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Results for “insertion sequence”

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PubMed1999

Regulation of human thioredoxin reductase expression and activity by 3'-untranslated region selenocysteine insertion sequence and mRNA instability elements.

Thioredoxin reductases function in regulating cellular redox and function through their substrate, thioredoxin, in the proper folding of enzymes and redox regulation of transcription factor activity. These enzymes are overexpressed in certain tumors and cancer cells and down-regulated in apoptosis and may play a role in regulating cell growth. Mammalian thioredoxin reductases contain a selenocysteine residue, encoded by a UGA codon, as the penultimate carboxyl-terminal amino acid. This amino acid has been proposed to carry reducing equivalents from the active site to substrates. We report expression of a wild-type thioredoxin reductase selenoenzyme, a cysteine mutant enzyme, and the UGA-terminated protein in mammalian cells and overexpression of the cysteine mutant and UGA-terminated proteins in the baculovirus insect cell system. We show that substitution of cysteine for selenocysteine decreases enzyme activity for thioredoxin by 2 orders magnitude, and that termination at the UGA codon abolishes activity. We further demonstrate the presence of a functional selenocysteine insertion sequence element that is highly active but only moderately responsive to selenium supplementation. Finally, we show that thioredoxin reductase mRNA levels are down-regulated by other sequences in the 3'-untranslated region, which contains multiple AU-rich instability elements. These sequences are found in a number of cytokine and proto-oncogene mRNAs and have been shown to confer rapid mRNA turnover.

3' Untranslated Regions↗

PubMed2000

Kinetics of the interaction of translation factor SelB from Escherichia coli with guanosine nucleotides and selenocysteine insertion sequence RNA.

The kinetics of the interaction of GTP and GDP with SelB, the specific translation factor for the incorporation of selenocysteine into proteins, have been investigated using the stopped-flow method. Useful signals were obtained using intrinsic (i.e. tryptophan) fluorescence, the fluorescence of methylanthraniloyl derivatives of nucleotides, or fluorescence resonance energy transfer from tryptophan to the methylanthraniloyl group. The affinities of SelB for GTP (K(d) = 0.74 micrometer) and GDP (K(d) = 13.4 micrometer) were considerably lower than those of other translation factors. Of functional significance is the fact that the rate constant for GDP release from its complex with SelB (15 s(-)(1)) is many orders of magnitude larger than for elongation factor Tu, explaining why a GDP/GTP exchange factor is not required for the action of SelB. In contrast, the rate of release of GTP is 2 orders of magnitude slower and not significantly faster than for elongation factor Tu. Using a fluorescently labeled 17-nucleotide RNA minihelix that represents a binding site for the protein and that is part of the fdhF selenocysteine insertion sequence element positioned immediately downstream of the UGA triplet coding for selenocysteine incorporation, the kinetics of the interaction were studied. The high affinity of the interaction (K(d) approximately 1 nm) appeared to be increased even further when selenocysteyl-tRNA(Sec) was bound to SelB, but to be independent of the presence or nature of the guanosine nucleotide at the active site. These results suggest that the affinity of SelB for its RNA binding site is maximized when charged tRNA is bound and decreases to allow dissociation and reading of codons downstream of the selenocysteine codon after selenocysteine peptide bond formation.

Bacterial Proteins↗

PubMed1987

DNA element of Corynebacterium diphtheriae with properties of an insertion sequence and usefulness for epidemiological studies.

The segment of DNA which is inserted within the tox gene of bacteriophage gamma and is responsible for its Tox- phenotype was found to be present and repeated approximately 30 times in the chromosome of Corynebacterium diphtheriae Belfanti 1030. Other C. diphtheriae strains contained a variable number of copies (1 to 25) of the same element. Sequence analysis showed that this repeated and interspersed DNA element was flanked by 9-base-pair direct repeats and that the 5' and 3' ends of the insertion contained sequences forming an imperfect inverted repeat. Therefore, the DNA segment here described has most of the typical structural features of a bacterial insertion sequence element. We show that different C. diphtheriae isolates derived from the same outbreak of diphtheria have an identical genomic distribution of this DNA element and that such DNA can be useful for epidemiological studies.

Base Sequence↗

PubMed1977

Sequence arrangements of the Escherichia coli chromosome and of putative insertion sequences, as revealed by electron microscopic heteroduplex studies.

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Base Sequence↗

PubMed1988

Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro.

We constructed SP6-human beta-globin derivative plasmids that included possible donor site (5' splice site) sequences at a specified position within the first intron. The runoff transcripts from these templates truncated in the second exon were examined for splicing in a nuclear extract from HeLa cells. In addition to the products from the authentic donor site, a corresponding set of novel products from the inserted, alternative donor site was generated. Thus, a short sequence inserted within an intron can be an active donor site signal in the presence of an authentic donor site. The active donor site sequences included a 9-nucleotide consensus sequence, 14- or 16-nucleotide sequences at the human beta-globin first or second donor, and those at simian virus 40 large T antigen or small t antigen donor. These included 3 to 8 nucleotides of an exon and 6 to 8 nucleotides of an intron. The activity of the inserted donor site relative to that of the authentic donor site depended on the donor sequence inserted. The relative activity also strongly depended on the concentrations of both KCl (40 to 100 mM) and MgCl2 (1.6 to 6.4 mM). At the higher KCl concentrations tested, all the inserted, or proximate, donor sites were more efficiently used. Under several conditions, some inserted donor sites were more active than was the authentic donor site. Our system provides an in vitro assay for donor site activity of a sequence to be tested.

Amino Acid Sequence↗

PubMed1990

Characterization of insertion sequence IS946, an Iso-ISS1 element, isolated from the conjugative lactococcal plasmid pTR2030.

The self-transmissible plasmid pTR2030 mobilized nonconjugative heterologous cloning vectors pGK12 (Cmr Emr) and pSA3 (Emr) at frequencies of 10(-5) to 10(-6) per input donor. Transconjugants harbored a 51- or 58-kilobase (kb) plasmid not found in the parental strains that cotransferred at high frequency with Cmr Emr and pTR2030-encoded phage resistance (Hsp+) in second-round matings (10(-1) per input donor). Restriction endonuclease mapping and DNA-DNA hybridization identified the 51- to 58-kb plasmids as pTR2030::vector cointegrates. Examination of four cointegrates indicated that pGK12 and pSA3 had inserted within two locations on pTR2030. Resolution of the cointegrates generated vector derivatives containing a 0.8-kb insert of pTR2030 DNA. Restriction analyses of several resolution plasmids indicated that the 0.8-kb element had inserted into various positions within pGK12 and pSA3 and in certain cases had inactivated the Cmr or Emr marker of pGK12. A conjugative mobilization assay demonstrated that the 0.8-kb element, designated IS946, mediated transpositional recombination. Nucleotide sequence determination identified IS946 as an 808-base-pair (bp) insertion sequence sharing ca. 96% homology with lactococcal insertion sequence ISS1. IS946 differed by 27 and 31 bp from ISS1S and ISS1T, respectively, and in 2 of 226 amino acids in the deduced sequence of the putative transposase. IS946 has perfect 18-bp terminal inverted repeats, identical to ISS1, and similarly generated 8-bp direct repeats of the target site upon insertion.

Amino Acid Sequence↗

PubMed1992

The 28S ribosomal RNA-encoding gene of Hymenoptera: inserted sequences in the retrotransposon-rich regions.

The genomes of two parasitoid wasps, Diadromus pulchellus and Eupelmus vuilleti, and the honey bee, Apis mellifera, contain few interspersed repeated sequences corresponding to transposons (Tn). This suggests that the genomic organisation of Hymenoptera could be due to the elimination of deleterious Tn in haploid males. We have used restriction-fragment length polymorphism analysis to show that nondeleterious Tn are present in the DNA (rDNA) encoding ribosomal RNA of twelve species of Hymenoptera. Sequence analysis of the 28S rDNA type-I and type-II insertion-rich regions of 80 species showed that this region is very highly conserved (95.8%). A consensus sequence and restriction map of the rDNA region were established. These sequence data were used to develop a strategy for detecting inserted elements in the rDNA fragments containing type-I or type-II insertion sites, and this strategy was used to screen twelve hymenopteran species and four non-Hymenoptera control species. The rDNA fragments from the Hymenoptera and control species contained inserted sequences in the area where type-I and type-II elements are inserted in the 28S rDNA retrotransposon-rich region of Diptera and Lepidoptera. The hymenopteran genomes therefore appear to contain repeated elements, the mobility and nature of which remain to be determined.

Animals↗

PubMed1983

IS200: a Salmonella-specific insertion sequence.

A new IS element (IS200) has been identified in Salmonella. The sequence was identified as an IS element by the following criteria: its insertion caused the mutation hisD984; six copies of the sequence are present in strain LT2 of S. typhimurium; and transposition of the sequence has been observed on several occasions. IS200 is found in almost all Salmonella species examined but is absent from most other enteric bacteria. The specificity of this element for Salmonella (and the absence of IS1-IS4 from Salmonella) suggest that transfer of insertion sequences between bacterial groups may be less extensive than is commonly believed. Alternatively, the distribution may suggest that these elements play a selectively important role in bacteria.

Base Sequence↗

PubMed1994

Nucleotide sequence of the afimbrial-adhesin-encoding afa-3 gene cluster and its translocation via flanking IS1 insertion sequences.

The afa gene clusters encode afimbrial adhesins (AFAs) that are expressed by uropathogenic and diarrhea-associated Escherichia coli strains. The plasmid-borne afa-3 gene cluster is responsible for the biosynthesis of the AFA-III adhesin that belongs to the Dr family of hemagglutinins. Reported in this work is the nucleotide sequence of the 9.2-kb insert of the recombinant plasmid pILL61, which contains the afa-3 gene cluster cloned from a cystitis-associated E. coli strain (A30). The afa-3 gene cluster was shown to contain six open reading frames, designated afaA to afaF. It was organized in two divergent transcriptional units. Five of the six Afa products showed marked homologies with proteins encoded by previously described adhesion systems that allowed us to attribute to each of them a putative function in the biogenesis of the AFA-III adhesin. AfaE was identified as the structural adhesin product, whereas AfaB and AfaC were recognized as periplasmic chaperone and outer membrane anchor proteins, respectively. The AfaA and AfaF products were shown to be homologous to the PapI-PapB transcriptional regulatory proteins. No function could be attributed to the AfaD product, the gene of which was previously shown to be dispensable for the synthesis of a functional adhesin. Upstream of the afa-3 gene cluster, a 1.2-kb region was found to be 96% identical to the RepFIB sequence of one of the enterotoxigenic E. coli plasmids (P307), suggesting a common ancestor plasmid. This region contains an integrase-like gene (int). Sequence analysis revealed the presence of an IS1 element between the int gene and the afa-3 gene cluster. Two other IS1 elements were detected and located in the vicinity of the afa-3 gene cluster by hybridization experiments. The afa-3 gene cluster was therefore found to be flanked by two IS1 elements in direct orientation and two in opposite orientations. The afa-3 gene cluster, flanked by two directly oriented IS1 elements, was shown to translocate from a recombinant plasmid to the E. coli chromosome. This translocation event occurred via IS1-specific recombination mediated by a recA-independent mechanism.

Adhesins, Escherichia coli↗

PubMed1981

Inserted sequences in bovine satellite DNA's.

The nucleotide sequence of the 1413-base-pair repeat unit of bovine 1.711a satellite DNA (density in cesium chloride, 1.711 grams per cubic centimeter) has been determined. The repeat unit contains two segments consisting of variants of a basic 23-base-pair sequence that is closely related to sequences of bovine 1.706 satellite DNA. A third segment of the repeat unit contains an unrelated 611-base-pair sequence that is not internally repetitive. This segment is flanked by inverted repeats of 8 base pairs and, on one side, by a direct repeat of the terminal sequence. A related segment is present in bovine 1.711b satellite DNA and is inserted into sequences derived from the 1.715 satellite. These nucleotide sequences suggest the timing of some of the stages in the evolution of these complex, closely related satellite DNA's and indicate the mechanisms inherent in their divergence from a common ancestor.

Animals↗

PubMed1989

Positive regulation of the Drosophila melanogaster G6PD gene by an insertion sequence.

In a previous study, we have shown that the three high-G6PD activity mutants are characterized by insertion of the Ins1 sequence consisting of a core sequence flanked by two defective P elements (KP and KP'; the 32nd base of the KP was replaced by guanine in the KP') in front of exonI of the G6PD gene and that the sequence responsible for positive regulation of the G6PD gene expression might be the core sequence but not the flanking KP and KP' elements. The core sequence is composed of either one or two identical units in each mutant. In this report we present evidence (1) that insertion of the Ins1 sequence gives rise to overproduction of G6PD mRNA, (2) that the length and the 5' end of G6PD mRNA do not differ in wild-type and three mutants, (3) that the insertion site of the Ins1 sequence is the same in the mutants, and (4) that each unit of the core sequence has the same in the mutants, and (4) that each unit of the core sequence has a pair of DNase I-hypersensitive sites. The possibility exists that the binding of some regulatory proteins to the DNase I-hypersensitive sites might accelerate the transcription rate of the G6PD gene.

Animals↗

PubMed1991

Isolation and genetic structure of IS112, an insertion sequence responsible for the inactivation of the SalI restriction-modification system of Streptomyces albus G.

IS112 is a transposable element identified in Streptomyces albus G by its frequent mutagenic insertion into the genes for the SalI restriction-modification system. IS112 is present in several copies in the genome of S. albus G. Homologous sequences were detected in other Streptomyces strains. Sequence analysis revealed that IS112 has a length of 883 bp with a GC content of 67.4%. The copy that was isolated contained imperfect inverted repeats (16/20 match) at its ends and was flanked by a 2 bp duplication at the target site, which was located within the gene (salIR) for the SalI endonuclease. A long open reading frame (ORF) encoding a putative polypeptide of 256-253 amino acids spans almost the entire sequence. Significant homology was detected between this polypeptide and that corresponding to ORFB of IS493, an insertion sequence recently isolated from Streptomyces lividans 66.

Amino Acid Sequence↗

PubMed1998

Target choice and orientation preference of the insertion sequence IS903.

We have examined the targeting preference of the bacterial insertion element IS903 by determining the sites of insertion of a large number of transposition events into the 55-kb conjugative plasmid pOX38. Despite the large target size, all the insertions were clustered in four small distinct regions associated with conjugal DNA transfer. Within these regions, many different sites were used for insertion; however, there were a few sites that IS903 inserted into more than once. Alignment of the insertion sites showed that there was no consensus sequence within the 9-bp target duplication but that there were preferred sequences located symmetrically on either side of the target. This is consistent with target recognition by a dimer or multimer of transposase, with either sequence-specific or structure-specific interactions on both sides of the target. We show further that when one of these preferred regions was cloned into a second conjugative plasmid, pUB307, it was still a preferred target, implying that all the sequences necessary for target selection are contained within this DNA segment. Also, we observed a very strong preference for insertion in a single orientation in pUB307. We examined the possibility that either DNA replication from the origin of vegetative replication, oriV, or the origin of transfer, oriT, might determine this orientation effect. We find that reversing the direction of vegetative replication had no effect on the orientation of transposon insertions; however, reversing the direction of DNA transfer abolished the orientation effect. This supports the idea that conjugal DNA transfer imparts a polarity on the target that is sensed by the transposon.

Base Sequence↗

PubMed1990

Site-specific transposition of insertion sequence IS630.

IS630 is a 1.15-kilobase sequence in Shigella sonnei that, unlike many mobile elements, seems not to mediate cointegration between different replicons. To assess its transposition, we constructed composite elements containing inverted copies of IS630 flanking a drug resistance gene. We found that these composite elements transposed to plasmid ColE1 in Escherichia coli. DNA sequencing showed that transposition was, in all cases, to the dinucleotide sequence 5'-TA-3'. There were two preferred insertion sites which corresponded to the TA sequences in the inverted repeats of a 13-base-pair stem region of the [rho]-dependent transcription terminator. IS630 is flanked by TA, and nucleotide substitution by in vitro mutagenesis at these ends did not affect transposition activity of a composite element or its ability to insert preferentially into TA within the 13-base-pair inverted repeat sequences or to duplicate the target sequence.

Base Composition↗

PubMed2006

Differentiation of carbazole catabolic operons by replacement of the regulated promoter via transposition of an insertion sequence.

The carbazole catabolic car operons from Pseudomonas resinovorans CA10 and Janthinobacterium sp. J3 have nearly identical nucleotide sequences in their structural and intergenic regions but not in their flanking regions. Transposition of ISPre1 from the anthranilate catabolic ant operon located an inducible promoter Pant upstream of the carCA10 operon, which is regulated by the AraC/XylS family activator AntR in response to anthranilate. The transposed Pant drives transcription of the carCA10 operon, which is composed of the car-AaAaBaBbCAcAdDFECA10 structural genes. Transcriptional fusion truncating Pant upstream of carAaCA10 resulted in constitutive luciferase expression. Primer extension analysis identified a transcription start point of the constitutive mRNA of the carCA10 operon at 385 nucleotides upstream of the carAaCA10 translation start point, and the PcarAa promoter was found. On the other hand, a GntR family regulatory gene carRJ3 is divergently located upstream of the carJ3 operon. The Pu13 promoter, required for inducible transcription of the carJ3 operon in the presence of carbazole, was identified in the region upstream of carAaJ3, which had been replaced with the Pant promoter in the carCA10 operon. Deletion of carRJ3 from a transcriptional fusion resulted in high level constitutive expression from Pu13. Purified CarRJ3 protein bound at two operator sequences OI and OII, showing that CarRJ3 directly represses Pu13 in the absence of its inducer, which was identified as 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoate, an intermediate of the carbazole degradation pathway.

Base Sequence↗

PubMed1995

Unique insertion sequence and pattern of CD4 expression in variants selected with immunotoxins from human immunodeficiency virus type 1-infected T cells.

To study the variability of human immunodeficiency virus type 1 (HIV-1), we used immunotoxins to select for variants within a population of H9 cells persistently infected with a molecular clone of HIV-1 designated NL4-3. Chimeric immunotoxin CD4-PE40 (a chimeric fusion protein consisting of the amino-terminal two domains of CD4 and the carboxy-terminal domains of Pseudomonas exotoxin A) was used to select for cells lacking cell surface expression of HIV Env (envelope proteins gp160, gp120, and gp41). The cells described here (A1, A7, C9, and E9) fail to express HIV proteins because they have markedly diminished transcription of the integrated provirus (A1, A7, and E9) or no HIV provirus (C9). Analysis demonstrated that two different cloned variants, A1 and E9, contain the complementary sequence of tRNA(3Lys) (45 bp) inserted 3' to the primer-binding site, following by a 169-bp deletion through the start of the gag gene. No HIV mRNA was detected by Northern (RNA) blotting, but PCR demonstrated the presence of the viral message. These variants were found very infrequently in the unselected H9/NL4-3 cell population, and they contained proviruses distinct from that found in the dominant population. In addition, all of these variants had similar patterns of CD4 surface expression that allowed them to escape reinfection within the tissue culture. The data are discussed with regard to mechanisms and errors of HIV reverse transcription, as well as the evolution of mutants within a population of persistently infected cells.

Base Sequence↗

PubMed1994

Functional analysis and modeling of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme.

To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we have constructed mutant proteins by inserting RGD-containing sequences flanked by two Cys residues between Val74 and Asn75 of human lysozyme. CRGDC-, CRGDSC-, and CGRGDSC-inserted mutant lysozymes were expressed in yeast, purified, and designated as Cys-RGD3, Cys-RGD4, and Cys-RGD5, respectively. In baby hamster kidney cells, these mutants were shown to possess high cell adhesion activity by interaction with vitronectin receptor (integrin alpha v beta 3), and this activity is 2-3-fold higher than that of the RGDS-inserted mutant lysozyme, RGD4. The mutant proteins also inhibited the binding of human fibrinogen to its receptor (integrin alpha IIb beta 3) at a lower concentration than the RGD4 protein. Peptide mapping and mass spectrometric analyses showed that the two inserted Cys residues in these mutants are linked to each other without any effects on the mode of the four disulfide bonds present in native human lysozyme. These results suggest that the introduction of a conformational constraint into the RGD region significantly increases the cell adhesion activity. The conformation of the RGD region in Cys-RGD4 was modeled by a Monte Carlo simulation. Most of the sampled conformations were grouped into three classes; the first is characterized by an extended Gly conformation, the second assumes a type II' beta turn, and the third has a salt bridge between Arg and Asp.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

The introduction of colonic-Bacteroides shuttle plasmids into Porphyromonas gingivalis: identification of a putative P. gingivalis insertion-sequence element.

Two Escherichia coli-Bacteroides plasmid-shuttle vectors pNJR5 and pNJR12 were introduced for the first time into Porphyromonas gingivalis W83 by conjugal transfer from E. coli. The transfer frequencies were comparable to those obtained when using colonic Bacteroides as recipients. Both plasmids were maintained in P. gingivalis W83 and could be isolated and introduced back into E. coli. Plasmid DNA extracted from one P. gingivalis W83 pNJR12 transconjugant had an additional 1.5 kb of inserted DNA. Southern-blot analysis of P. gingivalis W83 chromosomal DNA using this inserted DNA as a probe revealed the presence of multiple copies of this sequence on the chromosome. We propose that this DNA represents a P. gingivalis insertion sequence (IS) element and should be referred to as IS1126. This is the first IS element to be isolated from a Gram-negative oral anaerobic bacterium.

Blotting, Southern↗