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Results for “insertion, inversion”

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PubMed2000

Mobile elements and chromosomal evolution in the virilis group of Drosophila.

Species of the virilis group of Drosophila differ by multiple inversions and chromosome fusions that probably accompanied, or led to, speciation. Drosophila virilis has the primitive karyotype for the group, and natural populations are exceptional in having no chromosomal polymorphisms. We report that the genomic locations of Penelope and Ulysses transposons are nonrandomly distributed in 12 strains of D. virilis. Furthermore, Penelope and Ulysses insertion sites in D. virilis show a statistically significant association with the breakpoints of inversions found in other species of the virilis group. Sixteen newly induced chromosomal rearrangements were isolated from the progeny of D. virilis hybrid dysgenic crosses, including 12 inversions, 2 translocations, and 2 deletions. Penelope and Ulysses were associated with the breakpoints of over half of these new rearrangements. Many rearrangement breakpoints also coincide with the chromosomal locations of Penelope and Ulysses insertions in the parental strains and with breakpoints of inversions previously established for other species of the group. Analysis of homologous sequences from D. virilis and Drosophila lummei indicated that Penelope insertion sites were closely, but not identically, located at the nucleotide sequence level. Overall, these results indicate that Penelope and Ulysses insert in a limited number of genomic locations and are consistent with the possibility that these elements play an important role in the evolution of the virilis species group.

Animals↗

PubMed2004

Mutagenic insertion and chromosome engineering resource (MICER).

Embryonic stem cell technology revolutionized biology by providing a means to assess mammalian gene function in vivo. Although it is now routine to generate mice from embryonic stem cells, one of the principal methods used to create mutations, gene targeting, is a cumbersome process. Here we describe the indexing of 93,960 ready-made insertional targeting vectors from two libraries. 5,925 of these vectors can be used directly to inactivate genes with an average targeting efficiency of 28%. Combinations of vectors from the two libraries can be used to disrupt both alleles of a gene or engineer larger genomic changes such as deletions, duplications, translocations or inversions. These indexed vectors constitute a public resource (Mutagenic Insertion and Chromosome Engineering Resource; MICER) for high-throughput, targeted manipulation of the mouse genome.

Animals↗

PubMed1995

Generation of 28 sequence-tagged sites (STSs) from yeast artificial chromosome (YAC) clones anchored at human chromosome 21q22.1 region.

Twenty-eight sequence-tagged sites (STSs) were newly generated from the DNA sequences of vector-insert junctions from yeast artificial chromosomes (YACs) anchored at chromosome 21q22.1 region. The insert DNAs adjacent to vector arms were specifically amplified through inverse PCR method to clone into pUC19 vector for sequencing. Sixty DNA junctions from 44 CEPH YAC clones were cloned and sequenced. Of these DNA sequences of junctions between vector-arms and DNA inserts, twenty-eight STSs were finally obtained to show the accurate amplification, which is specific for human chromosome 21. The sets of 28 STSs were useful to build fine YAC contigs by STS-mediated YAC walking at the 21q22.1 region.

Animals↗

PubMed1999

Mechanisms of spiroplasma genome variation associated with SpV1-like viral DNA inferred from sequence comparisons.

Genomes of Spiroplasma citri strains have rearranged frequently during their evolution, partly due to multiple integrated sequences of spiroplasma viruses. To understand better the role of viral sequences in genome evolution, we examined available nucleotide sequences of viruslike elements in the S. citri chromosome. Comparison of integrated and nonintegrated sequences of spiroplasma virus SpV1-C74 DNA suggested that it is an encapsidated form of the circular transposition intermediate belonging to an insertion sequence (IS3) family member. One SpV1-C74 viral DNA fragment was identified as interrupting the remains of a DNA adenine modification methylase gene. A viral DNA insertion of SpV1-R8A2 B DNA had hallmarks of having suffered an internal deletion by a site-specific recombination system. Homologous recombination likely was responsible for several deletions within viral DNA. A homologous recombination event was inferred between part of a viral DNA insertion and a similar chromosomal sequence. Dispersed sequences from SpV1-like C4 open reading frames (ORFs) were identified as involved in a complex deletion-inversion event. Thus, SpV1-like sequences likely have altered spiroplasma genomes by inserting within active genes, destroying their function, by providing targets for site-specific recombination, by mediating deletions of sequences adjacent to their integration sites, and by providing targets for homologous recombination, leading to inversions.

Amino Acid Sequence↗

PubMed2001

Cardiopulmonary malformations in the inv/inv mouse.

The inv/inv mouse carries an insertional mutation in the inversin gene, (inv, for inversion of embryonic turning). Previously it had been reported that almost 100% of the homozygous offspring (inv/inv) were characterized by situs inversus totalis. In this report we identify the spectrum of cardiopulmonary anatomical abnormalities in inv/inv mice surviving to birth to determine whether the abnormalities seen are of the categories classically associated with human situs abnormalities. Stillborn mice, offspring that died unexpectedly (within 48 hr after birth), and neonates with phenotypic characteristics of situs inversus (right-sided stomachs, growth failure or jaundice) were processed for standard histological examination. Of 173 offspring, 34 (20%) neonates (11 stillborn, 9 unexpected deaths, and 14 mice with situs inversus phenotype) were examined, 27 of which were genotyped to be inv/inv. Interestingly, three inv/inv mice (11%) were found to have situs solitus. Twenty-four had situs inversus with normal, mirror-image cardiac anatomy (dextrocardia with atrioventricular concordance, ventriculoarterial concordance and a right aortic arch). The overall incidence of cardiovascular anomalies observed was 10 out of 27 (37%). The most frequent severe malformation, identified in 3 out of 27 animals, was a complex consisting of pulmonary infundibular stenosis/atresia with absence of pulmonary valve tissue and a ventricular septal defect. The pulmonary phenotype in inv/inv mice was situs inversus with occasional minor lobar abnormalities. We conclude that 1) cardiopulmonary malformations in inv/inv mice are not rare (37%), 2) the cardiopulmonary malformations observed in inv/inv specimens are not of the spectrum typically associated with human heterotaxia. In particular, inv/inv mice have a propensity for defects in the development of the right ventricular outflow tract and the interventricular septum, and 3) approximately one out of ten inv/inv mice is born with situs solitus and shows cardiac anomalies that correspond to those observed in inv/inv specimens with situs inversus. Our data therefore suggest that inversin, the product of the inv locus, may have specific roles in cardiac morphogenesis independent of its role in situs determination.

Animals↗

PubMed2003

Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome.

In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modified Ds transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.

Cloning, Molecular↗

PubMed1982

Structure and role of the herpes simplex virus DNA termini in inversion, circularization and generation of virion DNA.

The herpes simplex virus genome consists of two components, L and S, which invert relative to each other during viral replication. The a sequence is present at the genomic termini in direct orientation and at the L-S junction in inverted orientation. Previously, we showed that insertion of a fragment spanning the L-S junction into the viral genome causes additional inversions. In this study, we determine the nucleotide sequence of the genomic termini and show that insertion of either the free S terminus or the L terminus causes inversions in the viral genome. We conclude that the a sequence is the inversion-specific sequence, that linear unit-length molecules packaged in virions are generated by cleavage between adjacent copies of the a sequence, that cleavage produces 3' single-base extensions on the genomic termini and that the signal for cleavage is contained within the a sequence.

Animals↗

PubMed2002

Molecular genetic characterization of the EWS/CHN and RBP56/CHN fusion genes in extraskeletal myxoid chondrosarcoma.

Extraskeletal myxoid chondrosarcoma (EMC) is a soft-tissue neoplasm cytogenetically characterized by the translocations t(9;22)(q22;q11-12) or t(9;17)(q22;q11), generating EWS/CHN or RBP56/CHN fusion genes, respectively. In the present study, 18 EMCs were studied both cytogenetically and at the molecular level. Chromosomal aberrations were detected in 16 samples: 13 with involvement of 9q22 and 22q11-12, and three with rearrangements of 9q22 and 17q11. Fifteen cases had an EWS/CHN fusion transcript and three had an RBP56/CHN transcript. The most frequent EWS/CHN transcript (type 1; 10 tumors), involved fusion of EWS exon 12 with CHN exon 3, and the second most common (type 5; two cases) was fusion of EWS exon 13 with CHN exon 3. In all tumors with RBP56/CHN fusion, exon 6 of RBP56 was fused to exon 3 of CHN. By genomic XL PCR and sequence analyses, the breakpoints from 14 cases were mapped in the EWS, RBP56, and CHN genes. In CHN, 12 breakpoints were found in intron 2 and only two in intron 1. In EWS, the breaks occurred in introns 7 (one break), 12 (eight breaks), and 13 (one break), and in RBP56 in intron 6. Repetitive elements such as Alu and LINE sequences seem to have limited, if any, importance in the genesis of EWS/CHN and RBP56/CHN chimeras. Furthermore, there were no chi, chi-like, topoisomerase II, or translin consensus sequences in the introns harboring the translocation breakpoints, nor could the number of topo I sites in EWS, RBP56, and CHN introns explain the uneven distribution of the breakpoints among EWS or CHN introns. Additional genetic events, such as nucleotide insertions, homologies at the junction, deletions, duplications, and inversions, were found to accompany the translocations, indicating that the chromosomal translocations do not require sequence-specific recombinases or extensive homology between the recombined sequences.

Adult↗

PubMed1989

Characterization of the staphylococcal beta-lactamase transposon Tn552.

The staphylococcal beta-lactamase transposon Tn552 is a member of a novel group of transposable elements. The organization of genes in Tn552 resembles that of members of the Tn21 sub-group of Tn3 family transposons, which transpose replicatively by cointegrate formation and resolution. Thus, a possible resolution site ('resL') and a resolvase gene (tnpR or 'binL') have been identified. However, consistent with the fact that Tn552 generates 6 bp (rather than 5 bp) flanking direct repeats of target DNA, neither the putative transposase protein, nor the terminal inverted repeats of Tn552 are homologous to those of Tn3 elements. Tn552, like phage Mu and retroelements, is defined by the terminal dinucleotides 5' TG .. CA 3'. A naturally occurring staphylococcal plasmid, pI9789, contains a Tn552-derived resolution system ('resR-binR') that acts as a 'hotspot' for Tn552 transposition; insertion creates a segment of DNA flanked by inversely repeated resolution sites, one (resR) on pI9789 and the other (resL) on Tn552. The putative Tn552 resolvase, the most closely related of known resolvases to the homologous DNA invertases, initially was identified as a DNA invertase ('Bin') as a result of its ability to mediate efficient inversion of this segment in vivo.

Amino Acid Sequence↗

PubMed2005

3D needle-tissue interaction simulation for prostate brachytherapy.

This paper presents a needle-tissue interaction model that is a 3D extension of a prior work based on the finite element method. The model is also adapted to accommodate arbitrary meshes so that the anatomy can effectively be meshed using third-party algorithms. Using this model a prostate brachytherapy simulator is designed to help medical residents acquire needle steering skills. This simulation uses a prostate mesh generated from clinical data segmented as contours on parallel slices. Node repositioning and addition, which are methods for achieving needle-tissue coupling, are discussed. In order to achieve realtime haptic rates, computational approaches to these methods are compared. Specifically, the benefit of using the Woodbury formula (matrix inversion lemma) is studied. Our simulation of needle insertion into a prostate is shown to run faster than 1 kHz.

Brachytherapy↗

PubMed1996

The secG deletion mutation of Escherichia coli is suppressed by expression of a novel regulatory gene of Bacillus subtilis.

SecG, a membrane component of E. coli protein translocase, stimulates the translocation of proteins across the cell membrane through the cycle of topology inversion, which is coupled to the membrane-insertion and deinsertion cycle of SecA [Nishiyama et al. (1996) Cell 85, 71-81]. A gene of B. subtilis able to suppress the cold-sensitive phenotype of the secG deletion mutant of E. coli was cloned and found to encode a novel regulatory protein, ScgR. Similarity search revealed homology with known proteins such as GlnR of B. subtilis. Plasmid-encoded ScgR stimulated protein translocation in the deletion mutant. ScgR increased the proportion of cardiolipin at the expense of phosphatidylglycerol, but did not affect the composition of other lipid components of the cell, suggesting that the increased cardiolipin level compensates for the SecG function and thereby stimulates protein translocation.

Amino Acid Sequence↗

PubMed2003

Locating transposable element polymorphisms in bacterial genomes.

Although whole-genome sequencing is greatly extending our knowledge of the genetic capacity of those bacterial species, it is only directly informative for the particular strain sequenced. Many bacterial species exhibit more or less genetic polymorphism within their populations and characterising this variety is an extremely important way of elucidating the biology of these species. Often genomic polymorphisms are associated with multicopy elements, particularly transposable elements. We describe a novel method that efficiently characterises the sequences of such polymorphisms. We have optimised heminested inverse PCR (hINVPCR) to assess the diversity of insertional polymorphisms of a transposable element (IS6110) in clinical isolates of Mycobacterium tuberculosis. To increase the yield of information, genomic DNA was digested with different endonucleases (Bsp1286I, HaeII or PvuI), and primers based on both the 5' and 3' ends of IS6110 were used to amplify and determine the genomic sequence upstream (or downstream) of the transposable element. We found that both the choice of restriction enzyme and the use of primers at both ends of the transposable element significantly increased the diversity of the insertion sites identified. Band stabbing was incorporated into the method as an alternative to cloning in order to screen large number of isolates at a sequence level in a rapid and labour-efficient fashion. We describe some of the purposes to which such data can be put.

Cloning, Molecular↗

PubMed2004

Measurement of 15N csa/dipolar cross-correlation rates by means of Spin State Selective experiments.

We propose a method for the determination of (15)N csa/dipolar cross-correlation rates based on the measurement of the two apparent transverse (or longitudinal) relaxation rates associated with each component of the nitrogen doublet (N(alpha) and N(beta)). This is achieved by inserting a spin state selective scheme in conventional inverse Carr-Purcell-Meiboom-Gill (or inversion-recovery) pulse sequence which allows for the edition of a HSQC-type spectrum for each of the spin states. Transverse cross-correlation rates necessitate two independent sets of measurements (for N(alpha) and N(beta), respectively), whereas for longitudinal cross correlation rates, besides N(alpha) and N(beta) measurements, the method requires the knowledge of both the (15)N longitudinal auto-relaxation rate and the longitudinal two-spin order (2NzHz) auto-relaxation rate. These additional parameters are mandatory because of the non-exponential behavior of the N(alpha) and N(beta) longitudinal decays. Conversely, the present method does not require any complex manipulation of 2D spectra, the cross-correlation rates being obtained from the difference of the two (N(alpha) and N(beta)) apparent relaxation rates. This approach is applied to (15)N-labelled ubiquitin at two different magnetic fields (9.4 T and 14.1 T).

Magnetics↗

PubMed2001

The use of nonhuman primate models to improve gene transfer into haematopoietic stem cells.

Primitive haematopoietic progenitor and stem cells (HSC) have been pursued as highly desirable targets for genetic therapy as technology allowing safe and controllable transfer of exogenous genes into eukaryotic cells was developed a decade ago. Retroviral vectors have been used for the majority of preclinical and clinical studies directed at these cells, because these vectors have a number of the necessary properties, including chromosomal integration, helper-free production systems, and lack of toxicity. Until recently, however, results with these vectors in clinical trials and large animal models indicated efficiency of gene transfer as a major hurdle to be overcome. We have focused on using the rhesus macaque autologous transplantation model to optimize gene transfer to primitive haematopoietic cells, and investigate questions regarding in vivo stem cell behaviour, in a system with proven predictive value for human haematopoiesis. By optimization of transduction conditions using standard vectors, gene transfer efficiency to primitive repopulating cells has reached the clinically relevant range of 5-20% long-term. Alternative vector systems, have also yielded promising results. We have also found that relatively simple manipulation of cell cycle status prior to reinfusion of marked cells results in significantly improved engraftment of transduced cells: this finding may have an impact particularly in the nonablative setting. The high level marking has permitted insertion site analysis and clonal tracking in vivo. Inverse PCR and/or a ligation-mediated PCR procedure have demonstrated that a large number of transduced clones (over 50) contribute to multiple lineages in vivo for up to at least 2 years post-transplantation. Thus far we have little evidence for rapid clonal succession or lineage-restricted engraftment of transduced cells. These and other advances should result in successful gene therapy for a variety of acquired and congenital disorders affecting HSCs and their progeny lineages.

Animals↗

PubMed1997

prl mutations in the Escherichia coli secG gene.

SecG, an integral membrane component of the Escherichia coli preprotein translocase, contributes to the efficiency of the export process by undergoing cycles of topology inversion in the membrane, coupled with the insertion-deinsertion cycles of SecA. We have previously identified sec alleles of secG that cause a generalized secretion defect. In this study, by screening mutagenized secG libraries for suppressors of a malE signal sequence mutation, we isolated prl alleles of secG. By analogy with secY/prlA, secA/prlD, and secE/prlG, secG could therefore be called secG/prlH. The prlH mutations affect 13 codons distributed along the secG sequence, and none map to the codons affected by sec mutations. prlH suppressors suppress a variety of signal sequence mutations and they allow export of alkaline phosphatase lacking its entire signal sequence. Although secG was not identified in previous selections for prl mutants, several prlH alleles are as strong as the strongest known prlG alleles of secE. Some prlH alleles can also promote the export of alkaline phosphatase fused to predicted cytoplasmic domains of UhpT, an integral membrane protein. These results support the notion that SecG contributes to signal sequence recognition, and suggest that it may also contribute to the topology of integral membrane proteins.

ATP-Binding Cassette Transporters↗

PubMed2003

MCMC genome rearrangement.

MOTIVATION: As more and more genomes have been sequenced, genomic data is rapidly accumulating. Genome-wide mutations are believed more neutral than local mutations such as substitutions, insertions and deletions, therefore phylogenetic investigations based on inversions, transpositions and inverted transpositions are less biased by the hypothesis on neutral evolution. Although efficient algorithms exist for obtaining the inversion distance of two signed permutations, there is no reliable algorithm when both inversions and transpositions are considered. Moreover, different type of mutations happen with different rates, and it is not clear how to weight them in a distance based approach. RESULTS: We introduce a Markov Chain Monte Carlo method to genome rearrangement based on a stochastic model of evolution, which can estimate the number of different evolutionary events needed to sort a signed permutation. The performance of the method was tested on simulated data, and the estimated numbers of different types of mutations were reliable. Human and Drosophila mitochondrial data were also analysed with the new method. The mixing time of the Markov Chain is short both in terms of CPU times and number of proposals. AVAILABILITY: The source code in C is available on request from the author.

Algorithms↗

PubMed2001

Identification of chromosome inheritance modifiers in Drosophila melanogaster.

Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a "sensitized" minichromosome substrate (J21A) to screen approximately 3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.

Animals↗

PubMed1986

Prospective study of catheter replacement and other risk factors for infection of hyperalimentation catheters.

To determine risk factors for infection of hyperalimentation catheters, we prospectively studied 169 catheter systems (88 patients) by using a semiquantitative culture technique. Infection occurred in 24 (14%) catheters (16 patients), was inversely proportional to the number of previous catheters inserted by the operator (P less than .02), and was proportional to the interval between admission and catheter insertion (P less than .0005). Catheter replacement over a guidewire was no more likely to be associated with infection than was a de novo percutaneous insertion at another site (P = .6). Using a proportional hazards model, we estimated the risk of infection per day to be 1.3 times greater for a catheter if the patient had been hospitalized 50 days instead of seven days, and 3.8 times greater if the patient had a Swan-Ganz catheter at the time of insertion.

Adult↗