PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “mapping insertion sites”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed1992

A BlnI restriction map of the Salmonella typhimurium LT2 genome.

BlnI or AvrII (5'-CCTAGG) sites are very rare in the Salmonella typhimurium LT2 genome. BlnI was used to construct a physical map which was correlated with the genetic map by using three methods. First, Tn10 carries BlnI sites, and the extra restriction sites produced by 34 genetically mapped Tn10 insertions were physically mapped by using pulsed-field gel electrophoresis. Second, six genetically mapped Mud-P22 prophage insertions were used to assign BlnI fragments. Integration of Mud-P22 introduces 30 kb of DNA that can easily be detected by a "shift up" in all but the largest BlnI fragments. Finally, induced Mud-P22 insertions package more than 100 kb of genomic DNA adjacent to one side of the insertion. Some of the smaller BlnI fragments were localized by hybridization to a dot blot array of 52 lysates from induced Mud-P22 insertions. Of the 10 BlnI sites mapped, 6 probably occur in or near the 16S rRNA genes at about 55, 71, 83, 86, 88.5, and 89.5 min. There is one BlnI site in the 90-kb pSLT plasmid. Two additional BlnI fragments of about 7 and 4 kb have not been localized. The size of the genome was estimated as 4.78 Mb (+/- 0.1 Mb) excluding pSLT but including prophages Fels-1 and Fels-2. One BlnI fragment that maps between 55 and 59 min showed a 40-kb reduction in size in a strain cured of the approximately 40-kb Fels-2 prophage.

DNA Transposable Elements↗
PubMed2005

Phenotypic screening of Escherichia coli K-12 Tn5 insertion libraries, using whole-genome oligonucleotide microarrays.

Complete genome sequences in combination with global screening methods allow parallel analysis of multiple mutant loci to determine the requirement for specific genes in different environments. In this paper we describe a high-definition microarray approach for investigating the growth effects of Tn5 insertions in Escherichia coli K-12. Libraries of insertion mutants generated by a unique Tn5 mutagenesis system were grown competitively in defined media. Biotin-labeled runoff RNA transcripts were generated in vitro from transposon insertions in each population of mutants. These transcripts were then hybridized to custom-designed oligonucleotide microarrays to detect the presence of each mutant in the population. By using this approach, the signal associated with 25 auxotrophic insertions in a 50-mutant pool was not detectable following nine generations of growth in glucose M9 minimal medium. It was found that individual insertion sites could be mapped to within 50 bp of their genomic locations, and 340 dispensable regions in the E. coli chromosome were identified. Tn5 insertions were detected in 15 genes for which no previous insertions have been reported. Other applications of this method are discussed.

Chromosome Mapping↗
PubMed1993

Rapid physical mapping of YAC inserts by random integration of I-Sce I sites.

We have developed a novel strategy, based on the random insertion by homologous recombination of artificial I-Sce I sites within mammalian repetitive DNA sequences, which should greatly facilitate the high resolution physical mapping of large DNA fragments cloned in YAC. A set of transgenic yeast strains containing appropriately spaced I-Sce I sites within the YAC insert defines a series of nested physical intervals against which new genes, clones or DNA fragments can be mapped by simple hybridisation. Sequential hybridisation using such a series of nested YAC fragments as probes can also allow the rapid sorting of phage or cosmid libraries into contigs. This approach, which has been applied to a YAC containing a 460 kb insert from the mouse X chromosome, may also have applications for the restriction mapping of large genomic segments, mapping of exons and the search for homologous genes.

Animals↗
PubMed1991

H beta 58, an insertional mutation affecting early postimplantation development of the mouse embryo.

The generation and analysis of insertional mutations affecting mouse embryogenesis provides a powerful method to identify new genes that function in early development. In this paper, we describe an insertional mutation that interferes with postimplantation mouse development beginning at the time of gastrulation. Embryos homozygous for the H beta 58 transgenic insertion developed normally through the early postimplantation, egg cylinder stage (day 6.5 of development). At the primitive streak stage (day 7.5), however, they began to display characteristic abnormalities, including a retardation in the growth of the embryonic ectoderm (the earliest identifiable defect), and in some cases abnormalities of the amnion and chorion. Homozygotes continued to develop for 2-3 more days, reaching the size of a normal 8.5 day embryo, and formed tissues representative of all three germ layers, including several differentiated cell types. The site of insertion was mapped, by a combination of cytogenetic and genetic methods, to chromosome 10, and it appeared to define a new genetic locus. The inserted transgene provided a probe to clone and characterize the mutant locus, as well as the corresponding wild-type locus. In addition to an insertion of 10-20 copies of the transgene, the mutant locus contained a deletion of 2-3 kb of DNA found at the wild-type locus, and possibly an insertion of mouse repetitive DNA. However, genomic sequences on both sides of the insertion site remained co-linear in the wild-type and mutant genomes, and no chromosomal abnormalities could be detected. Five single copy DNA probes spanning the insertion site were tested for their ability to hybridize to RNA from 8.5 day embryos; one of the probes (located within the region deleted from the mutant chromosome) hybridized to a 2.7 kb mRNA encoded at the H beta 58 locus, thus identifying a gene whose disruption appears to be responsible for the mutant phenotype.

Animals↗
PubMed2006

Global mapping of transposon location.

Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.

Chromosome Mapping↗
PubMed1982

Two defective DNAs of human polyomavirus JC adapted to growth in human embryonic kidney cells.

Human polyomavirus JC (JCV) adapted to growth in human embryonic kidney (HEK) cells contains two or more species of shorter-length viral DNA even after two cycles of plaque purification in HEK cells. We have molecularly cloned JCV DNA from one plaque isolate and determined the physical map of its DNA. Using gel electrophoresis and electron microscopy, we found that the cloned DNA consisted of two classes of JCV DNA. One class of DNA had a deletion of 30% (between 0.7 and 1.0 map units from the EcoRI site) and an insertion of 1% at the same site in the late region. The other had a deletion of 35% (0.18 to 0.53 map units) and an insertion of 12% at the same site in the early region of the JCV genome. Both DNAs had added sequences near the origin of DNA replication. From their structure, the two DNAs appear to complement each other. These results indicate that the HEK-adapted JCV may be replicating by complementation between two defective mutants.

Cell Line↗
PubMed2003

The N-terminus of the human copper transporter 1 (hCTR1) is localized extracellularly, and interacts with itself.

We have used indirect immunofluorescense studies and glycosylation-site insertion and deletion mapping to characterize the topology of human copper transporter 1 (hCTR1), the putative human high-affinity copper-import protein. Both approaches indicated that hCTR1 contains three transmembrane domains and that the N-terminus of hCTR1, which contains several putative copper-binding sites, is localized extracellularly, whereas the C-terminus is exposed to the cytosol. Based on previous observations that CTR1 proteins form high-molecular-mass complexes, we investigated directly whether CTR1 proteins interact with themselves. Yeast two-hybrid studies showed that interaction of yeast, mouse, rat and human CTR1 occurs at the sites of their N-terminal domains, and is not dependent on the copper concentration in the growth media. Analysis of deletion constructs indicated that multiple regions in the N-terminus are essential for this self-interaction. In contrast, the N-terminal tail of the presumed low-affinity copper transporter, hCTR2, does not interact with itself. Taken together, these results suggest that CTR1 spans the membrane at least six times, permitting formation of a channel, which is consistent with its proposed role as a copper transporter.

Amino Acid Sequence↗
PubMedDate not supplied

Isolation and characterization of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus.

A Rhizobium loti gene required for effective invasion of the host Lotus pedunculatus has been identified by transposon Tn5 mutagenesis. Cosmids that complemented a previously isolated mutation (239) at this invasion (inv) locus were identified by in planta complementation and used to construct a physical map of the gene region. The insertion site of Tn5 in PN239 was mapped to a 7.5-kb EcoRI fragment, which complemented the mutation when subcloned into pLAFR1. Further Tn5 mutagenesis of the 7.5-kb fragment was carried out in Escherichia coli using bacteriophage lambda 467, and the mutations homogenotized into R. loti NZP2037. Three additional Fix- mutations were isolated, and these were found to map adjacent to the position of the original mutation in strain PN239. All the other Tn5 insertions isolated in the 7.5-kb fragment gave a Fix+ phenotype on L. pedunculatus. Electron microscopic examination of the L. pedunculatus nodules induced by the isolated Fix- mutants showed that bacteria were either blocked in release from the infection threads or were unable to undergo normal bacteroid development. The inv locus as defined by the Tn5 insertions was sequenced, and a single open-reading frame (ORF) of 576 bp, corresponding to a polypeptide of 21.3 kDa, was identified. The position and orientation of this ORF were consistent with those of the isolated Tn5 Fix- insertions.

Amino Acid Sequence↗
PubMed1998

Genotype-environment interaction at quantitative trait loci affecting sensory bristle number in Drosophila melanogaster.

The magnitude of segregating variation for bristle number in Drosophila melanogaster exceeds that predicted from models of mutation-selection balance. To evaluate the hypothesis that genotype-environment interaction (GEI) maintains variation for bristle number in nature, we quantified the extent of GEI for abdominal and sternopleural bristles among 98 recombinant inbred lines, derived from two homozygous laboratory strains, in three temperature environments. There was considerable GEI for both bristle traits, which was mainly attributable to changes in rank order of line means. We conducted a genome-wide screen for quantitative trait loci (QTLs) affecting bristle number in each sex and temperature environment, using a dense (3.2-cM) marker map of polymorphic insertion sites of roo transposable elements. Nine sternopleural and 11 abdominal bristle number QTLs were detected. Significant GEI was exhibited by 14 QTLs, but there was heterogeneity among QTLs in their sensitivity to thermal and sexual environments. To further evaluate the hypothesis that GEI maintains variation for bristle number, we require estimates of allelic effects across environments at genetic loci affecting the traits. This level of resolution may be achievable for Drosophila bristle number because candidate loci affecting bristle development often map to the same location as bristle number QTLs.

Animals↗
PubMed1992

Pattern of somatic transposition in a high copy Ac tomato line.

A transgenic tomato line containing between eight and ten copies per genome of an exceptionally active maize transposable element Ac has previously been described. Southern analyses indicated that these elements are somatically active in these plants. In order to characterize further the pattern of somatic transposition in this line, 24 independent Ac insertion events from a single plant were cloned. In 21 cases, Ac inserted into single copy genomic DNA while in three cases Ac inserted into sequences present at two to four copies per genome; none of the insertions occurred into more highly repetitive DNA. The chromosomal locations of 20 insertion sites were determined by RFLP mapping and a pattern of small dispersed clusters emerged. Thirteen of the 20 insertion sites were linked to at least one other insertion site but these were distributed over nine of the 12 tomato chromosomes. Only one Ac insertion was linked to the T-DNA locus. The structural integrity of these Ac elements was examined and no evidence of deletions or other rearrangements suggestive of Ds elements was found. The implications of these findings with respect to the use of Ac as a transposon tag in heterologous species are discussed.

Base Sequence↗
PubMed2006

Distribution and genomic location of active insertion sequences in the Burkholderia cepacia complex.

This study aimed firstly to establish the distribution and copy number within the Burkholderia cepacia complex of three insertion sequences (IS402, IS407 and IS1416) that possess the ability to activate transcription and hence influence gene expression. A second aim was to map the genomic insertion sites of one of the active insertion sequences (IS407) to establish putative links between insertion site and downstream gene activation. The resulting data revealed that all three insertion sequences were present in one-third of the 66 isolates tested. The three insertion sequences were prevalent across the nine B. cepacia complex species, although IS402 was absent from the 16 Burkholderia anthina strains tested and IS407 was absent from all 10 Burkholderia pyrrocinia strains. IS407 copies from six strains (two Burkholderia cenocepacia strains and one strain each of Burkholderia multivorans, Burkholderia stabilis, Burkholderia vietnamiensis and B. anthina) were mapped to the genome using hemi-nested inverse PCR. Insertions were found upstream of genes with wide-ranging functions. This study suggests that the abundance and distribution of these active insertion sequences is likely to affect genomic plasticity, and potentially gene transcription and pathogenicity.

Burkholderia Infections↗
PubMed2000

Combinatorial signaling through BMP receptor IB and GDF5: shaping of the distal mouse limb and the genetics of distal limb diversity.

In this study, we use a mouse insertional mutant to delineate gene activities that shape the distal limb skeleton. A recessive mutation that results in brachydactyly was found in a lineage of transgenic mice. Sequences flanking the transgene insertion site were cloned, mapped to chromosome 3, and used to identify the brachydactyly gene as the type IB bone morphogenetic protein receptor, BmprIB (ALK6). Expression analyses in wild-type mice revealed two major classes of BmprIB transcripts. Rather than representing unique coding RNAs generated by alternative splicing of a single pro-mRNA transcribed from one promoter, the distinct isoforms reflect evolution of two BmprIB promoters: one located distally, driving expression in the developing limb skeleton, and one situated proximally, initiating transcription in neural epithelium. The distal promoter is deleted in the insertional mutant, resulting in a regulatory allele (BmprIB(Tg)) lacking cis-sequences necessary for limb BmprIB expression. Mutants fail to generate digit cartilage, indicating that BMPRIB is the physiologic transducer for the formation of digit cartilage from the skeletal blastema. Expansion of BmprIB expression into the limb through acquisition of these distal cis-regulatory sequences appears, therefore, to be an important genetic component driving morphological diversity in distal extremities. GDF5 is a BMP-related signal, which is also required for proper digit formation. Analyses incorporating both Gdf5 and BmprIB(Tg) alleles revealed that BMPRIB regulates chondrogenesis and segmentation through both GDF5-dependent and -independent processes, and that, reciprocally, GDF5 acts through both IB and other type I receptors. Together, these findings provide in vivo support for the concept of combinatorial BMP signaling, in which distinct outcomes result both from a single receptor being triggered by different ligands and from a single ligand binding to different receptors.

Animals↗
PubMed2002

A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome.

We transposed Dissociation (Ds) elements from three start loci on chromosome 5 in Arabidopsis (Nossen ecotype) by using a local transposition system. We determined partial genomic sequences flanking the Ds elements and mapped the elements' insertion sites in 1,173 transposed lines by comparison with the published genomic sequence. Most of the lines contained a single copy of the Ds element. One-half of the lines contained Ds on chromosome 5; in particular, insertion "hot spots" near the three start loci were clearly observed. In the other lines, the Ds elements were transposed across chromosomes. We found other insertion hot spots at the tops of chromosomes 2 and 4, near nucleolus organizer regions 2 and 4, respectively. Another characteristic feature was that the Ds elements tended to transpose near the chromosome ends and rarely transposed near centromeres. The distribution patterns differed among the three start loci, even though they possessed the same Ds construct. More than one-half of the Ds elements were inserted irregularly into the genome; that is, they did not retain the perfect inverted repeat sequence of Ds nor leave perfect target site duplications. This precise analysis of distribution patterns will contribute to a comprehensive understanding of the transposing mechanism. From these Ds insertion sites, we have constructed a database for screening gene-knockout mutants in silico. In 583 of the 1,173 lines, the Ds elements were inserted into protein-coding genes, which suggests that these lines are gene-knockout mutants. The database and individual lines will be available freely for academic use from the RIKEN Bio-Resource Center (http://www.brc.riken.go.jp/Eng/index.html).

Arabidopsis↗
PubMed1995

Molecular and phenotypic characterization of a new mouse insertional mutation that causes a defect in the distal vertebrae of the spine.

We have identified and characterized the phenotype of a new insertional mutation in one line of transgenic mice. Mice carrying this mutation, which we have designated TgN(Imusd)370Rpw, display undulations of the vertebrae giving rise to a novel kinky-tail phenotype. Molecular characterization of the insertion site indicates that the transgene integration has occurred without any substantial alterations in the structure of the host sequences. Using probes that flank the insertion site, we have mapped the mutation to chromosome 5 near the semidominant mutation, thick tail (Tht). Thick tail does not complement the TgN(Imusd)370Rpw mutation; compound mutants containing one copy of each mutation display a more severe phenotype than either mutation individually.

Actins↗
PubMed2000

Quantitative trait loci affecting components of wing shape in Drosophila melanogaster.

Two composite multiple regression-interval mapping analyses were performed to identify candidate quantitative trait loci (QTL) affecting components of wing shape in Drosophila melanogaster defined by eight relative warp-based measures. A recombinant inbred line design was used to map QTL for the shape of two intervein regions in the anterior compartment of the wing, using a high resolution map of retrotransposon insertion sites between Oregon-R and Russian 2b. A total of 35 QTL representing up to 23 different loci were identified, many of which are located near components of the epidermal growth factor-Ras signal transduction pathway that regulates vein vs. intervein decision making and vein placement. Over one-half of the loci were detected in both sexes, and just under one-half were detected at two different growth temperatures. Different loci were found to affect aspects of shape in each intervein region, confirming that the shape of the whole wing should be regarded as a compound trait composed of several developmental units. In addition, a reciprocal backcross design was used to map QTL affecting shape in the posterior compartment of the wings of 831 flies, using a molecular map of 16 allele-specific oligohybridization single nucleotide polymorphism (SNP) markers between two divergent inbred lines. A total of 13 QTL were detected and shown to have generally additive effects on separable components of shape, in both sexes. By contrast, 8 QTL that affected wing size in these backcrosses were nearly dominant in their effects. The results confirm at the genetic level that wing shape is regulated independent of wing size and set up the hypothesis that wing shape is regulated in part through the regulation of the length and positioning of wing veins, involving quantitative regulation of the activity of secreted growth factors.

Animals↗
PubMed1997

Activation mechanism of the MAP kinase ERK2 by dual phosphorylation.

The structure of the active form of the MAP kinase ERK2 has been solved, phosphorylated on a threonine and a tyrosine residue within the phosphorylation lip. The lip is refolded, bringing the phosphothreonine and phosphotyrosine into alignment with surface arginine-rich binding sites. Conformational changes occur in the lip and neighboring structures, including the P+1 site, the MAP kinase insertion, the C-terminal extension, and helix C. Domain rotation and remodeling of the proline-directed P+1 specificity pocket account for the activation. The conformation of the P+1 pocket is similar to a second proline-directed kinase, CDK2-CyclinA, thus permitting the origin of this specificity to be defined. Conformational changes outside the lip provide loci at which the state of phosphorylation can be felt by other cellular components.

Binding Sites↗
PubMed1996

NuMA: a bipartite nuclear location signal and other functional properties of the tail domain.

Nuclear Mitotic Apparatus protein (NuMA) is a 238-kDa protein of the nuclear matrix in interphase that relocates to the spindle poles in mitosis. The globular tail domain (residues 1701 to 2115) contains the nuclear targeting sequence, the site for binding to the mitotic spindle as well as a site responsible for nuclear reformation. To more precisely map these sites, we inserted full-length human NuMA and 16 derivatives with increasing truncations of the tail domain into the pCMV5 vector and induced transient expression. NuMA was found in the interphase nucleus of all transfected BHK cells expressing either full-length NuMA or NuMA mutant proteins ending at or after residue 2005. In contrast, mutants ending at or before residue 2003 remained in the cytoplasm. In the full-length NuMA molecule, point mutations at position 1988 or 1989 or a double mutation at residues 2004 and 2005 cause NuMA to accumulate in the cytoplasm of both BHK and HeLa cells. The combined results indicate a bipartite nuclear location signal involving the sequences RKR (1987-1989) and KK (2004-2005) which are separated by 14 amino acid residues. In 30% of BHK cells transfected by the full-length clone, cytoplasmic aggregates of NuMA that colocalize with the centrosomes were documented in addition to the nuclear staining. In cells with large aggregates the cytoplasmic microtubular profile was disturbed. Observation of micronuclei formation suggests that a region important for normal nuclear reformation lies in the C-terminal 130 residues. Finally, NuMA mutant proteins ending at or after residue 1800 bound to the spindle poles of mitotic cells, while NuMA proteins ending at or before residue 1750 did not.

Amino Acid Sequence↗