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At least 127 records · Page 7Linked to original sources
PubMed1986

Molecular cloning of unintegrated closed circular DNA of porcine retrovirus.

Viral DNA of unintegrated closed circular form was isolated from a swine kidney cell line (SKL) which was infected with a porcine retrovirus Tsukuba-1 (PRetV) produced from a swine malignant lymphoma-derived cell line. Shimozuma-1 and cloned using a lambda phage vector, Charon 21A. One of ten independent clones contained the 8.3 kb DNA fragment as an insert, which was thought to be a full length of viral DNA molecule carrying a long terminal repeat (LTR) sequence. We have analyzed this insert by mapping the recognition sites of some restriction endonucleases by Southern blot hybridization with appropriate probes.

Animals↗
PubMed2004

An abundant placental transcript containing an IAP-LTR is allelic to mouse pregnancy-specific glycoprotein 23 (Psg23): cloning and genetic analysis.

Several families of endogenous retroviruses (ERVs) are expressed in mammalian placental tissues, and are implicated in aspects of placental development and function. We characterized the structure of abundant ERV-related transcripts in mouse placenta. In addition to the 7 kb full-length type I and 5 kb type I deleted intracisternal A-particle (IAP) transcripts, we identified and cloned an abundant 2 kb transcript encoding a novel member of the pregnancy-specific glycoprotein (Psg) gene family, which contains an IAP long terminal repeat (LTR) in the 3' untranslated region (UTR). The polyadenylation signal for the transcript is provided by the inserted LTR sequence. This sequence is allelic to Psg23 and is therefore denoted as Psg23(LTR). The transcript encodes a protein of 471 amino acids and has a domain organisation similar to previously described Psg proteins. Modelling of the protein N-domain produced a structure in good agreement with an existing crystalline structure for mouse sCEACAM1a. The LTR insertion is widely distributed among inbred mouse strains but is not found in 129/sv, CBA/2, or in wild mice. Cloning of the genomic region downstream of the LTR insertion site from the C57Bl/6J strain indicates that the insertion consists of a solo LTR without additional IAP sequence, and identified the original Psg23 polyadenylation signal sequence downstream of the insertion site. Psg23(LTR) was mapped to proximal chromosome 7 using the European collaborative interspecific mouse backcross (EUCIB) panel, and to yeast artificial chromosome (YAC) E072, which contains other members of the Psg gene family, by polymerase chain reaction (PCR). Northern blot analysis of RNA from adult and fetal mouse tissues and in situ hybridization to mid-gestation mouse embryos indicated that Psg23(LTR) is expressed predominantly in placental spongiotrophoblast. We detected a small, but statistically non-significant, bias in favour of transmission of Psg23(LTR) to the offspring of heterozygous parents. However, a larger study would be required to determine whether this allele is selectively advantageous to the developing embryo.

Alleles↗
PubMed1991

Transposon-facilitated DNA sequencing.

We describe here a transposon-based DNA sequencing strategy that allows the introduction of sequencing priming sites throughout a target sequence by bacterial mating. A miniplasmid was designed to select against transposon insertions into the vector. Sites of transposon insertion are mapped by the polymerase chain reaction with bacterial overnight cultures providing the templates. A small set of plasmids with transposons spaced several hundred base pairs apart can then be sequenced. Sequencing primers corresponding to the transposon ends allow sequencing in both directions. Thus, the entire sequence of both strands can be easily determined.

Base Sequence↗
PubMed1987

Determination of the single polyadenylation site of the human pro alpha 1(II) collagen gene.

Several cDNA clones for the human type II procollagen mRNA were isolated from a cartilage cDNA library. Six of the clones containing the longest inserts were subjected to restriction site mapping for alignment. All these six clones extended to the poly A tail. The longest clone, containing a 1470 bp insert, was named pHCAR3. Sequencing of pHCAR3 made it clear that neither of the two canonical AATAAA sequences of the human type II collagen gene is used as the polyadenylation signal. Two 60 bp stretches of high interspecies homology terminating in a hexanucleotide ATTAAA, located 23 nucleotides upstream of the poly A tail, apparently have an important role in determining the single polyadenylation signal for this gene. S1 protection experiments confirmed these observations.

Amino Acid Sequence↗
PubMed2004

Generation and characterization of a human bradykinin receptor B1 transgenic rat as a pharmacodynamic model.

Antagonists of the B1 bradykinin receptor (B1R) offer the promise of novel therapeutic agents for the treatment of inflammatory and neuropathic pain. However, the in vivo characterization of the pharmacodynamics of B1R antagonists is hindered by the low level of B1R expression in healthy tissue and the profound species selectivity exhibited by many compounds for the human B1R. To circumvent these issues, we generated a transgenic rat expressing the human B1R under the control of the neuron-specific enolase promoter. Membranes prepared from whole brain homogenates of heterozygous transgenic rats indicate a B1R expression level of 30 to 40 fmol/mg; there is no detectable B1R expression in control nontransgenic rats. The pharmacological profile of the B1R expressed in the transgenic rat matches that expected of the human, but not the rat receptor. The mapping of the transgene insertion site to rat chromosome 1 permitted the development of a reliable assay for the identification of homozygous transgenic rats. Significantly, homozygous transgenic rats express 2-fold more B1R than heterozygous animals. Autoradiographic analyses of tissue sections from transgenic rats reveal that the B1R is broadly expressed in both the brain and spinal cord. The human B1R expressed in the transgenic rat functions in an in vitro contractile assay and thus has the potential to elicit a functional response in vivo. Using the humanized B1R transgenic rat, an assay was developed that is suitable for the routine evaluation of a test compound's ability to occupy the human B1R in the central nervous system.

Animals↗
PubMed2006

Sarcoma derived from cultured mesenchymal stem cells.

To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole-body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain-specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon-related genetic abnormality, and p53 was not overexpressed. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.

Animals↗
PubMed1980

Translocation specificity of the Tn3 element: characterization of sites of multiple insertions.

247 independent events involving insertion of the TN3 transposable element into a 4 kb constructed plasmid (pTU4) of partially known DNA sequence were studied by restriction endonuclease mapping, and 65 of these insertion sites were examined further by DNA sequence analysis. Our results show that the previously proposed regional specificity for Tn3 insertion is associated with a strong preference for AT-rich segments as insertion sites. Moreover, multiple insertions of the Tn3 occurred at certain AT-rich nucleotide positions, and 23 of 26 independent insertion events at a single nucleotide position were found to be in the same orientation. A region of the recipient plasmid showing major homology with the terminal 18 bp of Tn3 was identified in the vicinity of an 11 nucleotide segment that included three insertional hot spots and 36 independent insertions. Our results indicate that the site and orientation of insertion of Tn3 are at least partly determined by the primary nucleotide sequence of the recipient genome, and suggest that insertional hot spots may result from the combined effects of AT richness plus homology of the recipient genome with the terminal sequences of Tn3.

Adenine↗
PubMed1992

A physical map of the Salmonella typhimurium LT2 genome made by using XbaI analysis.

XbaI digestion and pulsed-field gel electrophoresis of the genome of Salmonella typhimurium LT2 yields 24 fragments: 23 fragments (total size, 4,807 kb) are from the chromosome, and one fragment (90 kb) is from the virulence plasmid pSLT. Some of the 23 fragments from the chromosome were located on the linkage map by the use of cloned genes as probes and by analysis of strains which gain an XbaI site from the insertion of Tn10. Twenty-one of the fragments were arranged as a circular physical map by the use of linking probes from a set of 41 lysogens in which Mud-P22 was stably inserted at different sites of the chromosome; fragment W (6.6 kb) and fragment X (6.4 kb) were not located on the physical map. XbaI digestion of strains with Tn10 insertions allowed the physical locations of specific genes along the chromosome to be determined on the basis of analysis of new-fragment sizes. There is good agreement between the order of genes on the linkage map, which is based primarily on P22 joint transduction and F-mediated conjugation, and the physical map, but there are frequently differences in the length of the interval from the two methods. These analyses allowed the measurement of the amount of DNA packaged in phage P22 heads by Mud-P22 lysogens following induction; this varies from ca. 100 kb (2 min) to 240 kb (5 min) in different parts of the chromosome.

DNA Transposable Elements↗
PubMed1985

Tn5 insertions in the agrocin 84 plasmid: the conjugal nature of pAgK84 and the locations of determinants for transfer and agrocin 84 production.

The kanamycin-resistance transposon Tn5 was randomly introduced into pAgK84, a 47.7-kb plasmid coding for agrocin 84 production in Agrobacterium. Using such marked plasmids, pAgK84 was found to be conjugal. It could be transferred to several Agrobacterium strains including those harboring octopine- or nopaline-type Ti plasmids. Its presence has no effect on Ti plasmid functions such as opine utilization and tumorigenicity, but it does confer agrocin 84 immunity upon previously sensitive strains. The plasmid could also be conjugally transferred to a Nod+ Fix+ strain of Rhizobium meliloti. The production of agrocin 84 is expressed in all Agrobacterium and Rhizobium transconjugants tested. The agrocin plasmid could not be introduced into restrictionless Escherichia coli or Pseudomonas aeruginosa recipients by conjugation or transformation. The sites of 92 independent Tn5 insertions were mapped on pAgK84. These insertions are dispersed over the entire length of the plasmid. Analysis of the sites and effects of the Tn5 insertions has allowed us to construct a functional map of pAgK84. Forty-three of these insertions, spanning a 20-kb segment of the plasmid, abolished or greatly reduced the production of agrocin 84. The presence of two insertions within this segment having an effect on agrocin production suggests that at least three regions of the plasmid are involved in agrocin 84 biosynthesis. Fourteen of the Tn5 insertion derivatives are no longer conjugally transferable. These insertions all map to a single region of the plasmid and define about 3.5-kb as being associated with transfer functions.

Adenine Nucleotides↗
PubMed1991

Construction of a physical map of the chromosome of Shigella flexneri 2a and the direct assignment of nine virulence-associated loci identified by Tn5 insertions.

To establish the molecular basis of the chromosomal virulence genes of Shigella flexneri 2a (YSH6000), a Notl restriction map of the chromosome was constructed by exploiting Notl-linking clones, partial Notl digestion and DNA probes from various genes of Escherichia coli K-12. The map revealed at least three local differences in the placements of genes between YSH6000 and E. coli K-12. Using the additional Notl sites introduced by Tn5 insertion, nine virulence loci identified previously by random Tn5 insertions were physically mapped on the chromosome. To demonstrate the versatility of the Notl map in direct assignment of the virulence loci tagged by Tn5 to a known genetic region in E. coli K-12, the major class of avirulent mutants defective in the core structure of lipopolysaccharide (LPS) was examined for the sites of Tn5 insertions. The two Notl segments created by the Tn5 insertion in the Notl fragment were analysed by Southern blotting with two DNA probes for the 5' and 3' flanking regions of the rfa region, and shown to hybridize separately with each of them, confirming the sites of Tn5 in the rfa locus. This approach will facilitate direct comparison genetically mapped Tn5 insertion mutations of S. flexneri with genes physically determined in E. coli K-12.

Chromosome Mapping↗
PubMed1985

Nonrandom insertion of Tn5 into cloned human adenovirus DNA.

The bacterial transposable element Tn5 displays regional selectivity in target sites for transposition. To examine this integration specificity of Tn5, we have mapped 57 insertion events in a plasmid pXC1 containing a eukaryotic viral DNA fragment as a target for Tn5 insertional mutagenesis. We found a nonrandom distribution of integration sites in pXC1, suggesting preferred targets for transposition. However, DNA sequence analysis of seven mutants revealed no target site sequence specificity for Tn5 insertion. We demonstrated that the majority of these insertions mapped downstream from a fortuitous promoter sequence which was present and active in this cloned insert in pXC1. Furthermore, when this promoter region was removed, Tn5 was able to transpose into previously unused upstream target sequences. Our data suggest that transcriptional activity may influence Tn5 transposition.

Adenoviruses, Human↗
PubMed2000

A novel Drosophila alkaline phosphatase specific to the ellipsoid body of the adult brain and the lower Malpighian (renal) tubule.

Two independent Drosophila melanogaster P(GAL4) enhancer-trap lines revealed identical GAL4-directed expression patterns in the ellipsoid body of the brain and in the Malpighian (renal) tubules in the abdomen. Both P-element insertions mapped to the same chromosomal site (100B2). The genomic locus, as characterized by plasmid rescue of flanking DNA, restriction mapping, and DNA sequencing, revealed the two P(GAL4) elements to be inserted in opposite orientations, only 46 bp apart. Three genes flanking the insertions have been identified. Calcineurin A1 (previously mapped to 21E-F) lies to one side, and two very closely linked genes lie to the other. The nearer encodes Aph-4, the first Drosophila alkaline phosphatase gene to be identified; the more distant gene [l(3)96601] is novel, with a head-elevated expression, and with distant similarity to transcription regulatory elements. Both in situ hybridization with Aph-4 probes and direct histochemical determination of alkaline phosphatase activity precisely matches the enhancer-trap pattern reported by the original lines. Although the P-element insertions are not recessive lethals, they display tubule phenotypes in both heterozygotes and homozygotes. Rates of fluid secretion in tubules from c507 homozygotes are reduced, both basally, and after stimulation by CAP(2b), cAMP, or Drosophila leucokinin. The P-element insertions also disrupt the expression of Aph-4, causing misexpression in the tubule main segment. This disruption extends to tubule pigmentation, with c507 homozygotes displaying white-like transparent main segments. These results suggest that Aph-4, while possessing a very narrow range of expression, nonetheless plays an important role in epithelial function.

Alkaline Phosphatase↗
PubMed1998

Mapping the intercellular adhesion molecule-1 and -2 binding site on the inserted domain of leukocyte function-associated antigen-1.

By extensive mutagenic analysis of the inserted domain (I-domain) of the alpha-chain (CD11a) of the leukocyte function-associated antigen-1 (LFA-1), we have defined a putative binding surface for intercellular adhesion molecules 1 and 2 (ICAM-1 and -2). This analysis showed that individually mutating Leu-205 or Glu-241 to alanine completely abolished LFA-1 binding to ICAM-1 or -2 without affecting I-domain structure, as assayed by antibody binding. Mutating Thr-243 to alanine also had a profound effect on LFA-1 binding to ICAM-1 and -2, as seen by complete loss of binding to ICAM-1 and a significant reduction (70% decrease) in binding to ICAM-2. Mutating Glu-146 to alanine reduced LFA-1 binding to ICAM-1 or -2 by 70%, and mutating His-264 or Glu-293 to alanine reduced binding to ICAM-1 or -2 by about 30-40%. Mutating Thr-175 to alanine reduced binding to ICAM-1 by about 30% and binding to ICAM-2 by about 70%. Interestingly, mutating Lys-263 to alanine preferentially abolished LFA-1 binding to ICAM-2. Using these data, we have generated a model of the interface between the LFA-1 I-domain and residues in the first domain of ICAM-1 that have been shown to be critical for this interaction. In addition, this model, together with the ICAM-2 crystal structure, has been used to map residues that are likely to mediate LFA-1 I-domain binding to ICAM-2.

Amino Acid Sequence↗
PubMed1998

Ablation of manifest left free wall accessory pathways with polarity reversal mapping: ventricular approach.

Polarity reversal mapping for localization of the left free wall accessory pathway (AP) at the atrial insertion site has been shown to be effective for successful ablation, but this technique requires atrial septal puncture. We evaluated the safety, efficacy, and reproducibility of two dimensional polarity reversal mapping at the ventricular insertion site of the accessory pathway without atrial septal puncture in symptomatic patients with manifested left free wall AP. Polarity reversal mapping under the mitral annulus by transaortic approach was performed in 10 consecutive patients with conventional ablation catheter (6 French, 4 mm tip, 2 mm interelectrode distance), during sinus rhythm or atrial pacing. A low set high, bandpass filter (0.005-400Hz) was used. Radiofrequency (RF) ablation was performed at the site of ventricular electrocardiogram polarity reversal during sinus rhythm. Polarity reversal was identified in all patients at the ventricular side of the mitral annulus. Ablation was successful in all patients without complications. The procedure time was 86.0 +/- 21.1 min, the fluoroscopic exposure time was 16 +/- 12 min, the number of RF applications was 8 +/- 6, the power level 21 +/- 7 watts, and the time to initial AP block was 3.0 +/- 0.9 sec. Polarity reversal mapping is a safe and efficient technique at the ventricular insertion site. This technique might be complementary to the currently-utilized activation mapping technique.

Adult↗
PubMed1997

Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors.

Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.

Amino Acid Sequence↗
PubMed1991

Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification.

The structural gene, nirS, for the respiratory nitrite reductase (cytochrome cd1) from Pseudomonas stutzeri was identified by (i) sequencing of the N-terminus of the purified protein and partial sequencing of the cloned gene, (ii) immunoscreening of clones from a lambda gt11 expression library, (iii) mapping of the transposon Tn5 insertion site in the nirS mutant strain MK202, and (iv) complementation of strain MK202 with a plasmid carrying the insert from an immunopositive lambda clone. A mutation causing overproduction of cytochrome c552 mapped on the same 8.6 kb EcoRI fragment within 1.7 kb of the mutation affecting nirS. Two mutations affecting nirD, which cause the synthesis of an inactive cytochrome cd1 lacking heme d1, mapped 1.1 kb apart within a 10.5 kb EcoRI fragment contiguous with the fragment carrying nirS. Nir- mutants of another type that had low level synthesis of cytochrome cd1, had Tn5 insertions within an 11 kb EcoRI fragment unlinked to the nirS+ and nirD+ fragments. Cosmid mapping provided evidence that nirS and nirD, and the previously identified gene cluster for nitrous oxide respiration are closely linked. The nirS gene and the structural gene for nitrous oxide reductase, nosZ, are transcribed in the same direction and are separated by approximately 14 kb. Several genes for copper processing are located within the intervening region.

Amino Acid Sequence↗
PubMed1991

Completion of the IS map in E. coli: IS186 positions on the E. coli K12 chromosome.

Insertion sites of the transposable element IS186 were physically mapped in the genome of E. coli K12 strain BHB2600. This strain maintains four IS186 copies of which three, assigned to 0.3, 14.1 and 51.8 map min., share common map positions with the three IS186 copies in strains W3110 and HB101. The fourth, unique IS copy in BHB2600 maps at 49.3 min. The IS186 data complete the BHB2600 map for all chromosomal sites of known K12-associated IS types.

Blotting, Southern↗