A molecular mechanism for copper transportation to tyrosinase that is assisted by a metallochaperone, caddie protein

The Cu(II)-soaked crystal structure of tyrosinase that is present in a complex with a protein, designated “caddie,” which we previously determined, possesses two copper ions at its catalytic center. We had identified two copper-binding sites in the caddie protein and speculated that copper bound to caddie may be transported to the tyrosinase catalytic center. In our present study, at a 1.16–1.58 Å resolution, we determined the crystal structures of tyrosinase complexed with caddie prepared by altering the soaking time of the copper ion and the structures of tyrosinase complexed with different caddie mutants that display little or no capacity to activate tyrosinase. Based on these structures, we propose a molecular mechanism by which two copper ions are transported to the tyrosinase catalytic center with the assistance of caddie acting as a metallochaperone.     

Range expansion and lineage admixture of the Japanese evergreen tree Machilus thunbergii in central Japan

We investigated the range expansion histories of Machilus thunbergii populations in the Kinki region of central Japan on the basis of nuclear microsatellite data. In the Kinki region, M. thunbergii is typically found in the coastal area, with some fragmented populations inland, around Lake Biwa. Phylogenetic and Bayesian clustering analysis (STRUCTURE analysis) revealed that the inland populations have different genetic components between the west and east sides of Lake Biwa. The population located on the north side of the lake has an admixture of the two genetically differentiated lineages, contributing to an increase in the genetic diversity of the population. Populations around Lake Biwa had lost rare alleles and the F value obtained from STRUCTURE analysis was lower in the coastal populations than in the lake populations. These results suggest that populations around Lake Biwa experienced a bottleneck due to a founder effect during the initial migration to the lake and that glacial refugia of M. thunbergii in the Kinki region existed along the coast.     

Cheek-pouch dispersal of seeds by Japanese monkeys (Macaca fuscata yakui) on Yakushima Island, Japan

Seed dispersal by Japanese monkeys (Macaca fuscata yakui) via cheek-pouch was studied in a warm temperate evergreen forest on Yakushima Island. Plant list was compiled based on a study during 1986–1995, of which troops of monkeys have been habituated without artificial feeding. We followed the well-habituated monkeys in 1993 and 1994 to observe the feeding behavior and their treatments of fruits and seeds, and collected seeds dispersed by monkeys to record the distance carried from the mother trees. We checked the difference of germination ratio between seeds dispersed via cheek-pouch and seeds taken from mother trees by sowing experiments. Seeds and acorns of 22 species were observed to be dispersed via cheek-pouch of monkeys. Among them, three species with acorns were never dispersed via feces, and 15 species with drupes were seldom dispersed via feces. Plant species of which seeds are dispersed only via cheek-pouch had larger seeds than those of dispersed both via cheek-pouch and via feces, and typically had only one or two seeds in a fruit. As for one of cheek-pouch dispersal species,Persea thunbergii, the mean distance when seeds were carried from the mother trees via cheek-pouch was 19.7 m, and the maximum distance was as long as 105 m although more than 80% of seeds were dispersed within 30 m from mother trees. And 82% of seeds dispersed via cheek-pouch germinated. The easy separation of seeds from other parts of the fruit seems to facilitate cheek-pouch dispersal more than dispersal via feces. Cheek-pouch dispersal by monkeys has possibly enhanced the natural selection for larger seeds which bring forth larger seedlings with high shade-tolerance. In conclusion, cheek-pouch dispersal by monkeys is quite an important mode for trees in the mature stand in a warm temperate evergreen forest on Yakushima Island.     

Skewed male reproductive success and pollen transfer in a small fragmented population of the heterodichogamous tree <Emphasis Type="Italic">Machilus thunbergii</Emphasis>

Heterodichogamy is defined as the presence of two flower morphs that exhibit the male and female functions at different times among individuals within a population. Heterodichogamy is regarded as an adaptation to promote outcrossing through enhanced inter-morph mating, together with a 1:1 morph ratio. However, in highly fragmented populations, the morph ratio may be more likely to be biased by stochastic events. In such a situation, individuals of a minority morph within a population are expected to have higher reproductive success than those of a majority morph, which may suffer from pollen shortages of the minority morph. In this paper, we evaluated mating patterns and male reproductive success in a highly fragmented population of Machilus thunbergii, a putative heterodichogamous evergreen laurel tree. Results of paternity analysis indicated that the selfing rate was not clearly different between the two morphs. In contrast, the proportion of intra-morph mating was higher in the majority-morph (MM) mother trees than in the minority-morph (MF) mother trees. Bayesian estimated male reproductive success indicated that male reproductive success was higher in minority-morph (MF) than in majority-morph (MM) mother trees. These findings indicate that (1) the majority morph mothers, suffering a shortage of the opposite morph pollen, could partly compensate for the reduced reproductive success by intra-morph mating rather than by selfing, and (2) negative-frequency dependent selection may be involved in the maintenance of the two morphs.     

Distribution of the CD4 Alleles in Sus scrofa Demonstrates the Genetic Profiles of Western Breeds and Miniature Pigs

Widely used antipig CD4 monoclonal antibodies (mAbs) fail to recognize CD4 alleles characteristic of miniature pig lines such as the National Institutes of Health (NIH) miniature pigs and microminipigs. We surveyed polymorphisms in the coding sequence of the porcine CD4 gene among Western and Oriental pig breeds and Japanese wild boars and investigated their distribution. Of the 13 alleles that we identified among the 47 animals, 2 in group I and 3 in group II were found exclusively in Western breed pigs. Group IV alleles, which included mAb-nonbinding alleles, were found frequently in Oriental breed pigs, suggesting that the mAb-nonbinding allele arose from the gene pool of Oriental pigs. Group IV alleles were also found in Duroc and Large White pigs, suggesting genetic inflow from Oriental pig breeds into Western breeds. Comparison of the CD4 sequences of species in Cetartiodactyla suggested that the group IV alleles in Sus scrofa occurred before the divergence of this species from the other artiodactyls. The different antibody specificities of the various CD4 alleles may facilitate the discrimination of T-cell populations in transplantation studies using miniature pigs. The significance of the preservation of CD4 polymorphisms to immune function in pigs warrants further investigation.     

Geographic population structure and sequence divergence in the mitochondrial DNA control region of the Japanese wild boar (Sus scrofa leucomystax), with reference to those of domestic pigs

Mitochondrial DNA (mtDNA) control regions from 40 Japanese wild boars were examined by direct sequencing after amplification by PCR. From the DNA sequences obtained, we found eight haplotypes, whose differences arose via transitions. The geographical distribution of these different haplotypes indicated that wild boar populations inhabited limited areas and that there was some restricted gene flow between local populations. Eight mtDNA haplotypes from Eastern and Western domestic pigs and the Ryukyu wild boar were also analyzed as references to those from Japanese wild boars. The cluster analyses of the control-region sequences showed that those from Japanese wild boards belong to the Asian type as do those from Eastern domestic pigs and the Ryukyu wild boar, which differed from the European type (Western domestic pigs).     

Prediction of the Coding Sequences of Unidentified Human Genes. III. The Coding Sequences of 40 New Genes (KIAA0081-KIAA0120) Deduced by Analysis of cDNA Clones from Human Cell Line KG-1

We isolated full-length cDNA clones from size-fractionated cDNAlibraries of human immature myeloid cell line KG-1, and thecoding sequences of 40 genes were newly predicted. A computersearch of the GenBank/EMBL databases indicated that the sequencesof 14 genes were unrelated to any reported genes, while theremaining 26 genes carried some sequences with similaritiesto known genes. Significant transmembrane domains were identifiedin 17 genes, and protein motifs that matched those in the PROSITEmotif database were identified in 11 genes. Northern hybridizationanalysis with 18 different cells and tissues demonstrated that10 genes were apparently expressed in a cell-specific or tissue-specificmanner. Among the genes predicted, half were isolated from themedium-sized cDNA library and the other half from the small-sizedcDNA library, and their average sizes were 4 kb and 1.4 kb,respectively. As judged by Northern hybridization profiles,small-sized cDNAs appeared to be expressed more ubiquitouslyand abundantly in various tissues, compared with that of medium-sizedcDNAs.     

Assignment of Human Membrane-type Matrix Metalloproteinase-2 (MT2-MMP) Gene to 16q12 by FISH and PCR-based Human/Rodent Cell Hybrid Mapping Panel Analysis

A new group of matrixmetalloproteinase with a potential membranedomain was reported as membranetype matrixmetalloproteinases(MT-MMPs), and the gene coding for one of them, MT2-MMP, wasrecently cloned from a human lung cDNA library. To predict itsphysiological functions by the relation to the genetic disordersmapped on the chromosomes, the chromosomal location of the humanMT2-MMP gene was examined by fluorescence in situ hybridization(FISH) and PCR-based analysis with human/rodent hybrid cellmapping panels, and it was assigned to 16q12.     

Genetic variation and differentiation of the two river sculpins,Cottus nozawae andC. amblystomopsis,deduced from allozyme and restriction enzyme-digested mtDNA fragment length polymorphism analyses

Genetic differentiation of the two sibling species,Cottus nozawae andC. amblystomopsis, from the northern part of Japan (Hokkaido Island and the Tohoku District) was investigated using allozyme variations and restriction fragment length polymorphisms of mitochondrial DNA. Although the two species are morphologically very similar, previously being thought to be a single species, they have different life-cycles;C. nozawae has a fluvial life-cycle with a small number of large-sized eggs, whereasC. amblystomopsis is an amphidromous species with a large number of small-sized eggs. Four populations ofC. amblystomopsis from Hokkaido Island and 24 populations ofC. nozawae (22 from Hokkaido Island and 2 from the Tohoku District) were sampled and examined Intrapopulational differentiation in the two species was measured by examining several indexes, including proportion of polymorphic loci (P), mean heterozygosity (H) and nucleotide diversity (π). All measurements were higher in theC. amblystomopsis populations, suggesting that intrapopulational variation inC. nozawae was less than inC. amblystomopsis and reflecting the difference in effective population sizes between them. Cluster analyses were performed using the UPGMA method, based on the data matrices of genetic distance (D) and the net nucleotide difference (δ) between populations. TheC. nozawae andC. amblystomopsis populations from Hokkaido Island composed a large cluster (Hokkaido group), while theC. nozawae populations from the Tohoku District composed a different cluster (Tohoku group). Bootstrap probabilities deduced from 1000 bootstrap replications for presence or absence of restriction sites showed that the mtDNA haplotypes detected within the Tohoku Group occurred in 99.9% of the bootstrap replicates outside the mtDNA haplotypes of the Hokkaido group, while those within the Hokkaido group occurred in 3.5–64.9% of bootstrap replicates. Consequently, the Hokkaido populations of the two species (Hokkaido group) were genetically close to each other, whileC. nozawae from the Tohoku District (Tohoku group) were distant from the Hokkaido group. These results suggest that the ancestral populations of the two species on Hokkaido Island shared the same gene pool, even after becoming geographically isolated from the ancestral population ofC. nozawae in the Tohoku District by the formation of the Tsugaru Straits.     

Comparative Genome Mapping of the Ataxia–Telangiectasia Region in Mouse, Rat, and Syrian Hamster

Chromosomal locations of theAtm(ataxia–telangiectasia (AT)-mutated) andAcat1(mitochondrial acetoacetyl-CoA thiolase) genes in mouse, rat, and Syrian hamster were determined by direct R-banding FISH. Both genes were colocalized to the C-D band of mouse chromosome 9, the proximal end of q24.1 of rat chromosome 8, and qa4–qa5 of Syrian hamster chromosome 12. The regions in the mouse and rat were homologous to human chromosome 11q. Fine genetic linkage mapping of the mouse AT region was performed using the interspecific backcross mice.Atm, Acat1,andNpat,which is a new gene isolated from the AT region, and 12 flanking microsatellite DNA markers were examined. No recombinations were found among theAtm, Npat, Acat1,andD9Mit6loci, and these loci were mapped 2.0 cM distal toD9Mit99and 1.3 cM proximal toD9Mit102.Comparison of the linkage map of mouse chromosome 9 (MMU9) and that of human chromosome 11 (HSA11) indicates that there is a chromosomal rearrangement due to an inversion betweenEts1andAtm–Npat–Acat1and that the inversion of MMU9 originated from the chromosomal breakage at the boundary betweenGria4andAtm–Npat–Acat1on HSA11. This type of inversion appeared to be conserved in the three rodent species, mouse, rat, and Syrian hamster, using additional comparative mapping data with theRckgene.