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We have developed a terminal restriction fragment length polymorphism (T-RFLP) method for determining the structure and dynamics of the microbial gut community. In this paper, the improved T-RFLP method in combination with an analysis of the fecal 16S rDNA (ribosomal RNA gene) clone libraries from six individuals is described. A total of 418 different partial sequences of 16S rDNA were determined and subjected to a phylogenetic analysis and homology examination. We found that the sequences were roughly divided into six phylogenetic groups containing seven subgroups and were related to 71 known species with over 90% similarity. With the exception of a few cases, we found that in silico BslI-digestion of the sequences belonging to the same bacterial group or subgroup generated terminal restriction fragments of similar lengths. We concluded that human intestinal microbiota predominantly consists of the members of approximately ten phylogenetic bacterial groups and that these bacterial groups are effectively distinguished by our T-RFLP system.

The Japanese scallop (Mizuhopecten yessoensis) is one of the main fishery products in Japan, but with the expansion of culture operations of the Japanese scallop, various problems have been encountered including high mortality, poor growth, poor seed production, and so on. Moreover, there is concern that many years of cultivation may have affected the genetic structure of the scallop population. To approach these problems and concerns, we developed microsatellite markers as a molecular tool for population genetic studies. By using 4 microsatellite markers as well as a mitochondrial marker, we investigated the genetic structure of samples from the islands of Hokkaido (14 populations) and Honshu (Tohoku, 3 populations) in Japan, and south Primorye (4 populations) in Russia. All the populations sampled had high genetic diversity (average expected heterozygosity, 0.7011 to 0.7622; haplotype diversity, 0.6090 to 0.8848), and almost all showed a tendency of homozygote excess, which was significant in 2 populations. Hierarchical analysis of molecular variance tests based on the microsatellite and mitochondrial markers indicated that the 3 geographic regions were genetically divergent from one another, with little evidence of divergence within regions. Homogeneity in allele frequency distributions between natural and cultured scallops and allele frequency stability over a period of 2 decades indicated that the culturing operations have probably not had a substantial effect on the genetic structure of the populations.

Messenger RNA for yeast cytosolic polypeptide chain elongation factor 1 alpha (EF‐1 alpha) was partially purified from Saccharomyces cerevisiae. Double‐stranded complementary DNA (cDNA) was synthesized and cloned in Escherichia coli with pBR327 as a vector. Recombinant plasmid carrying yEF‐1 alpha cDNA was identified by cross‐hybridization with the E. coli tufB gene and the yeast mitochondrial EF‐Tu gene (tufM) under non‐stringent conditions. A yeast gene library was then screened with the EF‐1 alpha cDNA and several clones containing the chromosomal gene for EF‐1 alpha were isolated. Restriction analysis of DNA fragments of these clones as well as the Southern hybridization of yeast genomic DNA with labelled EF‐1 alpha cDNA indicated that there are two EF‐1 alpha genes in S. cerevisiae. The nucleotide sequence of one of the two EF‐1 alpha genes (designated as EF1 alpha A) was established together with its 5′‐ and 3′‐flanking sequences. The sequence contained 1374 nucleotides coding for a protein of 458 amino acids with a calculated mol. wt. of 50 300. The derived amino acid sequence showed homologies of 31% and 32% with yeast mitochondrial EF‐Tu and E. coli EF‐Tu, respectively.

Human T-celi leukemia virus type I (HTLV-I) contains the pX sequence which codes for the trans-activator of the long terminal repeat (LTR) and is thus postulated to be associated with leukemogenesis in adult T-cell leukemia. Overlapping open reading frames (ORF) in the pX sequence were recently found to code for p27x-III and p2l'x"h by ORF Ill, in addition to p40 coded for by ORF IV. The mechanism of expression of these newly identified proteins and their possible association with trans-activation were studied. On transfection of an expression plasmid that contains a cDNA sequence of thepX mRNA, products from both ORFs m and IV were detected in the cells. The RNA was synthesized in vitro from the cDNA clone by SP6 RNA polymerase and translated in a rabbit reticulocyte lysate. As translation products, two proteins, p27x-Z and p2lx-, were detected in addition to p4y0. Elimination of the first and second ATG codons in ORF III resulted in loss of the ability to code for p27x1u5 and p21x111, respectively, which indicated that the translations from these two ATG codons were independent. A mutant that lacked both ATG codons was fully active in trans-activation of chloramphenicol acetyltransferase gene expression directed by the LTR. These results indicate that a 2.1-kilobase pX mRNA of HTLV-I independently encodes three proteins, p40', p27x-h1, and p21x-11, by different ORFs and that the last two proteins are not involved in trans-activation of the unintegrated LTR. Human T-cell leukemia virus type I (HTLV-I) (19, 32) is an etiological agent of adult T-cell leukemia (ATL) (8, 9, 33) and an exogenous human retrovirus (21, 32). The viral genome has an extra sequence, pX, between the env gene and the 3' long terminal repeat (LTR) (23). Expression of the pX sequence was suggested to be associated with leukemogenesis by HTLV-I from the following findings: (i) the absence of a specific site for provirus integration in leukemic cells, disproving a cis-acting function of the LTR (22); (ii) frequent in vitro immortalization of infected T cells, suggesting direct involvement of viral products (18, 20); and (iii) the transacting function of the pX product, which activates viral gene expression (6, 30). The pX sequence contains four possible open reading frames (ORFs), I to IV (23) (Fig. 1A), and a product encoded by ORF IV was previously identified as p4WY (10, 14, 17, 28).

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To examine the genetic structure of Japanese scallop populations (Mizuhopecten yessoensis) in Hokkaido prefecture, Japan, and compare it with those in the Aomori prefecture, we applied a method for lineage analysis based on sequence variation in a mitochondrial DNA segment (NcR2). After showing that there was a low probability of doubly uniparental inheritance of mitochondrial DNA in the scallop, we sequenced the NcR2 regions of 914 individuals from 15 populations (13 in Hokkaido and 2 in Aomori). In total, 103 different haplotypes were detected. Results of homogeneity tests for pairwise populations and the fixation indices indicated that significant heterogeneity (P < 0.0005) and structuring (pairwise fixation index F(ST) = 0.1606-0.4444, P = 0.0000; fixation index among groups F(CT) = 0.1549, P = 0.0078) could be inferred between the Hokkaido and Aomori groups, but not among populations within the groups. Moreover, heterogeneity of the haplotype distribution between populations of the 1980s and 1990s or 2000s at the 4 culturing areas was not observed (P > 0.05), and the haplotype diversity between them was not significant (P = 0.05), suggesting that the culture operations had not imparted a significant effect on the genetic structure during these periods.