No abstract available.
Genomic Library* ; Helicobacter pylori* ; Helicobacter*

Microorganisms contain a large number of biocatalysts, which are of great potential in industrial applications. However, the traditional cultural approaches can obtain only less than 1% of microorganisms. As a culture-independent method, metagenomics is an advanced solution by means of extracting all microbial genomic DNAs in certain environmental habitat, constructing and screening metagenomic libraries to seek novel functional genes. It serves as an effective tool for studying these uncultured microorganisms. Therefore, mining novel biocatalysts from metagenome has drawn the attention of researchers in the world. In this paper, environment sample category, genomic DNA extraction, library construction and screening strategies were reviewed. Recent examples of isolated biocatalysts from metagenomic libraries were presented. Future research directions of metagenomics were also discussed.
Biocatalysis ; DNA ; Genomic Library ; Metagenomics ; trends

BamHI restriction patters and genomic library of Herpes simplex virus type 2 (HSV-2) stram G were constructed, and locations of the glycoproteins gB and gD, and it genes on the fragments were detected by Southern blot analysis. HISV-2 genomic DNAs were cleaved into twenty-seven fragments by BamHI enzyme in the range of 0.72 to 15.08 (total 150.44 kb), which were cloned into the BamHI site of pBluescript SK(+) to construct genome library of the HSV-2. The library was named by the order of the fragment size from smallest one to largest one. HSV-2 glycoprotein gD gene was located in PHLA2-21 and PHLA2-22 recombinant plasmids, gB gene in PHLA2-24 plasmic, and it gene in PHLA2-11 clone by Southern blot analysis.
Blotting, Southern ; Clone Cells ; DNA ; Genomic Library ; Glycoproteins* ; Herpes Simplex* ; Herpesvirus 2, Human* ; Phosphotransferases* ; Plasmids ; Simplexvirus*

BamHI restriction patters and genomic library of Herpes simplex virus type 2 (HSV-2) stram G were constructed, and locations of the glycoproteins gB and gD, and it genes on the fragments were detected by Southern blot analysis. HISV-2 genomic DNAs were cleaved into twenty-seven fragments by BamHI enzyme in the range of 0.72 to 15.08 (total 150.44 kb), which were cloned into the BamHI site of pBluescript SK(+) to construct genome library of the HSV-2. The library was named by the order of the fragment size from smallest one to largest one. HSV-2 glycoprotein gD gene was located in PHLA2-21 and PHLA2-22 recombinant plasmids, gB gene in PHLA2-24 plasmic, and it gene in PHLA2-11 clone by Southern blot analysis.
Blotting, Southern ; Clone Cells ; DNA ; Genomic Library ; Glycoproteins* ; Herpes Simplex* ; Herpesvirus 2, Human* ; Phosphotransferases* ; Plasmids ; Simplexvirus*

OBJECTIVETo investigate the genetic diversity and structure of Dendrobium huoshanense, a (CT)n enriched microsatellite library was constructed using a magnetic beads enrichment procedure.

METHODThe 3'-biotinylated oligonucleotide probe was used to hybridize with the digested D. huoshanense genomic DNA fragments whose both ends were ligated with adaptors. The hybridized complex was then combined with the streptavidin-coated magnetic beads. The captured microsatellite fragments were eluted, collected and cloned into pMD19-T vector. The recombinant plasmids were transformed into Escherichia coli DH5alpha competent cells. The clones that yielded two or more bands contained microsatellite fractions. Positive clones were screened and sequenced. Thirty pairs of primers were designed and synthesized. Polymorphism at each locus was determined using 24 individuals from a natural population from Huoshan county town in Anhui province.

RESULTTwelve polymorphic microsatellite loci from the microsatellite-enriched genomic library were newly developed across 24 D. huoshanense individuals. In total, 65 alleles were identified, and the number of alleles per locus ranged from 2 to 8. The mean observed and expected heterozygosities were 0.500 and 0.638, respectively. Two loci significantly deviated from Hardy-Weinberg equilibrium (P<0.05), which could be due to the presence of null alleles. Furthermore, three of twelve loci showed significant linkage disequilibrium (P<0.05).

CONCLUSIONThese results suggest that the identified polymorphic microsatellite markers will be useful in population genetic studies of D. huoshanense.

Porphyromonas gingivalis, a Gram negative, anaerobic, asaccharolytic rod, is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is both hemin and iron regulated has recently been purified and characterized in this oral pathogen. This study has identified a hemin-binding P. gingivalis protein by expression of a P. gingivalis genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. A library of genomic DNA fragments from P. gingivalis was constructed in plasmid pUC18, transformed into Escherichia coli strain DH5alpha, and screened for recombinant clones with heminbinding activity by plating onto hemin-containing agar. Of approximately 10,000 recombinant E. coli colonies screened on LB-amp-hemin agar, 10 exhibited a clearly pigmented phenotype. Each clone contained various insert DNA. The Hind III fragment transferred to the T7 RNA polymerase/promoter expression vector system produced a sligltly smaller (21 kDa) protein, a precursor form, immunoreactive to the antibody against the 24 kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 24 kDa heminbinding protein.
Agar ; Clone Cells ; DNA ; Escherichia coli ; Genomic Library ; Hemin ; Humans ; Iron ; Periodontal Diseases ; Phenotype ; Plasmids ; Porphyromonas gingivalis* ; Porphyromonas* ; RNA

The gene encoding the 66 kilodalton (kDa) protein of Borrelia hermsii HS1 was cloned and sequenced. Chromosomal DNA was prepared from purified B. hermsii and used in construction of genomic library. The library was screened for positive clones by 314 bp DIG-labeled probe synthesized on the basis of the part of the sequence of B. hermsii. Positive clone was subcloned into p2ErO vector and was designated as pBH11. pBH11 were subcloned into pBluscript vector and were designated as pBH11-1 (500 bp), pBH11-2 (800 bp), pBH11-3 (600 bp) and pBH11-4 (800 bp). The plasmids were sequenced and determined the nucleotide sequence of p66. The open reading frame of the p66 consisted of 1803 base pairs coding for 600 amino acid protein. The basic information on the p66 gene of B. hermsii HS1 obtained from this study will be useful for further analysis and experiment of pathogenesis of the borrelia.
Base Pairing ; Base Sequence ; Borrelia* ; Clinical Coding ; Clone Cells* ; Cloning, Organism* ; DNA ; Genomic Library ; Open Reading Frames ; Plasmids

We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F2 population of 107 plants with 320 RFLP, 136 AFLP, and 46 SSR markers. The resulting linkage map consists of 15 linkage groups covering 1,720 cM with an average map distance of 3.7 cM between framework markers. Most RFLP markers (80%) were pepper-derived clones and these markers were evenly distributed all over the genome. Genes for defense and biosynthesis of carotenoids and capsaicinoids were mapped on this linkage map. By using 30 primer combinations, AFLP markers were generated in the F2 population. For development of SSR markers in Capsicum, microsatellites were isolated from two small-insert genomic libraries and the GenBank database. This combined map provides a starting point for high-resolution QTL analysis, gene isolation, and molecular breeding.
Capsicum ; Carotenoids ; Chromosome Mapping* ; Clone Cells ; Databases, Nucleic Acid ; DNA Shuffling ; Genome* ; Genomic Library ; Microsatellite Repeats ; Polymorphism, Restriction Fragment Length

PURPOSE: We have previously cloned three enhancer factor genes encoding proteins that bind to long terminal repeats (LTRs) of Rous sarcoma virus. Among these genes, RSV- EF-I gene is expressed in rat hepatoma tissues and several proliferating cell lines but not in normal rat liver tissues. We have isolated the human homologue of RSV-EF-I gene and examined its expression in human hepatocellular carcinoma tissues. MATERIALS AND METHODS: We have screened the human genomic library and cDNA library of Hep G2 cell line derived from human hepatocellular carcinoma to isolate the human homologue of RSV-EF-I gene. RESULTS: We have isolated one cDNA clone containing about 1.5 kb insert and sequenced. Sequence analysis reveals that this human homologue of RSV-EF-I gene has a high similarities to human YB-1 mRNA, human DNA-binding protein B (dbpB) gene and other Y-box protein genes. It is expressed in human hepatocellular carcinoma but very slightly in normal human liver tissues in Northern blot analysis. CONCLUSION: Our data suggest that the human homologue of RSV-EF-I gene presumably belongs to Y-box protein family genes and plays a role in the transformation of the human hepatoma cells.
Animals ; Blotting, Northern ; Carcinoma, Hepatocellular* ; Cell Line ; Clone Cells ; DNA, Complementary ; Gene Library ; Genomic Library ; Hep G2 Cells ; Humans* ; Liver ; Rats ; RNA, Messenger ; Rous sarcoma virus* ; Sarcoma, Avian* ; Sequence Analysis ; Terminal Repeat Sequences

The polymerase chain reaction was used to develop a method for the detection of Helicobacter pylori, a causative agent of gastritis, as well as for the elucidation of its mode of transmission. A genomic library of Helicobacter pylori DNA in Escherichia coli JM109 was constructed by cloning Hind III-digested DNA fragments into plasmid vector pUC18. The nucleotide sequences from seven recombinant clones were determined and five sets of oligonucleotide primers were synthesized on the basis of the sequences from five clones (B4, B9, B10, C15 and I22). The PCR amplifications with these primers were performed using DNA samples from five strains of Helicobacter pylori, two Campylobacter spp. and eleven species of enteric bacteria. Amplifications of the target DNA fragments in all of 5 strains of Helicobacter pylori were observed from the PCR with primers derived from clone B4, B9, C15 and I22. When the specificity was checked with the DNA samples from 13 other bacteria as template DNA for the PCR, specific amplification that produced the correct size of the target DNA of Helicobacter pylori was shown only in the PCR with primers derived from clone B9 and C15. The detection limit in the PCR amplification, determined by the heat-lysis method, was 500 cells of Helicobacter pylori.
Base Sequence ; DNA, Bacterial/*analysis ; DNA, Recombinant ; Genomic Library ; Helicobacter Infections/diagnosis ; Helicobacter pylori/*genetics/isolation & purification ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Sensitivity and Specificity