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Results for “insertion, inversion”

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PubMed1983

Characterization of a viable, noninverting herpes simplex virus 1 genome derived by insertion and deletion of sequences at the junction of components L and S.

Earlier studies have shown that the DNA of herpes simplex virus 1 consists of two covalently linked components, L and S, each flanked by inverted repeats. The two components can invert, and viral DNA extracted from infected cells or virions consists of equimolar concentrations of four populations differing solely in the orientation of L and S components relative to each other. This paper describes a recombinant virus (1358) generated by an insertion of a chimeric thymidine kinase gene within the reiterated sequences of the S component and deletions that eliminated most of the internal inverted repeats at the junction between the L and S components. A characteristic of 1358 is that the L and S components are frozen in one (prototype) orientation. Inversion of L and S components is therefore not required for the replication of viral DNA.

Chromosome Inversion↗

PubMed1995

The dif resolvase locus of the Escherichia coli chromosome can be replaced by a 33-bp sequence, but function depends on location.

The dif locus (deletion-induced filamentation) of Escherichia coli is a resolvase site, located in the terminus region of the chromosome, that reduces chromosome multimers to monomers. In strains in which this site has been deleted, a fraction of the cells is filamentous, has abnormal nucleoid structure, and exhibits elevated levels of the SOS repair system. We have demonstrated that a 33-bp sequence, which is sufficient for RecA-independent recombination and which shows similarity to the cer site of pColE1, suppresses the Dif phenotype when inserted in the terminus region. Flanking sequences were not required, since suppression occurred in strains in which dif as well as 12 kb or 173 kb of DNA had been deleted. However, location was important, and insertions at a site 118 kb away from the normal site did not suppress the Dif phenotype. These sites were otherwise still functional, and they exhibited wild-type levels of RecA-independent recombination with dif-containing plasmids and recombined with other chromosomal dif sites to cause deletions and inversions. It is proposed that the functions expressed by a dif site depend on chromosome location and structure, and analysis of these functions provides a way to examine the structure of the terminus region.

Base Sequence↗

PubMed1999

A single negatively charged residue affects the orientation of a membrane protein in the inner membrane of Escherichia coli only when it is located adjacent to a transmembrane domain.

The orientation of membrane proteins is determined by the asymmetric distribution of charged residues in the sequences flanking the transmembrane domains. For the inner membrane of Escherichia coli, numerous studies have shown that an excess of positively charged residues defines a cytoplasmic domain of a membrane protein ("positive inside" rule). The role of negatively charged residues in establishing membrane protein topology, however, is not completely understood. To investigate the influence of negatively charged residues on this process in detail, we have constructed a single spanning chimeric receptor fragment comprising the N terminus and first transmembrane domain of the heptahelical G protein-coupled vasopressin V(2) receptor and the first cytoplasmic loop of the beta(2)-adrenergic receptor. When fused to alkaline phosphatase (PhoA), the receptor fragment inserted into the inner membrane of E. coli with its N terminus facing the cytoplasm (N(in)-C(out) orientation), although both membrane-flanking domains had rather similar topogenic determinants. The orientation of the receptor fragment was changed after the introduction of single glutamate residues into the N terminus. Orientation inversion, however, was found to be dependent on the location of the glutamate substitutions, which had to lie within a narrow window up to 6 residues distant from the transmembrane domain. These results demonstrate that a single negatively charged residue can play an active role as a topogenic determinant of membrane proteins in the inner membrane of E. coli, but only if it is located adjacent to a transmembrane domain.

Alkaline Phosphatase↗

PubMed1990

Genetic and molecular analysis of vgU and vgW: two dominant vg alleles associated with gene fusions in Drosophila.

In the absence of a vg+ gene, extensive cell death occurs in third instar imaginal discs, which results in a complete loss of adult wing margin structures. Essentially all molecularly characterized vg alleles are associated with deletions or insertions of DNA into the vg locus. These alterations reduce or eliminate a 3.8-kb vg-specific transcript, resulting in recessive loss of function alleles. We report here the analysis of two dominant vg alleles which have been identified (vgU and vgW). The vgU allele is associated with a chromosomal inversion which splits the vg locus, resulting in a gene fusion between vg and the mastermind (mam) neurogenic locus. Reversion analysis of vgU indicates that sequences from the mam locus are required for vgU dominance. The vgW allele is also the result of a chromosomal inversion, in this case resulting in a gene fusion between vg and the homeobox-containing invected (inv) gene. It is also associated with novel dominant homeotic transformations. Revertant analysis indicates that sequences from inv are required for the dominant wing and dominant homeotic effects of vgW. The vg dominance does not appear to be mediated through a reduction of vg expression or a novel fusion transcript in either vgU or vgW. The results are consistent with a model in which inappropriate expression of inv causes the dominant homeotic effects seen in vgW.

Alleles↗

PubMed1994

Endpoint bias in large Tn10-catalyzed inversions in Salmonella typhimurium.

A genetic strategy identified Salmonella typhimurium strains carrying large (> 40 kb) Tn 10-catalyzed inversions; the inverted segments were characterized by XbaI digestion and pulsed field gel electrophoresis. Two size classes of large inversions were found. More than half of the inversions extended 40-80 kb either clockwise or counterclockwise of the original Tn10 site. The remaining inversions extended up to 1620 kb (33% of the genome), but the distal endpoints of these inversions were not randomly scattered throughout the chromosome. Rather, each Tn10 repeatedly yielded similar (though not identical) inversions. The biased endpoint selection may reflect the limited search for target DNA sequences by the Tn10 transposase, and the spatial proximity of the donor and target regions in the folded S. typhimurium nucleoid. Using this interpretation, the data suggest that DNA sequences 40-80 kb clockwise and counterclockwise of the insertion site are in spatial proximity with the insertion, perhaps reflecting the organization of DNA into approximately 120-kb nucleoid domains. In addition, the data predict the spatial proximity of several distant DNA regions, including DNA sequences equidistant from the origin of DNA replication.

Base Sequence↗

PubMed2000

Aberrant splicing and altered spatial expression patterns in fruitless mutants of Drosophila melanogaster.

The fruitless (fru) gene functions in Drosophila males to establish the potential for male sexual behaviors. fru encodes a complex set of sex-specific and sex-nonspecific mRNAs through the use of multiple promoters and alternative pre-mRNA processing. The male-specific transcripts produced from the distal (P1) fru promoter are believed to be responsible for its role in specifying sexual behavior and are only expressed in a small fraction of central nervous system (CNS) cells. To understand the molecular etiology of fruitless mutant phenotypes, we compared wild-type and mutant transcription patterns. These experiments revealed that the fru(2), fru(3), fru(4), and fru(sat) mutations, which are due to P-element inserts, alter the pattern of sex-specific and sex-nonspecific fru RNAs. These changes arise in part from the P-element insertions containing splice acceptor sites that create alternative processing pathways. In situ hybridization revealed no alterations in the locations of cells expressing the P1-fru-promoter-derived transcripts in fru(2), fru(3), fru(4), and fru(sat) pharate adults. For the fru(1) mutant (which is due to an inversion breakpoint near the P1 promoter), Northern analyses revealed no significant changes in fru transcript patterns. However, in situ hybridization revealed anomalies in the level and distribution of P1-derived transcripts: in fru(1) males, fewer P1-expressing neurons are found in regions of the dorsal lateral protocerebrum and abdominal ganglion compared to wild-type males. In other regions of the CNS, expression of these transcripts appears normal in fru(1) males. The loss of fruitless expression in these regions likely accounts for the striking courtship abnormalities exhibited by fru(1) males. Thus, we suggest that the mutant phenotypes in fru(2), fru(3), fru(4), and fru(sat) animals are due to a failure to appropriately splice P1 transcripts, whereas the mutant phenotype of fru(1) animals is due to the reduction or absence of P1 transcripts within specific regions of the CNS.

Amino Acid Sequence↗

PubMed1994

IS6770, an enterococcal insertion-like sequence useful for determining the clonal relationship of clinical enterococcal isolates.

Enterococci expressing resistance to antimicrobial agents are increasingly important nosocomial pathogens. Effective strategies to prevent or abort outbreaks of resistant enterococcal infection will rely on an accurate understanding of the mechanisms by which these organisms spread. A 1065-bp insertion-like sequence (IS6770) is present in varying copy numbers in > 90% of enterococcal strains thus far examined. Hybridization patterns resulting from hybridization of enterococcal genomic DNA with an internal IS6770 probe vary considerably between unrelated strains and correlate well with results of pulsed-field gel electrophoresis and field-inversion gel electrophoresis in identifying clonal relationships among enterococcal isolates. IS6770 analysis of several outbreaks of resistant enterococci has confirmed the spread of single resistant clones rather than the emergence of resistance within the resident flora. These results suggest that IS6770 hybridization will be a useful tool for tracing the epidemiology of nosocomial enterococcal infections.

Amino Acid Sequence↗

PubMed2001

Orientational control of fimE expression in Escherichia coli.

Phase-variable expression of type 1 fimbriae is, in part, controlled by site-specific DNA inversion of the fim switch in Escherichia coli. Of the two fim recombinases (FimB and FimE) that catalyse the inversion reaction, FimE exhibits a strong bias for phase switching from the ON to the OFF orientation. The specificity associated with fimE is the result of two different mechanisms: (i) FimE exhibits a preference for the invertible element in the ON orientation as substrate for recombination; (ii) the invertible element in the OFF orientation acts in cis to inhibit recombinase activity (orientational control). We show here that the invertible element negatively regulates fimE, even though expression of a fimE-lacZYA transcriptional fusion is unaffected by orientational control. The fimE transcript extends into the invertible region and hence switch ON-specific and switch OFF-specific mRNA contain different sequences. Furthermore, we show that orientational control is suppressed by the insertion of a structured RNA (tRNA(Gly)) between fimE and the fim switch, indicating that the switch OFF-specific mRNA is inactivated by 3' to 5' degradation. Analysis of the fim switch reveals that it contains two inhibitory elements that exert orientational control independently.

Bacterial Proteins↗

PubMed2005

Engrailed-ZmOCL1 fusions cause a transient reduction of kernel size in maize.

ZmOCL1 is the founding member of the ZmOCL (Outer Cell Layer) family encoding putative transcription factors of the HD-ZIP IV class. It is expressed in the L1 cell layer of the embryo and several other tissues of maize. After determination of the intron/exon structure a mutator insertion was isolated in the upstream region. No notable phenotypes and wildtype levels of ZmOCL1 transcript were observed in homozygous mutant plants. In contrast transgenic plants carrying a fusion of the repressor domain of the Drosophila Engrailed gene with the DNA binding and dimerisation domains of ZmOCL1 showed a transient reduction of embryo, endosperm and kernel size that was most obvious around 15 DAP. An inverse relationship was observed between the degree of size reduction and the expression level of the transcript. In reciprocal crosses the size reduction was only observed when the transgenic plants were used as females and no expression of male transmitted transgenes was detected. Smaller kernels resembled younger kernels of wild-type siblings indicating that interference with ZmOCL1 function leads to an overall slow-down of early kernel development. Based on marker gene analysis ZmOCL1 may act via a modification of gibberellin levels. Phylogenetic analyses based on the intron/exon structure and sequence similarities of ZmOCL1 and other HD-ZIP IV proteins from maize, rice and Arabidopsis helped to identify orthologues and suggested an evolution in the function of individual genes after the divergence of monocots and dicots.

Binding Sites↗

PubMed1982

Studies on the expression of outer membrane protein 2 in escherichia coli.

The relative level of protein 2 expressed in the outer membrane of strains of Escherichia coli K-12 lysogenized with bacteriophage PA-2 was found to be influenced by both the growth temperature and lc+ gene dosage. An increase in either of these parameters was accompanied by an increase in the level of protein 2 up to an apparent saturation level. Any increase in the amount of protein 2 was accompanied by a concomittant decrease in the amount of OmpF and OmpC porins. This inverse relationship led to the maintenance of an approximately constant protein mass per unit of peptidoglycan. Our results are discussed in light of recent genetic studies on the regulation of the OmpF and OmpC porins and can be explained through the competition of these three matrix proteins for a common export or insertion site.

Bacterial Outer Membrane Proteins↗

PubMed2003

Genotype and phenotype of haemophilia A in Thai patients.

To study genotype and phenotype correlation of haemophilia A in Thai patients, molecular defects of the factor VIII (FVIII) gene were examined and their correlation with clinical phenotypes were evaluated. The molecular pathologies of FVIII in Thai patients were found to be heterogeneous. The most common mutation was FVIII intron 22 inversion accounting for about 30% of the severe cases while gene deletion was rare. Sixteen point mutations were identified, comprising two nonsense mutations (R-5X and R1966X), five missense mutations (T233I, D542Y, G1850V, W2229S and G2325C), five nucleotide deletions (1145delT, 1187-8delACAC, 1191-4delA, 1458delGA and 1534delA), three nucleotide insertions (1439-41insA, 1934insTA and 2245insACTA) and one splicing defect (IVS15+1G>T). Nine mutations (T233I, D542Y, 1145delT, 1458delGA, 1534delA, 1934insTA, W2229S, 2245insACTA and G2325C) were novel, firstly identified in Thai patients. The genotypes were found to correlate with clinical phenotypes in a majority of cases. However, in five patients the molecular defects did not correlate with clinical severity and FVIII:C level. Cellular and molecular mechanisms were proposed to be responsible in amelioration of clinical severity caused by deleterious mutations. Carrier detection by direct mutation analysis was also demonstrated.

DNA Mutational Analysis↗

PubMed1998

Rapid discrimination of sequences flanking and within T-DNA insertions in the Arabidopsis genome.

An improvement to previous methods for recovering Arabidopsis thaliana genomic DNA flanking T-DNA insertions is presented that allows for the avoidance of some of the cloning difficulties caused by the concatameric nature of T-DNA inserts. The principle of the procedure is to categorize by size restriction fragments of mutant DNA, produced in separate digestions with NdeI and Bst1107I. Given that the sites for these two enzymes are contiguous within the pGV3850:1003 T-DNA construct, the restriction fragments obtained fall into two categories: those showing identical size in both digestions, which correspond to sequences internal to T-DNA concatamers; and those of different sizes, that contain the junctions between plant DNA and the T-DNA insert. Such a criterion makes it possible to easily distinguish the digestion products corresponding to internal T-DNA parts, which do not deserve further attention, and those which presumably include a segment of the locus of interest. Discrimination between restriction fragments of genomic mutant DNA can be made on rescued plasmids, inverse PCR amplification products or bands in a genomic blot.

Arabidopsis↗

PubMed2006

Genome-wide mapping of foamy virus vector integrations into a human cell line.

Integration-site selection by retroviruses and retroviral vectors has gained increased scientific interest. Foamy viruses (FVs) constitute a unique subfamily (Spumavirinae) of the family Retroviridae, for which the integration pattern into the human genome has not yet been determined. To accomplish this, 293 cells were transduced with FV vectors and the integration sites into the cellular genome were determined by a high-throughput method based on inverse PCR. For comparison, a limited number of murine leukemia virus (MLV) and human immunodeficiency virus (HIV) integration sites were analysed in parallel. Altogether, 628 FV, 87 HIV and 141 MLV distinct integration sites were mapped to the human genome. The sequences were analysed for RefSeq genes, promoter regions, CpG islands and insertions into cellular oncogenes. Compared with the integration-site preferences of HIV, which strongly favours active genes, and MLV, which favours integration near transcription-start regions, our results indicate that FV integration has neither of these preferences. However, once integration has occurred into a transcribed region of the genome, FVs tend to target promoter-close regions, albeit with less preference than MLV. Furthermore, our study revealed a palindromic consensus sequence for integration, which was centred on the virus-specific, four-base-duplicated target site. In summary, it is shown that the integration pattern of FVs appears to be unique compared with those of other retroviral genera.

Cell Line↗

PubMed1999

Contributions of genome sequencing to understanding the biology of Helicobacter pylori.

About half of the world's population carries Helicobacter pylori, a gram-negative, spiral bacterium that colonizes the human stomach. The link between H. pylori and, ulceration as well as its association with the development of both gastric cancer and mucosa-associated lymphoid tissue lymphoma in humans is a serious public health concern. The publication of the genome sequences of two stains of H. pylori gives rise to direct evidence on the genetic diversity reported previously with respect to gene organization and nucleotide variability from strain to strain. The genome size of H. pylori strain 26695 is 1,6697,867 bp and is 1,643,831 bp for strain J99. Approximately 89% of the predicted open reading frames are common to both of the strains, confirming H. pylori as a single species. A region containing approximately 45% of H. pylori strain-specific open reading frames, termed the plasticity zone, is present on the chromosomes, verifying that some strain variability exists. Frequent alteration of nucleotides in the third position of the triplet codons and various copies of insertion elements on the individual chromosomes appear to contribute to distinct polymorphic fingerprints among strains analyzed by restriction fragment length polymorphisms, random amplified polymorphic DNA method, and repetitive element-polymerase chain reaction. Disordered chromosomal locations of some genes seen by pulsed-field gel electrophoresis are likely caused by rearrangement or inversion of certain segments in the genomes. Cloning and functional characterization of the genes involved in acidic survival, vacuolating toxin, cag-pathogenicity island, motility, attachment to epithelial cells, natural transformation, and the biosynthesis of lipopolysaccharides have considerably increased our understanding of the molecular genetic basis for the pathogenesis of H. pylori. The homopolymeric nucleotide tracts and dinucleotide repeats, which potentially regulate the on- and off-status of the target genes by the strand-slipped mispairing mechanism, are often found in the genes encoding the outer-membrane proteins, in enzymes for lipopolysaccharide synthesis, and within DNA modification/restriction systems. Therefore, these genes may be involved in the H. pylori-host interaction.

Amino Acid Sequence↗

PubMed1997

Evolution of the black muntjac (Muntiacus crinifrons) karyotype revealed by comparative chromosome painting.

The black muntjac (Muntiacus crinifrons) has an unusual karyotype of 2n = 8 in females and 2n = 9 in males. We have studied the evolution of this karyotype by hybridising chromosome-specific paints derived from flow-sorted chromosomes of the Chinese muntjac (M. reevesi, 2n = 46) to chromosomes of the black muntjac. The hybridisation pattern allowed us to infer chromosomal homologies between these two species. Tandem and centromeric fusions, reciprocal translocations, and insertions are involved in the reduction of the diploid number from 2n = 46 to 2n = 8, 9. The painting patterns further show complex chromosomal rearrangements in the male black muntjac which involve more than half the karyotype, including both sex chromosomes. Since early meiosis is reported to be normal without any visible inversion loops of the synaptonemal complex, the observed chromosomal rearrangements would lead to heterosynapsis and, therefore, leave a large fraction of the male black muntjac karyotype balanced between the two sexes.

Animals↗

PubMedDate not supplied

Plantaris tendon reconstruction of the lateral ankle ligaments.

Numerous procedures have evolved to operatively stabilize the ankle suffering from chronic inversion instability. The use of the plantaris tendon to anatomically reconstruct the lateral collateral ankle ligaments is among those that avoid the complication of restriction of subtalar joint range of motion. The authors present a modified technique of reconstructing the calcaneofibular and anterior talofibular ligaments using a plantaris tendon that maintains its insertion. A retrospective study of eight patients that elected the procedure for chronic ankle instability was conducted. The average follow-up was 12.7 months. Significant improvement in functional score (p less than 0.01) and talar tilt (p less than 0.05) was noted following the procedure. No restriction of subtalar joint motion was observed in any patient after the procedure. The advantages and disadvantages of the procedure are discussed. The authors conclude that the procedure appears to be a desirable alternative in achieving ankle stability while maintaining normal hindfoot function.

Adolescent↗

PubMed1995

Mechanism of HIV persistence: implications for vaccines and therapy.

Periodic infusion of autologous HIV-antigen presenting cells (APCs), that stimulate the cytotoxic (CTL) response, while being incapable of producing virus, should lower viral burden and boost CD4+ count in HIV-seropositive individuals. Viral burden reasserts itself after antiviral therapy ceases or is interrupted for long. Therapy, therefore, would have to continue for life. These are predictions from a computer model of HIV-immune kinetics. The model equations describe the interactive kinetics of viral burden, CD4+ cell decline, neutralization of free virus by antibodies, infection of cells, and killing of infected cells by CTL. The computed trajectories of the kinetic equations reproduce the typical course of an HIV infection and the model yields several predictions that are not intuitively obvious, among them: (a) Persistence of HIV infection (failure of the immune system to clear infection) is an intrinsic property of the kinetics of the HIV-immune interaction. (b) The chronic state of infection is inherently stable, which means that the infection rebounds to the determined steady state, whenever antiviral therapy stops. (c) CTL is chronically activated, and the level correlates inversely with the avidity of neutralizing antibodies. (d) APCs have to be infused at a rate such as to boost and maintain the CTL response above the chronic level. Other therapies include CTL stimulation, via the macrophage route, by erythrocytes, into which MHC binding HIV-CTL epitope polypeptide fragments have been inserted; passive immunization, virion-trapping by CD4 analogs or CD4 expressing erythrocytes; and combination therapies with AZT, IL-2. These are also analyzed. Concerning HIV etiology, the model assumes that cells other than CD4+ cells (such as macrophages/monocytes) become infected, and contribute to the viral burden, and that infectible cells remain available even as CD4+ cells become exhausted. The model further assumes that CD4+ cells decline not only through direct killing by HIV and CTL, but by dysregulation and excess apoptosis caused by the presence of virus. The model predicts that persistence of HIV infection does not depend upon latently infected cells or escape mutants, as has been suggested.(ABSTRACT TRUNCATED AT 400 WORDS)

AIDS Vaccines↗

PubMed2005

An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1, near SOX3, causes X-linked recessive hypoparathyroidism.

X-linked recessive hypoparathyroidism, due to parathyroid agenesis, has been mapped to a 906-kb region on Xq27 that contains 3 genes (ATP11C, U7snRNA, and SOX3), and analyses have not revealed mutations. We therefore characterized this region by combined analysis of single nucleotide polymorphisms and sequence-tagged sites. This identified a 23- to 25-kb deletion, which did not contain genes. However, DNA fiber-FISH and pulsed-field gel electrophoresis revealed an approximately 340-kb insertion that replaced the deleted fragment. Use of flow-sorted X chromosome-specific libraries and DNA sequence analyses revealed that the telomeric and centromeric breakpoints on X were, respectively, approximately 67 kb downstream of SOX3 and within a repetitive sequence. Use of a monochromosomal somatic cell hybrid panel and metaphase-FISH mapping demonstrated that the insertion originated from 2p25 and contained a segment of the SNTG2 gene that lacked an open reading frame. However, the deletion-insertion [del(X)(q27.1) inv ins (X;2)(q27.1;p25.3)], which represents a novel abnormality causing hypoparathyroidism, could result in a position effect on SOX3 expression. Indeed, SOX3 expression was demonstrated, by in situ hybridization, in the developing parathyroid tissue of mouse embryos between 10.5 and 15.5 days post coitum. Thus, our results indicate a likely new role for SOX3 in the embryonic development of the parathyroid glands.

Animals↗