Light- and Electron- microscopic and Immunohistochemical Studies of Human Rhabdomyosarcomas. Comparisons Among Primary Tumors, Heterotransplants in Nude Mice, and Cultured Cells from 13 Patients

Eighteen human rhabdomyosarcomas (RMS) were transplanted into the sub-cutaneous space on the back of nude mice. Thirteen of the tumors gave rise to transplantable tumors that were further examined morphologically and immuno-histochemically. The morphology of the transplanted tumors was similar to that of the primary tumors. Immunohistochemically, five primary tumors and six transplanted tumors were reacted with both desmin and myoglobin. However, in eleven cases cultured cells derived from the transplanted tumors, which showed elongated to strap-spindle-shaped cytoplasm resembling myotubules, reacted more intensely with both myoglobin and desmin. On ultrastructural examination, six primary tumors and seven transplanted tumors were found to have myofilaments or Z-bands. However, cultured cells showed myofilaments or Z-bands in their cytoplasm in all cases examined. We concluded that, on xeno-grafting, the histologic characteristics of the primary tumor are essentially con-served, and that tumor cells under culture conditions undergo an increased differentiation of skeletal muscle. These human RMS strains in nude mice and in cell lines will provide an excellent model system for investigating the biology of RMS and for further study of the molecular events underlying the genesis of this tumor

Isolation and characterization of salmonid telomeric and centromeric satellite DNA sequences

Satellite DNA clones with a 37 bp repeat unit were obtained from BglII-digested genomic DNA of Masu salmon (Oncorhynchus masou) and Chum salmon (O. keta). Fluorescence in situ hybridization (FISH) analysis with the isolated clones as a probe showed that these repetitive sequences were localized in the telomeric regions of chromosomes in both species. Southern and dot blot analyses suggested conservation of homologous sequences with similar repeat unit in other salmonids including the species of the genus Oncorhynchus and Salvelinus, but lack or scarcity of such sequences in the genus Hucho and Salmo. Similarly, polymerase chain reaction (PCR)-based cloning of satellite DNA referring to a reported Rainbow trout (O. mykiss) centromeric sequence was successful for the Oncorhynchus, Salvelinus and Hucho species. The obtained satellite DNA clones were localized with FISH in the centromeric regions of chromosomes of the species from these three genera. Although PCR cloning of the centromeric satellite DNA had failed in the Salmo species due to some base changes in the priming sites, dot blot hybridization analysis suggested conservation of homologous satellite DNA in the genus Salmo as in the other three genera. In the neighbor-joining tree of cloned centromeric satellite DNA sequences, the genus Oncorhynchus and Salvelinus formed adjacent clades, and the clade of the genus Hucho included the reported centromeric sequence of the genus Salmo. Conservation pattern and molecular phylogeny of the telomeric and centromeric satellite DNA sequences isolated herein support a close phylogenetic relationship between the genus Oncorhynchus and Salvelinus and between the Salmo and Hucho

Kin-related Social Organization in a Winter Population of the Vole Clethrionomys rufocanus

Kinship among gray-sided voles, Clethrionomys rufocanus, was investigated during the winter of 1992/93 in a 3-ha enclosure using both molecular and catch-mark-release techniques. Forty-six adult voles (22 males and 24 females) having high heterozygosities, which were collected from several natural populations, were released into the enclosure on 29 September 1992. Most fall-born individuals of both sexes stayed in their natal site during the non-breeding period (December-March), although reproductively active females dispersed during the fall breeding season (September-November). These philopatric individuals aggregated and formed an maternal family in the winter. Several females which failed to reproduce were solitary in this season. Some individuals which were derived from several families also aggregated into a mixed lineage group. Survival rate of fall-born voles from earlier litters was higher than that from later ones. Maternal families broke up quickly after the onset of spring reproduction. Most females established a territory near the wintering site and made a kin-cluster, in which close relatives neighbored each other. Maternal families in winter bring about female kin-clusters in spring, which may influence reproductive output in the breeding season

Phylogeography of Neptune whelk (Neptunea arthritica) suggests sex-biased impact of tributyltin pollution and overfishing around northern Japan

The Neptune whelk, Neptunea arthritica, is a sublittoral snail from Pacific waters that is a food resource and supports a commercially important fishery in northern Japan. This species showed a severe decline during the 1970s and 1980s, possibly because of overfishing, imposex caused by tributyltin (TBT) pollution and parasite infection. In the present study, we investigated genetic variation among the populations of N. arthritica from eight localities in northern Japan, including Hokkaido and Aomori, using a mitochondrial DNA (mtDNA) marker, a partial sequence of the cytochrome c oxidase subunit I (COI) gene. We also addressed the evolutionary history of N. arthritica and human impact on the population genetic profiles of this species. The parsimony network showed 14 COI haplotypes separated into two groups (Groups A and B), with an intermediate haplotype connecting both groups. Among eight populations, six were fixed for only one or two haplotypes, and no geographic-genetic correlation was found; they were probably affected by random drift. These results contrasted with those from previous microsatellite analysis, which indicated that geographic structure was the result of restricted gene flow between populations. Our results suggested that N. arthritica diverged into Groups A and B during the Pliocene; however, recent TBT pollution and size-selective fishing pressure have reduced genetic diversity and concealed the natural population structure. The present study also suggested that human impact may cause longstanding and possibly irreversible modification of ecosystems, particularly for species forming discrete and relatively small local populations, such as N. arthritica. Thus, the combined use of mtDNA and microsatellite genetic data provides a powerful tool to investigate the health of biodiversity in molluscs

The genetic population structure of lacustrine sockeye salmon, Oncorhynchus nerka, in Japan as the endangered species

Lacustrine sockeye salmon (Oncorhynchus nerka) are listed as an endangered species in Japan despite little genetic information on their population structure. In order to clarify the genetic diversity and structure of Japanese populations for evaluating on the bottleneck effect and an endangered species, we analyzed the ND5 region of mitochondrial DNA (mtDNA) and 45 single nucleotide polymorphisms (SNPs) in 640 lacustrine sockeye salmon in Japan and 80 anadromous sockeye salmon in Iliamna Lake of Alaska. The genetic diversity of the Japanese population in both mtDNA and SNPs was significantly less than that of the Iliamna Lake population. Moreover, all Japanese populations had SNP loci deviating from the HWE. In spite of low genetic diversity, the SNP analyses resulted that the Japanese population was significantly divided into three groups. These suggest that Japanese sockeye salmon populations should be protected as an endangered species and genetically disturbed by the hatchery program and transplantations

Genetic variation among Japanese populations of chum salmon inferred from the nucleotide sequences of the mitochondrial DNA control region

We examined the nucleotide sequences of 500 bp variable portion from the 5' end of mitochondrial (mt) DNA control region in about 500 individuals from 12 populations that were captured in 11 rivers, six in Hokkaido and five in Honshu, Japan. Comparison of the sequences showed 10 variable sites, defining a total of 12 haplotypes in the examined individuals. All the 12 haplotypes occurred in seven Hokkaido populations, whereas only six haplotypes were found in the five Honshu populations. Among these haplotypes, two were common in all the Hokkaido and Honshu populations. The AMOVA analysis inferred a genetic differentiation among three geographic regions, i.e. Hokkaido, Pacific Ocean coast in Honshu, and Japan Sea coast in Honshu. Haplotype diversity was higher in the populations of Hokkaido than those of Honshu, indicating a greater genetic variation in the Hokkaido than the Honshu populations. The estimates of painwise population F_[ST] suggested that the regional differentiation was mostly ascribed to the divergence between populations in Hokkaido and the Pacific coast in Honshu

Induction of viable gynogenetic progeny using eggs and UV-irradiated sperm from the Chinese tetraploid loach, Misgurnus anguillicaudatus

When eggs from the Chinese tetraploid loach that had 100 chromosomes were fertilized with UV-irradiated sperm, we obtained viable gynogenetic progeny without any additional treatment for the duplication of maternal chromosomes, which survived beyond first feeding towards adult stage of development. Gynogenetic progeny were determined to be diploid since they possessed 50 chromosomes, along with two chromosomes bearing nucleolar organizing regions (NORs), detected by silver nitrate staining (Ag-NORs), chromomycin-A3 (CMA3)-positive sites and fluorescence in situ hybridization (FISH) signals for rDNA loci. In contrast, when gynogens were induced using eggs from diploid loach fertilized by UV-irradiated sperm, but without chromosome doubling, we found that all resultant progeny were non-viable haploid gynogens with 25 chromosomes, along with one NOR-bearing chromosome detected by Ag-NORs, CMA3 and FISH. These observations demonstrate the true genetic tetraploid nature of the Chinese loach possessing 100 chromosomes, and the potential use of this tetraploid as a source of functional diploid gametes for further ploidy manipulation experiments

The origin of natural tetraploid loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) inferred from meiotic chromosome configurations.

In the loach, or Oriental weatherfish Misgurnus anguillicaudatus (Teleostei: Cobitidae), diploid (2n = 50) and tetraploid individuals (4n = 100) are often sympatric in central China. The evolutionary mechanism of this tetraploidization was analyzed with the observation of meiotic behavior of chromosomes in both the germinal vesicles of mature oocytes and the primary spermatocytes in diploid and tetraploid loaches. Whereas diploid specimens usually showed 25 bivalents in meiotic cells, tetraploid loaches exhibited 0–6 quadrivalents and 38–50 bivalents in both sexes, with the modal number of quadrivalents as three in females and four in males. In the diploid specimens, the two largest metacentric chromosomes bearing nucleolar organizing regions (NORs) identified by chromomycin A3 staining and fluorescence in situ hybridization with a 5.8S + 28S rDNA probe formed one bivalent with terminal association. In the tetraploids, four NOR-bearing chromosomes never formed a quadrivalent, but were organized into two terminally-associated bivalents. These findings suggest an autotetraploid origin of the natural tetraploid loach and subsequent rediploidization of whole genome. The latter process, however, seems still in progress as inferred from the concurrence of up-to several quadrivalents and the majority of bivalents