Chromatin-induced Spindle Assembly Plays an Important Role in Metaphase Congression of Silkworm Holocentric Chromosomes

The kinetochore plays important roles in cell cycle progression. Interactions between 4 chromosomes and spindle microtubules allow chromosomes to congress to the middle of the 5 cell and to segregate the sister chromatids into daughter cells in mitosis. The chromosome 6 passenger complex (CPC), composed of the Aurora B kinase and its regulatory subunits 7 INCENP, Survivin, and Borealin, plays multiple roles in these chromosomal events. In the 8 genome of the silkworm, Bombyx mori, which has holocentric chromosomes, the CPC 9 components and their molecular interactions were highly conserved. In contrast to 10 monocentric species, however, the silkworm CPC co-localized with the chromatin-driven 11 spindles on the upper side of prometaphase chromosomes without forming bipolar mitotic 12 spindles. Depletion of the CPC by RNAi arrested the cell cycle progression at prometaphase 13 and disrupted the microtubule network of the chromatin-driven spindles. Interestingly, 14 depletion of mitotic centromere-associated kinesin (MCAK) recovered formation of the 15 microtubule network but did not overcome the cell cycle arrest at prometaphase. These 16 results suggest that the CPC modulates the chromatin-induced spindle assembly and 17 metaphase congression of silkworm holocentric chromosomes

Establishment of Monitoring System to Detect Single Copy DNA Included in One Genome but not in Another Using Representational Difference Analysis

Polyrnerase chain reaction (PCR) -coupled subtractive procedure, representa-tional difference analysis (RDA) , is an efficient method to find the differences between two complex genomes. RDA has been applied to detect genetic lesions in cancer, the identification of unknown pathogens from the genomes, and the isolation of polymorphic markers. However, characterization of various clones obtained by RDA is time consuming and laborious work, and it is of great impor-tance to monitor whether RDA really works. To establish a monitoring system to detect single copy target DNA, we studied whether RDA could detect four fragments of non-human DNA which were added in one genome but not in another. We were able to successfully detect the target DNAs which were mixed at the ratio of single and ten copies per haploid genome using RDA with some modification of the original protocol. We confirmed that RDA was sensi-tive and effective enough to detect such genetic lesions as occurred in cancer cells. The target DNA used in this model could be utilized as a positive control in other applications of RDA

Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA

The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair

W-derived BAC probes as a new tool for identification of the W chromosome and its aberrations in Bombyx mori

We isolated four W chromosome-derived bacterial artificial chromosome (W-BAC) clones from Bombyx mori BAC libraries by the polymerase chain reaction and used them as probes for fluorescence in situ hybridization (FISH) on chromosome preparations from B. mori females. All four W-BAC probes surprisingly highlighted the whole wild-type W sex chromosome and also identified the entire original W-chromosomal region in W chromosome-autosome translocation mutants. This is the first successful identification of a single chromosome by means of BAC-FISH in species with holokinetic chromosomes. Genomic in situ hybridization (GISH) by using female-derived genomic probes highlighted the W chromosome in a similar chromosome-painting manner. Besides the W, hybridization signals of W-BAC probes also occurred in telomeric and/or subtelomeric regions of the autosomes. These signals coincided well with those of female genomic probes except one additional GISH signal that was observed in a large heterochromatin block of one autosome pair. Our results support the opinion that the B. mori W chromosome accumulated transposable elements and other repetitive sequences that also occur, but scattered, elsewhere in the respective genome. Edited by: E.R. Schmid

The diversity of Plasmodium falciparum isolates from asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo

Background:Understanding Plasmodium falciparum population diversity and transmission dynamics provides information on the intensity of malaria transmission, which is needed for assessing malaria control interventions. This study aimed to determine P. falciparum allelic diversity and multiplicity of infection (MOI) among asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo (DRC).Methods:A total of 438 DNA samples (248 asymptomatic and 190 symptomatic) were characterized by nested PCR and genotyping the polymorphic regions of pfmsp1 block 2 and pfmsp2 block 3.Results:Nine allele types were observed in pfmsp1 block2. The K1-type allele was predominant with 78% (229/293) prevalence, followed by the MAD20-type allele (52%, 152/293) and RO33-type allele (44%, 129/293). Twelve alleles were detected in pfmsp2, and the 3D7-type allele was the most frequent with 84% (256/304) prevalence, followed by the FC27-type allele (66%, 201/304). Polyclonal infections were detected in 63% (95% CI 56, 69) of the samples, and the MOI (SD) was 1.99 (0.97) in P. falciparum single-species infections. MOIs significantly increased in P. falciparum isolates from symptomatic parasite carriers compared with asymptomatic carriers (2.24 versus 1.69, adjusted b: 0.36, (95% CI 0.01, 0.72), p = 0.046) and parasitaemia > 10,000 parasites/μL compared to parasitaemia < 5000 parasites/μL (2.68 versus 1.63, adjusted b: 0.89, (95% CI 0.46, 1.25), p < 0.001).Conclusion:This survey showed low allelic diversity and MOI of P. falciparum, which reflects a moderate intensity of malaria transmission in the study areas. MOIs were more likely to be common in symptomatic infections and increased with the parasitaemia level. Further studies in different transmission zones are needed to understand the epidemiology and parasite complexity in the DRC

Prediction of p53 target genes based on integrative analysis of chromatin immunoprecipitated and sequenced tags，by using Galaxy，a web-based interactive platform for large-scale genome analysis

Chromatin immunoprecipitation (ChIP) followed by sequencing of immunoprecipitated DNA fragments is the high throughput method for identifying transcription factor binding sites. In one such method, ChIP PET, paired end ditags (PETs) derived from both ends of the immunoprecipitated DNA fragments are sequenced and mapped to the genome. We report here the prediction of p53 target genes by meta analyzing tags of p53 ChIP PET and by combining with other genomic annotations, using Galaxy, a web based platform for large scale genome analysis. We found 327 of p53 binding sites on the genome of 5-fluorouracil (5-FU)-treated HCT116 colon cancer cells by searching the total 65,509 PETs for PET clusters. The search for p53 target gene, which focused on PET clusters with computationally-predicted p53 binding motif, identified 20 of putative p53 target genes as well as 11 of known p53 targets. Another search for p53 target genes, which focused on PET clusters located within 50-kb flanking regions of transcription start sites of genes, identified 278 of Refseq genes, 79 of non-coding RNAs and 5 of microRNAs as p53 targets which included lots of known validated targets. Our results indicate that sequencing-based ChIP analysis combined with the existing genome annotation is effective method to predict p53 binding loci and target genes, and also show that the Galaxy platform is well-suited for multiple-type analyses and visualization of ChIP data, leading to functional annotation of transcription factor binding sites

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

ノウ ノ ハクブツカン ニ シュウゾウ サレテイル コウチュウモク タンメイ タイプ

農の博物館に収蔵されている甲虫目の担名タイプのすべてを,それぞれの学名,タイプの種類,性別とともに報告した。原著論文の記述によると,ハネカクシ科,クワガタムシ科,コガネムシ科,シバンムシ科,カミリキムシ科,ヒゲナガゾウムシ科に含まれる合計395種18亜種の担名タイプが保管されているはずだが,1950年以前に澤田玄正博士が記載したコガネムシ科の担名タイプは,第二次世界大戦の戦火と終戦後の混乱で失われたと考えられる。All the name-bearing types of Coleoptera deposited in the Museum of Agriculture, Tokyo University of Agriculture (Atsugi Campus) are listed. According to the original descriptions, name-bearing types of 395 species and 18 subspecies in the families Staphylinidae, Lucanidae, Scarabaeidae, Anobiidae, Cerambycidae, and Anthribidae should be housed in the museum. However, none of the holotypes and syntypes of Scarabaeidae described before 1951 by Dr. H. Sawada can been found there. It is most probable that they were lost during or after the Second World War

Pathobiological implications of mucin (MUC) expression in the outcome of small bowel cancer.

Mucins have been associated with survival in various cancer patients, but there have been no studies of mucins in small bowel carcinoma (SBC). In this study, we investigated the relationships between mucin expression and clinicopathologic factors in 60 SBC cases, in which expression profiles of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC6 and MUC16 in cancer and normal tissues were examined by immunohistochemistry. MUC1, MUC5AC and MUC16 expression was increased in SBC lesions compared to the normal epithelium, and expression of these mucins was related to clinicopathologic factors, as follows: MUC1 [tumor location (p = 0.019), depth (p = 0.017) and curability (p = 0.007)], MUC5AC [tumor location (p = 0.063) and lymph node metastasis (p = 0.059)], and MUC16 [venous invasion (p = 0.016) and curability (p = 0.016)]. Analysis of 58 cases with survival data revealed five factors associated with a poor prognosis: poorly-differentiated or neuroendocrine histological type (