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PubMed1980

[Transposon-induced genetic rearrangements in the chromosome of Vibrio El Tor and their application to the construction of the first genetic map of a cholera vibrio].

The specificity of integration of chloramphenicol resistance transposon (Tn9) into Vibrio El Tor chromosome was studied. As shown by the isolation of auxotrophs offerring various nitritional requirements, Tn9 displayed fairly low specificity in its interaction with the chromosome. When inserted into cholera vibrio chromosome, Tn9 induced secondary rearrangements of genetic material. Nutritional requirements were identified for the polyauxotrophic clones which resulted from the mutations in chromosome region adjacent to the insertion sites of Tn9 and Tn10. Based on these observations a preliminary map was constructed for certain regions of V. eltor chromosome.

Chromosome Mapping↗

PubMed1976

Prophage map of converting corynebacteriophage beta.

A prophage map for corynebacteriophage beta consisting of seven markers has been constructed and compared with the vegetative map. The mapping system utilizes heteroimmune double lysogens and capitalizes on the fact that these double lysogens are very unstable and throw off monolysogenic segregants. The prophage map, produced by characterizing the recombinant phage in these monolysogenic segregants, appears to be a cyclic permutation of the vegetative map with the gene for toxin at one end of the prophage map and the gene for phage immunity at the other. This permutation is in accord with the Campbell model for insertion of lambda phage if a site between the toxin and immunity genes in the vegetative map is designated as the phage attachment site. The position of the gene for toxin in the prophage map suggests that converting phages may have originated as specialized transducing phages for this gene.

Bacteriophages↗

PubMed1990

Recovery and characterization of hybrid dysgenesis-induced mei-9 and mei-41 alleles of Drosophila melanogaster.

X-Linked methyl methanesulfonate (MMS)-sensitive mutations were induced with hybrid dysgenesis using four P strains: pi 2, Harwich, T-007 and OK-1. Mutations were identified after two generations of backcrosses to M strain females to replace the autosomes. Among 51,471 X-chromosomes examined 10 carried stable MMS-sensitive mutations representing 8 independent events. Males of the mutant strains failed to induce gonadal dysgenesis in crosses to Oregon-R females at 28.5 degrees C. Complementation tests showed that 3 of the induced mutations were mei-9 alleles, 2 were mei-41 alleles, 1 was a mus102 allele, and 2 were alleles at a newly identified MMS-sensitive locus, mus112 (map position: 1-32.8). As assayed by in situ hybridization on polytene chromosomes, each X-chromosome had no more than four P element insertions. 4 of the 8 mutations recovered in this study proved to have P element insertions at or very close to sites to which MMS sensitivity has been mapped. Hybrid dysgenesis-induced reversion of 2 mutants, mei-9RT1 and mei-41RT2, is associated with the loss of the P element from regions 4B and 14C respectively.

Alleles↗

PubMed1985

Restriction endonuclease mapping of the hepatitis B viral genome isolated from Taiwan.

The Eco RI fragment of hepatitis B virus (HBV) DNA isolated from human blood plasma Dane particles were inserted into plasmid pUC8 Eco RI site and transformed into E. coli JM103 host. Two recombinants pTWL1 and pTWL2 were found to carry 3.2 kbp fragment and proved to have HBV genome by Southern hybridization method. The 1.4 kbp Bam HI fragment which carried the hepatitis B viral surface antigen (HBsAg) gene, obtained via Bam HI digestion of Dane particles DNA which was made fully double stranded by endogenous DNA polymerase reaction, was also inserted into plasmid pUC8 Bam HI site. Four recombinant clones, pTWS1, pTWS2, pTWS3, and pTWS4 were found. Only one of the clones pTWS1 carried the HBsAg gene in a correct orientation with respect to the lac promoter sequence. The physical mapping of HBV DNA was performed with several restriction endonucleases. Our results indicated that the HBV DNA insert contains unique XbaI and HpaI cleavage sites and lacks the cleavage sites for the HindIII, SmaI, KpnI, SalI, and SstI endonucleases. The locations of Bam HI, BglII, and HincII endonucleases cleavage sites within the cloned HBV DNA of the pTWL1 plasmid were similar to that HBV DNA of adw and adw2 subtypes.

Chromosome Mapping↗

PubMed1987

Construction of an ordered overlapping library of bacteriophage P1 DNA in phage vector lambda D69.

A library of bacteriophage P1 DNA was constructed in the phage vector lambda D69. The DNA of some 150 randomly chosen lambda-P1 hybrid phages containing P1 DNA fragments 5-10 kb in size was analyzed by restriction endonuclease digestion using enzymes EcoRI, BglII, and BamHI that cleave P1 DNA at known positions on the physical map of P1. Approximately one third of the phages contained P1 DNA inserts having two or more restriction sites for any one of these enzymes, thus allowing the location of the insert to be determined with respect to the physical map. Genetic tests allowed detection of lambda-P1 hybrid phages possessing inserts with functional P1 ban and CmR genes. A subset of 18 phages was analyzed in more detail; their P1 DNA inserts comprise an ordered collection of overlapping P1 DNA fragments that cover almost 98% of the P1 genome.

Bacteriophage lambda↗

PubMed1984

The structural gene for AMP nucleosidase. Mapping, cloning, and overproduction of the enzyme.

A mutant of Escherichia coli which contained no detectable AMP nucleosidase activity (EC 3.2.2.4) was produced by treatment with nitrosoguanidine and identified by a colorimetric assay for AMP nucleosidase in individual colonies from agar plates. Conjugation experiments indicated a close linkage between the AMP nucleosidase locus (amn) and his. Assays for AMP nucleosidase in E. coli strains with deletions in the his region established that amn is not located between attP2H and mgl . Transduction experiments with bacteriophage P1 were used to construct a linkage map in the his and amn region of the 100-min chromosome map of E. coli, which places amn at 43.3 min. A Rec- strain of E. coli ( FP4102 ) which carries the uvrC to his region (42.1-44.1 min) as an F' episome was used to confirm this location. The episome from FP4102 was used as the source of DNA for cloning amn. BamHI restriction fragments of the episome were inserted into the homologous site of pBR322 and used to transform the Amn- strain of E. coli to Amn+. The transforming plasmid contained a 9-kb (kilobase) insert. Partial restriction of plasmid with ClaI and religation gave a plasmid with a 6-kb insert which retains the amn gene. Restriction mapping of the plasmid has identified ClaI and PstI sites which appear to be within the amn locus. E. coli (Rec-) which contains the 9-kb plasmid of pBR322 containing amn overproduces AMP nucleosidase when grown in the presence of ampicillin. The specific activity of AMP nucleosidase increases from 0.016 mumol/min/mg of protein to 0.32 mumol/min/mg of protein in extracts of the wild type and the plasmid-bearing strains, respectively. A simple purification procedure yields 10 mg of homogeneous AMP nucleosidase from 25 g of packed cells.

Bacteriophage lambda↗

PubMed1990

Characterization of RAD4 gene required for ultraviolet-induced excision repair of Saccharomyces cerevisiae propagated in Escherichia coli without inactivation.

The previously isolated RAD4 gene designated as pPC1 from the genomic library of Saccharomyces cerevisiae (Yoon et al., 1985, Korean J. Genetics 7, 97-104) appeared to propagate in Escherichia coli and yet retained its complementing activity to rad4 mutants without inactivation. The subcloned RAD4 gene was found to be localized within a 2.5 kb DNA fragment flanking Bg1II and BamHI sites in the insert DNA, and was shown to have the same restriction map as a yeast chromosomal DNA, as determined by Southern hybridization. Tetrad analysis and pulse-field chromosome mapping have revealed that the cloned RAD4 gene can be mapped and integrated into the yeast chromosome V, the actual site of this gene. DNA-tRNA hybridization has shown that the isolated RAD4 gene did not contain a suppressor tRNA gene. These results have indicated that the pPC1 is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplification in E. coli.

Chromosome Mapping↗

PubMed1990

Use of a novel cassette to label phenotypically a cryptic plasmid of Bacillus subtilis and map loci involved in its stable maintenance.

In order to facilitate studies on the maintenance of cryptic plasmids from Gram-positive bacteria we have constructed a novel cassette cAPG1000 (5.0 kb) which carries both a selectable marker (chloramphenicol resistance from Staphylococcus aureus plasmid pC194) and a screenable marker (the xylE gene from the TOL plasmid of Pseudomonas putida expressed from a cloned promoter of Bacillus phage SPO2) and which is flanked by terminators to prevent transcription from the cassette activating or inhibiting loci adjacent to the site of insertion. To demonstrate the usefulness of this cassette we have mapped loci required for stable maintenance of an 8.6 kb cryptic plasmid endogenous to Bacillus subtilis (pPOD2000) from the properties of cAPG1000 insertion and insertion/deletion derivatives. We have identified the replication region as well as separate regions required for segregational and structural stability. The segregational mechanism is very efficient since it allows no detectable loss despite the fact that bacteria carrying the plasmid have a greatly increased mean generation time.

Bacillus subtilis↗

PubMed1992

Polymerase chain reaction based mapping of rye involving repeated DNA sequences.

A novel type of polymerase chain reaction (PCR) marker was developed for the mapping of cereal rye (Secale cereale). Primer pairs were synthesized targeting the insertion sites of three individual copies of the R173 family of rye specific repeated DNA sequences. While one primer was derived from a sequence within the respective R173 element, the second primer corresponded to a flanking region. The complex banding patterns obtained in rye allowed not only the mapping of the three R173 elements to certain chromosome regions of 1RS (the short arm of rye chromosome 1) but also the mapping of an additional 3-10 easily identifiable bands per primer pair to other rye chromosomes. Linkage mapping of a polymorphic 1R band derived from three rye cultivars demonstrated the presence of nonallelic, dominant markers in two independent crosses. Because of the high copy number of the R173 family (15,000 copies per diploid rye genome), its dispersion over the entire length of all chromosomes and the high number of markers obtained per primer pair, PCR markers based on the R173 family provide an almost unlimited source for well-spaced markers in rye mapping.

Base Sequence↗

PubMed1997

Zinc(II) tolerance in Escherichia coli K-12: evidence that the zntA gene (o732) encodes a cation transport ATPase.

A transposon (Tn 10dCam) insertion mutant of Escherichia coli K-12 was isolated that exhibited hypersensitivity to zinc(II) and cadmium(II) and, to a lesser extent, cobalt(II) and nickel (II). The mutated gene, located between 75.5 and 76.2 min on the chromosome, is named zntA (for Zn(II) transport or tolerance). The metal-sensitive phenotype was complemented by a genomic DNA clone mapping at 3677.90-3684.60 kb on the physical map. Insertion of a kanamycin resistance (KnR) cassette at a SalI site in a subcloned fragment generated a plasmid that partially complemented the zinc(II)-sensitive phenotype. DNA sequence analysis revealed that the KnR cassette was located within the putative promoter region of an ORF (o732 or yhhO) predicted to encode a protein of 732 amino acids, similar to cation transport P-type ATPases in the Cpx-type family. Inverse PCR and sequence analysis revealed that the Tn 10dCam element was located within o732 in the genome of the zinc(II)-sensitive mutant. The zntA mutant had elevated amounts of intracellular and cell surface-bound Zn(II), consistent with the view that zntA+ encodes a zinc(II) efflux protein. Exposure of the zntA mutant to cobalt(II) and cadmium(II) also resulted in elevated levels of intracellular and cell surface-bound metal ions.

Adenosine Triphosphatases↗

PubMed2001

[Fragile X mental retardation protein interacts with human NDK/Nm23-H2].

OBJECTIVE: To investigate the physiological role of fragile X mental retardation protein (FMRP) and screen the proteins interacting with FMRP in human fetal hippocampus cDNA library. METHODS: Human fetal hippocampus cDNA library was constructed in yeast two-hybrid DAD vector pGAD10. Quality of the library was measured by picking up random colonies as templates for PCR testing. Proteins interacting with FMRP were screened by yeast two-hybrid system. Furthermore, the interaction site of FMRP was mapped in yeast. RESULTS: The average length of inserts of the two-hybrid library was 1.5 kb, and the ratio of recombinant colonies was about 90%. Human NDK/Nm23-H2 was found interacting with FMRP. NDK/Nm23-H2 interacted with FMRP exon 1-12, as well as FMRP isoforms without exon 12, and exons 14-17. NDK/Nm23-H2 couldn't interact with FMRP exon 1-6 and exon 2-7 fragments. CONCLUSIONS: Human NDK/Nm23-H2 can bind FMRP directly. The interaction site of FMRP is located at its exon 1-11. This interaction in vitro might alter the intracellular distribution of NDK/Nm23-H2, and even regulates the transcription and expression of FMRP.

Fragile X Messenger Ribonucleoprotein 1↗

PubMed2005

KaPPA-view: a web-based analysis tool for integration of transcript and metabolite data on plant metabolic pathway maps.

The application of DNA array technology and chromatographic separation techniques coupled with mass spectrometry to transcriptomic and metabolomic analyses in plants has resulted in the generation of considerable quantitative data related to transcription and metabolism. The integration of "omic" data is one of the major concerns associated with research into identifying gene function. Thus, we developed a Web-based tool, KaPPA-View, for representing quantitative data for individual transcripts and/or metabolites on plant metabolic pathway maps. We prepared a set of comprehensive metabolic pathway maps for Arabidopsis (Arabidopsis thaliana) and depicted these graphically in Scalable Vector Graphics format. Individual transcripts assigned to a reaction are represented symbolically together with the symbols of the reaction and metabolites on metabolic pathway maps. Using quantitative values for transcripts and/or metabolites submitted by the user as Comma Separated Value-formatted text through the Internet, the KaPPA-View server inserts colored symbols corresponding to a defined metabolic process at that site on the maps and returns them to the user's browser. The server also provides information on transcripts and metabolites in pop-up windows. To demonstrate the process, we describe the dataset obtained for transgenic plants that overexpress the PAP1 gene encoding a MYB transcription factor on metabolic pathway maps. The presentation of data in this manner is useful for viewing metabolic data in a way that facilitates the discussion of gene function.

Computational Biology↗

PubMed1992

Isolation of anonymous DNA markers for human chromosome 22q11 from a flow-sorted library, and mapping using hybrids from patients with DiGeorge syndrome.

DiGeorge syndrome (DGS) is a human developmental defect of the structures derived from the third and fourth pharyngeal pouches. It apparently arises due to deletion of 22q11. We describe a strategy for the isolation of DNA probes for this region. A deleted chromosome 22, which includes 22q11, was flow-sorted from a lymphoblastoid cell line of a patient with cat eye syndrome and used as the source of DNA. A DNA library was constructed from this chromosome by cloning into the EcoR1 site of the vector Lambda gt10. Inserts were amplified by PCR and mapped using a somatic cell hybrid panel of this region. Out of 32 probes, 14 were mapped to 22q11. These probes were further sublocalised within the region by dosage analysis of DGS patients, and by the use of two new hybrid cell lines which we have produced from DGS patients. One of these lines (7939B662) contains the altered human chromosome segregated from its normal homologue. This chromosome 22 contains an interstitial deletion in 22q11, and will be useful for localising further probes to the DGS region.

Base Sequence↗

PubMed1978

Mapping of regions on cloned Saccharomyces cerevisiae 2-mum DNA coding for polypeptides synthesized in Escherichia coli minicells.

Saccharomyces cerevisiae 2-mum DNA and some of its restriction fragments were integrated in vector pCR1 ,pBR313 or pBR322 and their expression in Escherichia coli P678-54 minicells was analyzed. 2mum DNA inserted at the EcoRI site of pCR1 or pBR313 and at the PstI site of pBR322 promoted the synthesis of polypeptides of 48,000, 37,000, 35,000 and 19,000 daltons. The DNA regions coding for these polypeptides were mapped on the 2-mum DNA molecule by insertion of single EcoRI or HindIII restriction fragments and comparison of the polypeptides produced. For the synthesis of the 37,000 dalton polypeptide, intact sites RIB and H3 were required. The disappearance of the 37,000 dalton polypeptide on interruption of one of these sites by insertion of the vector, was correlated with the appearance of a polypeptide of 22,000 or 23,500 daltons respectively. The DNA sequence coding for the 37,000 daltons polypeptide, therefore, has to be located in the S-loop region close to or overlapping with the site RIB and H3. Assuming that the 22,000 and the 23,500 dalton polypeptides are truncated forms of the 37,000 dalton polypeptide, the last polypeptide can be exactly mapped. The polypeptide of 48,000 daltons was mapped to that half of the L-loop segment containing the sites H1 and H2. If, however, HindIII fragment H1-H2 was expressed, the 48,000 dalton polypeptide was lost and concomitantly a 43,000 dalton polypeptide appeared. We assume that this polypeptide results from early termination of the polypeptide of 48,000 daltons. The 35,000 and 9,000 dalton polypeptides were mapped to the S-loop region. The integrated inverted repeat sequence of yeast 2-mum Dna did not induce any detectable insert-specific polypeptide synthesis.

Base Sequence↗

PubMed1996

Rapid mapping of genomic P1 clones: the mouse L-isoaspartyl/D-aspartyl methyltransferase gene.

We report the mapping of the gene for the murine protein-L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1. 77) from a 129 mouse strain. This gene encodes an enzyme present in all tissues that can catalyze the first step of a repair reaction in which age-damaged proteins containing abnormal l-isoaspartyl (or d-aspartyl) residues can be converted to forms containing normal l-aspartyl residues. We first mapped the restriction sites from a genomic P1 clone using a rapid method generally applicable to all bacteriophage P1 clones containing large DNA inserts. We show that a single pulsed-field electrophoresis blot can be used to map an entire 89-kb P1 clone insert for eight restriction endonucleases with an error of no more than 2% of the length of the fragment, or 1 kb at the middle of the insert. In this method, we combine complete restriction endonuclease digestion at rare sites within the P1 vector with partial restriction endonuclease digestion within the insert. After size separation by pulsed-field gel electrophoresis and blotting, the fragments are detected by Southern hybridization with probes to the vector. This method is potentially useful for restriction mapping other large DNA clones such as artificial chromosomes. We then determined the positions of the exons of the methyltransferase gene by restriction mapping of long PCR fragments. The previously unidentified exon 8, which encodes the -DEL C-terminus of the more acidic isozyme II, was sequenced and mapped 5. 3 kb from the end of exon 7.

Animals↗

PubMed2004

Mu dI1(Ap lac) mutagenesis of Yersinia pestis plasmid pFra and identification of temperature-regulated loci associated with virulence.

The F1 capsule of Yersinia pestis, encoded by the 100 kb plasmid pFra, is often assumed to be essential for full virulence of Y. pestis. However, virulent strains of Y. pestis that are F1- and either pFra+ or pFra- have been reported. To assess the role of pFra-encoded factors in virulence, mutants in pFra with insertions of the defective transposing bacteriophage Mu dl(Ap lac) were obtained, by using the wild type (wt) and the pLcr-cured derivative of strain C092. Mutants that exhibited temperature regulation of lactose fermentation and retarded electrophoretic mobility of pFra were selected. A total of 15 insertion mutants were isolated in the wt strain (12 of which had a single insertion in the genome, in pFra); and 24 mutants in the isogenic pLcr- derivative. Four of the pLcr+ mutants, and none of the pLcr- mutants, were F1-. All F1- mutants were decreased in virulence for mice compared to the wt parent; and five of the F1+ mutants also were significantly attenuated in mice. Fusion end-joints of insert DNA were cloned into Escherichia coli by using pMLB524, a vector for rescuing operon fusions of lacZ. Recombinants were obtained which contained pFra inserts ranging from < 2kb to approximately 36 kb, and the insertions occurred at several sites on pFra. All of the four F1- mutants tested mapped within the F1 capsule operon (caf1). The remaining five attenuated mutants sequenced were F1+ and mapped outside of but near the operon. Sequencing and complete analysis of the pFra insertions mutants could facilitate identification of new potential virulence factors.

Animals↗

PubMed1997

Entrainment mapping in patients with sustained atrioventricular nodal reentrant tachycardia: insights into the sites of conduction slowing in the slow atrioventricular nodal pathway.

The inferoposterior region of the triangle of Koch is hypothesized to be the location of the atrial insertion of the slow atrioventricular (AV) nodal pathway. However, the actual site of conduction slowing in the slow AV nodal pathway is unknown. Entrainment mapping during AV nodal reentry can localize the reentrant pathway as follows: the AH interval measured from the mapping catheter = A'H (where A' is the exit site of the reentrant circuit) minus A'A (the conduction time from A' to the site of mapping); the SH interval during entrainment = SA' (the conduction time from stimulus into the reentry circuit) plus A'H. Thus, in all cases, the SH interval should be greater than or equal to the AH interval, and the deltaAH-SH should increase as distance and conduction time (SA' and A'A) from the reentry circuit increases. Fourteen patients with typical AV nodal reentry (cycle length 346 +/- 62 ms) and 1 with fast-slow (cycle length 430 ms) underwent activation and entrainment mapping from 8 to 12 sites in the triangle of Koch and coronary sinus. Pacing was performed at 2 to 3 mA above threshold, at a cycle length 10 ms shorter than tachycardia. A mapping site was defined as being in close proximity to the circuit if the deltaAH-SH was within 120% of the shortest 20th percentile deltaAH-SH value from all measured sites. In the 14 typical cases, 45 of 83 sites (54%) in the anatomic slow pathway region fulfilled criteria for close proximity to the reentry circuit compared with 13 of 50 sites (26%) outside of this region (p = 0.005). For these patients, the shortest SH interval measured from any entrainment site was 294 +/- 58 ms (89 +/- 10% of tachycardia cycle length, range 70% to 119%), indicating that the site of slow conduction in the slow pathway during AV nodal reentrant tachycardia was distal to all mapped sites. Thus, during typical AV nodal reentry, the "slow" pathway does not conduct slowly, and its insertion is located at or within the inferoposterior or midseptal regions in most cases.

Adult↗

PubMed1983

IS2 insertion is a major cause of spontaneous mutagenesis of the bacteriophage P1: non-random distribution of target sites.

Insertion mutations arising spontaneously in the P1 prophage and affecting vegetative phage reproduction were screened for the presence of insertion sequence 2 (IS2). Filter hybridization identified 28 out of 44 independent insertions as IS2. Their target specificity is not random. A region that amounts to < 2% of the phage genome had trapped 15 of the 28 IS2 elements. However, precise mapping of nine mutants in this hot spot segment revealed no preferred insertion site. Rather, the nine IS2 are distributed over the whole target segment and IS2 are found in both orientations. Sequence data indicate that at least two sequence variants of IS2 participated in mutagenesis of the phage genome. The detectable transposition of IS2 from the host chromosome to the prophage occurs with a frequency of 3 x 10(-5) per cell per generation under the particular experimental conditions. It is concluded that IS2, a natural resident of Escherichia coli K12 strains, is an important agent for spontaneous mutagenesis and exerts this action non-randomly along the genome.

Bacteriophage P1↗