The influence of climatic oscillations during the Quaternary Era on the genetic structure of Asian black bears in Japan.

The Asian black bear (Ursus thibetanus) inhabits two of the main islands, Honshu and Shikoku, in Japan. To determine how climatic oscillations during the Quaternary Era affected the genetic structure of the black bear populations in Japan, we examined their phylogeographic relationships and compared their genetic structure. We analysed an approximately 700-bp sequence in the D-loop region of mitochondrial DNA collected from 589 bears in this study with 108 bears from a previous study. We observed a total of 57 haplotypes and categorized them into three clusters (Eastern, Western and Southern) based on the spatial distribution of the haplotypes. All but 2 of the 41 haplotypes in the Eastern cluster were distributed locally. Genetic diversity was generally low in northern Japan and high in central Japan. Demographic tests rejected the expansion model in northern populations. Haplotypes of the Western and Southern clusters were unique to local populations. We conclude that the extant genetic structure of the Asian black bear populations arose as follows: first, populations became small and genetic drift decreased genetic diversity in the northern area during the last glacial period, whereas large continuous populations existed in the southern part of central Japan. These patterns were essentially maintained until the present time. In western and southern Japan, the effects of climatic oscillations were smaller, and thus, local structure was maintained.

Bottlenecks, drift and differentiation: the population structure and demographic history of sika deer (Cervus nippon) in the Japanese archipelago.

We assessed genetic differentiation and diversity in 14 populations of sika deer (Cervus nippon) from Japan and four populations of sika deer introduced to the UK, using nine microsatellite loci. We observed extreme levels of differentiation and significant differences in diversity between populations. Our results do not support morphological subspecies designations, but are consistent with previous mitochondrial DNA analyses which suggest the existence of two genetically distinct lineages of sika deer in Japan. The source of sika introduced to the UK was identified as Kyushu. The underlying structure of Japanese populations probably derives from drift in separate glacial refugia and male dispersal limited by distance. This structure has been perturbed by bottlenecks and habitat fragmentation, resulting from human activity from the mid-nineteenth century. Most current genetic differentiation and differences in diversity among populations probably result from recent drift. Coalescent model analysis suggests sika on each of the main Japanese islands have experienced different recent population histories. Hokkaido, which has large areas of continuous habitat, has maintained high levels of gene flow. In Honshu the population is highly fragmented and is likely to have been evolving by drift alone. In Kyushu there has been a balance between gene flow and drift but all the populations have experienced high levels of drift. Habitat fragment size was not significantly associated with genetic diversity in populations but there was a significant correlation between habitat fragment size and effective population size.

Genetic Variations Revealed by Microsatellite Markers ina Small Population of the Sika Deer (Cervus nippon)on Kinkazan Island, Northern Japan.

Genetic variations within a population of the Japanese sika deer, Cervus nippon, on Kinkazan Island were studied by microsatellite analysis. Seventeen pairs of polymerase chain reaction primers designed for several species of ungulates successfully amplified polymorphic microsatellite DNA in sika deer. About 20% of the Kinkazan population was sampled and genotyped for nine diagnostic microsatellite loci. Alleles at those loci in the Kinkazan population were found to be under the Hardy-Weinberg equilibrium. To determine whether the Kinkazan deer have a reduced level of genetic variability, an average heterozygosity in the population was calculated and compared with the values determined for other populations from Hyogo, Yamaguchi, Shimane, Tsushima, and Nagasaki. Neither the observed nor the expected heterozygosity in the Kinkazan deer significantly differed from that in the other populations. Our result indicated that, despite its small population size, the Kinkazan deer preserve extensive microsatellite variations.

Pedigree Analysis of the Sika Deer (Cervus nippon) using Microsatellite Markers.

The usefulness of microsatellite markers in pedigree analysis of the sika deer (Cervus nippon) was tested in a herd in which the maternal lineages were recorded. Eighteen sets of microsatellite primers originally designed for bovine, ovine, and cervine loci successfully amplified polymorphic DNA in the deer. The numbers of alleles per locus ranged from two to seven, and the observed heterozygosity ranged from 0.350 to 0.900. The resolution power of the markers in paternity testing was then determined by calculating exclusion probabilities and paternity indices. Parentages of the study population were efficiently discriminated by genotyping 17 microsatellite loci. The microsatellite data were also used to calculate the genetic relatedness between individuals, which significantly correlated with coancestry coefficients for the pairs. Our results demonstrate that the microsatellite markers are efficient tools in studying the social structure and behavior of the sika deer, as well as in monitoring the inbreeding status.

Partial nuclear localization of a bovine phosphoprotein, BCNT, that includes a region derived from a LINE repetitive sequence in Ruminantia.

BCNT, named after Bucentaur, is a protein that contains a 324-amino-acid region derived from part of a long interspersed DNA sequence element (LINE) in Ruminantia. However, the unique portion is completely missing in human and mouse BCNTs. Since no significant information on their function has been obtained by homology search, we at first examined cellular localization and biochemical characteristics of bovine BCNT to get a hint on its function. Subcellular fractionation and immunohistochemical analyses using a normal bovine epithelial cell line and bovine brain revealed that a significant amount of bovine BCNT is localized in the nuclei, while the major portion is present in the cytosol. Furthermore, it was shown that bovine BCNT is a phosphoprotein and that both bovine and human BCNTs are phosphorylated by casein kinase II in vitro. These results show that BCNTs consist of a unique family, probably a substrate of casein kinase II, which may contribute further to the understanding of gene evolution.

Two genetically distinct lineages of the sika deer, Cervus nippon, in Japanese islands: comparison of mitochondrial D-loop region sequences.

To investigate genetic diversity among populations of the sika deer, Cervus nippon, nucleotide sequences (705-824 bases) of the mitochondrial D-loop regions were determined in animals from 13 localities in the Japanese islands. Phylogenetic trees constructed by the sequences indicated that the Japanese sika deer is separated into two distinct lineages: the northern Japan group (the Hokkaido island and most of the Honshu mainland) and the southern Japan group (a part of the southern Honshu mainland, the Kyushu island, and small islands around the Kyushu island). All sika deer examined in this study shared four to seven units of repetitive sequences (37 to 40 bases each) within the D-loop sequences. The number of tandem repeats was different among the populations, and it was specific to each population. Six or seven repeats occurred in populations of the northern Japan group, while four or five repeats occurred in populations of the southern Japan group. Each repeat unit included several nucleotide substitutions, compared with others, and 26 types were identified from 31 animals. Sequences of the first, second, and third units in arrays were clearly different between the northern and the southern groups. Based on these D-loop data, colonization and separation of the sika deer populations in the Japanese islands were estimated to have occurred less than 0.5 million years before present. Our results provide an invaluable insight into better understanding the evolutionary history, phylogeny, taxonomy, and population genetics of the sika deer.

Assessment of genetic variations within populations of Sika deer in Japan by analysis of randomly amplified polymorphic DNA (RAPD).

Random amplified polymorphic DNA (RAPD) was exploited as a genetic marker to assess the level of genetic variation in populations of Sika deer, Cervus nippon, in Japan. DNA samples were collected from three local populations in Japan, namely, Kinkazan, Goyozan and Ashoro populations. Four arbitrary primers, when used individually, amplified an average of five RAPD fragments in the polymerase chain reaction (PCR). The number of polymorphic bands was scored to calculate band-sharing coefficients within populations. Average band-sharing coefficients revealed a higher degree of homogeneity in the Kinkazan population. Samples collected from larger populations, namely, Ashoro and Goyozan, revealed greater polymorphism than samples from the Kinkazan deer. Our data suggest that RAPD is useful as a marker for detecting genetic variations in populations of Sika deer with reduced levels of genetic diversity.

Mitochondrial DNA polymorphism in subspecies of the Japanese Sika deer, Cervus nippon.

Variations in the mitochondrial DNA (mtDNA) of Sika deer (Cervus nippon) from seven localities in Japan were examined to assess the degree of genetic differentiation among subspecies. The entire mtDNA of C. n. centralis, which is about 16,600 base pairs in length, was cloned into plasmids. Using various clones as hybridization probes, we constructed restriction maps of mtDNA for 59 deer and identified six different mtDNA genotypes. The genotypes observed in the subspecies centralis from Honshu Island (the mainland) were distinct from that of the same subspecies from the Tsushima Islands. By contrast, the subspecies yesoensis, which is distributed on Hokkaido Island, shared at least two mtDNA genotypes with the mainland centralis. Analysis of genetic distances showed that Sika deer on the Japanese islands consist of two distinct clusters, namely, the Hokkaido-Honshu (yesoensis-mainland centralis) and the Yakushima-Tsushima (yakushimae-Tsushima centralis) populations. The molecular phylogenetic data obtained for these subspecies do not reflect the present taxonomic status of these subspecies that is based on comparative morphometry. Genetic differentiation of Sika deer in the Japanese islands is a possible example of vicariance.

Overproduction and translational regulation of rp49 ribosomal protein mRNA in transgenic Drosophila carrying extra copies of the gene.

During Drosophila early development the translation of ribosomal protein mRNAs is regulated specifically and coordinately. In this study we assayed for changes of ribosomal protein rp49 gene expression in flies transformed with extra copies of the gene. RNA blot analysis revealed that the rp49 transcript was overrepresented in most of the transformed fly lines: flies carrying three times more genes than the wild type contained up to seven times more mRNA. The abundance of other ribosomal protein mRNAs was not affected. Despite the large differences in rp49 mRNA content, the proportion of the rp49 mRNA which was associated with polysomes during oogenesis and early embryogenesis did not differ significantly from the wild type, implying that rp49 protein is overproduced in the transgenic flies. The results indicate that the basis for coordinate r-protein gene expression lies in the intrinsic properties of r-protein genes, rather than in a dynamic system that separately modulates the expression of individual genes.

Levels of tyrosinase and its mRNA in coat-color mutants of C57BL/10J congenic mice: effects of genic substitution at the agouti, brown, albino, dilute, and pink-eyed dilution loci.

The levels of tyrosinase activities, tyrosinase cross-reacting material (TY-CRM), and tyrosinase mRNA were determined for skins from five congenic strains of mice (Mus musculus) with different coat colors. The dopa oxidase activity and melanin formation activity were directly proportional to the abundance of TY-CRM in all of the mutants. The levels of the enzyme activities and TY-CRM were increased in brown (b/b) and dilute (d/d) mice, while they were reduced in agouti (A/A) and pink-eyed dilution (p/p) animals. In albino (c/c) mice, the tyrosinase activities and melanin formation activity were scarcely detectable, and no TY-CRM was observed. Contrary to these, the levels of tyrosinase mRNA in agouti and albino mutants were almost the same as that in black mice, and those in brown and dilution mutants were 20-30% higher than that in black mice. These results indicate that the mutations at the brown and dilute loci exert their influence on the tyrosinase mRNA level, presumably by affecting the transcription of the tyrosinase gene, and that the mutations at the agouti and albino loci exert their influence on the TY-CRM level by regulating translation of tyrosinase mRNA and/or post-translation modification of the enzyme.

Effects of the lethal yellow (Ay) and recessive yellow (e) genes on the population of epidermal melanocytes in newborn mice.

Very few melanocytes can be detected by the DOPA reaction in the dorsal epidermis of newborn lethal yellow mice (Ay/a). Nevertheless, the epidermis contains a considerable number of melanoblasts (cells positive for the combined DOPA-premelanin reaction). On the other hand, numerous melanocytes as well as melanoblasts are found in the dorsal epidermis of black mice (a/a). The number of epidermal melanoblasts is smaller in (Ay/a than in a/a mice even though the same number of melanocytes is found in the dermis of these animals. It seems probable that the product of the A y gene suppresses either the differentiation or the proliferation of epidermal melanoblasts. The number of melanoblasts plus melanocytes in day-17 embryos from a cross between Ay/a and a/a mice shows a bimodal distribution. It seems possible that half of the embryos were Ay/a and possessed a reduced number of melanoblasts and melanocytes. This result seems to suggest that the Ay gene is active at this embryonic stage. In contrast to the case for the epidermis from Ay/a mice, numerous DOPA-positive melanocytes were detected in the epidermis from e/e mice. However, the total number of melanoblasts plus melanocytes in e/e epidermis did not differ from that in Ay/a epidermis, suggesting that the mode of action of the e gene in the epidermis is different from that of the Ay gene.

Immunological quantitation of tyrosinase from wild-type and albino mutant mice.

The relationship between gene dosage, enzyme activity, and level of immunologically cross-reacting material (CRM) was examined in mammalian tyrosinase (EC 1.14.18.1) by rocket immunoelectrophoresis. Skin extracts from mice heterozygous (C/c) and homozygous (c/c) for the albino locus contain 46% and 0% of CRM, respectively, as compared with wild-type (C/C) animals. Enzyme activity and CRM level were directly proportional in these genotypes, suggesting that the albino locus controls the quantity of tyrosinase produced in melanocytes.

Action of the e locus of mice in the response of phaeomelanic hair follicles to alpha-melanocyte-stimulating hormone in vitro.

Dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic AMP) induced eumelanin synthesis in hair bulb melanocytes of recessive yellow (e/e) mice in vitro, whereas alpha-melanocyte-stimulating hormone (alpha-MSH) did not. In contrast, the melanocytes of lethal yellow (Ay/a) mice produced eumelanin in response to both dibutyryl cyclic AMP and alpha-MSH. These results suggest that the e locus controls a mechanism that determines the function of an alpha-MSH receptor.