RESUMO
We constructed a bacterial artificial chromosome (BAC)-based physical map of chromosomes 2 and 3 of Drosophila melanogaster, which constitute 81% of the genome. Sequence tagged site (STS) content, restriction fingerprinting, and polytene chromosome in situ hybridization approaches were integrated to produce a map spanning the euchromatin. Three of five remaining gaps are in repeat-rich regions near the centromeres. A tiling path of clones spanning this map and STS maps of chromosomes X and 4 was sequenced to low coverage; the maps and tiling path sequence were used to support and verify the whole-genome sequence assembly, and tiling path BACs were used as templates in sequence finishing.
Assuntos
Mapeamento de Sequências Contíguas , Drosophila melanogaster/genética , Genoma , Animais , Centrômero/genética , Cromatina/genética , Cromossomos Bacterianos/genética , Clonagem Molecular , Impressões Digitais de DNA , Eucromatina , Biblioteca Gênica , Genes de Insetos , Marcadores Genéticos , Vetores Genéticos , Hibridização In Situ , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Análise de Sequência de DNA , Sitios de Sequências Rotuladas , Telômero/genéticaRESUMO
A 30-fold redundant human bacterial artificial chromosome (BAC) library with a large average insert size (178 kb) has been constructed to provide the intermediate substrate for the international genome sequencing effort. The DNA was obtained from a single anonymous volunteer, whose identity was protected through a double-blind donor selection protocol. DNA fragments were generated by partial digestion with EcoRI (library segments 1--4: 24-fold) and MboI (segment 5: sixfold) and cloned into the pBACe3.6 and pTARBAC1 vectors, respectively. The quality of the library was assessed by extensive analysis of 169 clones for rearrangements and artifacts. Eighteen BACs (11%) revealed minor insert rearrangements, and none was chimeric. This BAC library, designated as "RPCI-11," has been used widely as the central resource for insert-end sequencing, clone fingerprinting, high-throughput sequence analysis and as a source of mapped clones for diagnostic and functional studies.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Biblioteca Genômica , Análise de Sequência de DNA/métodos , Clonagem Molecular/métodos , Mapeamento de Sequências Contíguas , DNA Satélite/genética , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Rearranjo Gênico , Marcadores Genéticos/genética , Projeto Genoma Humano , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Recombinação Genética/genética , Mapeamento por Restrição , Sitios de Sequências RotuladasRESUMO
Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9-14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, approximately 1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research.