CENP-I is essential for centromere function in vertebrate cells

We identified a novel essential centromere protein, CENP-I, which shows sequence similarity with fission yeast Mis6 protein, and we showed that CENP-I is a constitutive component of the centromere that colocalizes with CENP-A, -C, and -H throughout the cell cycle in vertebrate cells. To determine the precise function of CENP-I, we examined its role in centromere function by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-I, cells arrested at prometaphase with misaligned chromosomes for long periods of time. Eventually, cells exited mitosis without undergoing cytokinesis. Immunocytochemical analysis of CENP-I-deficient cells demonstrated that both CENP-I and CENP-H are necessary for localization of CENP-C but not CENP-A to the centromere.     

Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis

The specific activity of subtilisin E, an alkaline serine protease of Bacillus subtilis, was substantially increased by optimizing the amino acid residue at position 31 (Ile in the wild-type enzyme) in the vicinity of the catalytic triad of the enzyme. Eight uncharged amino acids (Cys, Ser, Thr, Gly, Ala, Val, Leu, and Phe) were introduced at this site, which is next to catalytic Asp32, using site-directed mutagenesis. Mutant enzymes were expressed in Escherichia coli and were prepared from the periplasmic space. Only the Val and Leu substitutions gave active enzyme, and the Leu31 mutant was found to have a greatly increased activity compared to the wild-type enzyme. The other six mutant enzymes showed a marked decrease in activity. This result indicates that a branched-chain amino acid at position 31 is essential for the expression of subtilisin activity and that the level of the activity depends on side chain structure. The purified Leu31 mutant enzyme was analyzed with respect to substrate specificity, heat stability, and optimal temperature. It was found that the Leu31 replacement caused a prominent 2-6-fold increase in catalytic efficiency (kcat/Km) due to a larger kcat for peptide substrates.     

Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system

The relative quantities of 26 known transfer RNAs of Escherichia coli have been measured previously (Ikemura, 1981). Based on this relative abundance, the usage of cognate codons in E. coli genes as well as in transposon and coliphage genes was examined. A strong positive correlation between tRNA content and the occurrence of respective codons was found for most E. coli genes that had been sequenced, although the correlation was less significant for transposon and phage genes. The dependence of the usage of isoaccepting tRNA, in E. coli genes encoding abundant proteins, on tRNA content was especially noticeable and was greater than that expected from the proportional relationship between the two variables, i.e. these genes selectively use codons corresponding to major tRNAs but almost completely avoid using codons of minor tRNAs. Therefore, codon choice in E. coli genes was considered to be largely constrained by tRNA availability and possibly by translational efficiency. Based on the content of isoaccepting tRNA and the nature of codon-anticodon interaction, it was then possible to predict for most amino acids the order of preference among synonymous codons. The synonymous codon predicted in this way to be the most preferred codon was thought to be optimized for the E. coli translational system and designated as the “Optimal codon”. E. coli genes encoding abundant protein species use the optimal codons selectively, and other E. coli genes, such as amino acid synthesizing genes, use optimal and “non-optimal” codons to a roughly equal degree. The finding that the frequency of usage of optimal codons is closely correlated with the production levels of individual genes was discussed from an evolutionary viewpoint.     

Recombination in the class III region of the mouse major histocompatibility complex

The sites of meiotic recombination in the class II region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. To search for hotspots in the class III region, we mapped combiantional break-points of 79 Ab: H2-D recombinants with 11 DNA markers; these included Tnx, the gene for an extracellular matrix protein, tenascin X, the Notch-related Int3 gene, and a microsatellite marker, D17Mit13, none of which had previously been mapped precisely. The results gave the gene order Eb-61.11-Int3-Tnx-Cyp21/C4-Bf-Hsp68c-D17Mit13-Tnfa/Tnfb-D. The crossover sites in 40 of the 79 recombinants were cofiend within the Eb/Int3:Tnx/Cyp21 interval. The result demonstrated that an unequal distribution of recombination is a general feature of the mouse MHC, suggesting the presence of a recombinational hotsopt within the Int3:Tnx interval.     

cDNA Cloning of the Human Homologues of the MouseKe4andKe6Genes at the Centromeric End of the Human MHC Region

cDNA clones corresponding to theHKE4andHKE6genes at the centromeric end of the HLA region on human chromosome 6p21.3 were isolated and characterized. The predicted amino acid sequences of HKE4 and HKE6 exhibited 81.5 and 85.6% identity to the mouse homologues, Ke4 and Ke6, respectively.HKE4may encode a membrane protein with histidine-rich charge clusters. HKE6 possesses remarkable amino acid sequence conservation with several bacterial proteins with oxidoreductase function and also shows significant homology with the two unique functional domains containing the nucleotide cofactor binding site and the consensus motif characteristic of the members of the superfamily of short-chain alcohol dehydrogenases such as human and rat steroid and prostaglandin dehydrogenases.     

Nucleotide Sequencing Analysis of the 146-Kilobase Segment around theIkBLandMICAGenes at the Centromeric End of the HLA Class I Region

To elucidate the complete gene structure and to identify new genes involved in the development ofHLAclass I antigen-associated diseases in the class I region of the human major histocompatibility complex on chromosome 6, a YAC clone (745D12) covering the 146-kb segment around theIkBLandMICAloci was isolated from a YAC library constructed from the B-cell line, BOLETH. A physical map of this region was constructed by isolation of overlapping cosmid clones derived from 745D12. Of these, five contiguous cosmids were chosen for DNA sequencing by the shotgun strategy to give a single contig of 146,601 bp from 2.8 kb telomeric of theIkBLgene to exon 6 ofMICA.This region was confirmed to contain five known genes,IkBL, BAT1, MICB, P5-1,andHLA-X(class I fragment), from centromere to telomere, and their exon–intron organizations were determined. The3.8-1homologue gene (3.8-1-hom) showing 99.7% identity with the3.8-1cDNA clone, which was originally isolated using the 3.8-kbEcoRI fragment between theHLA-54/Hand theHLA-Ggenes, was detected betweenMICAandMICBand was suggested to represent the cognate3.8-1genomic sequence from which the cDNA clone was derived. No evidence for the presence of expressed new genes could be obtained in this region by homology and EST searches or coding and exon prediction analyses. One TA microsatellite repeat spanning 2545 bases with as many as 913 repetitions was found on the centromeric side of theMICAgene and was indicated to be a potential hot spot for genetic recombination. The two segments of approximately 35 kb upstream of theMICAandMICBgenes showed high sequence homology (about 85%) to each other, suggesting that segmental genome duplication including theMICAandMICBgenes must have occurred during the evolution of the humanMHC.     

Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes

Recent awareness that most microorganisms in the environment are resistant to cultivation has prompted scientists to directly clone useful genes from environmental metagenomes. Two screening methods are currently available for the metagenome approach, namely, nucleotide sequence-based screening and enzyme activity-based screening. Here we have introduced and optimized a third option for the isolation of novel catabolic operons, that is, substrate-induced gene expression screening (SIGEX). This method is based on the knowledge that catabolic-gene expression is generally induced by relevant substrates and, in many cases, controlled by regulatory elements situated proximate to catabolic genes. For SIGEX to be high throughput, we constructed an operon-trap gfp-expression vector available for shotgun cloning that allows for the selection of positive clones in liquid cultures by fluorescence-activated cell sorting. The utility of SIGEX was demonstrated by the cloning of aromatic hydrocarbon-induced genes from a groundwater metagenome library and subsequent genome-informatics analysis.     

Antitumor activity and action mechanisms of Lactobacillus casei through the regulation of immune responses

Lactobacillus casei strain Shirota (LcS) has been demonstrated to have beneficial effects in numerous disease models, especially murine autoimmune disease and carcinogenesis models, via host immune modulation. It has also been reported that LcS induced recovery of the host immune responses that were decreased by treatment with carcinogens, and that augmented the natural killer activity and T cell functions of host immune cells. After LcS is ingested by the host, it is incorporated into M cells in Peyer's patches (PP) and digested to form active components. In PP, macrophages or dendritic cells that phagocytosed LcS gained ability to produce tumor necrosis factor-alpha. The components of LcS digested in PP were then recognized through toll-like receptor 2 in macrophages, resulting in the production of several cytokines which elicit varied responses in host immune cells. Also, it was observed by 2D-PAGE analyses that the expression level and/or the phosphorylation of some proteins in PP and mesenteric lymph nodes were definitely altered after the ingestion of LcS, providing more evidence of cellular responses.     

Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells

CENP-C is an evolutionarily conserved centromere protein that is thought to be an important component in kinetochore assembly in vertebrate cells. However, the functional role of CENP-C in cell cycle progression remains unclear. To further understand CENP-C function, we developed a method incorporating the hyper-recombinogenic chicken B lymphocyte cell line DT40 to create several temperature-sensitive CENP-C mutants in DT40 cells. We found that, under restrictive conditions, one temperature-sensitive mutant, ts4-11, displayed metaphase delay and chromosome missegregation but proceeded through the cell cycle until arrest at G₁ phase. Furthermore, ts4-11 cells were transfected with a human HeLa cell cDNA library maintained in a retroviral vector, and genes that suppressed the temperature-sensitive phenotype were identified. One of these suppressor genes encodes SUMO-1, which is a ubiquitin-like protein. This finding suggests that SUMO-1 may be involved in centromere function in vertebrate cells. The novel strategy reported here will be useful and applicable to a wide range of proteins that have general cell-autonomous function in vertebrate cells.