End-sequencing and characterization of silkworm (Bombyx mori) bacterial artificial chromosome libraries

<p>Abstract</p> <p>Background</p> <p>We performed large-scale bacterial artificial chromosome (BAC) end-sequencing of two BAC libraries (an <it>Eco</it>RI- and a <it>Bam</it>HI-digested library) and conducted an <it>in silico </it>analysis to characterize the obtained sequence data, to make them a useful resource for genomic research on the silkworm (<it>Bombyx mori</it>).</p> <p>Results</p> <p>More than 94000 BAC end sequences (BESs), comprising more than 55 Mbp and covering about 10.4% of the silkworm genome, were sequenced. Repeat-sequence analysis with known repeat sequences indicated that the long interspersed nuclear elements (LINEs) were abundant in <it>Bam</it>HI BESs, whereas DNA-type elements were abundant in <it>Eco</it>RI BESs. Repeat-sequence analysis revealed that the abundance of LINEs might be due to a GC bias of the restriction sites and that the GC content of silkworm LINEs was higher than that of mammalian LINEs. In a BLAST-based sequence analysis of the BESs against two available whole-genome shotgun sequence data sets, more than 70% of the BESs had a BLAST hit with an identity of ≥ 99%. About 14% of <it>Eco</it>RI BESs and about 8% of <it>Bam</it>HI BESs were paired-end clones with unique sequences at both ends. Cluster analysis of the BESs clarified the proportion of BESs containing protein-coding regions.</p> <p>Conclusion</p> <p>As a result of this characterization, the identified BESs will be a valuable resource for genomic research on <it>Bombyx mori</it>, for example, as a base for construction of a BAC-based physical map. The use of multiple complementary BAC libraries constructed with different restriction enzymes also makes the BESs a more valuable genomic resource. The GenBank accession numbers of the obtained end sequences are <ext-link ext-link-type="gen" ext-link-id="DE283657">DE283657</ext-link>–<ext-link ext-link-type="gen" ext-link-id="DE378560">DE378560</ext-link>.</p

A BAC-Based Integrated Linkage Map of the Silkworm \u3cem\u3eBombyx mori\u3c/em\u3e

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

A single amino acid mutation in an ABC transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, \u3cem\u3eBombyx mori\u3c/em\u3e

Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93, which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis

Repression of tyrosine hydroxylase is responsible for the sex-linked chocolate mutation of the silkworm, \u3cem\u3eBombyx mori\u3c/em\u3e

Pigmentation patterning has long interested biologists, integrating topics in ecology, development, genetics, and physiology. Wild-type neonatal larvae of the silkworm, Bombyx mori, are completely black. By contrast, the epidermis and head of larvae of the homozygous recessive sex-linked chocolate (sch) mutant are reddish brown. When incubated at 30 °C, mutants with the sch allele fail to hatch; moreover, homozygous mutants carrying the allele sch lethal (schl) do not hatch even at room temperature (25 °C). By positional cloning, we narrowed a region containing sch to 239,622 bp on chromosome 1 using 4,501 backcross (BC1) individuals. Based on expression analyses, the best sch candidate gene was shown to be tyrosine hydroxylase (BmTh). BmTh coding sequences were identical among sch, schl, and wild-type. However, in sch the ∼70-kb sequence was replaced with ∼4.6 kb of a Tc1-mariner type transposon located ∼6 kb upstream of BmTh, and in schl, a large fragment of an L1Bm retrotransposon was inserted just in front of the transcription start site of BmTh. In both cases, we observed a drastic reduction of BmTh expression. Use of RNAi with BmTh prevented pigmentation and hatching, and feeding of a tyrosine hydroxylase inhibitor also suppressed larval pigmentation in the wild-type strain, pnd+ and in a pS (black-striped) heterozygote. Feeding L-dopa to sch neonate larvae rescued the mutant phenotype from chocolate to black. Our results indicate the BmTh gene is responsible for the sch mutation, which plays an important role in melanin synthesis producing neonatal larval color

Phosphorylation of clock protein PER1 regulates its circadian degradation in normal human fibroblasts.

Recent advances suggest that the molecular components of the circadian clock generate a self-sustaining transcriptional-translational feedback loop with a period of approx. 24 h. The precise expression profiles of human clock genes and their products have not been elucidated. We cloned human clock genes, including per1, per2, per3, cry2 and clock, and evaluated their circadian mRNA expression profiles in WI-38 fibroblasts stimulated with serum. Transcripts of hPer1, hPer2, hPer3, hBMAL1 and hCry2 (where h is human) underwent circadian oscillation. Serum-stimulation also caused daily oscillations of hPER1 protein and the apparent molecular mass of hPER1 changed. Inhibitor studies indicated that the CKI (casein kinase I) family, including CKIepsilon and CKIdelta, phosphorylated hPER1 and increased the apparent molecular mass of hPER1. The inhibition of hPER1 phosphorylation by CKI-7 [ N -(2-aminoethyl)-5-chloro-isoquinoline-8-sulphonamide], a CKI inhibitor, disturbed hPER1 degradation, delayed the nuclear entry of hPER1 and allowed it to persist for longer in the nucleus. Furthermore, proteasome inhibitors specifically blocked hPER1 degradation. However leptomycin B, an inhibitor of nuclear export, did not alter the degradation state of hPER1 protein. These findings indicate that circadian hPER1 degradation through a proteasomal pathway can be regulated through phosphorylation by CKI, but not by subcellular localization

Construction of a Single Nucleotide Polymorphism Linkage Map for the Silkworm, Bombyx mori, Based on Bacterial Artificial Chromosome End Sequences

We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide polymorphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female × an F(1) (p50T × C108T) male, we analyzed segregation patterns of 534 SNPs between p50T and C108T, detected among 3840 PCR amplicons, each associated with a p50T BAC end sequence. This enabled us to construct a linkage map composed of 534 SNP markers spanning 1305 cM in total length distributed over the expected 28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used to construct the linkage map, 89 were associated with 107 different ESTs. Since each of the SNP markers is directly linked to a specific genomic BAC clone and to whole-genome sequence data, and some of them are also linked to EST data, the SNP linkage map will be a powerful tool for investigating silkworm genome properties, mutation mapping, and map-based cloning of genes of industrial and agricultural interest

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