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At least 991 records · Page 55Linked to original sources
PubMed1997

Physical map of the Clostridium difficile chromosome.

Clostridium difficile is a causative agent in antibiotically induced diarrhea and pseudomembraneous colitis. The ability of strains of C. difficile to cause disease depends upon the presence of two toxin genes and their corresponding proteins, designated toxin A and toxin B. Previous studies conducted in this laboratory indicated that toxigenic strains of C. difficile possess both toxin genes, whereas non-toxigenic strains do not. Likewise, the studies showed that toxigenic and non-toxigenic strains of C. difficile differ significantly in chromosomal organization by ribotype analysis. Therefore, the chromosomal organization of a reference strain was investigated. Pulsed field gel electrophoresis was utilized to generate a physical map of the chromosome of the toxigenic Clostridium difficile strain ATCC 43594. Restriction digestions of whole chromosomes with the enzymes NruI and SacII generated consistent macrofragment profiles. NruI digestion resulted in 14 discernible bands containing 16 fragments of DNA. SacII digestions resulted in 14 discernible bands containing 15 fragments of DNA. Restriction digestions with both SacII and NruI resulted in 21 discernible bands containing 31 fragments of DNA. Probing of single and double digests with an extensive set of NruI and SacII single- and double-digest bands clarified the location of individual fragments in relation to one another, resulting in a restriction map of the chromosome. PCR-generated probes of five loci of C. difficile were used to map the location of seven genes on the chromosome. Finally, the addition of all fragments from NruI, SacII and NruI/SacII digestions resulted in an approximate chromosome size of 4.4 Mb.

Chromosomes, Bacterial↗
PubMed1994

Rapid mapping of NotI linking clones with differential hybridization and Alu-PCR.

For construction of a NotI restriction map of the human genome, the isolation and mapping of unique NotI linking clones represent important and critical steps. Recently we have shown that an Alu-PCR approach can be used for isolation of NotI linking clones from defined regions of the chromosomes. This represents a useful method for isolating and analyzing a small number of clones, but it would be laborious to use it for mapping many NotI linking clones simultaneously. Here we suggest another modification of Alu-PCR for rapid concurrent mapping of many NotI linking clones. The results clearly demonstrate the utility of this approach. Seventy-one random NotI linking clones were analyzed. Among them, 65 clones (91.5%) were correctly selected and mapped using this approach. With differential hybridization and Alu-PCR, a significant portion of all human NotI linking clones (> 30%) can be rapidly mapped to particular chromosomes or to defined regions of these chromosomes.

Animals↗
PubMed1998

Homozygous deletion and frequent allelic loss of the 21q11.1-q21.1 region including the ANA gene in human lung carcinoma.

The frequent occurrence of 21q deletions in human non-small cell lung carcinoma (NSCLC) indicates the presence of a tumor suppressor gene on this chromosome arm. Since the ANA (Abundant in Neuroepithelium Area) gene, a member of an antiproliferative gene family, was mapped to 21q11.2-q21.1, we searched for genetic alterations of the ANA gene in human lung cancers. The gene was homozygously deleted in a human NSCLC cell line, Ma17. The gene was mapped in the 0.33 Mb Not1 fragment at 21q21.1 of the Not1 restriction map for 21q. Loss of heterozygosity (LOH) at this locus was detected in 24/47 (51.1%) of NSCLC, and the frequency of LOH in brain metastases was significantly higher than that in stage I-II primary tumors (P = 0.018). These results suggested that the homozygously deleted region harbors a novel tumor suppressor gene involved in NSCLC progression. Since mutation of the ANA gene was not detected in other lung cancer cell lines and fresh lung tumors with LOH at this locus, it is unlikely that the ANA gene is a target gene inactivated by two mutational events in this chromosomal region. Physical mapping of the homozygously deleted region showed that the deletion had occurred interstitially at 21q11.1-q21.1 and the size of the deletion was estimated as being more than 3 Mb. Our mapping results will facilitate further efforts to identify a tumor suppressor gene on 21q.

Aged↗
PubMed1989

The acid phosphatase genes PHO10 and PHO11 in S. cerevisiae are located at the telomeres of chromosomes VIII and I.

Of the three regulated acid phosphatase genes in S. cerevisiae (PHO5, PHO10 and PHO11) two have previously been cloned (PHO5 and PHO11). We have now identified PHO10 and show by restriction mapping that it is highly homologous to PHO11. This homology includes not only the coding sequence but also a stretch of about 2 kb upstream and 2.2 kb downstream of the genes. Analysis of strains in which either gene had been disrupted shows that the two genes are located at the telomeres of two different chromosomes. PHO10 3.6 kb from the end of a chromosome I. This makes PHO11 the gene closest to the end of a chromosome that has been physically mapped so far in S. cerevisiae. The organization of the two genes varies strongly from strain to strain consistent with a high incidence of telomere rearrangement. In one of twenty transformants examined a conversion event could be directly demonstrated that resulted in a chromosome VIII which had acquired a copy of the telomere from chromosome I.

Acid Phosphatase↗
PubMed1990

Branch capture reactions: effect of recipient structure.

Branch capture reactions (BCR) contain two DNA species: (i) a recipient restriction fragment terminating in an overhang and (ii) a displacer-linker duplex terminating in a displacer tail complementary to the overhang as well as contiguous nucleotides within the recipient duplex. Branched complexes containing both species are captured by ligation of the linker to the recipient overhang. Specificity depends upon branch migration and is increased by substitution of bromodeoxycytidine for deoxycytidine in the displacer. BCR rates and specificities were determined for recipient overhangs that were (i) 5' and 3', (ii) 3 and 4 nucleotides long, and (iii) 0-100% G+C. Model systems permitted independent determination of G+C and branching effects on ligation rates and verification of rapid equilibrium between the branched complex and its component species. With all 4-base overhangs, recipient duplexes permitting extensive branch migration became saturated with displacer-linker duplexes. With increasing G+C, increasing ligation at competing sites led to decreased BCR specificity. BCR may be used to label a DNA fragment prior to electrophoresis, mark a fragment for affinity chromatography, or introduce a new overhang sequence compatible with a restriction endonuclease site in a cloning vector. A protocol was confirmed for mapping restriction sites in cloned DNA.

Base Sequence↗
PubMed1994

Two major groups of vitellogenin cDNA clones from Oreochromis aureus (Steindachner).

cDNA libraries were constructed using livers of male and female Oreochromis aureus that were stimulated with estradiol. Restriction map analyses of vitellogenin (Vg) cDNA clones indicated 4 subgroups with related restriction patterns, represented by pOAVg2 (from female) and pOAVg50, 71 & 87 (from male) fish. With the exception of pOAVg2, all other clones obtained from female fish resembled pOAVg50. There are no internal repeats within these cDNA clones. Southern blot cross-hybridisations distinguished pOAVg87 (group A) from pOAVg2, 50 and 71 (group B). The existence of these 2 major groups of Vg cDNAs was further confirmed by dot blot hybridisations and hybrid melting assays under varying stringencies. The difference of melting temperature, Tm between the 2 groups suggests a 7% intraspecific divergence in the Vg gene family.

Animals↗
PubMed1981

Linear mitochondrial deoxyribonucleic acid from the yeast Hansenula mrakii.

The mitochondrial deoxyribonucleic acid (mtDNA) from a petite-negative yeast, Hansenula mrakii, was studied. A linear restriction map was constructed with 11 restriction enzymes. The linearity of the genome was confirmed by direct end labeling of the molecule, followed by restriction analysis. The molecular weight of the DNA was found to be 55,000 base pairs. This is the first linear mtDNA found in yeast species. Using specific gene probes obtained from Saccharomyces cerevisiae mtDNA, we have constructed a gene map of H. mrakii mtDNA. The arrangement of genes in this linear genome was very different from the circular mtDNA of other known yeasts.

Chromosome Mapping↗
PubMed1980

Histone genes from Xenopus laevis: molecular cloning and initial characterization.

Histone DNA sequences, were detected in Eco RI fragments of total Xenopus laevis DNA, by hybridization with 32P-labeled h22-DNA, a histone gene repeat unit of the sea urchin Psammechinus miliaris. The about 6 kb-size class, which was found to hybridize, was subsequently integrated into the E. coli plasmid pCR1. A clone was isolated that contains a 5.8 kb EcoRI fragment hybridizing with h22-DNA. A physical map was constructed using the restriction endonucleases BamHI, PstI, HincII, BglII, XbaI, PvuII, XhoI, AvaI, SmaI, HinfI and HpaII. The fragment was not cleaved by KpnI, AvaI, SalI and HindIII. Using this restriction map we were able to determine the gene order by hybridization with purified gene probes derived from h22-DNA. The gene order was found to be H3, H4, H2A and H2B. The localization of the H1 gene was not possible, probably due to its greater evolutionary divergence. Part of the sequence of the H3-gene is presented providing unambiguous evidence on the identity, map position and polarity of this gene.

Animals↗
PubMed1979

A restriction endonuclease cleavage map of bacteriophage P2.

A restriction endonuclease cleavage map of phage P2 was constructed. The enzymes used and, within parenthesis, the number of their cleavage sites on the P2 lg cc DNA molecule were: AvaI(3), BalI(1), BAMI(3), BglII(3), HaeIII (more than 40; only three were mapped), HindIII(0), HpaI(10), KpnI(3), PstI(3), SalI(2) and SmaI(2). The EcoRI cleavage sites (3), as determined earlier, were used as reference points for this study. The DNAs of a variety of P2 mutants carrying chromosomal aberrations (dell, del2, del3, del6, vir22, vir37(2), vir79 and vir94) were also similarly examined.

Chromosome Aberrations↗
PubMed1995

Construction of two YAC contigs in human Xp11.23-p11.22, one encompassing the loci OATL1, GATA, TFE3, and SYP, the other linking DXS255 to DXS146.

We have constructed two YAC contigs in the Xp11.23-p11.22 interval of the human X chromosome, a region that was previously poorly characterized. One contig, of at least 1.4 Mb, links the pseudogene OATL1 to the genes GATA1, TFE3, and SYP and also contains loci implicated in Wiskott-Aldrich syndrome and synovial sarcoma. A second contig, mapping proximal to the first, is estimated to be over 2.1 Mb and links the hypervariable locus DXS255 to DXS146, and also contains a chloride channel gene that is responsible for hereditary nephrolithiasis. We have used plasmid rescue, inverse PCR, and Alu-PCR to generate 20 novel markers from this region, 1 of which is polymorphic, and have positioned these relative to one another on the basis of YAC analysis. The order of previously known markers within our contigs, Xpter-OATL1-GATA-TFE3-SYP-DXS255146- Xcen, agrees with genomic pulsed-field maps of the region. In addition, we have constructed a rare-cutter restriction map for a 710-kb region of the DXS255-DXS146 contig and have identified three CPG islands. These contigs and new markers will provide a useful resource for more detailed analysis of Xp11.23-p11.22, a region implicated in several genetic diseases.

Animals↗
PubMed1995

Construction of 110 cosmid markers and a 4.5-Mb YAC contig on human chromosome 8p12-q11.

Microcell hybrids containing various regions of human chromosome 8 were formed by microcell-mediated transfer of neo-tagged chromosome 8 into the cells derived from severe combined immunodeficiency (SCID) mouse. Thus, 110 cosmid markers were isolated from SV40-transformed SCID fibroblast cell line (SCVA) containing a p12-q11.1 region of human chromosome 8 and were assigned to eight regions in 8p12-q11.1, using a microcell-hybrid panel. For positional cloning of a human gene that restores the DNA-repair defect in a mouse with SCID on 8p11.1-q11.1 (SCID region), we constructed a yeast artificial chromosome (YAC) contig of about 4.5 Mb. Overlapping YACs were further aligned by restriction mapping, using rare-cutting restriction endonucleases. The cosmids and YAC contig should facilitate isolation of the SCID gene and other genes, such as the Werner syndrome-responsible gene in or near this region.

Animals↗
PubMed1980

A physical map of the permuted genome of bacteriophage T1.

A restriction map has been constructed for the DNA of coliphage T1 which locates the cleavage sites of the restriction endonucleases, BglI (6 cuts), BglII (16 cuts), EcoRI (2 cuts), HindIII (2 cuts) and PstI (2 cuts). Digestions with BglI and BglII reveal fragments which are present in sub-molar quantities. Two methods, one using the selective removal of molecular ends with exonuclease III and the other involving the comparison of digestion patterns of concatemeric and virion DNA, have shown that the submolar fragments are at or close to the ends of the molecules. Digestions with BglI show that one terminal fragment has a very precise molecular weight whereas all the others are of heterogenous molecular weight. These results are consistent with the model for DNA packaging in which maturation is initiated at a precise site on a concatemeric precursor and proceeds by the encapsidation of up to four successive 'headfuls' of 1.065 genome equivalents (MacHattie and Gill 1977).

Chromosome Mapping↗
PubMed1997

Restriction enzyme and DNA hybridization analysis of cellulolytic Streptomyces isolates of different origin.

Streptomyces rochei A2 endoglucanase (eglS) and beta-glucosidase (bgs1) genes were used as probes to survey their distribution among 16 Streptomyces strains isolated from different sources and characterized for their cellulolytic activities. The eglS probe hybridized to the genomic DNA of 12 strains with a restriction pattern different from that of S. rochei A2. The DNA from all strains, except one, hybridized with the bgs1 probe and one strain showed the same restriction pattern as seen in S. rochei A2. The sequence localized by the eglS probe in S. thermoviolaceus and the one localized by the bgs1 probe in strain EC1 were cloned and expressed in E. coli in plasmids pTAE and pCSF203, respectively. The restriction maps showed that the cloned genes were identical to eglS and bgs1. The restriction enzyme analysis and genomic DNA from all the strains identified nine different groups, each characterized by a distinctive pattern and in agreement with the results of the hybridization experiments.

DNA, Bacterial↗
PubMed1996

Sequence-specific DNA modification in Acetobacter xylinum.

Two cryptic plasmids have been discovered in Acetobacter xylinum B42 and in its derivative PEA-1, a cellulose defective mutant. These two plasmids were designated pAX1 and pAX2 (50 and 105 kb in size, respectively). A restriction map was constructed for pAX1. Attempts to cure these plasmids were unsuccessful. Enzyme restriction analysis showed that these plasmids contain protected EcoRI and ApoI sites. Using Southern blot and hybridization techniques, the protection was extended to chromosomal DNA. Enzyme restriction analysis of several plasmids, from different origins and containing different incompatibility groups, isolated from strain PEA-1 also showed EcoRI and ApoI protection. The presence of modifications on specific sequences was not found in A. xylinum 8747. These results strongly suggest the presence of a modification system in A. xylinum B42 that recognizes the tetranucleotide 5'-AATT.

Base Sequence↗
PubMed1997

Physical mapping shows that the unstable oxytetracycline gene cluster of Streptomyces rimosus lies close to one end of the linear chromosome.

A restriction map of the 8 Mb linear chromosome of Streptomyces rimosus R6-501 was constructed for the enzymes Asel (13 fragments) and Dral (7 fragments). Linking clones for all 12 Asel sites and 5 of the 6 Dral sites were isolated. The chromosome has terminal inverted repeats of 550 kb, which are the longest yet reported for a Streptomyces species. The oxytetracycline gene cluster lies about 600 kb from one end, which might account for its frequent spontaneous amplification and deletion. Several other markers were localized on the chromosome (dnaA and recA, the rrn operons, the attachment site for pSAM2 and prophages RP2 and RP3). Comparison of the conserved markers with the map of Streptomyces coelicolor A3(2) suggested there are differences in genome organization between the two species.

Chromosome Mapping↗
PubMed1989

Computers: the best friends a human genome ever had.

Mapping and sequencing the human genome will generate large amounts of data, which must be sorted, analyzed, and stored for rapid retrieval to complete this enormous task. Computers and their software programs provide the most important tool to the molecular biologist today. A discussion of current capabilities and future needs in computer hardware and software for the human genome project is the topic of this paper. The use of computer programs to generate restriction maps, manage clone libraries, manage sequence projects, and generate consensus sequences is presented. The use of computers to communicate useful information rapidly to scientific colleagues is also mentioned. The role of both GenBank and BIONET is central to the dissemination and analysis of sequence information. The capabilities of electronic communication worldwide for assisting this project is available on the BIONET National Computer Resource, using existing networks.

Base Sequence↗
PubMed1991

Mapping DNA by stochastic relaxation: schedule for optimal annealing.

The analysis of the various cooling schedules for the simulated annealing algorithm is made towards the restriction map construction. Algorithm behaviour under control of three different cost functions is considered and the discrete cost function is found to handle successfully experimental data with realistic error sizes. A program using this function has been derived from the algorithm to construct linear and circular DNA maps.

Algorithms↗
PubMed1995

Physical map of the region containing the gene for Batten disease (CLN3).

CLN3 has been mapped genetically to 16p12, to the interval between D16S288 and D16S383, a sex-averaged genetic distance of 2.1 cM. Analysis of disease haplotypes for four microsatellite markers in this interval, D16S288, D16S299, D16S298, and SPN, has shown significant allelic association between one allele at each of these loci and CLN3. All four of the associated markers were used as nucleation sites in the isolation of genomic clones (YACs). A contig was assembled which contains 3 of the 4 associated markers and which confirmed the relative order of these markers. Marker D16S272 has been located on the physical map between D16S288 and D16S299. Restriction mapping has demonstrated the location of possible CpG islands. One gene, STP, has been localised on the YAC contig proximal to D16S298 and is therefore a candidate for CLN3. Other genes, including IL4R, SGLT2, and UQCRC2, have been excluded from this region.

Alleles↗