Interdigitated arrangement of two oligo(A)-terminated DNA sequences in Drosophila.

A cluster of repeated sequences composed of three distinguishable units has been isolated from Drosophila melanogaster, and characterized. The region, cloned as pDmI 158, contains a segment that is homologous to the type 1 ribosomal insertions, a member of the F family of transposable sequences, and a newly described repeated sequence that we have named G. F elements are transposable sequences that lack terminal repeats, generate target site duplications at the point of insertion, and contain an oligo(A) stretch at one end. G sequences are structurally similar though non-homologous to F in that they also carry an oligo(A) stretch. The structure of the 158 region of the genome is best explained by assuming three consecutive events. An F element did insert into a ribosomal insertion-like sequence, followed by the introduction of a G sequence into F. Subsequently, a DNA segment comprising a portion of G and F was tandemly triplicated to yield the arrangement observed. The nested interspersion of repeated sequence elements may be a common feature of eukaryotic genomes.     

A new family of interspersed repetitive DNA sequences in the mouse genome

Repetitive DNA sequences near immunoglobulin genes in the mouse genome (Steinmetz et al., 1980a,b) were characterized by restriction mapping and hybridization. Six sequences were determined that turned out to belong to a new family of dispersed repetitive DNA. From the sequences, which are called R1 to R6, a 475 base-pair consensus sequence was derived. The R family is clearly distinct from the mouse B1 family (Krayev et al., 1980). According to saturation hybridization experiments, there are about 100,000 R sequences per haploid genome, and they are probably distributed throughout the genome. The individual R sequences have an average divergence from the consensus sequence of 12.5%, which is largely due to point mutations and, among those, to transitions. Some R sequences are severly truncated. The R sequences extend into A-rich sequences and are flanked by short direct repeats. Also, two large insertions in the R2 sequence are flanked by direct repeats. In the neighbourhood of and within R sequences, stretches of DNA have been identified that are homologous to parts of small nuclear RNA sequences. Mouse satellite DNA-like sequences and members of the B1 family were also found in close proximity to the R sequences. The dispersion of R sequences within the mouse genome may be a consequence of transposition events. The possible role of the R sequences in recombination and/or gene conversion processes is discussed.     

Identification and characterization of piggyBac-like elements in the genome of domesticated silkworm, Bombyx mori

piggyBac is a short inverted terminal repeat (ITR) transposable element originally discovered in Trichoplusia ni. It is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in insects and mammals. Many piggyBac-like sequences have been found in the genomes of phylogenetically species from fungi to mammals. We have identified 98 piggyBac-like sequences (BmPBLE1-98) from the genome data of domesticated silkworm (Bombyx mori) and 17 fragments from expressed sequence tags (ESTs). Most of the BmPBLE1-98 probably exist as fossils. A total of 21 BmPBLEs are flanked by ITRs and TTAA host dinucleotides, of which 5 contain a single ORF, implying that they may still be active. Interestingly, 16 BmPBLEs have CAC/GTG not CCC/GGG as the characteristic residues of ITRs, which is a surprising phenomenon first observed in the piggyBac families. Phylogenetic analysis indicates that many BmPBLEs have a close relation to mammals, especially to Homo sapiens, only a few being grouped with the T. ni piggyBac element. In addition, horizontal transfer was probably involved in the evolution of the piggyBac-like elements between B. mori and Daphnia pulicaria. The analysis of the BmPBLEs will contribute to our understanding of the characteristic of the piggyBac family and application of piggyBac in a wide range of insect species.     

Bacterial interspersed mosaic elements (BIMEs) are present in the genome of Klebsiella

Bacterial interspersed mosaic elements (BIMEs) constitute a family of highly repetitive sequences containing palindromic units (PUs), also called repetitive extragenic palindromes (REPs). BIMEs were originally described in Escherichia coli and Salmonella typhimurium. We show here, by determining the nucleotide sequence of two intergenic regions of Klebsiella pneumoniae, by computer searches, and by hybridization, that sequences with similar characteristics are found in the genome of several Klebsiella species. This reinforces the idea that BIMEs play general and important roles in enterobacteria such as in the organization of the bacterial chromosome.     

The genome sequence of silkworm, Bombyx mori.

We performed threefold shotgun sequencing of the silkworm (Bombyx mori) genome to obtain a draft sequence and establish a basic resource for comprehensive genome analysis. By using the newly developed RAMEN assembler, the sequence data derived from whole-genome shotgun (WGS) sequencing were assembled into 49,345 scaffolds that span a total length of 514 Mb including gaps and 387 Mb without gaps. Because the genome size of the silkworm is estimated to be 530 Mb, almost 97% of the genome has been organized in scaffolds, of which 75% has been sequenced. By carrying out a BLAST search for 50 characteristic Bombyx genes and 11,202 non-redundant expressed sequence tags (ESTs) in a Bombyx EST database against the WGS sequence data, we evaluated the validity of the sequence for elucidating the majority of silkworm genes. Analysis of the WGS data revealed that the silkworm genome contains many repetitive sequences with an average length of <500 bp. These repetitive sequences appear to have been derived from truncated transposons, which are interspersed at 2.5- to 3-kb intervals throughout the genome. This pattern suggests that silkworm may have an active mechanism that promotes removal of transposons from the genome. We also found evidence for insertions of mitochondrial DNA fragments at 9 sites. A search for Bombyx orthologs to Drosophila genes controlling sex determination in the WGS data revealed 11 Bombyx genes and suggested that the sex-determining systems differ profoundly between the two species.     

Aligning the proteome and genome of the silkworm, Bombyx mori

A technology of mass spectrometry (MS) was used in this study for the large-scale proteomic identification and verification of protein-encoding genes present in the silkworm (Bombyx mori) genome. Peptide sequences identified by MS were compared with those from an open reading frame (ORF) library of the B. mori genome and a cDNA library, to validate the coding attributes of ORFs. Two databases were created. The first was based on a 9× draft sequence of the silkworm genome and contained 14,632 putative proteins. The second was based on a B. mori pupal cDNA library containing 3,187 putative proteins of at least 30 amino acid residues in length. A total of 81,000 peptide sequences with a threshold score of 60% were generated by the MS/MS analysis, and 55,400 of these were chosen for a sequence alignment. By searching these two databases, 6,649 and 250 proteins were matched, which accounted for approximately 45.4% and 7.8% of the peptide sequences and putative proteins, respectively. Further analyses carried out by several bioinformatic tools suggested that the matches included proteins with predicted transmembrane domains (1,393) and preproteins with a signal peptide (976). These results provide a fundamental understanding of the expression and function of silkworm proteins.     

家蚕组织蛋白酶基因家族的鉴定及表达特征分析

家蚕是鳞翅目完全变态昆虫,在其变态过程中伴随着巨大的形态变化,包括旧组织的解离和新组织的形成,在这过程中有多种组织蛋白酶参与。组织蛋白酶是一类细胞内蛋白酶,广泛存在于各个物种中,包括组织蛋白酶B、H、L等几个亚家族。对家蚕组织蛋白酶的研究将有利于阐明家蚕变态发育的详细过程。通过对家蚕基因组数据库进行筛选,共在家蚕中鉴定到13种组织蛋白酶,并对这13种组织蛋白酶的基本信息和表达模式进行了分析。另外,利用家蚕基因芯片数据和荧光定量PCR分析,鉴定编号为BGIBMGA004622的基因为卵巢特异表达的组织蛋白酶L亚家族基因。该基因全长1 209 bp,编码402个氨基酸。经过序列分析,该酶与其他物种的组织蛋白酶L具有较高的同源性,其活性位点高度保守,且与鳞翅目的组织蛋白酶L在进化上聚为一支。同时,对该基因进行克隆并原核表达,结果显示重组蛋白以包涵体的形式表达。定量PCR结果显示,该酶在蛹发育初期表达量逐渐升高,至蛹3 d达到最高值,推测其可能参与卵巢与卵母细胞的发育过程。     

Characterization of Argonaute family members in the silkworm,Bombyx mori

Abstract The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified based on the genome sequence of Bombyx mori. Based on their sequence similarity, BmAgo1 and BmAgo2 belong to the Ago subfamily, while BmAgo3 and BmPiwi are in the Piwi subfamily. Phylogenetic analysis reveals that silkworm Argonaute family members are conserved in insects. Conserved amino acid residues involved in recognition of the 5′ end of the small RNA guide strand and of the conserved (aspartate, aspartate and histidine [DDH]) motif present in their PIWI domains suggest that these four Argonaute family members may have conserved slicer activities. The results of microarray expression analysis show that there is a low expression level for B. mori Argonaute family members in different tissues and different developmental stages, except for BmPiwi. All four B. mori Argonaute family members are upregulated upon infection with B. mori nucleopolyhedrovirus. The complete coding sequence of BmPiwi, the homolog of Drosophila piwi, was cloned and its expression occurred mainly in the area where spermatogonia and spermatocytes appear. Our results provide an overview of the B. mori Argonaute family members and suggest that they may have multiple roles. In addition, this is also the first report, to our knowledge, of the response of RNA silencing machinery to DNA virus infection in insects.     

Genome-wide analysis of the ATP-binding cassette (ABC) transporter gene family in the silkworm, Bombyx mori

The ATP-binding cassette (ABC) superfamily is a larger protein family with diverse physiological functions in all kingdoms of life. We identified 53 ABC transporters in the silkworm genome, and classified them into eight subfamilies (A-H). Comparative genome analysis revealed that the silkworm has an expanded ABCC subfamily with more members than Drosophila melanogaster, Caenorhabditis elegans, or Homo sapiens. Phylogenetic analysis showed that the ABCE and ABCF genes were highly conserved in the silkworm, indicating possible involvement in fundamental biological processes. Five multidrug resistance-related genes in the ABCB subfamily and two multidrug resistance-associated-related genes in the ABCC subfamily indicated involvement in biochemical defense. Genetic variation analysis revealed four ABC genes that might be evolving under positive selection. Moreover, the silkworm ABCC4 gene might be important for silkworm domestication. Microarray analysis showed that the silkworm ABC genes had distinct expression patterns in different tissues on day 3 of the fifth instar. These results might provide new insights for further functional studies on the ABC genes in the silkworm genome.     

Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori

The sex chromosomes of the silkworm, Bombyxmori, are designated ZW(XY) for females and ZZ(XX) for males. The W chromosome of B. mori does not recombine with the Z chromosome and autosomes and no genes for morphological characters have been mapped to the W chromosome as yet. Furthermore, femaleness is determined by the presence of a single W chromosome, regardless of the number of autosomes or Z chromosomes. To understand these interesting features of the W chromosome, it is necessary to analyze the W chromosome at the molecular biology level. Initially to isolate DNA sequences specific for the W chromosome as randomly amplified polymorphic DNA (RAPD) markers, we compared the genomic DNAs between males and females by PCR with arbitrary 10-mer primers. To the present, we have identified 12 W-specific RAPD markers, and with the exception of one RAPD marker, all of the deduced amino acid sequences of these W-specific RAPD markers show similarity to previously reported amino acid sequences of retrotransposable elements from various organisms. After constructing a genomic DNA lambda phage library of B. mori we obtained two lambda phage clones, one containing the W-Kabuki RAPD sequence and one containing the W-Samurai RAPD sequence and found that these DNA sequences comprised nested structures of many retrotransposable elements. To further analyze the W chromosome, we obtained 14 W-specific bacterial artificial chromosome (BAC) clones from three BAC libraries and subjected these clones to shotgun sequencing. The resulting assembly of sequences did not produce a single contiguous sequence due to the presence of many retrotransposable elements. Therefore, we coupled PCR with shotgun sequencing. Through these analyses, we found that many long terminal repeat (LTR) and non-LTR retrotransposons, retroposons, DNA transposons and their derivatives, have accumulated on the W chromosome as strata. These results strongly indicate that retrotransposable elements are the main structural component of the W chromosome.