Human BAC ends

The Human BAC Ends database includes all non-redundant human BAC end sequences (BESs) generated by The Institute for Genomic Research (TIGR), the University of Washington (UW) and California Institute of Technology (CalTech). It incorporates the available BAC mapping data from different genome centers and the annotation results of each end sequence for the contents of repeats, ESTs and STS markers. For each BAC end the database integrates the sequence, the phred quality scores, the map and the annotation, and provides links to sites of the library information, the reports of GenBank, dbGSS and GDB, and other relevant data. The database is freely accessible via the web and supports sequence or clone searches and anonymous FTP. The relevant sites and resources are described at http://www.tigr.org/ tdb/humgen/bac_end_search/bac_end_intro.html     

Human BAC ends quality assessment and sequence analyses

End sequences from bacterial artificial chromosomes (BACs) provide highly specific sequence markers in large-scale sequencing projects. To date, we have generated >300,000 end sequences from >186,000 human BAC clones with an average read length of >460 bp for a total of 141 Mb covering approximately 4.7% of the genome. Over 60% of the clones have BAC end sequences (BESs) from both ends representing more than fivefold coverage of the human genome by the paired-end clones. Our quality assessments and sequence analyses indicate that BESs from human BAC libraries developed at The California Institute of Technology (CalTech) and Roswell Park Cancer Institute have similar properties. The analyses have highlighted differences in insert size for different segments of the CalTech library. Problems with the fidelity of tracking of sequence data back to physical clones have been observed in some subsets of the overall BES dataset. The annotation results of BESs for the contents of available genomic sequences, sequence tagged sites, expressed sequence tags, protein encoding regions, and repeats indicate that this resource will be valuable in many areas of genome research.     

Construction of a BAC library and generation of BAC end sequence-tagged connectors for genome sequencing of the African malaria mosquito Anopheles gambiae

A Bacterial Artificial Chromosome (BAC) genomic DNA library of Anopheles gambiae, the major human malaria vector in sub-Saharan Africa, was constructed and characterized. This library (ND-TAM) is composed of 30,720 BAC clones in eighty 384-well plates. The estimated average insert size of the library is 133 kb, with an overall genome coverage of approximately 14-fold. The ends of approximately two-thirds of the clones in the library were sequenced, yielding 32,340 pair-mate ends. A statistical analysis (G-test) of the results of PCR screening of the library indicated a random distribution of BACs in the genome, although one gap encompassing the white locus on the X-chromosome was identified. Furthermore, combined with another previously constructed BAC library (ND-1), ~2,000 BACs have been physically mapped by polytene chromosomal in situ hybridization. These BAC end pair mates and physically mapped BACs have been useful for both the assembly of a fully sequenced A. gambiae genome and for linking the assembled sequence to the three polytene chromosomes. This ND-TAM library is now publicly available at both http://www.malaria.mr4.org/mr4pages/index.html/ and http://hbz.tamu.edu/, providing a valuable resource to the mosquito research community.     

Parameters influencing high-efficiency transfection of bacterial artificial chromosomes into cultured mammalian cells

Although bacterial artificial chromosomes (BACs) provide a well-characterized resource for the analysis of large chromosomal domains, low transfection rates have proven a significant limitation for their use in cell culture models. Using TP53 BAC clones that contain expression cassettes for enhanced green fluorescent protein or red fluorescent protein, we have examined conditions that promote BAC transfection in hamster, human, and mouse cell lines. Atomic force microscopy shows that BAC transfection efficiency correlates with the generation of small, highly condensed but dispersed lipid: BAC DNA transfection complexes. BAC DNA purity and concentration are critical for good transfection; debris from purification columns induces the formation of large aggregates that do not gain entry into the cell, and DNA concentrations must be optimized to promote intramolecular condensation rather than intermolecular linking, which also causes aggregation and diminished transfection efficiency. The expression of both markers and genes within BACs initially occurs at lower levels than observed with plasmids, requiring 3-5 days to evaluate the transfection results. We also show that BACs can be co-transfected with other BACs, which provides for increased experimental flexibility.     

RESOURCERER: a database for annotating and linking microarray resources within and across species

Microarray expression analysis is providing unprecedented data on gene expression in humans and mammalian model systems. Although such studies provide a tremendous resource for understanding human disease states, one of the significant challenges is cross-referencing the data derived from different species, across diverse expression analysis platforms, in order to properly derive inferences regarding gene expression and disease state. To address this problem, we have developed RESOURCERER, a microarray-resource annotation and cross-reference database built using the analysis of expressed sequence tags (ESTs) and gene sequences provided by the TIGR Gene Index (TGI) and TIGR Orthologous Gene Alignment (TOGA) databases [now called Eukaryotic Gene Orthologs (EGO)].     

以绵羊MHC区段BAC克隆酶切片段为探针杂交筛选绵羊混合组织cDNA文库

在已知中国美利奴羊MHC(Major histocompatibility complex)区段BAC(Bacterial artificial chromosome)克隆序列信息和预测的基因注释前提下,用位于中国美利奴羊基因组BAC文库MHC区段的6个BAC克隆酶切片段为探针,以噬菌斑原位杂交筛选法筛选中国美利奴羊混合组织cDNA文库(库库杂交),对分离到的cDNA阳性克隆进行全序列测定,并与相应的已知序列信息和基因注释的BAC克隆比对以及在NCBI Blastn数据库中序列相似性检索,旨在验证基因注释结果的准确性和对基因(序列)功能的初步分析。实验中,经过两轮杂交共筛选出27个cDNA阳性克隆(序列),并发现这些序列均可定位到相应的BAC克隆上,且25条序列处在注释基因的外显子部分;在NCBI数据库中经Blastn序列相似性检索发现,23条序列与牛基因的序列相似性最高,且与免疫功能密切相关。     

Sequencing,annotation and comparative analysis of nine BACs of giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.     

General method for the modification of different BAC types and the rapid generation of BAC transgenic mice

Most genome projects have relied on the sequencing of bacterial artificial chromosomes (BACs), which encompass 100-300 kb of genomic DNA. As a consequence, several thousand BAC clones are now mapped to the human and mouse genome. It is therefore possible to identify in silico a BAC clone that carries a particular gene and obtain it commercially. Given the large size of BACs, most if not all regulatory sequences of a gene are present and can be used to direct faithful and tissue-specific expression of heterologous genes in vitro in cell cultures and in vivo in BAC-transgenic mice. We describe here an optimized and comprehensive protocol to select, modify, and purify BACs in order to generate BAC-transgenic mice. Importantly, this protocol includes a method to generate, within 2 days, complex plasmid cassettes required to modify BACs, and to efficiently modify different types of BACs selected from the two major BAC libraries available. Altogether, using a combination of genomic database analysis, overlap PCR cloning, and BAC recombination in bacteria, our approach allows for the rapid and reliable generation of "pseudo knockin" mice. genesis 38:39-50, 2004.     

A BAC contig map over the proximal approximately 3.3 Mb region of horse chromosome 21

A total of 207 BAC clones containing 155 loci were isolated and arranged into a map of linearly ordered overlapping clones over the proximal part of horse chromosome 21 (ECA21), which corresponds to the proximal half of the short arm of human chromosome 19 (HSA19p) and part of HSA5. The clones form two contigs - each corresponding to the respective human chromosomes - that are estimated to be separated by a gap of approximately 200 kb. Of the 155 markers present in the two contigs, 141 (33 genes and 108 STS) were generated and mapped in this study. The BACs provide a 4-5x coverage of the region and span an estimated length of approximately 3.3 Mb. The region presently contains one mapped marker per 22 kb on average, which represents a major improvement over the previous resolution of one marker per 380 kb obtained through the generation of a dense RH map for this segment. Dual color fluorescence in situ hybridization on metaphase and interphase chromosomes verified the relative order of some of the BACs and helped to orient them accurately in the contigs. Despite having similar gene order and content, the equine region covered by the contigs appears to be distinctly smaller than the corresponding region in human (3.3 Mb vs. 5.5-6 Mb) because the latter harbors a host of repetitive elements and gene families unique to humans/primates. Considering limited representation of the region in the latest version of the horse whole genome sequence EquCab2, the dense map developed in this study will prove useful for the assembly and annotation of the sequence data on ECA21 and will be instrumental in rapid search and isolation of candidate genes for traits mapped to this region.     

Recombining overlapping BACs into a single larger BAC

Background  

BAC clones containing entire mammalian genes including all the transcribed region and long range controlling elements are very useful for functional analysis. Sequenced BACs are available for most of the human and mouse genomes and in many cases these contain intact genes. However, large genes often span more than one BAC, and single BACs covering the entire region of interest are not available. Here we describe a system for linking two or more overlapping BACs into a single clone by homologous recombination.     

Efficient recombination-based methods for bacterial artificial chromosome fusion and mutagenesis

The availability of genomic sequence information and extensive bacterial artificial chromosome (BAC) libraries for both the mouse and human genomes is ushering in a new era in biological research and disease modeling. To facilitate the study of large mammalian genes in vivo, we have developed: i) a simple lambda bacteriophage-based methodology for recombining overlapping bacterial artificial chromosomes (BACs) into a single larger BAC, and ii) a new methodology for targeting "seamless" mutations into BACs. In the first method, overlapping sequence from one BAC is cloned alongside a selectable marker and placed between unique sequence arms from the terminus of the other BAC to create a targeting construct. Two rounds of recombination-based cloning are then performed. The robustness of this methodology is demonstrated herein by using it to obtain a 254 kb BAC containing the entire human androgen receptor (hAR) gene. In the second method, transient expression of three lambda bacteriophage genes to 'pop-in' a targeting cassette is followed by RecA expression from the targeting vector itself to 'pop-out' the vector backbone. This new "hybrid recombineering" method combines the strengths of the lambda bacteriophage and RecA systems, while avoiding their major weaknesses. Application of this method for introduction of a 162 CAG repeat expansion into the hAR 254kb BAC is shown. With "hybrid recombineering", we believe that the power and utility of the classical 'pop-in/pop-out' approach -- so commonly and efficiently employed in yeast for decades -- can now be achieved with BACs.     

Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: use of a cosmid and a bacterial artificial chromosome library

The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum.     

Five-color-based high-information-content fingerprinting of bacterial artificial chromosome clones using type IIS restriction endonucleases

We have developed a high-information-content fingerprinting (HICF) system for bacterial artificial chromosome (BAC) clones using a Type IIS restriction endonuclease, HgaI, paired with a Type II restriction endonuclease, RsaI. In the method described, unknown five-base overhangs generated with HgaI are partially or fully sequenced by modified fluorescent dideoxy terminators. Using an in-lane size standard labeled with a fifth dye, fragments are characterized by both the size and the sequence of its terminal one to five bases. The enhanced information content associated with this approach significantly increases the accuracy and efficiency of detecting shared fragments among BAC clones. We have compared data obtained from this method to predicted HICF patterns of 10 fully sequenced BACs. We have further applied HICF to 555 BAC clones to assemble contigs spanning 16p11.2 to 16p13.1 of human chromosome 16.     

A BAC-based integrated linkage map of the silkworm Bombyx mori

Background

In 2004, draft sequences of the model lepidopteran Bombyx mori were reported using whole-genome shotgun sequencing. Because of relatively shallow genome coverage, the silkworm genome remains fragmented, hampering annotation and comparative genome studies. For a more complete genome analysis, we developed extended scaffolds combining physical maps with improved genetic maps.

Results

We mapped 1,755 single nucleotide polymorphism (SNP) markers from bacterial artificial chromosome (BAC) end sequences onto 28 linkage groups using a recombining male backcross population, yielding an average inter-SNP distance of 0.81 cM (about 270 kilobases). We constructed 6,221 contigs by fingerprinting clones from three BAC libraries digested with different restriction enzymes, and assigned a total of 724 single copy genes to them by BLAST (basic local alignment search tool) search of the BAC end sequences and high-density BAC filter hybridization using expressed sequence tags as probes. We assigned 964 additional expressed sequence tags to linkage groups by restriction fragment length polymorphism analysis of a nonrecombining female backcross population. Altogether, 361.1 megabases of BAC contigs and singletons were integrated with a map containing 1,688 independent genes. A test of synteny using Oxford grid analysis with more than 500 silkworm genes revealed six versus 20 silkworm linkage groups containing eight or more orthologs of Apis versus Tribolium, respectively.

Conclusion

The integrated map contains approximately 10% of predicted silkworm genes and has an estimated 76% genome coverage by BACs. This provides a new resource for improved assembly of whole-genome shotgun data, gene annotation and positional cloning, and will serve as a platform for comparative genomics and gene discovery in Lepidoptera and other insects.