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PubMed2005

Insertion- and deletion-associated genetic diversity of Mycobacterium tuberculosis phospholipase C-encoding genes among 106 clinical isolates from Turkey.

Bacterial phospholipase C has been reported to play a role in the pathogenesis of many bacteria. In order to gain a better understanding of the potential role of Mycobacterium tuberculosis phospholipase C in the pathogenesis of human tuberculosis, we investigated the genetic diversity of the four M. tuberculosis phospholipase C-encoding genes (plcA, plcB, plcC, and plcD) resulting from the IS6110 insertion and associated deletion, among 106 clinical isolates obtained from Turkey, by using PCR, Southern hybridization, and DNA sequencing. Two sequenced M. tuberculosis strains, H37Rv and CDC1551, were used as the references in the comparison. Sixty-six (62.3%) of the 106 isolates had an intact plcD gene, and 40 (37.7%) showed an interruption of the gene. Of the latter 40 isolates, 19 (47.5%) had an IS6110 insertion with no associated deletion in the plcD gene, 2 (5%) had an IS6110 insertion and an associated deletion within the plcD gene, 15 (37.5%) had an IS6110 insertion in the plcD gene that was associated with a partial deletion of the plcD gene and its right forward adjacent region, and 4 (10%) had a complete deletion of the plcD gene. The proportions of the isolates with an interrupted plcA, plcB, or plcC gene were 3.8, 1.9, and 3.8%, respectively. The data indicate that there is a much higher frequency of IS6110 insertion and deletion in the plcD gene than in the plcA, plcB, and plcC genes of M. tuberculosis.

Bacterial Proteins↗
PubMed1996

Retroviral insertional activation in a herpesvirus: transcriptional activation of US genes by an integrated long terminal repeat in a Marek's disease virus clone.

Insertional activation of host proto-oncogenes has been recognized as a basic mechanism by which nonacute retroviruses induce cancer. Our previous work has demonstrated that retroviruses can efficiently integrate into DNA virus genomes. Specifically, coinfection of cultured fibroblasts with a chicken herpesvirus, Marek's disease virus (MDV), and a chicken retrovirus results in frequent stable retroviral insertions into the herpesvirus genome. Such insertions could alter the expression of herpesvirus genes, possibly resulting in novel phenotypic properties. In this article, we report the characterization of a replication-competent clone of MDV with integrated retroviral sequences. This virus was isolated from a chicken following injection of fibroblasts coinfected with MDV and the retrovirus, reticuloendotheliosis virus. Transcripts originating from the reticuloendotheliosis virus long terminal repeat promoters were found to encode the adjoining MDV genes, SORF2, US1, and US10. This virus replicates well in culture but has an unusual phenotype in chickens, characterized by an attenuated virulence which produces no nerve lesions but, rather, severe thymic atrophy. While the causal relationship between the insertion and the observed phenotypes remains to be established, our data provide the first evidence of retroviral insertional activation of herpesvirus genes.

Amino Acid Sequence↗
PubMed1998

Insertion of a sequence encoding light chain 3 of microtubule-associated proteins 1A and 1B in a pestivirus genome: connection with virus cytopathogenicity and induction of lethal disease in cattle.

Pestiviruses represent the first RNA viruses for which recombination with cellular protein-coding sequences has been reported. As a result of such recombinations cytopathogenic (cp) pestiviruses can develop from noncytopathogenic (noncp) viruses. In the case of bovine viral diarrhea virus (BVDV), the generation of cp mutants is linked to the induction of the lethal syndrome mucosal disease (MD) in cattle. The cp BVDV JaCP was isolated from an animal which had come down with MD. The genome of JaCP contains a novel kind of cellular insertion (LC3*) which is flanked by duplicated pestivirus sequences. Neither insertion nor duplication is present in the genome of the accompanying noncp virus JaNCP. As part of the viral polyprotein, the insertion in the JaCP genome is translated into a polypeptide almost identical to a fragment of light chain 3, a subunit of the microtubule-associated proteins 1A and 1B from the rat. Transient-expression studies revealed that the LC3* sequence is able to induce an additional cleavage of the viral polyprotein. The respective cleavage occurs directly downstream of the LC3*-encoded sequence and is not dependent on the NS3 serine protease. Insertion of LC3* into an infectious noncp pestivirus cDNA clone without duplicated viral sequences resulted in recovery of a defective cp virus able to replicate only in the presence of a noncp helper virus. In contrast, introduction of both insertion and duplication led to an autonomously replicating cp virus.

Amino Acid Sequence↗
PubMed2002

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by a two-amino-acid insertion in HIV-1 Vif from a nonprogressing mother and child.

We studied a 15-year-old girl, patient X, who has maintained consistently low plasma loads of human immunodeficiency virus type 1 (HIV-1) RNA, as well as normal and stable CD4(+) T-cell concentrations. She has presented no clinical manifestations of AIDS, despite having only received zidovudine monotherapy for a part of her life. Patient X's HIV-positive mother (patient Y) has also not progressed to AIDS and has never been treated with antiretroviral agents. HIV-1 isolated from patient X replicated poorly in human peripheral blood mononuclear cells (PBMC). In order to map the determinant of the poor growth of patient X's isolate, viral sequences from patient X were determined and examined for insertion or deletion mutations. These sequences contained a two-amino-acid insertion mutation in the Vif gene, which was also observed in uncultured PBMC acquired at different times. Furthermore, Vif sequences harbored by patient Y contained the identical mutation. These observations suggest that polymorphic HIV-1 was transmitted to patient X perinatally 15 years previously and has been maintained since that time. Recombinant HIV-1, engineered with Vif sequences from patient X, replicated in PBMC to levels approximately 20-fold lower than that of wild type. Removal of the insertion mutation from this recombinant restored replication efficiency to wild-type levels, while introduction of the insertion mutation into wild-type Vif sequences resulted in greatly decreased replication. Furthermore, Vif protein from patient X's HIV-1 was aberrantly cleaved, suggesting a mechanism for loss of Vif function. Since HIV-1 containing these sequences replicates poorly, the implication is that the two-amino-acid insertion mutation in Vif contributes significantly to the nonprogressor status of this mother and child. Further studies of these sequences might provide information regarding contributions of Vif structure and/or function to HIV-1 virulence.

Adolescent↗
PubMed1999

Use of a recombination reporter insert to define meiotic recombination domains on chromosome III of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, meiotic recombination is initiated by DNA double-strand breaks (DSBs). DSBs usually occur in intergenic regions that display nuclease hypersensitivity in digests of chromatin. DSBs are distributed nonuniformly across chromosomes; on chromosome III, DSBs are concentrated in two "hot" regions, one in each chromosome arm. DSBs occur rarely in regions within about 40 kb of each telomere and in an 80-kb region in the center of the chromosome, just to the right of the centromere. We used recombination reporter inserts containing arg4 mutant alleles to show that the "cold" properties of the central DSB-deficient region are imposed on DNA inserted in the region. Cold region inserts display DSB and recombination frequencies that are substantially less than those seen with similar inserts in flanking hot regions. This occurs without apparent change in chromatin structure, as the same pattern and level of DNase I hypersensitivity is seen in chromatin of hot and cold region inserts. These data are consistent with the suggestion that features of higher-order chromosome structure or chromosome dynamics act in a target sequence-independent manner to control where recombination events initiate during meiosis.

Argininosuccinate Lyase↗
PubMed2005

Tandem insertions of Alu elements.

Alu elements are non-autonomous, non-LTR retroposons that represent the most abundant mobile elements in the human genome (1.1 x 10(6) copies/genome). They preferentially insert adjacent to existing Alu elements. It has been proposed that Alu elements utilize LINE-1 machinery for their retroposition. The LINE-1 endonuclease cleaves at a loose consensus sequence. We have utilized a bioinformatics approach to show the order of insertion of pairs of young (Y) and old (S or J) Alu subfamily members. Our data suggest that the consensus LINE-1 endonuclease cleavage site used for insertion of the old Alu elements can be reused for integration of the younger ones inserting adjacent to them. However, there is also a preference at the 3' end of Alu into a non-ideal cleavage site that may represent unique properties of the A-tail for integration. Alu elements inserting adjacent to one another may suggest the saturation of the optimal integration sites with existing Alu elements, rather than any innate preference for Alu elements to integrate adjacent to other Alus.

Alu Elements↗
PubMed1995

Deletion and insertion mutations in short tandem repeats in the coding regions of human genes.

In vitro studies in bacterial, yeast and eukaryotic systems have demonstrated the existence of deletion and insertion 'hot-spots' involving repetitive sequences. Slipped-strand mispairing (SSM) has been suggested to be the mechanism involved. Progress in human molecular genetics has allowed the identification of many mutations causing diseases. Analysis of sequences involved in these mutations provides an opportunity to investigate the contribution of short tandem repeats to the naturally occurring mutations in coding regions of human genes. We have analyzed the sequences surrounding 625 disease-causing mutations in the coding regions of three genes: the cystic fibrosis transmembrane conductance regulator, beta globin and factor IX. Altogether, 134 (21%) insertion and deletion mutations of 4 base pairs or less were identified. In 47% of these mutations, the deletions and insertions occurred within a unit repeated tandemly 2- to 7-fold. These were classified as SSM mutations. The proportion of SSM mutations was significantly higher than expected by chance. The estimated net proportion of deletion and insertion mutations attributed to SSM was 27%. These results indicate that very short repetitive sequences contribute significantly to the generation of deletion and insertion mutations in human genes, and to the evolution of diversity of their coding regions.

Base Composition↗
PubMed2002

A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction.

BACKGROUND: The jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. RESULTS: We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, alphas. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. CONCLUSION: This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.

Cell Line↗
PubMed2003

Novel prion protein insert mutation associated with prolonged neurodegenerative illness.

BACKGROUND: Mutations in the prion protein gene (PRNP) are found in approximately 13 to 15% of persons classified as dying from a transmissible spongiform encephalopathy. Point and octapeptide repeat insert and deletion mutations are described in the open reading frame (ORF) of PRNP. The authors present a clinicopathologic study of a patient with a family history of a lengthy and progressive neurodegenerative disorder associated with a novel large octapeptide repeat insert mutation. METHODS: Neuropathologic examination, including immunohistochemistry for the prion protein, was undertaken. The ORF of PRNP was amplified by PCR, cloned, and sequenced. Homogenate of cerebral tissue underwent Western blot analysis for the prion protein before and after proteinase K treatment. RESULTS: The proband died after a 16-year illness commencing at age 29 years. Confident premortem clinical diagnosis was not achieved despite a brain biopsy. Autopsy examination of the brain confirmed a spongiform encephalopathy. Prion protein immunohistochemistry revealed occasional granular deposits in the cerebellar granular layer. The proband was found to harbor a novel PRNP 168 base pair (bp) insert mutation. CONCLUSION: The authors have identified a novel 168 bp octapeptide repeat insert mutation. Prion protein immunohistochemistry differs from previous cases harboring seven octapeptide repeat and other long insert mutations. Optimization of PRNP analysis, especially PCR conditions, is essential to avoid overlooking this type of mutation and delay the correct molecular genetic diagnosis.

Adult↗
PubMed1992

Insertional mutagenesis in Drosophila. II. P element mediated transformation of Drosophila yakuba.

Drosophila yakuba, a member of melanogaster subgroup being free of P element, acquired resistance to an antibiotic neomycin by the transformation utilizing P element. In this species, the transformation frequency was comparable to that of D. melanogaster. Further, the occurrence of 8 base pairs duplication upon the insertion of the element was confirmed. These facts suggest that the P element could be inserted into the genome in the same manner, even in D. yakuba. Any consensus for preferential insertion could not be found on the nucleotide sequence as in D. melanogaster. However, it is noticeable that a series of the short palindromic stretches was common around the insertion sites in both species. It suggests that a structural feature of DNA plays a role as a landmark for P element insertion.

Animals↗
PubMed2004

Insertions of mini-Tn10 transposon T-POP in Salmonella enterica sv. typhi.

We have mutagenized a clinical strain of Salmonella enterica sv. typhi with mini-transposon Tn10dTet (T-POP) to obtain conditional lethal (tetracycline-dependent) mutants with T-POP insertions upstream of essential genes. Generalized transducing phage P22 was used to introduce T-POP from a S. typhimurium donor into a S. typhi recipient. Chromosomal DNA was purified from the mutagenized donor strains, fragmented, and then electroporated into S. typhi to backcross the original T-POP insertions. Four tetracycline-dependent mutants with two distinct terminal phenotypes were found among 1700 mutants with T-POP insertions. When grown in the absence of tetracycline, two of the four tetracycline-dependent mutants arrest at a late stage in the cell cycle, can be rescued by outgrowth in media with tetracycline, and define a reversible checkpoint late in the cell cycle. One of these insertions creates an operon fusion with a gene, yqgF, that is conserved among gram-negative bacteria and likely encodes an essential Holliday junction resolvase. T-POP insertions can be used not only to identify essential S. typhi genes but also to reveal novel phenotypes resulting from the depletion of their products.

Bacteriophage P22↗
PubMed2002

B cell development arrest upon insertion of a neo gene between JH and Emu: promoter competition results in transcriptional silencing of germline JH and complete VDJ rearrangements.

Previous targeting experiments within the IgH locus have shown that V(D)J recombination was affected by an insertion of a neo gene within E(mu) upstream of the core enhancer, but not by insertions downstream of the enhancer. Similarly, class switch recombination to a given (C) gene was affected only by interposition of neo in between that gene and the 3' IgH enhancers. Here we show that insertion of neo upstream E(mu) only marginally impairs V(D)J recombination, but results in an altered D and J(H) gene usage and completely blocks transcription of the germline J(H) region and the rearranged VDJ segments. Although transcriptional silencing of J(H) occurs upstream of the insertion and results in the lack of mature B cells in homozygous mutant animals, IgH transcription is maintained downstream of the insertion together with neo transcription and can be up-regulated by LPS stimulation or upon fusion with plasmacytoma cells. Altogether these data argue for a polarized "neo effect" involving promoter competition and further show that V(D)J rearrangement can be uncoupled from transcription.

Alleles↗
PubMed1999

[Mutation by deletion-insertion in BRCA-1 gene in three unrelated French breast/ovarian cancer families: possible implication of a mobile element].

A new type of mutation by deletion-insertion in BRCA-1 gene is found in three unrelated French breast/ovarian cancer families. Surprisingly, deletion and insertion occurred at the same nucleotide position at the end of exon 11 (3958del5ins4), thus generating a truncated protein. This original mutation consists in a deletion of 5 bp (CTCAG) and in an insertion of 4 different bp (AGGC). Here, we proposed two hypothesis to explain this phenomenom. The first hypothesis is the formation of a hairpin stem-loop structure comprising the mutational site and the sequence corresponding to the duplication insertion 2 nucleotides before the mutation. The second hypothesis, more speculative, consists in an abortive integration of a human mobile element as a human transposon (tigger 1) which involved a deletion of 5 bp during its excision and an insertion of 4 bases corresponding to the 5' extremity of the transposon.

Adult↗
PubMed1981

A comparison of the initial pain response following insertion of the Copper 7 and combined Multiload Copper 250-short IUDs.

The immediate postinsertion and 3 minute postinsertion pain response in nulliparous women undergoing insertion of either the Copper 7 (Cu 7) or the Multiload Copper 250-short (ML Cu 250-short) IUD has been studied. 35 women were fitted with a Cu 7 device and 23 with the ML Cu 250-short. The immediate postinsertion pain scores were significantly higher in the ML Cu 250-short group than in the Cu 7 group. There was no significant difference between the 3 minutes postinsertion scores for the 2 groups. The 3 minute pain scores were significantly lower than the immediate pain scores for each individual device group. Immediate postinsertion IUD pain may be related to the presenting diameter and insertional mechanism of the device. This pain is of short duration and after 3 minutes, the level of pain is independent of the type of device which has been inserted. The rapid diminution of pain at 3 minutes postinsertion indicates that the routine use of certain analgesic techniques, e.g., paracervical block before IUD insertion, is not justified. Pain following IUD insertion may provide a useful model for the study of various types of peripheral and centrally acting analgesics.

Chemical Phenomena↗
PubMed2003

[An insertion mutant of LeuRS with 116 amino acid residues has full activity].

Escherichia coli leucyl-tRNA synthetase (LeuRS) belongs to class I aminoacyl-tRNA synthetases. It consists of 860 amino acid residues and catalyzes the leucylation of tRNA(Leu). An insertion of its 253-368 peptide fragment between 368 to 369 in CP1 domain of this enzyme was shown to maintain the activity of the enzyme, and the insertion mutant was named as LeuRS-C. Because the insertion mutant of LeuRS was sensitive to operation of the purification, a plasmid containing the gene encoding LeuRS with His(6)-tag at its N-terminus was constructed to facilitate the purification of His(6)-LeuRS-C through one-step affinity chromatography on Ni(2+)-NTA column. The purified His(6)-LeuRS-C had full activity as the native LeuRS with His-tag at the N-terminus (His(6)-LeuRS), although the mutant enzyme had an insertion of 116 amino acid residues. The kinetic parameters of His(6)-LeuRS-C were determined. The secondary structure estimated by CD spectrum and thermal stability of the insertion mutant was compared with those of His(6)-LeuRS, respectively.

Circular Dichroism↗
PubMed1992

Disruption of the APC gene by a retrotransposal insertion of L1 sequence in a colon cancer.

The APC gene is responsible for familial adenomatous polyposis and is considered to be a tumor suppressor gene associated with development of sporadic colorectal tumors. Here we report the disruption of the APC gene caused by somatic insertion of a long interspersed repetitive element (LINE-1 sequence) into the last exon of the APC gene in a colon cancer. The inserted sequence was composed of a 3' portion of the LINE-1 consensus sequence and nearly 180 base pairs of polyadenylate tract. Furthermore, since an 8-base pair target site duplication was observed, retrotranscriptional insertion of an active LINE-1 sequence is suspected as the cause of this insertion event. This is the first report of the disruption of a tumor suppressor gene caused by somatic insertion of a mobile genetic element.

Adenomatous Polyposis Coli↗
PubMed1985

A comparison of the actual and expected pain response following insertion of an intrauterine contraceptive device.

The expected, immediate, and three-minute pain response following IUCD insertion was evaluated in 84 nulliparous women undergoing a first IUCD insertion. Expected pain was significantly higher than immediate pain and both were significantly higher than three-minute pain. Immediate pain following IUCD insertion was positively related to expected pain but the relationship was not strong enough to be of clinical value. The pain was significantly related to the degree of cervical resistance and this relationship was made much more obvious by the use of the expected pain parameter which is a valuable additional clinical measurement in pain research. IUCD insertion pain consists of a short cervical (less than 3 minutes) and longer fundal component. Avoiding excessive uterine manipulation during device insertion results in lower immediate and later pain response scores for a given device when comparisons are made with other studies, where standard techniques were used.

Adolescent↗
PubMed1981

Pain response following insertion of a Gravigard (Copper-7) intrauterine contraceptive device in nulliparous women.

A total of 33 nulliparous women aged between 18 and 35 have been observed for related symptoms following insertion of an intrauterine contraceptive device. Seventy-five percent suffered some abdominal pain initially but this was reduced to 1% by the end of the week. The symptoms of cramp and backache showed a similar trend. There was no relationship between symptoms and the day of the cycle on which insertion was performed. It is concluded that careful insertion of the Copper-7 in this group of patients is well tolerated irrespective of the day of cycle on which insertion is performed. The initial dynamic response following IUCD insertion may be an additional factor in the assessment of the performance of an individual intrauterine contraceptive device.

Abdomen↗