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PubMed1987

Insertion forces with intrauterine devices: implications for uterine perforation.

The force required to insert a Copper 7, Multiload Copper or Nova T IUD was measured in 197 successful and 25 unsuccessful insertion attempts. These forces were compared with the forces required to perforate freshly obtained uterine specimens with a metal uterine sound and Dalkon shield device, as well as with the clinically tested devices. The mean insertion forces for the Copper 7, Nova T and Multiload Copper devices were 1.502 N, 2.134 N and 4.041 N respectively, while the mean insertion pressures (N/mm2) were 0.203, 0.209 and 0.122 respectively. The mean in vitro fundal perforation forces with metal sounds was 20.7 N and with the Dalkon shield 31.6 N. The Copper 7, Multiload Copper and Nova T IUDs achieved mean in vitro forces of 5.75 N, 9.2 and 8.1 N respectively, without causing perforation. Primary uterine perforation at the time of insertion of these devices appears unlikely.

Adolescent↗
PubMed1992

Analysis of the bovine viral diarrhea virus genome for possible cellular insertions.

Mucosal disease is the most severe disease resulting from bovine viral diarrhea virus (BVDV) infection in cattle. Two biotypes of BVDV may be isolated from animals with mucosal disease: cytopathic (cp) and noncytopathic (ncp). These "pairs" of cp/ncp viruses are often closely related and it has been suggested that the cp virus arises from a ncp virus by insertion of cellular RNA in the p125 region of the BVDV genome. We have used four pairs of cp/ncp BVDV isolated from cattle with mucosal disease, to examine the genomic sequence of the region of the genome coding for the nonstructural protein p125 (processed to p54/p80 in cp viruses) by PCR analysis and sequencing. We did not detect any cellular gene insertions in any of the four ncp viruses; however, we found a large duplication of the p80 gene and a ubiquitin gene insertion in three of the four cp isolates. Our results suggest that cellular RNA insertions in the p125 region may contribute significantly to the cytopathogenicity of BVDV. However, this does not appear to be the only mechanism of cytopathogenicity as we did not detect any insertions or duplications in one of the cp viruses. Comparison of the DNA sequence in the p80 region revealed greater homology within the "pairs" than to NADL, which lend further support to the hypothesis that a cp virus is originated from a ncp virus.

Amino Acid Sequence↗
PubMed1992

Genomic structure of the locus associated with an insertional mutation in line 4 transgenic mice.

In line 4 transgenic mice, six to eight copies of a 50-kb lambda recombinant clone are arranged in a head-to-tail tandem array on chromosome 3. Embryos homozygous for the transgene become arrested in their development on Day 5 of gestation shortly after implantation. The insertion site was cloned using a small segment of the transgene as a probe. Comparison of the insertion site with the wildtype locus indicates that a 22-kb deletion of host DNA has occurred in line 4 mice. Restriction enzyme analyses showed that neither the tandem array nor the flanking chromosomal DNA had any detectable rearrangements. Sequencing of the junctions between host and foreign DNA, however, revealed insertions of small fragments of DNA of unknown origin as well as an insertion of a DNA segment derived from another region of the transgene. Therefore, disruption of an essential gene in the line 4 transgenic mouse may have been caused by the insertion of 300-400 kb of foreign DNA or a deletion of sequences in the host genome.

Animals↗
PubMed2004

Insertional inactivation of oprD in clinical isolates of Pseudomonas aeruginosa leading to carbapenem resistance.

Recently, a Texas, USA hospital isolated seven Pseudomonas aeruginosa strains displaying dual resistance to fluoroquinolones and imipenem. These isolates were resistant to the fluoroquinolones through overexpression of the MexXY efflux pump and/or QRDR mutations and resistant to imipenem through downregulation of oprD transcription. The purpose of this study was to evaluate the molecular events responsible for decreased transcriptional expression of oprD in these strains. Expression of oprD could only be detected in two of the strains, but expression was very low as indicated by the high number of RT-PCR cycles required to amplify the product. PCR was performed to amplify the oprD gene using primers upstream of the promoter and downstream of the structural gene. Amplified products were sequenced, and sequences were compared to wild-type P. aeruginosa strain PAO1. Two isolates provided PCR products of the predicted size of 1586 bp, but sequencing revealed a single base change within the structural gene resulting in a premature stop codon. The other five isolates provided PCR products that were 1.3-1.6 kb larger than expected, suggesting the presence of large inserts. Sequence analysis indicated these inserts were novel insertion sequence elements transposed into different locations within oprD. In summary, loss of OprD in all seven isolates was associated with mutations or insertions within oprD. Although the point mutations that resulted in premature stop codons would explain the loss of the OprD protein in two isolates. This observation does not explain the observed decrease in transcriptional expression. This is the first report of carbapenem resistance occurring through insertional inactivation of the oprD gene by IS elements.

Amino Acid Sequence↗
PubMed2006

Whole genome computational comparative genomics: A fruitful approach for ascertaining Alu insertion polymorphisms.

Alu elements are the most active and predominant type of short interspersed elements (SINEs) in the human genome. Recently inserted polymorphic (for presence/absence) Alu elements contribute to genome diversity among different human populations, and they are useful genetic markers for population genetic studies. The objective of this study is to identify polymorphic Alu insertions through an in silico comparative genomics approach and to analyze their distribution pattern throughout the human genome. By computationally comparing the public and Celera sequence assemblies of the human genome, we identified a total of 800 polymorphic Alu elements. We used polymerase chain reaction-based assays to screen a randomly selected set of 16 of these 800 Alu insertion polymorphisms using a human diversity panel to demonstrate the efficiency of our approach. Based on sequence analysis of the 800 Alu polymorphisms, we report three new Alu subfamilies, Ya3, Ya4b, and Yb11, with Yb11 being the smallest known Alu subfamily. Analysis of retrotransposition activity revealed Yb11, Ya8, Ya5, Yb9, and Yb8 as the most active Alu subfamilies and the maintenance of a very low level of retrotransposition activity or recent gene conversion events involving S subfamilies. The 800 polymorphic Alu insertions are characterized by the presence of target site duplications (TSDs) and longer than average polyA-tail length. Their pre-integration sites largely follow an extended "NT-AARA" motif. Among chromosomes, the density of Alu insertion polymorphisms is positively correlated with the Alu-site availability and is inversely correlated with the densities of older Alu elements and genes.

Alu Elements↗
PubMed2006

Mutational patterns associated with the 69 insertion complex in multi-drug-resistant HIV-1 reverse transcriptase that confer increased excision activity and high-level resistance to zidovudine.

Human immunodeficiency virus type 1 (HIV-1) strains having dipeptide insertions in the fingers subdomain and other drug resistance-related mutations scattered throughout their reverse transcriptase (RT)-coding region show high-level resistance to zidovudine (AZT) and other nucleoside analogues. Those phenotypic effects have been correlated with their increased ATP-dependent phosphorolytic activity on chain-terminated primers. Mutations T69S and T215Y and a dipeptide insertion (i.e. Ser-Ser) between positions 69 and 70 are required to achieve low-level resistance to thymidine analogues. However, additional amino acid substitutions are necessary to achieve the high-level phenotypic resistance to AZT shown by clinical HIV isolates carrying a dipeptide insertion in their RT-coding region. In order to identify those mutations that contribute to resistance in the sequence context of an insertion-containing RT derived from an HIV clinical isolate (designated as SS RT), we expressed and purified a series of chimeric enzymes containing portions of the wild-type or SS RT sequences. ATP-mediated excision activity measurements using AZT- and stavudine (d4T)-terminated primers and phenotypic assays showed that molecular determinants of high-level resistance to AZT were located in the fingers subdomain of the polymerase. Further studies, using recombinant RTs obtained by site-directed mutagenesis, revealed that M41L, A62V and in a lesser extent K70R, were the key mutations that together with T69S, T215Y and the dipeptide insertion conferred high levels of ATP-dependent phosphorolytic activity on AZT and d4T-terminated primers. Excision activity correlated well with AZT susceptibility measurements, and was consistent with phenotypic resistance to d4T. Structural analysis of the location of the implicated amino acid substitutions revealed a coordinated effect of M41L and A62V on the positioning of the beta3-beta4 hairpin loop, which plays a key role in the resistance mechanism.

Adenosine Triphosphate↗
PubMed2005

Tumor model-specific proviral insertional mutagenesis of the Fos/Jdp2/Batf locus.

Retroviral activation of the AP-1/ATF super family member Jdp2 was recently reported to be a common event in M-MLV-induced T cell lymphoma in p27-null C57x129 mice as compared to wild type-inoculated mice but has not been found important in other models. On the basis of retroviral tag retrieval from 1190 individual Akv- and SL3-3-induced lymphomas, we here report that insertional mutagenesis into the 250-kb Fos/Jdp2/Batf locus is associated with SL3-3 MLV-induced T but not Akv-induced B cell lymphomas of NMRI and SWR mice. Integration pattern and clonality analyses suggest that Jdp2 participates in SL3-3-induced tumorigenesis distinctly as compared to the M-MLV setting. Northern blot analysis showed Jdp2 to be alternatively spliced in various normal tissues as well as MLV-induced lymphomas. Interestingly, in some tumors, proviral insertion seems to activate different mRNA sub-species. Whereas elevated mRNA levels of the Fos gene could not be correlated with provirus presence, in one case, Northern blot analysis as well as quantitative real-time PCR indicated proviral activation of the AP-1 super family member Batf, a gene not previously reported to be a target of insertional mutagenesis. A novel integration cluster between Jdp2 and Batf apparently did not influence the expression level of either gene, underscoring the importance of addressing expression effects to identify target genes of insertion. Altogether, such distinct insertion patterns point to different mechanism of activation of specific proto-oncogenes and are consequently of importance for the understanding of proviral activation mechanisms as well as the specific role of individual oncogenes in tumor development.

3T3 Cells↗
PubMed1992

Interactions between intrauterine contraceptive device use and breast-feeding status at time of intrauterine contraceptive device insertion: analysis of TCu-380A acceptors in developing countries.

OBJECTIVE: This paper is a reassessment of earlier findings from a preliminary analysis of data from a multicenter international trial regimen on breast-feeding and non-breast-feeding women in which events related to insertion, expulsion, and removal of the TCu-380A intrauterine contraceptive device (ParaGard 380) were investigated. STUDY DESIGN: Performance of the TCu-380A through 12 months after insertion was compared with life-table rate analysis, chi 2, Fisher exact test, or Student's t test. Variables were events reported during intrauterine contraceptive device insertion and events throughout the 12 months of study participation by breast-feeding status. RESULTS: Breast-feeding among intrauterine contraceptive device users was associated with fewer insertion-related complaints and lower removal rates for bleeding and pain. No uterine perforations were reported throughout the study. CONCLUSION: Differences in the performance of the TCu-380A intrauterine contraceptive device suggest physiologic effects associated with lactational amenorrhea. The TCu-380A intrauterine contraceptive device is a viable option for women breast-feeding at the time of intrauterine contraceptive device insertion.

Adult↗
PubMed1998

Insertion of a bovine SMT3B gene in NS4B and duplication of NS3 in a bovine viral diarrhea virus genome correlate with the cytopathogenicity of the virus.

Bovine viral diarrhea virus (BVDV) infection in cattle can lead to the development of a fatal syndrome called mucosal disease (MD). The induction of MD requires co-existence of two biotypes of BVDV: cytopathic (cp) and non-cytopathic (ncp). Studies have shown that in some cases, cp viruses are generated from ncp viruses by cellular gene insertions, duplications or rearrangements in the viral genome. Cellular ubiquitin gene is the most frequently reported insert in BVDV 1. Here we report the finding of a novel cellular gene insertion (termed cSNCINS) in the NS4B gene accompanied by the duplication of NS3 in a cpBVDV genome. The amino acid sequence of the insert is identical to that of the human SMT3B protein and is 98% similar to that of the human SMT3A protein. Both SMT3B and SMT3A proteins are homologues of the yeast SMT3 protein. The cSNCINS protein is encoded by an open reading frame located on a 1150-bp bovine endometrial cDNA fragment. Our results indicate that the cSNCINS is a bovine homologue of the human SMT3B gene and that insertion of the BSMT3B gene together with duplication of NS3 in the viral genome may account for the cytopathogenicity of this virus.

Amino Acid Sequence↗
PubMed2000

Insertion with long target duplication: a mechanism for gene mobility suggested from comparison of two related bacterial genomes.

The complete genome sequences of two closely related organisms--two Helicobacter pylori strains--have recently become available. Comparison of these genomes at single base pair level has suggested the presence of a mechanism for bacterial gene mobility--insertion with long target duplications. This mechanism is formally similar to classical transposon insertion, but the duplication is much longer, often in the range of 100bp. Restriction and/or modification enzyme genes are often within or adjacent to the insertion. A similar process may have mediated insertion of the cag(+) pathogenicity island in H. pylori. A similar structure was identified in comparisons between Neisseria meningitidis and Neisseria gonorrhoeae genomes. We hypothesize that this mechanism, as well as two other types of polymorphism linked with restriction-modification genes (insertion accompanied by target deletion and a tripartite structure composed of substitution/inversion/deletion), have resulted from attack by restriction enzymes on the chromosome.

Base Sequence↗
PubMed1996

The use of an improved transposon mutagenesis system for DNA sequencing leads to the characterization of a new insertion sequence of Streptomyces lividans 66.

A DNA sequencing strategy was developed based on the tetracycline resistance transposon Tn1721. A universal M13 primer binding site (UP) for DNA sequencing and restriction sites for mapping were inserted near one end of Tn1721 and the new derivative, Tn5491, introduced onto a conjugative F' plasmid. The target sequence is inserted between two inverted resolution sites (res) of Tn1721 present on the high-copy plasmid pJOE2114. Due to the inviability of long palindromic sequences in Escherichia coli insertions between the inversely orientated res sites of pJOE2114 are positively selected. Transposition of Tn5491 into the target sequence is selected by cointegrate formation of Tn5491 during transposition, mating and transfer of the nonconjugative sequencing vector. After cointegrate resolution, the additional res sites in the vector result in a second site-specific recombination removing most of the transposon (except of 136 bp) and part of the target sequence. The reduced plasmid sizes and the use of the universal primer improved the quality of the sequencing results obtained on an automated fluorescent sequencer. A 3.35-kb EcoRI fragment from the 30-kb terminal inverted repeats (TIR) of the Streptomyces lividans chromosome was sequenced by this method. A 1304-bp sequence was found on this fragment with the features of insertion elements. The element called IS1372 had 27-bp IR and two potential open reading frames. The predicted gene products had similar sizes and high similarity to gene products encoded by insertion sequences of the IS3 family. Furthermore, a potential signal stimulating ribosomal shifts and typical for members of the IS3 family was identified. Five to seven copies of IS1372 were found in different strains of S. lividans but none in other Streptomyces species tested.

Amino Acid Sequence↗
PubMed1997

Insertional mutagenesis by a modified in vitro Ty1 transposition system.

Transposable elements are useful tools for insertional mutagenesis and have many potential applications in the characterization of complex genomes. Here we describe a system which facilitates the construction of large transposon insertion libraries useful for genome sequencing and functional genomic analysis. We developed two transposons, TyK and TyK'GFP+, which can be introduced into target DNAs by Ty1-mediated transposition in vitro, and several modifications which decrease the frequency of false transposition events and direct the recovery of transpositions into passenger rather than vector DNA. Insertions of TyK'GFP+ additionally may yield fusions to the Aequorea green fluorescent protein (GFP), useful in studies of gene expression and protein targeting. Transposition in vitro was obtained into target DNAs of up to 50 kb in size, restriction mapping showed insertion to be relatively random, and the sequence of 55 insertion sites showed neither strong site nor base compositional preference. Our data suggest that TyK-based artificial transposons will be suitable for a variety of genetic applications in many organisms.

DNA Transposable Elements↗
PubMed2002

A P-insertion screen identifying novel X-linked essential genes in Drosophila.

The recent determination and annotation of the entire euchromatic sequence of the Drosophila melanogaster genome predicted the existence of about 13600 different genes (Science 287 (2000) 2185; http://www.fruitfly.org/annot/index.html). In parallel, the Berkeley Drosophila Genome Project (BDGP) has undertaken systematic P-insertion screens, to isolate new lethals and misexpressing lines. To date, however, the genes of the X chromosome have been under-represented in the screens performed. In order both to characterize several X-linked genes of prime interest to our laboratories and contribute to the collection of lethal P-insertions available to the community, we performed a P-insertion mutagenesis of the X chromosome. Using the PlacW and PGawB P-elements as mutagens, we generated two complementary sets of enhancer-trap lines, l(1)(T)PL and l(1)(T)PG, respectively, which both contain a reporter gene whose developmental expression can be monitored when driven by nearby enhancer sequences. We report here the characterization of 260 new insertions, mapping to 133 different genes or predicted CGs. Of these, 83 correspond to genes for which no lethal mutation had yet been reported. For 64 of those, we could confirm that lethality was solely due to the P-element insertion. The primary molecular data, reporter gene expression patterns (observed in embryos, third instar larvae and adult ovaries) and proposed CG assignment for each strain can be accessed and updated on our website at the following address: http://www-cbd.ups-tlse.fr:8080/screen.

Animals↗
PubMed1993

Cooperative stabilization of Escherichia coli ribonuclease HI by insertion of Gly-80b and Gly-77-->Ala substitution.

The insertion of a Gly residue (designated as Gly-80b) between the C-cap of the alpha II-helix (Gln-80) and the N-cap of the alpha III-helix (Trp-81) in Escherichia coli ribonuclease HI enhances the protein stability by 0.4 kcal/mol in delta G (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., & Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Another mutation within the alpha II-helix, Gly-77-->Ala, reduces the stability by 0.9 kcal/mol. Simultaneous introduction of these mutations enhances the stability by 0.8 kcal/mol, indicating that the effects of these mutations are cooperative and not simply independent. We determined the crystal structures of these three mutant proteins (G80b-, A77-, and A77/G80b-RNase H) to investigate this cooperative mechanism of the protein stabilization. The structures revealed that the inserted Gly-80b assumes a left-handed helical conformation in both the G80b- and the A77/G80b-RNase H. This inserted glycine residue allows the formation of a "paperclip", which is a common motif at the C-termini of alpha-helices. Accompanying the formation of the paperclip motif, two intrahelical hydrogen bonds are formed between the backbone atoms (O78-N80b and O80b-N84). The stabilization caused by the insertion of Gly-80b can be ascribed to the formation of these hydrogen bonds. The Gly-77-->Ala substitution destabilizes the protein due to the deformed packing interactions in the hydrophobic core around Ala-77 and the stress in the wedged indole ring of Trp-81. These effects are alleviated by the insertion of Gly-80b, which relaxes the backbone structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed2003

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome.

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5'-upstream regions, coding sequences and 3'-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5'-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.

Arabidopsis↗
PubMed2006

Tumor suppressor gene identification using retroviral insertional mutagenesis in Blm-deficient mice.

Retroviral insertional mutagenesis preferentially identifies oncogenes rather than tumor suppressor (TS) genes, presumably because a single retroviral-induced mutation is sufficient to activate an oncogene and initiate a tumor, whereas two mutations are needed to inactivate a TS gene. Here we show that TS genes can be identified by insertional mutagenesis when the screens are performed in Blm-deficient backgrounds. Blm-deficient mice, like Bloom syndrome patients, have increased frequencies of mitotic recombination owing to a mutation in the RecQ protein-like-3 helicase gene. This increased mitotic recombination increases the likelihood that an insertional mutation in one allele of a TS gene will become homozygoused by non-sister chromatid exchange and the homozygosity of the insertion provides a marker for identifying the TS gene. We also show that known as well as novel TS genes can be identified by insertional mutagenesis in Blm-deficient mice and identify two JmjC family proteins that contribute to genome stability in species as evolutionarily diverse as mammals and Caenorhabditis elegans.

Adenosine Triphosphatases↗
PubMed1996

Linker insertion scanning of regA, an activator of exotoxin A production in Pseudomonas aeruginosa.

RegA is a transcriptional activator that controls exotoxin A (ETA) production in Pseudomonas aeruginosa. To date, functional assays performed with the purified protein have not clearly defined the molecular mechanism of action of RegA. In this study, we sought to identify important coding regions of regA by analysing the sequences around linker insertion mutations in regA that affected toxA transcription. First, we constructed a strain with the regAB locus deleted from the chromosome, PA103 delta regAB::Gm. toxA transcription was obliterated in strain PA103 delta regAB::Gm, demonstrating that the regAB locus is essential for ETA production. Next, we constructed a series of 6 bp linker insertion mutations distributed throughout regA. These regA linker insertion mutants were sequenced and screened in PA103 delta regAB::Gm for their effects on regulation of ETA production. Six linker insertion mutations occurring between amino acids (aa) 53 and 163 of RegA were isolated that resulted in depression of toxA transcription to varying levels relative to the parental regAB locus. One of these linker insertion mutations (pTR53), resulted in a lack of iron-regulated ETA production and occurred directly upstream from a predicted transmembrane alpha-helix. The other five linker mutations (pTR88, pTR124, pTR132, pTR132-2 and pTR163) occurred within or flanked a region of RegA between aa 87-142 with similarity to the transcriptional activation domains of ToxR, VirG and OmpR. These data suggest the presence of a previously unidentified transcriptional activation domain in RegA between aa 87-142 and implicate the predicted transmembrane alpha-helix in the N-terminus as being involved in sensory transduction.

ADP Ribose Transferases↗
PubMed1996

Isolation and characterization of insertional mutations in flagellin genes in the archaeon Methanococcus voltae.

Methanococcus voltae is a flagellated member of the Domain Archaea that has four flagellin genes arranged in two transcriptional units. One transcriptional unit encodes only flaA while the second is a multi-cistronic unit encoding three flagellin genes (flaB1, flaB2, and flaB3) as well as at least seven other open reading frames downstream. The polymerase chain reaction was used to amplify an internal fragment of the flaA gene which was subsequently cloned into an insertion vector developed for M. voltae. Transformation of protoplasts with this vector led to the isolation of mutant strains that had insertions in flaA or flaB2. Mutant strains carrying insertions in flaA had flagelia that were similar to wild-type cells in both number and appearance when viewed using the electron microscope. In addition, some of these mutant strains had profiles identical to the wild type in immunoblots developed with antisera raised against the 31 kDa flagellin of M. voltae. All flaA mutant strains and the wild-type cells showed immuno-cross-reactive bands at 33 and 31 kDa (corresponding to purified flagellins) as well as at 18 kDa. Some flaA mutant strains also showed an immuno-cross-reactive band at 27 kDa which probably represents a truncated flagellin produced by the insertion vector. However, both types of flaA mutant strains were less motile than the wild type in semi-swarm plate experiments. The mutant strain with an insertion in flaB2 was non-flagellated when examined by electron microscopy and it was non-motile in semi-swarm plate experiments. It represents the first structural mutant strain isolated in a methanogen. This mutant strain lacked the 33, 31, and 18 kDa immuno-cross-reactive bands observed in the wild type and flaA mutant strains, and instead had a novel band at 20 kDa. This band may represent an unmodified flagellin which still has an attached leader peptide. If so, then one of the downstream genes in the multi-cistronic transcriptional unit may encode a leader peptidase for the flagellin system.

Archaeal Proteins↗