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PubMed1989

High efficiency vectors for cosmid microcloning and genomic analysis.

We describe the construction and use of cosmid vectors designed for microcloning, gene isolation and genomic mapping starting from submicrogram amounts of eukaryotic DNA. These vectors contain (1) multiple cos sites to allow for simple and efficient cloning using non size-selected DNA; (2) bacteriophage T3 and T7 promoter sequences flanking the cloning site to allow for the synthesis of end-specific probes for chromosome walking; (3) a selectable gene for immediate gene transfer of cosmid DNA into mammalian cells; (4) recognition sequences for specific oligodeoxyribonucleotides to allow rapid restriction mapping; (5) unique NotI, SacII or SfiI sites flanking the cloning site to allow for removal of the cloned DNA insert from the vector. These cosmid vectors allow the construction of high quality genomic libraries in situations where the quantity of purified DNA is extremely limited, such as when using DNA prepared from purified mammalian chromosomes isolated by fluorescence-activated cell sorting.

Bacteriophages↗
PubMed1992

Radiation-reduced hybrids for the myotonic dystrophy locus.

The myotonic dystrophy (DM) gene maps to the long arm of human chromosome 19 and is flanked by markers ERCC1 and D19S51. Also mapping to this region is the polio virus receptor gene (PVS). To produce more markers for this interval, we have constructed radiation-reduced hybrids by selecting for the retention of ERCC1 and for the loss of PVS. One of the cell lines produced has been characterized extensively and contains about 2 Mb of human DNA derived exclusively from chromosome 19, and includes ERCC1 and D19S51. Phage libraries constructed from DNA of this cell line have been screened and several new markers identified, including two for which cDNAs have been isolated. These represent candidate genes for DM. The new markers have also been used to extend the long-range restriction map of this region.

Base Sequence↗
PubMed1990

Physical and genetic mapping of polymorphic loci in Xq28 (DXS15, DXS52, and DXS134): analysis of a cosmid clone and a yeast artificial chromosome.

Sequences corresponding to the Xq28 loci DXS15, DXS52, DXS134, and DXS130 were shown to be present in a 140-kb yeast artificial chromosome (YAC XY58, isolated by Little et al.). This YAC clone appears to contain a faithful copy of this genomic region, as shown by comparison with human DNA and with a cosmid clone that contains probes St14c (part of the DXS52 sequences) and cpX67 (DXS134). cpX67 and St14c are contained in 11 kb and detect the same MspI RFLP polymorphism. A comparison of the YAC restriction map and pulsed-field gel electrophoresis data leads us to propose the following order of loci: DXS52(VNTR)-DXS33-DXF22S3-DXS130-DXS134 -DXS52-DXS15-DXS52, this whole cluster being comprised within 575 kb. The physical proximity of the DXS15, DXS52, and DXS134 loci led us to reinvestigate recombination events that had been reported between these loci in families from the Centre d'Etude du Polymorphisme Humain. Our results do not support the assumption that this region shows increased recombination.

Chromosomes, Fungal↗
PubMed1989

Physical fine mapping of the choroideremia locus using Xq21 deletions associated with complex syndromes.

Characterization of several male-viable deletions and duplications with 20 random DNA probes has enabled us to subdivide the Xq21 region into seven discernible intervals. Almost all of the deletions spanning part of Xq21 are associated with choroideremia and mental retardation, with deafness being another common feature. The gene locus for choroideremia was assigned to interval 3 spanning the loci DXS95, DXS165, and DXS233. Genes for X-linked deafness and mental retardation were tentatively assigned to interval 2. Deletions of intervals 4 through 7 were not associated with any clinical abnormality. We have constructed a preliminary long-range restriction map of intervals 2 and 3 using field-inversion gel electrophoresis. The DXS232, DXS121, and DXS233 loci are located on the same SfiI fragment, whereas the DXS165 and DXS95 loci could not be linked to this cluster using SfiI and SalI.

Chromosome Deletion↗
PubMed1990

The gene for human acidic fibroblast growth factor encodes two upstream exons alternatively spliced to the first coding exon.

We have isolated two cDNA clones encoding human acidic fibroblast growth factor (aFGF) which represent the utilization of alternative upstream exons in aFGF mRNA. Isolation and sequence analysis of genomic clones spanning the first coding exon and each of the upstream sequences confirms that the divergent 5' sequences are separate exons, spliced alternatively to the first coding exon 34 nucleotides upstream of the initiator AUG codon. Restriction mapping of the genomic clones provides a minimum size estimate of 45 kilobase pairs for the aFGF locus.

Base Sequence↗
PubMed1989

Shared surface epitopes among trypanosomes of the same serodeme expressing different variable surface glycoprotein genes.

African trypanosomes evade the immune response of the mammalian host by undergoing antigenic variation, caused by sequence changes in a variable surface glycoprotein (VSG). The majority of trypanosome clones analyzed thus far are not known to share surface exposed epitopes or express appreciably homologous VSGs. We show here that four clones of Trypanosoma brucei from the same serodeme express different VSGs and share exposed epitopes to varying degrees, as defined by monoclonal antibodies. Rabbit antiserum against any one of the four VSGs recognizes epitopes present on all four trypanosomes in live cell immunofluorescence assay. The expressed VSGs are partially homologous at the N-terminus with multiple point substitutions of amino acids which distinguish each of the four VSGs. The genes coding for these VSGs are members of one gene family and an expression-linked copy with a unique restriction map is present in each trypanosome. Analysis of the ontogeny of the expressed genes should reveal mechanisms of evolution in trypanosome variable antigen repertoires.

Amino Acid Sequence↗
PubMed1992

Evidence and characterization of temperate bacteriophage in Streptococcus salivarius subsp. thermophilus St18.

Lysogenic strains of Streptococcus salivarius subsp. thermophilus were studied using induction with mitomycin C (MC). The induction and presence of temperate phage were investigated carrying out tests on sensitive strains, electron microscopy and phage DNA analysis. Forty-five Str. salivarius subsp. thermophilus strains were subjected to induction with MC and growth of the various cultures was evaluated. Only one strain of those tested showed lysis after adding MC, thus indicating a possible lysogenic state, 0.5 micrograms MC/ml being the optimal dose. Two phi 18 phage-sensitive strains out of 45 were isolated in which this phage behaved as virulent, causing lysis of the culture in broth, but no lysis plaques on agar medium were detected. The St18 strain was cured by u.v. irradiation but no mutants sensitive to the phi 18 phage were found among the clones non-inducible by MC. The presence of phages having a hexagonal isometric head and a long non-contractile tail in the lysate obtained after inducing the St18 strain was confirmed by examination under the electron microscope. Study of the phage DNA showed a genome size of 40.9 +/- 0.5 kb without cohesive end fragments. In addition, the restriction map of the phage genome was constructed. This study has demonstrated lysogeny in Str. salivarius subsp. thermophilus and also that several phage infections of Str. salivarius subsp. thermophilus starters may have an 'endogenous' origin.

Bacteriophages↗
PubMed1998

Escherichia coli CoIV plasmid pRK100: genetic organization, stability and conjugal transfer.

Uropathogenic Escherichia coli strains express chromosomal and plasmid-encoded virulence-associated factors such as specific adhesins, toxins and iron-uptake systems. A CoIV plasmid (pRK100) of a uropathogenic strain and its host KS533 were studied. The host strain encodes the K1 capsule, and P and S fimbriae, but neither haemolysin nor the cytotoxic-necrotic factor CNF1, indicating that this strain does not harbour a larger pathogenicity island. A restriction map of pRK100 was constructed on the basis of hybridization experiments and nucleotide sequencing. pRK100 harbours CoIV, the conserved replication region RepFIB, the aerobactin-uptake system, a RepFIC replicon and additionally Colla as well as transposon Tn5431. The location of the RepFIC replicon was similar to that in plasmid F. CoIV plasmids and F thus share a region spanning more than half the length of plasmid F. Even though their replication and transfer regions are homologous, CoIV plasmids are found only in E. coli strains. Among the four other species tested, conjugal transfer of pRK100 was demonstrated, with low frequency, only to Klebsiella pneumoniae, suggesting that a natural barrier effectively bars transfer. In vitro stability of the plasmid with integration into the chromosome to ensure maintenance in the presence of an incompatible plasmid was demonstrated.

Bacterial Capsules↗
PubMed1989

Genetic and antigenic comparison of Streptococcus mutans fructosyltransferase and glucan-binding protein.

The genes for fructosyltransferase (ftf) and glucan-binding protein (gbp) of Streptococcus mutans strain Ingbritt have been cloned in E. coli using bacteriophage and plasmid vectors. A single ftf gene appears to be responsible for the appearance of several FTF of different electrophoretic mobilities. Despite the fact that both ftf and gbp proteins synthesise fructans from sucrose in S. mutans and shown closely similar electrophoretic mobilities and isoelectric points, the restriction maps of the two genes are distinct, their DNAs do not hybridise and there is no immunological cross-reaction between the two proteins.

Antigens, Bacterial↗
PubMed2000

Characterization of the 16S rRNA gene V2 region and the rrn operons of Gardnerella vaginalis.

Ribosomal RNA (rRNA) gene polymorphism was apparent when Gardnerella vaginalis DNA restriction profiles were hybridized with nonradioactively labeled total rRNA isolated from this bacterium. In contrast, use of a polymerase chain reaction (PCR)-based 16S rRNA gene V2 region resulted in a 118-bp V2-PCR amplicon that was specific and common in all 30 tested G. vaginalis isolates. In addition to providing a G. vaginalis-specific fingerprint, when the V2-PCR amplicon along with total rRNA were utilized as probes, a partial rRNA gene restriction map could be constructed. G. vaginalis contains two rrn operons with an EcoRI fragment of 1.6 kb common to both.

DNA Fingerprinting↗
PubMed1989

Gene conversion in Escherichia coli: the recF pathway for resolution of heteroduplex DNA.

The independent repair of mismatched nucleotides present in heteroduplex DNA has been used to explain gene conversion and map expansion after general genetic recombination. We have constructed and purified heteroduplex plasmid DNAs that contain heteroallelic 10-base-pair insertion-deletion mismatches. These DNA substrates are similar in structure to the heteroduplex DNA intermediates that have been proposed to be produced during the genetic recombination of plasmids. These DNA substrates were transformed into wild-type and mutant Escherichia coli strains, and the fate of the heteroduplex DNA was determined by both restriction mapping and genetic tests. Independent repair events that yielded a wild-type Tetr gene were observed at a frequency of approximately 1% in both wild-type and recB recC sbcB mutant E. coli strains. The independent repair of small insertion-deletion-type mismatches separated by 1,243 base pairs was found to be reduced by recF, recJ, and ssb single mutations in an otherwise wild-type genetic background and reduced by recF, recJ, and recO mutations in a recB recC sbcB genetic background (the ssb mutation was not tested in the latter background). Independent repair of small insertion-deletion-type mismatched nucleotides that were as close as 312 nucleotides apart was observed. There was no apparent bias in favor of the insertion or deletion of mutant sequences.

Bacterial Proteins↗
PubMed1992

Ar+ plasma-induced damage to DNA in bacteriophage lambda: implications for the arrangement of DNA in the phage head.

Bacteriophage lambda was bombarded with low-energy Ar+ ions with the goal of determining whether particular regions of the DNA genome are found preferentially in the outer portion of the packaged DNA mass. The strategy was to fragment the DNA selectively near the surface of the virus by exposing intact phage to Ar+ ions energetic enough to break covalent chemical bonds in DNA but not energetic enough to penetrate deeply beneath the viral capsid shell. Broken DNA was then isolated, and its genomic origin was identified by Southern hybridization to mapped restriction fragments of lambda DNA. Analysis of such Southern blots revealed that all regions of the lambda genome were represented among the small DNA fragments generated during all times of Ar+ bombardment examined. Depending on the duration of exposure, however, particular regions of the genome were found to be enriched in the small-fragment population. After short periods of exposure, sequences from the leftmost 10% and from the right half of the standard genetic map were enriched in the broken-DNA fraction. Among sequences in the right half of the genome, the enrichment was progressively more pronounced beginning in the middle of the genetic map and proceeding toward the right end. In phage bombarded for longer periods of time, rightward sequences were preferentially depleted in the small-fragment population. In contrast, when Ar+ bombardment was carried out with free lambda DNA rather than intact phage, small DNA fragments arose uniformly from all regions of the genome at all times of exposure examined. The results indicate that in the intact phage, DNA sequences from the right half and from the very leftmost regions of the genome have a tendency to lie closer to the capsid than does the remainder of the genome. Since DNA is packaged into the prohead beginning at the left end, our results suggest that packaging occurs in such a way that newly entering DNA tends to be disposed externally to that packaged at earlier times.

Argon↗
PubMed1992

Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci.

Degenerate oligonucleotides based on the published Escherichia coli glutamate decarboxylase (GAD) protein sequence were used in a polymerase chain reaction to generate a DNA probe for the E. coli GAD structural gene. Southern blots showed that there were two cross-hybridizing GAD genes, and both of these were cloned and sequenced. The two GAD structural genes, designated gadA and gadB, were found to be 98% similar at the nucleotide level. Each gene encoded a 466-residue polypeptide, named, respectively, GAD alpha and GAD beta, and these differed by only five amino acids. Both GAD alpha and GAD beta contain amino acid residues which are highly conserved among pyridoxal-dependent decarboxylases, but otherwise the protein sequences were not homologous to any other known proteins. By restriction mapping and hybridization to the Kohara miniset library, the two GAD genes were located on the E. coli chromosome. gadA maps at 4046 kb and gadB at 1588 kb. Neither of these positions is in agreement with the current map position for gadS as determined by genetic means. Analysis of Southern blots indicated that two GAD genes were present in all E. coli strains examined, including representatives from the ECOR collection. However, no significant cross-hybridizing gene was found in Salmonella species. Information about the DNA sequences and map positions of gadA and gadB should facilitate a genetic approach to elucidate the role of GAD in E. coli metabolism.

Amino Acid Sequence↗
PubMed1998

Mapping an endometrial cancer tumor suppressor gene at 10q25 and development of a bacterial clone contig for the consensus deletion interval.

Frequent loss of chromosome 10q sequences in endometrial cancers suggests the involvement of a tumor suppressor gene. Previous loss-of-heterozygosity (LOH)studies have pointed to the 10q25-q26 region as the likely site of a tumor suppressor involved in endometrial tumorigenesis (S. L. Peiffer et al., 1995, Cancer Res. 55: 1922-1926; S. Nagase et al., 1996, Br. J. Cancer 74: 1979-1983; S. Nagase et al.,1997, Cancer Res. 57: 1630-1633). In an attempt to define further the localization of a tumor suppressor gene at 10q25, we screened a panel of 123 endometrioid adenocarcinomas for loss of heterozygosity of 10q25.3 sequences. Forty-three (35%) revealed LOH at one or more loci. The observed patterns of allelic loss define a minimum consensus region of deletion between D10S221 and D10S610. A sequence-ready bacterial clone contig and a long-range restriction map for a 1-Mb interval spanning the deletion region were developed as the first step in experiments directed toward the discovery the 10q25 tumor suppressor.

Carcinoma, Endometrioid↗
PubMed1994

Hepatic lipase mRNA is expressed in rat and human steroidogenic organs.

Rat and human steroidogenic organs contain an enzyme activity that is indistinguishable from hepatic lipase present in liver. Using primers that recognize exons 5 and 8 of the rat and human HL gene, a 596-bp product was found by RT-PCR in rat liver, adrenal, ovaries and testes, but not in heart and kidney. A similar product was also observed with human hyperplastic adrenocortical tissue. Identity of this product with part of the HL cDNA was confirmed by restriction mapping and internal re-amplification. Our results indicate that the HL gene is transcribed in steroidogenic tissues that also contain HL protein.

Adrenal Glands↗
PubMed1989

Plasmid-mediated chloramphenicol resistance in Staphylococcus hyicus.

A small plasmid of 3.95 kb, encoding resistance to chloramphenicol (Cm) was detected in three of 33 Staphylococcus hyicus strains. The plasmid in each of the three strains was indistinguishable by Southern-blot hybridization and restriction enzyme analysis. It was shown by curing and by transformation to specify resistance to Cm. A preliminary restriction map of the plasmid, designated pSC2, is presented. Chloramphenicol acetyltransferase was demonstrated by enzyme assay and by SDS-PAGE of cell-free lysates of pSC2 transformants.

Animals↗
PubMedDate not supplied

[Cloning and expression of the Bacillus stearothermophilus neutral proteinase gene in Bacillus subtilis cells].

Gene of thermostable Bacillus stearothermophilus metalloprotease was cloned and expressed in mesophilic Bacillus subtilis cells. It was demonstrated that this gene is closely related by its restriction map to B. stearothermophilus T metalloprotease gene cloned earlier. Thermostability level and thermal optimum of activity of metalloprotease, the product of cloned gene expression, were estimated.

Bacillus subtilis↗
PubMed1996

Gene localization and expression of thienamycin cyclase gene in Streptomyces lividans TK24.

Transformants of S. lividans TK24 were obtained by transforming a recombinant plasmid p6BC12 harboring the thienamycin cyclase gene into it. An antibacterial substance could be detected by the conversion of fermentation broth of the Y3 block mutant and the purified Y3 mutant intermediate with a cell-free extract of S. lividans TK24 transformant. Paper chromatographic analysis showed that the conversion product of Y3 fermentation broth with cell-free extract of S. lividans TK24 was thienamycin and an unstable antibacterial substance was a product of Y3 intermediate with cell-free extract of the transformants. This result indicated that the thienamycin cyclase gene was expressed in S. lividans TK24 and complemented the deficiency in the Y3 block mutant. Restriction analysis of p6BC12 was carried out and the restriction map was constructed. The thienamycin cyclase gene was localized on a 0.9 kb PstI-HinCII fragment from a bioconversion result. The 1.0 kb IPNS homologous DNA fragment in plasmid p6BC12 was excluded from the cyclase activity.

Genes, Bacterial↗