A comprehensive BAC resource

The Human Genome Project has generated extensive map and sequence data for a large number of Bacterial Artificial Chromosome (BAC) clones. In order to maximize the efficient use of the data and to minimize the redundant work for the research community, The Institute for Genomic Research (TIGR) comprehensive BAC resource (cBACr) (http://www.tigr.org/tdb/BacResource/BAC_resourc e_intro. html) was built as an expansion of the TIGR human BAC ends database. This resource collects, integrates and reports the information on library, maps, sequence, annotation and functions for each human and mouse BAC. The current database contains 635 016 human BACs and 265 617 mouse BACs that were characterized by various approaches, among which 22 705 human clones and 1000 mouse clones have sequence and annotation data.     

Prediction of the coding sequences of mouse homologues of KIAA gene: II. The complete nucleotide sequences of 400 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have accumulated information of the coding sequences of uncharacterized human genes, which are known as KIAA genes, and the number of these genes exceeds 2000 at present. As an extension of this sequencing project, we recently have begun to accumulate mouse KIAA-homologous cDNAs, because it would be useful to prepare a set of human and mouse homologous cDNA pairs for further functional analysis of the KIAA genes. We herein present the entire sequences of 400 mouse KIAA cDNA clones and 4 novel cDNA clones which were incidentally identified during this project. Most of clones entirely sequenced in this study were selected by computer-assisted analysis of terminal sequences of the cDNAs. The average size of the 404 cDNA sequences reached 5.3 kb and that of the deduced amino acid sequences from these cDNAs was 868 amino acid residues. The results of sequence analyses of these clones showed that single mouse KIAA cDNAs bridged two different human KIAA cDNAs in some cases, which indicated that these two human KIAA cDNAs were derived from single genes although they had been supposed to originate from different genes. Furthermore, we successfully mapped all the mouse KIAA cDNAs along the genome using a recently published mouse genome draft sequence.     

Sample sequencing of a Salmonella typhimurium LT2 lambda library: comparison to the Escherichia coli K12 genome

As part of the ongoing sequencing of the complete Salmonella typhimurium LT2 genome, a partly ordered set of 416 lambda clones has been developed, representing over 90% of the genome. The average insert size is 17 kb. Sequences were obtained from both ends of each clone in this set. A total of over 600 kb of sequence has been deposited in the genome survey sequence section of GenBank. This resource of clones is available from the Salmonella Genome Stock Center. A preliminary comparison with the Escherichia coli K12 genome indicates that there are likely to be many hundred insertion deletion events, encompassing more than one gene, that distinguish these genomes. Fully 30% of the S. typhimurium sequences have no close homologs in the GenBank database.     

Database for exchangeable gene trap clones: Pathway and gene ontology analysis of exchangeable gene trap clone mouse lines

Gene trapping in embryonic stem (ES) cells is a proven method for large‐scale random insertional mutagenesis in the mouse genome. We have established an exchangeable gene trap system, in which a reporter gene can be exchanged for any other DNA of interest through Cre/mutant lox‐mediated recombination. We isolated trap clones, analyzed trapped genes, and constructed the database for Exchangeable Gene Trap Clones (EGTC) [ http://egtc.jp ]. The number of registered ES cell lines was 1162 on 31 August 2013. We also established 454 mouse lines from trap ES clones and deposited them in the mouse embryo bank at the Center for Animal Resources and Development, Kumamoto University, Japan. The EGTC database is the most extensive academic resource for gene‐trap mouse lines. Because we used a promoter‐trap strategy, all trapped genes were expressed in ES cells. To understand the general characteristics of the trapped genes in the EGTC library, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analysis and found that the EGTC ES clones covered a broad range of pathways. We also used Gene Ontology (GO) classification data provided by Mouse Genome Informatics (MGI) to compare the functional distribution of genes in each GO term between trapped genes in the EGTC mouse lines and total genes annotated in MGI. We found the functional distributions for the trapped genes in the EGTC mouse lines and for the RefSeq genes for the whole mouse genome were similar, indicating that the EGTC mouse lines had trapped a wide range of mouse genes.     

Regions of the terminal repetitions of the herpes simplex virus type 1 genome. Relationship to immunoglobulin switch-like DNA sequences

Several recombinant clones isolated from a mouse genomic library were previously shown to hybridize with a SmaI fragment located in the terminal repetition of the S component of herpes simplex virus DNA. We report here the nucleotide sequence of the related regions in two mouse clones, TGL19 and TGL35, as well as that of the SmaI fragment of HSV-1. The mouse DNA clones have a core of repetitive sequences 80% homologous to a tandem repeat (reiteration II) in the viral fragment. The regions of homology are in turn related to immunoglobulin class-switch sequences, due mostly to the presence of the pentamer TGGG(G), involved in class-switch recombination. These results suggest that the HSV genome has recombination sequences identical to those of the host cell and provide a possible explanation for the high frequency of recombination events observed in this region of the viral genome.     

MagnaportheDB: a federated solution for integrating physical and genetic map data with BAC end derived sequences for the rice blast fungus Magnaporthe grisea

We have created a federated database for genome studies of Magnaporthe grisea, the causal agent of rice blast disease, by integrating end sequence data from BAC clones, genetic marker data and BAC contig assembly data. A library of 9216 BAC clones providing >25-fold coverage of the entire genome was end sequenced and fingerprinted by HindIII digestion. The Image/FPC software package was then used to generate an assembly of 188 contigs covering >95% of the genome. The database contains the results of this assembly integrated with hybridization data of genetic markers to the BAC library. AceDB was used for the core database engine and a MySQL relational database, populated with numerical representations of BAC clones within FPC contigs, was used to create appropriately scaled images. The database is being used to facilitate sequencing efforts. The database also allows researchers mapping known genes or other sequences of interest, rapid and easy access to the fundamental organization of the M.grisea genome. This database, MagnaportheDB, can be accessed on the web at http://www.cals.ncsu.edu/fungal_genomics/mgdatabase/int.htm.     

Construction and characterization of two BAC libraries from Brachypodium distachyon, a new model for grass genomics.

Brachypodium is well suited as a model system for temperate grasses because of its compact genome and a range of biological features. In an effort to develop resources for genome research in this emerging model species, we constructed 2 bacterial artificial chromosome (BAC) libraries from an inbred diploid Brachypodium distachyon line, Bd21, using restriction enzymes HindIII and BamHI. A total of 73,728 clones (36,864 per BAC library) were picked and arrayed in 192,384-well plates. The average insert size for the BamHI and HindIII libraries is estimated to be 100 and 105 kb, respectively, and inserts of chloroplast origin account for 4.4% and 2.4%, respectively. The libraries individually represent 9.4- and 9.9-fold haploid genome equivalents with combined 19.3-fold genome coverage, based on a genome size of 355 Mb reported for the diploid Brachypodium, implying a 99.99% probability that any given specific sequence will be present in each library. Hybridization of the libraries with 8 starch biosynthesis genes was used to empirically evaluate this theoretical genome coverage; the frequency at which these genes were present in the library clones gave an estimated coverage of 11.6- and 19.6-fold genome equivalents. To obtain a first view of the sequence composition of the Brachypodium genome, 2185 BAC end sequences (BES) representing 1.3 Mb of random genomic sequence were compared with the NCBI GenBank database and the GIRI repeat database. Using a cutoff expectation value of E<10-10, only 3.3% of the BESs showed similarity to repetitive sequences in the existing database, whereas 40.0% had matches to the sequences in the EST database, suggesting that a considerable portion of the Brachypodium genome is likely transcribed. When the BESs were compared with individual EST databases, more matches hit wheat than maize, although their EST collections are of a similar size, further supporting the close relationship between Brachypodium and the Triticeae. Moreover, 122 BESs have significant matches to wheat ESTs mapped to individual chromosome bin positions. These BACs represent colinear regions containing the mapped wheat ESTs and would be useful in identifying additional markers for specific wheat chromosome regions.     

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。     

Recent segmental and gene duplications in the mouse genome

Background

The high quality of the mouse genome draft sequence and its associated annotations are an invaluable biological resource. Identifying recent duplications in the mouse genome, especially in regions containing genes, may highlight important events in recent murine evolution. In addition, detecting recent sequence duplications can reveal potentially problematic regions of the genome assembly. We use BLAST-based computational heuristics to identify large (≥ 5 kb) and recent (≥ 90% sequence identity) segmental duplications in the mouse genome sequence. Here we present a database of recently duplicated regions of the mouse genome found in the mouse genome sequencing consortium (MGSC) February 2002 and February 2003 assemblies.

Results

We determined that 33.6 Mb of 2,695 Mb (1.2%) of sequence from the February 2003 mouse genome sequence assembly is involved in recent segmental duplications, which is less than that observed in the human genome (around 3.5-5%). From this dataset, 8.9 Mb (26%) of the duplication content consisted of 'unmapped' chromosome sequence. Moreover, we suspect that an additional 18.5 Mb of sequence is involved in duplication artifacts arising from sequence misassignment errors in this genome assembly. By searching for genes that are located within these regions, we identified 675 genes that mapped to duplicated regions of the mouse genome. Sixteen of these genes appear to have been duplicated independently in the human genome. From our dataset we further characterized a 42 kb recent segmental duplication of Mater, a maternal-effect gene essential for embryogenesis in mice.

Conclusion

Our results provide an initial analysis of the recently duplicated sequence and gene content of the mouse genome. Many of these duplicated loci, as well as regions identified to be involved in potential sequence misassignment errors, will require further mapping and sequencing to achieve accuracy. A Genome Browser database was set up to display the identified duplication content presented in this work. This data will also be relevant to the growing number of investigators who use the draft genome sequence for experimental design and analysis.

     

Development of an integrative database with 499 novel microsatellite markers for Macaca fascicularis

Background

Cynomolgus macaques (Macaca fascicularis) are a valuable resource for linkage studies of genetic disorders, but their microsatellite markers are not sufficient. In genetic studies, a prerequisite for mapping genes is development of a genome-wide set of microsatellite markers in target organisms. A whole genome sequence and its annotation also facilitate identification of markers for causative mutations. The aim of this study is to establish hundreds of microsatellite markers and to develop an integrative cynomolgus macaque genome database with a variety of datasets including marker and gene information that will be useful for further genetic analyses in this species.

Results

We investigated the level of polymorphisms in cynomolgus monkeys for 671 microsatellite markers that are covered by our established Bacterial Artificial Chromosome (BAC) clones. Four hundred and ninety-nine (74.4%) of the markers were found to be polymorphic using standard PCR analysis. The average number of alleles and average expected heterozygosity at these polymorphic loci in ten cynomolgus macaques were 8.20 and 0.75, respectively.

Conclusion

BAC clones and novel microsatellite markers were assigned to the rhesus genome sequence and linked with our cynomolgus macaque cDNA database (QFbase). Our novel microsatellite marker set and genomic database will be valuable integrative resources in analyzing genetic disorders in cynomolgus macaques.     

Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova.

In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome.     

Genomic structure and chromosomal mapping of the mouse hic-5 gene that encodes a focal adhesion protein

The hic-5 gene encodes a focal adhesion protein that has striking similarity to paxillin. Genomic clones of the mouse hic-5 gene were isolated, and included 10 exons that covered the whole mouse mRNA sequence. Comparison of the sequence with those in the expressed sequence tag database suggested that the hic-5 gene contained an extra exon (named exon 1') located about 1kb upstream of exon 1, and mouse cells seemed to express two alternatively spliced forms of mRNA. All the exon-intron boundaries followed the GT/AG rule. Physical mapping and fluorescent in situ hybridization analysis indicated that the hic-5 gene is located on mouse chromosome 7, 60. 0cM from the centromere.     

Sequence organization of cloned intracisternal A particle genes

Seven recombinant DNA clones containing mouse intracisternal A particle genes were isolated and analyzed by restriction enzyme digestion, Southern blot analysis and heteroduplex mapping. The sequence organization of the individual genes was found to differ, with one end of the gene region being most variable, while a central segment of 1.8 kb was missing from two of the clones. A third region, common to all the clones and containing the 3' end of the gene, is present in about 1800 copies per haploid genome, but the central portion is found in only 650 copies. The same reiteration frequency is found in both myeloma tumor and mouse liver DNA. The most abundant intracisternal A particle RNA in two different myeloma lines was found to be 3.5 kb, and RNA/DNA hybrids show that the RNA is homologous to all but a small internal segment of one of the clones.     

Looking for relationships between the most repeated dispersed DNA sequences in the mouse: small R elements are found associated consistently with long MIF repeats.

Four highly-repeated dispersed DNA sequence families have been described in the mouse genome. These are the three small elements B1, B2 and R and the large (6 kb) MIF element. Together these comprise approximately 10% of the mouse genome. Possible relationships between these families are pertinent to the genome as a whole. We report here that the B1s, B2s and Rs are all randomly organized in the genome with respect to each other. Surprisingly though, the R and MIF families are found together consistently in a set of random genomic clones and in selected clones. We find Rs often located on one end of the MIF at a consistent site and conclude that a minority of Rs are an integral part of MIF while the majority of Rs are not associated with MIFs. We propose that isolated R elements are truncated forms of MIF. Also we speculate on the mechanism of dispersal of these elements through the mouse genome.