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Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice.

ABSTRACT: A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for beta-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with beta-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.

SUBMITTER: Walter J

PROVIDER: S-EPMC1087578 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice.

Walter Jens J Mangold Marco M Tannock Gerald W GW

Applied and environmental microbiology 20050501 5

A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for beta-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with beta-glucanase activity were detected. The inserts ...[more]

PMID: 15870321

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Project description:BACKGROUND: Efficient screening of bacterial artificial chromosome (BAC) libraries with polymerase chain reaction (PCR)-based markers is feasible provided that a multidimensional pooling strategy is implemented. Single nucleotide polymorphisms (SNPs) can be screened in multiplexed format, therefore this marker type lends itself particularly well for medium- to high-throughput applications. Combining the power of multiplex-PCR assays with a multidimensional pooling system may prove to be especially challenging in a polyploid genome. In polyploid genomes two classes of SNPs need to be distinguished, polymorphisms between accessions (intragenomic SNPs) and those differentiating between homoeologous genomes (intergenomic SNPs). We have assessed whether the highly parallel Illumina GoldenGate Genotyping Assay is suitable for the screening of a BAC library of the polyploid Brassica napus genome. RESULTS: A multidimensional screening platform was developed for a Brassica napus BAC library which is composed of almost 83,000 clones. Intragenomic and intergenomic SNPs were included in Illumina's GoldenGate Genotyping Assay and both SNP classes were used successfully for screening of the multidimensional BAC pools of the Brassica napus library. An optimized scoring method is proposed which is especially valuable for SNP calling of intergenomic SNPs. Validation of the genotyping results by independent methods revealed a success of approximately 80% for the multiplex PCR-based screening regardless of whether intra- or intergenomic SNPs were evaluated. CONCLUSIONS: Illumina's GoldenGate Genotyping Assay can be efficiently used for screening of multidimensional Brassica napus BAC pools. SNP calling was specifically tailored for the evaluation of BAC pool screening data. The developed scoring method can be implemented independently of plant reference samples. It is demonstrated that intergenomic SNPs represent a powerful tool for BAC library screening of a polyploid genome.

Project description:BACKGROUND: Bacterial artificial chromosome (BAC) libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. RESULTS: Here, we report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink (Neovison vison). The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library. These included candidate genes for coat coloring, hair growth and length, coarseness, and some receptors potentially involved in viral diseases in mink. The extensive screening yielded positive results for 19 of these genes. Thirty-five clones corresponding to 19 genes were sequenced using 454 Roche, and large contigs (184 kb in average) were assembled. Knowing the complete sequences of these candidate genes will enable confirmation of the association with a phenotype and the finding of causative mutations for the targeted phenotypes.Additionally, 1577 BAC clones were end sequenced; 2505 BAC end sequences (80% of BACs) were obtained. An excess of 2 Mb has been analyzed, thus giving a snapshot of the mink genome. CONCLUSIONS: The availability of the CHORI-321 American mink BAC library will aid in identification of genes and genomic regions of interest. We have demonstrated how the library can be used to identify specific genes of interest, develop genetic markers, and for BAC end sequencing and deep sequencing of selected clones. To our knowledge, this is the first report of 454 sequencing of selected BAC clones in mammals and re-assures the suitability of this technique for obtaining the sequence information of genes of interest in small genomics projects. The BAC end sequences described in this paper have been deposited in the GenBank data library [HN339419-HN341884, HN604664-HN604702]. The 454 produced contigs derived from selected clones are deposited with reference numbers [GenBank: JF288166-JF288183 &JF310744].

Dataset Information

Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice.

Publications

Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice.

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