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H Shizuya

Publications and source records attributed to H Shizuya.

At least 37 records · Page 2Linked to original sources
PubMed1996

Framework for a physical map of the human 22q13 region using bacterial artificial chromosomes (BACs).

Detailed physical maps of entire chromosomes based on combined genetic, cytogenetic, and structural information are essential components for positional cloning and genomic sequencing. Despite the wealth of genetic information of the known diseases in the chromosome 22q13, the construction of a detailed physical map of the terminal region is difficult due to the sparsity of the genetic markers. We present here a map of bacterial artificial chromosome (BAC) contigs that cover a number of genetic loci in the 22q13 region. One hundred thirty-six BACs with an average insert size of 140 kb are assembled into 35 contigs defined by 64 markers in 22q13-qter. Twenty-three anonymous markers are now linked to the previously mapped genetic anchor points.

Chromosome Mapping↗
PubMed1996

Cloning of contiguous genomic fragments from human chromosome 21 harbouring three trefoil peptide genes.

A group of small peptides with a typical cysteine-rich domain (termed trefoil motif or P-domain) is abundantly expressed at mucosal surfaces of specific normal and neoplastic tissues. Their association with the maintenance of surface integrity was suggested. The first known human trefoil peptide (pS2) was isolated from breast cancer cells (MCF7). Its oestrogen-inducible gene, and the human homologue to the porcine spasmolytic peptide gene (hSP/SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be localised at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene were described recently. By using suitable oligonucleotide primers and PCR and isolating large (110-250 kb) genomic recombinants cloned in the bacterial artificial chromosome (BAC) system, we present a genomic region from chromosome band 21q22.3 cloned in contiguous sequences and encoding all three members of human P-domain/trefoil peptides proving a physical linkage of all three trefoil peptide genes. Such genomic sequences will provide useful experimental material for analysis of gene regulation, for gene modification experiments and for establishing transgenic cells or animals.

Base Sequence↗
PubMed1996

The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds.

Breast carcinoma is the most common malignancy among women in developed countries. Because family history remains the strongest single predictor of breast cancer risk, attention has focused on the role of highly penetrant, dominantly inherited genes in cancer-prone kindreds (1). BRCA1 was localized to chromosome 17 through analysis of a set of high-risk kindreds (2), and then identified four years later by a positional cloning strategy (3). BRCA2 was mapped to chromosomal 13q at about the same time (4). Just fifteen months later, Wooster et al. (5) reported a partial BRCA2 sequence and six mutations predicted to cause truncation of the BRCA2 protein. While these findings provide strong evidence that the identified gene corresponds to BRCA2, only two thirds of the coding sequence and 8 out of 27 exons were isolated and screened; consequently, several questions remained unanswered regarding the nature of BRCA2 and the frequency of mutations in 13q-linked families. We have now determined the complete coding sequence and exonic structure of BRCA2 (GenBank accession #U43746), and examined its pattern of expression. Here, we provide sequences for a set of PCR primers sufficient to screen the entire coding sequence of BRCA2 using genomic DNA. We also report a mutational analysis of BRCA2 in families selected on the basis of linkage analysis and/or the presence of one or more cases of male breast cancer. Together with the specific mutations described previously, our data provide preliminary insight into the BRCA2 mutation profile.

BRCA2 Protein↗
PubMed1996

Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.

We have cloned and mapped a circular 630-kb human extrachromosomal structure (termed amplisome) using the bacterial artificial chromosome (BAC) cloning system. Twenty-one BACs were isolated from an amplisome-enriched library by colony hybridization. The insert sizes range from 25 to 143 kb, with an average size of 82 kb. The coverage of the amplisome in clones is approximately 2.7-fold. To construct a physical map of the amplisome, we used three different but complementary methods: hybridization, STS content mapping, and fingerprinting. In addition, we compared the advantages and the drawbacks of these techniques in mapping the amplisomal BACs. The 21 BACs were grouped into two contigs and the two small gaps (3.5 and 26.5 kb) were filled by screening of a human genomic BAC library. The organization of the amplisome revealed by the BAC-based physical map is consistent with the long-range restriction map reported previously. Our results demonstrate that a 630-kb region can be rapidly cloned and mapped into contigs by use of the BAC system. Because of the low frequency (<0.1%) of chimerism and rearrangement, these BAC clones are ready for DNA sequencing and functional analysis.

Chromosome Mapping↗
PubMed1996

Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon.

One potential approach for characterizing uncultivated prokaryotes from natural assemblages involves genomic analysis of DNA fragments retrieved directly from naturally occurring microbial biomass. In this study, we sought to isolate large genomic fragments from a widely distributed and relatively abundant but as yet uncultivated group of prokaryotes, the planktonic marine Archaea. A fosmid DNA library was prepared from a marine picoplankton assemblage collected at a depth of 200 m in the eastern North Pacific. We identified a 38.5-kbp recombinant fosmid clone which contained an archaeal small subunit ribosomal DNA gene. Phylogenetic analyses of the small subunit rRNA sequence demonstrated it close relationship to that of previously described planktonic archaea, which form a coherent group rooted deeply within the Crenarchaeota branch of the domain Archaea. Random shotgun sequencing of subcloned fragments of the archaeal fosmid clone revealed several genes which bore highest similarity to archaeal homologs, including large subunit ribosomal DNA and translation elongation factor 2 (EF2). Analyses of the inferred amino acid sequence of archaeoplankton EF2 supported its affiliation with the Crenarchaeote subdivision of Archaea. Two gene fragments encoding proteins not previously found in Archaea were also identified: RNA helicase, responsible for the ATP-dependent alteration of RNA secondary structure, and glutamate semialdehyde aminotransferase, an enzyme involved in initial steps of heme biosynthesis. In total, our results indicate that genomic analysis of large DNA fragments retrieved from mixed microbial assemblages can provide useful perspective on the physiological potential of abundant but as yet uncultivated prokaryotes.

Amino Acid Sequence↗
PubMed1996

A third P-domain peptide gene (TFF3), human intestinal trefoil factor, maps to 21q22.3.

Small peptides displaying a cysteine-rich module (termed P-domain or trefoil motif) form a recently increasing group of peptides abundantly expressed at mucosal surfaces of specific tissues and are associated with the maintenance of surface integrity. The estrogen-inducible pS2 gene (BCEI) and the human homolog to the porcine spasmolytic peptide (hsP) gene (SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be located at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene (TFF3) were described recently. By PCR analysis of a somatic cell hybrid panel and FISH using two large genomic recombinants (110 kb, 210 kb) cloned in the Bacterial Artificial Chromosome (BAC) system, we show that this gene coding for the new member of human P-domain/trefoil peptides also maps to chromosome region 21q22.3 suggesting a physical linkage of all three trefoil peptide genes.

Amino Acid Sequence↗
PubMed1995

Characterization of a human chromosome 22 enriched bacterial artificial chromosome sublibrary.

Selection of chromosomal sublibraries from total human genomic libraries is critical for chromosome-based physical mapping approaches. We have previously reported a method of screening total human genomic library using flow sorted chromosomal DNA as a hybridization probe and selection of a human chromosome 22-enriched sublibrary from a total human bacterial artificial chromosome (BAC) library (Nucleic Acids Res 1995; 23: 1838-39). We describe here further details of the method of construction as well as characterization of the chromosome 22-enriched sublibrary thus constructed. Nearly 40% of the BAC clones that have been mapped by fluorescence in situ hybridization (FISH) analysis were localized to chromosome 22. By screening the sublibrary using chromosome 22-specific hybridization probes, we estimated that the sublibrary represents at least 2.5 x coverage of chromosome 22. This is in good agreement with the results from FISH mapping experiments. FISH map data also indicate that chromosome 22-specific BACs in the sublibrary represent all the subregions of chromosome 22.

Animals↗
PubMed1995

Construction and utility of a human chromosome 22-specific Fosmid library.

We have previously demonstrated the capability of the Fosmid vector based on Escherichia coli F-factor replicon to stably propagate cosmid-sized human genomic DNA fragments. Using the Fosmid vector, we have constructed and arrayed a 10 x human chromosome 22-specific library, partly by picking human positive clones from a total Fosmid library constructed using DNA from human-hamster hybrid cell line containing human chromosome 22, and partly by using flow-sorted chromosomal DNA. The clones and physical contig maps of the clones in the library will serve as a valuable resource for detailed analysis of the chromosome by providing reliable materials for high resolution mapping and sequencing. In order to efficiently build physical maps for the chromosomal regions of interest spanning several hundred kilobases to a megabase, it is necessary to rapidly identify subsets of the Fosmid clones from the library that cover such regions. In this report, we describe a method of using random amplification products derived from YAC clones to rapidly identify a subset of Fosmid clones that cover a specific genomic subregion.

Animals↗
PubMed1994

Selection of chromosome 22-specific clones from human genomic BAC library using a chromosome-specific cosmid library pool.

A new approach to rapidly identify chromosome-specific subsets of clones from a total human genomic library is described. We report here the results of screening a human bacterial artificial chromosome (BAC) library using the total pool of clones from a chromosome 22-specific cosmid library as a composite probe. The human BAC library was gridded on filters at high density and hybridized with DNA from the pooled chromosome 22-specific Lawrist library under suppressive conditions. In a single hybridization, we picked 280 candidates from the BAC library representing over 30,000 clones (or 1.2x coverage of human genome). This subset contained more than 60% of the chromosome 22-specific BAC clones that were previously found to be present in the original BAC library. In principle, this approach can be applied to select a subset of clones from other global libraries with relatively large inserts using a pool from a regional library as a composite probe. It is important to note that the target and probe libraries must be based on vectors that share no homology with each other.

Chromosomes, Bacterial↗
PubMed1994

A new bacteriophage P1-derived vector for the propagation of large human DNA fragments.

We have designed a P1 vector (pCYPAC-1) for the introduction of recombinant DNA into E. coli using electroporation procedures. The new cloning system, P1-derived artificial chromosomes (PACs), was used to establish an initial 15,000 clone library with an average insert size of 130-150 kilobase pairs (kb). No chimaerism has been observed in 34 clones, by fluorescence in situ hybridization. Similarly, no insert instability has been observed after extended culturing, for 20 clones. We conclude that the PAC cloning system will be useful in the mapping and detailed analysis of complex genomes.

Bacteriophage P1↗
PubMed1994

A P1-based physical map of the region from D17S776 to D17S78 containing the breast cancer susceptibility gene BRCA1.

BRCA1, a breast and ovarian cancer susceptibility locus, has been isolated and maps to 17q21. A physical map of the BRCA1 region which extended from the proximal boundary at D17S776 to the distal boundary at D17S78 was constructed and consists of 51 sequence tagged sites (STSs) from P1 and YAC ends, nine new short-tandem repeat (STR) polymorphic markers, and eight identified genes. The contig, which spans the estimated 2.3 Mb region, contains 29 P1s, 11 YACs, two BACs, and one cosmid. Based on key recombinants in two linked families, BRCA1 was further localized to a region bounded by D17S1321 on the proximal side and D17S1325 on the distal side. Within this estimated 600 kb region, the contig was composed completely of P1s and BACs ordered by STS-content mapping and confirmed by DNA restriction fragment fingerprinting.

Base Sequence↗
PubMed1992

Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector.

A bacterial cloning system for mapping and analysis of complex genomes has been developed. The BAC system (for bacterial artificial chromosome) is based on Escherichia coli and its single-copy plasmid F factor. It is capable of maintaining human genomic DNA fragments of greater than 300 kilobase pairs. Individual clones of human DNA appear to be maintained with a high degree of structural stability in the host, even after 100 generations of serial growth. Because of high cloning efficiency, easy manipulation of the cloned DNA, and stable maintenance of inserted DNA, the BAC system may facilitate construction of DNA libraries of complex genomes with fuller representation and subsequent rapid analysis of complex genomic structure.

Cloning, Molecular↗
PubMed1992

Stable propagation of cosmid sized human DNA inserts in an F factor based vector.

Instability of complex mammalian genomic DNA inserts is commonplace in cosmid libraries constructed in conventional multicopy vectors. To develop a means to construct stable libraries, we have developed a low copy number cosmid vector based on the E. coli F factor replicon (Fosmid). We have tested relative stability of human DNA inserts in Fosmids and in two conventional multicopy vectors (Lawrist 16 and Supercos) by comparing the frequency of changes in restriction patterns of the inserts after propagating randomly picked human genomic clones based on these vectors. We found that the clones based on Fosmid vector undergo detectable changes at a greatly reduced frequency. We also observed that sequences that undergo drastic rearrangements and deletions during propagation in a conventional vector were stably propagated when recloned as Fosmids. The results indicate that Fosmid system may be useful for constructing stable libraries from complex genomes.

Cloning, Molecular↗
PubMed1984

Heat-labile alkaline phosphatase from Antarctic bacteria: Rapid 5' end-labeling of nucleic acids.

A heat-labile alkaline phosphatase has been purified to near homogeneity from HK47, a bacterial strain isolated from Antarctic seawater. The active form of the enzyme has a molecular weight of 68,000 and is uniquely monomeric. The optimal temperature for the enzymatic activity is 25 degrees C. Complete and irreversible thermal inactivation of the enzyme occurs in 10 min at 55 degrees C. By using this heat-labile enzyme for dephosphorylation followed by a 10-min heat treatment, rapid end-labeling of nucleic acids by T4 polynucleotide kinase has been achieved.

Journal Article↗
PubMed1984

Bacterial plasmids in antarctic natural microbial assemblages.

Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid incidence (20%). Plasmids were significantly more frequent in the strains which had been first isolated from low-nutrient media (46%) than in the strains which had been isolated from high-nutrient media (25%). Multiple forms of plasmids were observed in two-thirds of the plasmid-carrying strains. A majority of the plasmids detected were estimated to have a mass of 10 megadaltons or less. Among 48 plasmid-carrying strains, 7 showed antibiotic resistance. It is concluded that bacterial plasmids are ubiquitous in natural microbial assemblages of the pristine marine ecosystem of Antarctica.

Journal Article↗
PubMed1981

Host requirements for growth of lambda-P22 hybrid in Escherichia coli.

The requirements for growth of bacteriophage lambda containing the deoxyribonucleic acid replication region from Salmonella phage P22 were determined in a burst size experiment. The products of genes dnaE, dnaJ, dnaK, dnaY, dnaZ, and seg were required, but not the products of genes dnaA, dnaB, dnaC, and dnaX. This lambda-P22 hybrid phage was also dependent on polA for growth at 32 degrees C.

Bacteriophage lambda↗