens recognize a MHC isoform in the same tissues that are enriched for the mRNA. These insertions appear to be a mechanism for generating additional MHC-B isoforms specific to the nervous system.

Amino Acid Sequence↗

[Effect of poly(dA).poly(dT) sequence inserted into EcoR1 site of pBr322 plasmid on the expression of tet and bla genes].

The 99 base pair poly(dA).poly(dT) stretch was inserted into EcoRI site of pBR plasmid. The effect of this insertion on tet-gene expression from P2 promoter, and bla-gene expression from P1 and P3 promoters was studied. The insertion results in a slight enhancement of the resistance of the cells to tetracycline and a decreased sensitivity to ampicillin. It is suggested that these findings reflect the effect of insertion on the transcription of the corresponding genes.

Ampicillin Resistance↗

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Previous genetic analyses indicated that translational frameshifting in the--1 direction occurs within the run of six adenines in the sequence 5'-TTAAAAAACTC-3' at nucleotide positions 305-315 in IS 1, where the two out-of-phase reading frames insA and B'-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84-87. IS 1 mutants with a 1 bp insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84-87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-Lys-Lys-Leu. An IS 1 mutant with the DNA segment 5'-CTTAAAAACTC-3' at positions 305-315 carrying the termination codon TAA in the B'-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The beta-galactosidase activity specified by several IS 1-lacZ fusion plasmids, in which B'-insB is in-frame with lacZ, showed that the region 292-377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu-Lys-Lys-Leu encoded by the DNA segment 5'-TTAAAAAACTC-3', indicating that--1 frameshifting does occur within the run of adenines.

Adenine↗

IS2-43 and IS2-44: new alleles of the insertion sequence IS2 which have promoter activity.

The sequence of two new IS2 alleles with promoter activity (IS2-43 and IS2-44) is reported. The alleles are identical and are formed by a 17 bp tandem duplication in an AT-rich region of IS2. This created a new RNA polymerase binding site. A mutation was found that increased the frequency of formation of these 17 bp duplications but not of another class of duplications, the "mini-insertions". This suggested that the mechanisms of formation of the two classes of duplications are different.

Alleles↗

[Immunity to repeated transposition of the insertion sequence IS21].

The ability of pBR325 derivatives carrying a copy of IS21-element to accept the second copy of this element from plasmid pRP19.6, a temperature-sensitive for replication mutant of RPI containing the duplicated IS21 was studied. It was shown that the frequency of IS21 transposition into plasmids pBR32S::IS21 differing by localization IS21 was lower by two orders of magnitude as compared to that of pBR325. The restriction endonuclease analysis revealed that the insertion of the second copy of IS21 resulted in the formation of pBR325 derivatives carrying the tandem repeated copies of IS21. It was also shown that the plasmids pBR325::IS21 were capable of increasing the frequency of pRP19.6 insertion into the bacterial chromosome from 3-9 to 200-300 times depending on IS21 localization. On the basis of the results obtained and literature data the possible mechanism of the transposition immunity is discussed.

Chromosome Mapping↗

Structure of a processed gene of mouse cytoplasmic gamma-actin transposed into a BAM5 sequence: insertion has created 13 base-pair direct repeats.

We have cloned a processed gene related to cytoplasmic gamma-actin from a phage library of mouse brain DNA. The gene lacks introns, carries a poly(dA) tract of 19-base pairs at the 3' end and is flanked by 13-base-pair direct repeats. Southern blotting experiments indicated that this segment is embedded in highly repetitive and strongly methylated sequences. Direct comparison of the flanking sequences with those of mouse repetitive DNAs revealed that this processed gene is integrated into a BAM5 family, which is one of the repetitive sequences of the mouse genome. In addition, it was shown that the direct repeats were created by a duplication of the corresponding 13-bp stretch of the BAM5 DNA as a consequence of the integration of this segment. These results strongly suggest that the processed gamma-actin gene has been conveyed to this locus through a poly(A)+ RNA intermediate by a transposon-like mechanism.

Actins↗

Homology between an endogenous viral LTR and sequences inserted in an activated cellular oncogene.

Recently, some of us reported the detection and molecular cloning of a rearranged cellular oncogene, designated rc-mos, from a non-virally-induced mouse myeloma, XRPC24. Recombinant lambda phage DNA containing the rc-mos gene was active in transforming NIH 3T3 cells in a transfection assay, whereas recombinant DNA containing the unrearranged c-mos gene was not. In rc-mos, coding sequences from the 5' end of c-mos were found to have been displaced by a novel cellular element whose nucleotide sequence was reported. We now document the fact that a 349-base pair (bp) segment of the novel DNA immediately adjacent to the retained c-mos sequences in rc-mos has close homology with the long terminal repeat (LTR) of a known intracisternal A-particle gene. This homology was mentioned in Nature recently after it had been brought to the attention of the editors (N. Hozumi and R. Hawley, personal communication).

Base Sequence↗