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 307 records · Page 17Linked to original sources

PubMed1986

Spacing between simian virus 40 early transcriptional control sequences is important for regulation of early RNA synthesis and gene expression.

We have analyzed the effect of insertion mutants between the simian virus 40 (SV40) 21-base pair (bp) repeats and the early-early (EE) TATA sequence. Insertion of 4, 42, or 90 bp of DNA at the SV40 NcoI site (map position 37) has been analyzed for its effect on expression of the SV40 early gene and positioning of the RNA 5' ends. Insertion of 4 bp reduced SV40 early promoter-dependent chloramphenicol acetyltransferase (CAT) expression by six- to eightfold. Increasing the size of the insertion to 42 or 90 bp resulted in a further drop in early gene expression to basal levels. At the RNA level, the 4-bp insertion reduced EE RNA synthesis approximately 10-fold. No concomitant increase in late-early (LE) RNA synthesis was observed. Insertion of 42 or 90 bp of DNA resulted in a decrease of EE RNA synthesis and a stimulation of LE RNA synthesis. Deletion of the SV40 72-bp repeats from the insertion mutants demonstrated that some, but not all, of the LE RNA depends upon the presence of these sequences. These studies suggest that the ability of RNA polymerase II to utilize the EE (TATA-directed) transcriptional control sequence requires an interaction with the upstream 21-bp repeats or the 72-bp repeats or both. That LE RNA levels in pJI1-in42 CAT and pJI1-in90 CAT were equivalent to the level of EE RNA in pJI1-CAT, yet the level of CAT gene expression was decreased greater than 10-fold, suggests that LE mRNA is under translational control and probably prefers a 5' initiation codon proximal to that of the CAT gene.

Base Sequence↗

PubMed2005

Acinar cell apoptosis in Serpini2-deficient mice models pancreatic insufficiency.

Pancreatic insufficiency (PI) when left untreated results in a state of malnutrition due to an inability to absorb nutrients. Frequently, PI is diagnosed as part of a larger clinical presentation in cystic fibrosis or Shwachman-Diamond syndrome. In this study, a mouse model for isolated exocrine PI was identified in a mouse line generated by a transgene insertion. The trait is inherited in an autosomal recessive pattern, and homozygous animals are growth retarded, have abnormal immunity, and have reduced life span. Mice with the disease locus, named pequeño (pq), exhibit progressive apoptosis of pancreatic acinar cells with severe exocrine acinar cell loss by 8 wk of age, while the islets and ductal tissue persist. The mutation in pq/pq mice results from a random transgene insertion. Molecular characterization of the transgene insertion site by fluorescent in situ hybridization and genomic deletion mapping identified an approximately 210-kb deletion on Chromosome 3, deleting two genes. One of these genes, Serpini2, encodes a protein that is a member of the serpin family of protease inhibitors. Reintroduction of only the Serpini2 gene by bacterial artificial chromosome transgenic complementation corrected the acinar cell defect as well as body weight and immune phenotypes, showing that deletion of Serpini2 causes the pequeño phenotype. Dietary supplementation of pancreatic enzymes also corrected body size, body weight, and immunodeficiency, and increased the life span of Serpini2-deficient mice, despite continued acinar cell loss. To our knowledge, this study describes the first characterized genetic animal model for isolated PI. Genetic complementation of the transgene insertion mutant demonstrates that Serpini2 deficiency directly results in the acinar cell apoptosis, malabsorption, and malnutrition observed in pq/pq mice. The rescue of growth retardation, immunodeficiency, and mortality by either Serpini2 bacterial artificial chromosome transgenic expression or by pancreatic enzyme supplementation demonstrates that these phenotypes are secondary to malnutrition in pq/pq mice.

Animals↗

PubMed1990

Bronze-2 gene of maize: reconstruction of a wild-type allele and analysis of transcription and splicing.

The maize Bronze-2 (Bz2) gene, whose product acts late in the anthocyanin biosynthetic pathway, has been cloned and its transcript has been mapped. We have developed a general procedure for reconstructing wild-type alleles from transposable element-induced mutants. An existing transposon-containing clone, bz2::mu1 [McLaughlin, M., and Walbot, V. (1987). Genetics 117, 771-776], was modified by replacing the region of bz2::mu1 containing the transposon with the corresponding polymerase chain reaction-amplified sequence from the progenitor allele that has no Mu insertion. Particle gun delivery of the reconstructed Bz2 gene to embryonic scutellar tissue lacking a functional Bz2 gene complemented the bz2 mutant phenotype, as demonstrated by the production of purple spots. Having cloned the wild-type allele, we then analyzed the Bz2 transcript, whose features include an 82-nucleotide 5'-untranslated leader, one small intron (78 base pairs) within the coding region, and multiple polyadenylation sites. Four Mutator transposon insertions that eliminate gene function were mapped within the 850-nucleotide transcription unit. We found that variable levels of unspliced Bz2 RNA are present in purple husk tissue; this finding may indicate that the expression of Bz2 is regulated in part at the level of transcript processing.

Alleles↗

PubMed2003

Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility.

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and activates host inflammatory responses. In this study, we examine the nature of the TLR5-flagellin interaction. With deletional, insertional and alanine-scanning mutagenesis, we precisely mapped the TLR5 recognition site on flagellin to a cluster of 13 amino acid residues that participate in intermolecular interactions within flagellar protofilaments and that are required for bacterial motility. The recognition site is buried in the flagellar filament, and monomeric flagellin, but not the filamentous molecule, stimulated TLR5. Finally, flagellin coprecipitated with TLR5, indicating close physical interaction between the molecules. These studies demonstrate the exquisite ability of the innate immune system to precisely target a conserved site on flagellin that is essential for bacterial motility.

Alanine↗

PubMed1995

Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora.

A 16 kb transcript of the ams region, which is essential for biosynthesis of amylovoran, the acidic exopolysaccharide of Erwinia amylovora, was detected by Northern hybridization analysis. The positive regulator RcsA enhanced transcription of the large mRNA from the ams operon. The nucleotide sequence of this area revealed 12 open reading frames (ORFs), which are all transcribed in the same direction. Five ORFs corresponded to the previously mapped genes amsA to amsE. Sequence analysis of the insertion sites of several Tn5 mutations confirmed these data. Tn5 or site-directed mutagenesis of the ORFs 477, 377, 144, and 743 revealed an amylovoran-deficient phenotype, and the newly identified genes were named amsG, amsH, amsI, and amsF, respectively. The predicted amino acid sequence of AmsG is highly homologous to galactosyl-1-phosphate undecaprenylphosphate transferases. AmsB and AmsD are similar to other glycosyl transferases, and AmsH may be related to BexD. A significant homology to mammalian phosphatases was observed for AmsI. AmsA shows characteristic motifs for membrane association and ATP binding. AmsF carries a secretory signal sequence in the N-terminus and could be involved in periplasmic processing of the repeating units. Complementation experiments located a promoter region required for gene expression as far as 500 bp upstream of amsG. It is preceded by a typical transcriptional termination sequence. A mutation upstream of the terminator did not affect amylovoran synthesis. Partial nucleotide sequences further upstream of the ams region showed homology to genes mapped at 45 min on the Escherichia coli chromosome. A termination sequence was also found downstream of the ams operon at a distance of 16 kb from the promoter. Between amsF and this terminator, three additional ORFs were detected.

Amino Acid Sequence↗

PubMed1985

Cloning determinants of pathogenesis from Pseudomonas syringae pathovar syringae.

Transposon mutagenesis and a cosmid genomic library of DNA from the bean pathogen Pseudomonas syringae pathovar syringae were used to identify and isolate sequences essential for pathogenesis. Strain PS9021, derived by Tn5 mutagenesis, was determined to be nonpathogenic on Phaseolus vulgaris cultivar Red Mexican and incapable of inducing a hypersensitive response in Nicotiana tabacum. This mutant also produced fluidal rather than firm colonies on selected agar media. A Tn5-containing EcoRI fragment from PS9021 was cloned and used to probe 1500 members of a genomic library constructed with DNA from the pathogenic parent strain and the wide host range cosmid pVK102. One member that hybridized to the probe contained a cosmid with a 30-kilobase-pair insert (pOSU3101) that complemented the mutant phenotypes when mobilized into PS9021. A restriction endonuclease cleavage site map of pOSU3101 was constructed and sequences essential for pathogenesis were determined by subcloning. Approximately 8.5 kilobase pairs of the insert were essential for restoration by complementation of pathogenesis and hypersensitive response and wild-type colony morphology in strain PS9021.

Journal Article↗

PubMed1996

Chloroplastic aspartate aminotransferase from Arabidopsis thaliana: an examination of the relationship between the structure of the gene and the spatial structure of the protein.

A clone encoding a plastid isoenzyme of aspartate amino-transferase (AAT5) was isolated from an Arabidopsis genomic library and its complete sequence determined. The gene for AAT5 (asp5) contains an open reading frame of 2447 bp comprising 11 exons separated by introns ranging in length from 74 to 207 bp. The upstream regulatory region contains a putative TATA box and multiple copies of two sequence motifs, CTCTT and AAAGAT, previously associated with nodule-specific gene activity in legumes. The deduced primary amino acid sequence of the protein product of asp5 was used to generate a three-dimensional structure of the AAT5 protein by using the computer program Sybyl: Biopolymer Composer and known AAT structures on the protein databases. Both the mature protein and its precursor protein containing a putative N-terminal transit peptide were modelled. The resulting structure of the precursor protein indicated that the transit peptide might also inhibit dimerization of the protein until after its translocation across the chloroplast membrane. The derived structure of the mature protein was then analysed in terms of its component elements of secondary structure, and the positions on the polypeptide back-bone corresponding to intron insertion sites were determined. It is observed that the introns tend to map to regions between structural subdomains of the protein and also map to sites on the surface of the molecule. The asp5 gene in Arabidopsis is thus consistent with Gilbert's exon-shuffling theory of gene evolution [Gilbert (1985) Science 228, 823-824]. A high degree of conservation of intron insertion sites between AAT genes from different plants and animals is observed, particularly within the part of the gene encoding a large beta-sheet structure that forms the structural and functional core of the protein. This beta-sheet structure is thus believed to compromise an ancient and very highly conserved moiety of the molecule.

Amino Acid Sequence↗

PubMed1986

Tn1721 derivatives for transposon mutagenesis, restriction mapping and nucleotide sequence analysis.

New derivatives of the tetracycline-resistance transposon Tn1721 that carry resistances to chloramphenicol, tetracycline, kanamycin and streptomycin are described. These elements are provided on various plasmid vehicles and as chromosomal insertions to extend the range of targets for Tn mutagenesis. Single EcoRI sites at the ends of these transposons proved most useful for physical mapping, for the generation of new EcoRI sites in cloning experiments, for end-labelling and for sequencing of DNA adjacent to an insertion.

Base Sequence↗

PubMed1992

Reconstitutional mutagenesis of the maize P gene by short-range Ac transpositions.

The tendency for Ac to transpose over short intervals has been utilized to develop insertional mutagenesis and fine structure genetic mapping strategies in maize. We recovered excisions of Ac from the P gene and insertions into nearby chromosomal sites. These closely linked Ac elements reinserted into the P gene, reconstituting over 250 unstable variegated alleles. Reconstituted alleles condition a variety of variegation patterns that reflect the position and orientation of Ac within the P gene. Molecular mapping and DNA sequence analyses have shown that reinsertion sites are dispersed throughout a 12.3-kb chromosomal region in the promoter, exons and introns of the P gene, but in some regions insertions sites were clustered in a nonrandom fashion. Transposition profiles and target site sequence data obtained from these studies have revealed several features of Ac transposition including its preference for certain target sites. These results clearly demonstrate the tendency of Ac to transpose to nearby sites in both proximal and distal directions from the donor site. With minor modifications, reconstitutional mutagenesis should be applicable to many Ac-induced mutations in maize and in other plant species and can possibly be extended to other eukaryotic transposon systems as well.

Alleles↗

PubMed1979

Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus.

We have constructed a hybrid plasmid by insertion of the thymidine kinase (TK) gene of Herpes simplex virus (HSV) type I at the BamHI site on Escherichia coli plasmid pBR322. The restriction endonuclease cleavage site map for the viral DNA fragment was determined for ten nucleases, and the insert in the recombinant plasmid has the same restriction nuclease digestion pattern as bona fide viral DNA. This result indicates that the plasmid contains an accurate copy of the viral DNA. The viral TK gene carried on the plasmid can be introduced into mammalian cells where it is expressed. This source of DNA with a selectable marker should be of considerable practical use in gene-transfer experiments in mammalian cells.

Chromosome Mapping↗

PubMed1997

P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: correlation of physical and cytogenetic maps in chromosomal region 86E-87F.

We have established a collection of 2460 lethal or semi-lethal mutant lines using a procedure thought to insert single P elements into vital genes on the third chromosome of Drosophila melanogaster. More than 1200 randomly selected lines were examined by in situ hybridization and 90% found to contain single insertions at sites that mark 89% of all lettered subdivisions of the Bridges' map. A set of chromosomal deficiencies that collectively uncover approximately 25% of the euchromatin of chromosome 3 reveal lethal mutations in 468 lines corresponding to 145 complementation groups. We undertook a detailed analysis of the cytogenetic interval 86E-87F and identified 87 P-element-induced mutations falling into 38 complementation groups, 16 of which correspond to previously known genes. Twenty-one of these 38 complementation groups have at least one allele that has a P-element insertion at a position consistent with the cytogenetics of the locus. We have rescued P elements and flanking chromosomal sequences from the 86E-87F region in 35 lines with either lethal or genetically silent P insertions, and used these as probes to identify cosmids and P1 clones from the Drosophila genome projects. This has tied together the physical and genetic maps and has linked 44 previously identified cosmid contigs into seven "super-contigs" that span the interval. STS data for sequences flanking one side of the P-element insertions in 49 lines has identified insertions in the alphagamma element at 87C, two known transposable elements, and the open reading frames of seven putative single copy genes. These correspond to five known genes in this interval, and two genes identified by the homology of their predicted products to known proteins from other organisms.

Alleles↗

PubMed1989

Cloning and genetic analysis of tra cistrons of the Tra 2/Tra 3 region of plasmid RP1.

Transfer-defective mutants of the 10.4-kb Tra 2/Tra 3 region of RP1 were identified by their ability to be complemented by clones carrying all or part of this region. The respective mutations occurred in six cistrons whose order (traA, B, E, R, P, Q) and location were determined by deletion and insertion mapping. The cistrons occupy a minimum of 5.5 kb with the most distal, traA, spanning the 28.0-kb map position and traR the KpnI site at map position 24.1 kb. Each cistron is expressed independently, as Tn5 or Tn504 insertions in any one cistron do not affect the other five. The phenotypes controlled by each cistron suggest that all contribute to pilus biosynthesis/function while three (traB, R, and P) also contribute to surface exclusion. Given the occurrence of tra cistrons in the "silent" region between Tra 2 and Tra 3 we propose that the epithet "Tra 2" should be used to describe this entire region.

Chromosome Deletion↗

PubMed1986

Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi.

The gene coding for the lipase of Pseudomonas fragi was cloned into Escherichia coli JM83 by inserting Sau3A-generated DNA fragments into the BamH I site of pUC9. The plasmid isolated, pKKO, was restriction mapped and the position of the lipase gene on the 2.0 kb insert was pinpointed by subcloning. DNA sequencing revealed that the open reading frame comprises 405 nucleotides and gives a preprotein of 135 amino acids with a predicted Mr of 14643. By comparing the putative lipase amino acid sequence with porcine pancreatic, rat lingual and Staphylococcus hyicus lipases the amino acid sequence around the reactive serine was found to be common among the types of lipase which have been reported.

Amino Acid Sequence↗

PubMed1998

Construction and validation of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease cleavage.

RecA-assisted restriction endonuclease (RARE) cleavage is an "Achilles' heel" approach to restriction mapping whereby a RecA-protein-oligodeoxynucleotide complex protects an individual restriction site from methylation, thus limiting subsequent digestion to a single, predetermined site. We have used RARE cleavage to cut yeast artificial chromosomes (YACs) at specific EcoRI sites located within or adjacent to sequence-tagged sites (STSs). Each cleavage reaction produces two YAC fragments whose sizes are a direct measure of the position of the STS in the YAC. In this fashion, we have positioned 45 STSs within a contig of 19 independent YACs and constructed a detailed RARE-cleavage map that represents 8.4 Mbp of human chromosome 6p21.3-22. By comparing maps of overlapping YACs, we were able to detect seven internal deletions that ranged from approximately 75 kbp to approximately 1 Mbp in size. Thirteen pairs of EcoRI sites were targeted for double RARE cleavage in uncloned total human DNA. The excised fragments, up to 2 Mbp in size, were resolved by pulsed-field gel electrophoresis and were detected by hybridization. In general, the genomic RARE-cleavage results support the YAC-based map. In one case, the distance in uncloned DNA between the two terminal EcoRI sites of a YAC insert was approximately 1 Mbp larger than the YAC itself, indicating a major deletion. The general concept of RARE-cleavage mapping as well as its applications and limitations are discussed.

Chromosome Mapping↗

PubMed1983

Intracisternal A-particle genes as movable elements in the mouse genome.

We analyzed two functionally defective mouse kappa light chain gene variants previously shown to contain novel insertions of repetitive DNA in their intervening sequences [Hawley, R. G., Shulman, M. J., Murialdo, H., Gibson, D. M. & Hozumi, N. (1982) Proc. Natl. Acad. Sci. USA 79, 7425-7429]. Heteroduplex analysis of the cloned genes shows that the insertions consist of intracisternal A-particle (IAP) genetic elements. Each insertion includes an IAP 5' long terminal repeat (LTR) sequence and extends to a characteristic IAP internal BamHI site where the IAP sequence is interrupted because the mutant genes were cloned from complete BamHI digests of the cellular DNAs. Restriction enzyme mapping indicates that the 5' LTR boundaries of the inserted IAP elements correspond closely to the previously determined rearrangement sites in the mutant genes. The IAP insertions in the two mutants can be distinguished by restriction-site differences and by the fact that one of them contains a deletion that is absent in the other. Nucleotide sequence data are presented for the LTRs of one full-length IAP gene copy randomly selected from a mouse genomic DNA library. These LTRs show many features typical of known integrated retroviral terminal repeat units, and the entire gene is bracketed by short direct repeats within the adjacent cellular DNA. Thus, the findings show that IAP genetic elements can appear in new locations in mouse cellular DNA and suggest that this may occur through a process of proviral insertion.

Animals↗

PubMed2003

Common sites of retroviral integration in mouse hematopoietic tumors identified by high-throughput, single nucleotide polymorphism-based mapping and bacterial artificial chromosome hybridization.

Retroviral insertional mutagenesis in mouse hematopoietic tumors provides a powerful cancer gene discovery tool. Here, we describe a high-throughput, single nucleotide polymorphism (SNP)-based method, for mapping retroviral integration sites cloned from mouse tumors, and a bacterial artificial chromosome (BAC) hybridization method, for localizing these retroviral integration sites to common sites of retroviral integration (CISs). Several new CISs were identified, including one CIS that mapped near Notch1, a gene that has been causally associated with human T-cell tumors. This mapping method is applicable to many different species, including ones where few genetic markers and little genomic sequence information are available. It can also be used to map endogenous proviruses.

Animals↗

PubMed1991

Molecular cloning of lin-29, a heterochronic gene required for the differentiation of hypodermal cells and the cessation of molting in C.elegans.

The lin-29 gene product of C.elegans activates a temporal developmental switch for hypodermal cells. Loss-of-function lin-29 mutations result in worms that fail to execute a stage-specific pattern of hypodermal differentiation that includes exist from the cell cycle, repression of larval cuticle genes, activation of adult cuticle genes, and the cessation of molting. Combined genetic and physical mapping of restriction fragment length polymorphisms (RFLPs) was used to identify the lin-29 locus. A probe from the insertion site of a Tc1 (maP1), closely linked and to the left of lin-29 on the genetic map, was used to identify a large set of overlapping cosmid, lambda and yeast artificial chromosome (YAC) clones assembled as part of the C.elegans physical mapping project. Radiolabeled DNA from one YAC clone identified two distinct allele-specific alterations that cosegregated with the lin-29 mutant phenotype in lin-29 intragenic recombinants. lin-29 sequences were severely under-represented in all cosmid and lambda libraries tested, but were readily cloned in a YAC vector, suggesting that the lin-29 region contains sequences incompatible with standard prokaryotic cloning techniques.

Animals↗

PubMed1994

Construction and characterization of a human chromosome 2-specific BAC library.

We have constructed a human chromosome 2-specific bacterial artificial chromosome (BAC) library using DNA from the somatic cell hybrid GM10826. The average size of the clones is about 63 kb. The coverage and distribution of the library were estimated by screening with known polymorphic genetic markers and fluorescence in situ hybridization (FISH). Twenty-one markers tested positive when DNA pools prepared from approximately one-sixth of the library were screened with 33 known markers. This is consistent with the theoretical calculation of 63% coverage at one genomic equivalent. This suggested that the coverage of the library is approximately 5-6x. FISH analysis with 54 BACs revealed single site hybridization to chromosome 2, and the clones were distributed randomly on the chromosome. We have also performed direct sequencing of the BAC insert ends to generate sequence-tagged sites suitable for mapping and chromosome walking. This is the first reported human chromosome 2-specific BAC library and should provide a resource for physical mapping and disease searching for this chromosome.

Base Sequence↗