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Staphylococcal enterotoxins (SEs) have repeatedly caused food poisoning incidents worldwide. Some of the challenges associated with food poisoning outbreaks are that traditional detection methods are expensive and require long processing times and trained technicians. Microchannel devices represent a potential detection method by which these difficulties can be overcome. In this paper, we propose that immuno-pillar devices may represent a rapid, highly sensitive, and low-cost analytical system for the simultaneous detection of staphylococcal enterotoxin types A, B, and D (SEA, SEB, and SED) in milk. To prepare milk samples simulating food contaminated with SEs, commercial milk was spiked with equal amounts of SEA, SEB, and SED. A quantitative analysis of the milk samples was performed within 15 min by using the microchannel device. The analysis required only 0.5 μL of untreated milk sample. The resultant limit of detection was 15.6 pg mL−1 for each SE, and the total assay time and sensitivity were markedly shorter and higher, respectively, than those for commercially available assay kits. The detection range of each enterotoxin using these devices was estimated as 15.6 pg mL−1 to 100 ng mL−1, which completely covers the SE concentrations that can lead to foodborne diseases based on the U.S. Food and Drug Administration's criterion for the infectious SE dose in SE poisoning (1 μg SE). Using our devices, frequent assessment of food potentially contaminated with SE is possible.

1. The E3 ubiquitin protein ligase 1 (WWP1) gene, the mutation of which causes muscular dystrophy in chickens, is expressed not only in the pectoral muscle, but also in a number of tissues such as the kidney. Therefore, this study examined some parameters related to kidney function in muscular dystrophic (MD) chickens. 2. Plasma osmolality, Na+ and K+ concentrations, aldosterone levels, and the expression of aquaporin (AQP) 2, AQP3, and α subunits of the amiloride-sensitive epithelial sodium channel (αENaC) were analysed in the kidneys of 5-week-old MD chickens and White Leghorn (WL) chickens under physiological conditions or after one day of water deprivation. 3. Plasma osmolality, Na+ concentrations, and plasma aldosterone levels were significantly higher in MD chickens than in WL chickens. αENaC mRNA expression levels were lower in MD chickens than in WL chickens. AQP2 and AQP3 mRNA expression levels were similar in the two strains of chickens. 4. Plasma osmolality correlated with aldosterone levels and AQP2 and αENaC mRNA levels in WL chickens. In MD chickens, plasma osmolality correlated with AQP2 mRNA levels, but not with plasma aldosterone or αENaC mRNA levels. 5. These results suggest that neither water reabsorption nor the expression of AQP2 and AQP3 is impaired in MD chickens and that a WWP1 gene mutation may or may not directly induce an abnormality in Na+-reabsorption in the kidneys of MD chickens, potentially through αENaC.

During early development in vertebrates, pluripotent cells are generated from the neural crest and migrate according to their presumptive fate. In birds and mammals, one of the progeny cells, melanoblasts, generally migrate through a dorsolateral route of the trunk region and differentiate to melanocytes. However, Silky is an exceptional chicken in which numerous melanoblasts travel via a ventral pathway and disperse into internal organs. Finally, these ectopic melanocytes induce heavy dermal and visceral melanization known as Fibromelanosis (Fm). To identify the genetic basis of this phenotype, we confirmed the mode of inheritance of Fm as autosomal dominant and then performed linkage analysis with microsatellite markers and sequence-tagged site markers. Using 85 backcross progeny from crossing Black Minorca chickens (BM-C) with F1 individuals between White Silky (WS) and BM-C Fm was located on 10.2–11.7 Mb of chicken chromosome 20. In addition, we noticed a DNA marker that all Silky chickens and the F1 individuals showed heterozygous genotyping patterns, suggesting gene duplication in the Fm region. By quantitative real-time PCR assay, Silky line-specific gene duplication was detected as an ∼130-kb interval. It contained five genes including endothelin 3 (EDN3), which encoded a potent mitogen for melanoblasts/melanocytes. EDN3 with another three of these duplicated genes in Silky chickens expressed almost twofold of those in BM-C. Present results strongly suggest that the increase of the expression levels resulting from the gene duplication in the Fm region is the trigger of hypermelanization in internal organs of Silky chickens.

The electrophoretic variation in bovine hemoglobin-beta (HBB) is one of the most investigated genetic markers. The presence of a unique HBB variant, HBB(X), in Southeast Asian cattle has been reputed as a sign of gene-flow from wild bovine species. In this study, we analyzed the DNA sequences of HBB genes in domestic and wild bovine species to verify this belief. Isoelectric focusing of HBB chain revealed that the HBB(X) in domestic cattle had dimorphism and was separated into HBB(X1) and HBB(X2). The HBB(X1) had the same DNA sequence of the common HBB variant in gayal (Bos gaurus frontalis), while some of the HBB(X2) were identical with that of Cambodian banteng (Bos javanicus birmanicus). As a result, we confirmed that the bovine HBB variants can be a good indicator of introgression between wild and domestic cattle. The HBB(X1) was always predominant to HBB(X2) in the continental populations, suggesting that the gaur had contributed to the gene pool of domestic cattle in this region much more than the banteng. On the other hand, the mitochondrial DNA analysis could not detect gene-flow from wild species. Autosomal markers that can trace the phylogeny between alleles are suitable for the assessment of bovine interspecific introgression.

In the current study, milk protein variation was examined in cattle (Bos indicus), mithun (Bos frontalis), yak (Bos grunniens) and their hybrid populations in Bhutan to estimate genetic variability, conduct genetic characterization and assess the possibility of gene flow between mithun and cattle. Isoelectric focusing of 372 milk samples from 11 populations detected four molecular types of β-lactoglobulin (A, B, E and M), five molecular types of α(S1) -casein (A, B, C, E and X) and three molecular types of k-casein (A, B and X). Mithun and yak shared alleles but were found to exhibit different allele frequencies for the proteins studied. The degree of genetic variability within populations was measured by average heterozygosity and ranged from 24-40% in cattle, 26% for yak and 33% for mithun. We also resolved the traditional mithun and cattle hybridization system via principal component analysis. Our results suggested secondary introgression of mithun genes to the village Thrabum population, and a close genetic relationship between Bhutanese indigenous cattle and Indian cattle.

Six polymorphic sites of the bovine prion protein gene (PRNP) were genotyped in 569 animals of Asian native cattle, Japanese breeds, purebred mythun (Bos frontalis), and mythun x cattle composite animals. At the 23-bp indel site, a deletion (23-) allele was a major allele in all populations except mythun. At the 12-bp indel site, an insertion (12+) allele was a major allele in all populations. The 14-bp indel site was polymorphic in all Asian native cattle. In the octapeptide repeat region, a six-repeat allele was a major allele in all populations, and 5/5 and 4/6 genotypes were detected in Japanese Black and Mongolian cattle and in mythun, respectively. Two nonsynonymous single nucleotide polymorphisms (SNPs) (K3T and S154N) were detected in Asian native cattle and mythun. Haplotype analysis using the genotypes of the six sites estimated 33 different haplotypes. The haplotype 23- 12- K 6 S 14+ was found in all populations.

The objective of the present study was to conduct the genetic characterization of nine experimental chicken lines based on multilocus microsatellite analysis. Commercial chicken lines were also analyzed in order to compare their levels of genetic uniformity with those of the experimental lines. In six experimental lines, more than 80% of genotyped loci showed fixed allele for all individuals in each line, whereas only 17.5% of genotyped loci were fixed in commercial lines, at the maximum. One of experimental lines (GSN/1) was categorized as a highly inbred line on the basis of all individuals having the same, single allele at every microsatellite locus. Genetic information obtained from the present study should be helpful for the utilization and management of experimental chicken resources.

Polymorphisms in the prion protein gene (PRNP) are known to be associated with transmissible spongiform encephalopathies in human, sheep and goats. There is tentative association between PRNP promoter polymorphism and bovine spongiform encephalopathy (BSE) susceptibility in cattle. In this study, we genotyped for six bovine PRNP polymorphic sites including a 23-bp indel in the promoter, a 12-bp indel in the intron 1, two nonsynonymous single nucleotide polymorphisms (SNPs), octapeptide repeats in the coding region and a 14-bp indel in the 3'-untranslated region in 178 animals representing Japanese Brown, Kuchinoshima feral, Mishima, Japanese Shorthorn and Holstein. In 64 Japanese Brown cattle, three indel sites were polymorphic. All of the six sites were monomorphic in Kuchinoshima. The 23-bp and 12-bp indel sites were polymorphic in Mishima cattle. The 23-bp and 14-bp indel sites were polymorphic in Japanese Shorthorn cattle. Both SNP sites were monomorphic in all cattle examined in this study. At the 23-bp indel site, the genotype frequencies of Japanese Brown and Holstein breeds were similar to that of BSE affected cattle. We estimated 12 different haplotypes from these genotypic data. A '23-12-K6S14+' haplotype was the major haplotype in all populations, whose frequencies ranged from 0.50 to 1.00.

The phylogenetic positions of the Bayanbulak sheep in China and the Sipsu sheep in Bhutan in the northern Asian sheep group were determined on the basis of allele frequency data for five informative and polymorphic loci of blood protein and nonproteins, such as transferrin (TF), arylesterase (ES), hemoglobin-beta (HB-beta), X-protein (XP), and potassium transport (KE), using different electrophoretic and ion-densitometric techniques. Based on Nei's genetic distance, clustering analysis by the UPGMA method showed that the Bayanbulak sheep is clustered in the northern Asian sheep group. Furthermore, the Bayanbulak sheep belongs to a subgroup containing the Khalkhas and Hu sheep of the Mongolian sheep group, which is distinguished from another subgroup of the small-tailed Han, Tan, Tong, and Wadi sheep. The Bayanbulak sheep was closest to the Hu sheep, despite a morphological difference in the fat deposits. In addition to these findings, the Sipsu sheep was verified to belong to the Baruwal sheep.

Four complete amino acid sequences of hemoglobin β chains were determined for the swamp and the river types of the Asiatic water buffalo (Bubalus bubalis) and two species of the subgenus Anoa in Bubalus; B. (A.) depressicornis (H. Smith, 1827), the lowland anoa, and B. (A.) quarlesi (Ouwens, 1910), the mountain anoa. The two types of the bubalis were identical in the 145 amino acid residues of the β chains and, compared to this sequence, the two residues were substituted in the depressicornis (β49Thr Ser and 134Ala Thr) and the five were in the quarlesi (β53Val Ile, 74Met Ile, 111Val Ile, 115Arg His and 134Ala Thr). While both Anoa species diverged from the bubalis by the β134Ala Thr, they differed from each other by the five substitutions. The Anoa species are endemic to Sulawesi of Indonesia. Their speciation and the present coexistence were discussed with reference to probable immigrations of two ancestral Anoa species to Sulawesi at so long interval that had caused a reproductive isolation between the two wild animals. The earlier immigrants were postulated to be ancestral to the quarlesi and the later ones to the depressicornis.

Two bovine haemoglobin beta chains, electrophoretically identical with the beta A chain of Herefords, were obtained from Ongole and Banteng, Bos javanicus, cattle. The amino acid residue differences of the two beta chains were compared by electrophoresis, cation-exchange and reverse-phase chromatography, amino acid analyses, and Edman degradation in comparison with beta A chain. The results showed that two beta chains differed from the beta A chain of the Hereford breed by the substitution of serine with threonine at the beta 43 position. No other difference was found between the two chains and beta A. This new beta chain type was termed beta A Zebu, which forms a possible evolutionarily transitional type between the beta A and the rare variant beta D Zambia found previously in African zebu cattle. The beta A Zebu differentiates from the previous beta B by at least four amino acid substitutions involving five codon-base changes.

Histocompatibility was assessed in the RIR-Y8/NU, BL-E, YL, and WL-G chicken closed colonies by hemagglutination test using anti-red blood cell (RBC) antibodies (HT), skin transplantation test (STT), and formation of isohemagglutinins (FIHs) during STT. The YL individuals all showed the survival of skingrafts for more than 17 days with no FIHs in STT and no RBC antigenic variations in HT, indicating a histocompatible nature together with high homogeneity at serological loci. The BL-E as well as WL-G closed colonies were also found to be histocompatible in the STT with no FIHs, although the HT showed heterogeneities at serological locus/loci other than the B and C blood group loci which have significant effects on histocompatibility or FIHs in chicken. In the RIR-Y8/NU closed colonies, one individual in 6 reciprocal combinations of the STT showed early skingraft rejection with positive FIHs caused by different B locus alleles, and the HT suggested relatively high heterogeneities at the other serological loci too. The closed colonies of YL, BL-E, and WL-G will be useful avian materials for transplantation or related experiments, but RIR-Y8/NU needs further pedigree selection for serological homogeneity.

In this study, we analyzed DNA sequence of mitochondrial DNA (mtDNA) control regions on the 130 native domestic pigs and eight wild boars in the mainland South and South-east Asian countries including Bhutan, Cambodia, Laos, Myanmar, and Vietnam. Forty-four haplotypes were found in the 138 individuals, 41 were in the domestic and four were in wild boars. Only one haplotype was shared by domestic and wild population in Bhutan. In other cases, mtDNA of wild boars did not show close affinity to that of the domestic pigs in the same location, indicating that the native domestic pigs in these countries did not originate in the present habitat. Phylogenetic analyzes of mtDNA haplotypes recapitulated several major clusters identified in other studies, but 11 haplotypes were grouped in a new cluster we named MTSEA. In most cases, more than one lineage group were present in a sampling station, indicating that the present indigenous domestic pigs may have multiple origins. The MTSEA haplotypes were present in relatively high frequencies in domestic pigs in the mountainous area of mainland South-east Asia (Cambodia and Laos), with a few found in Myanmar and Bhutan. The distributions of MTSEA haplotypes are in great conformity with the distribution of present-day Mon-Khmer language and indicated the existence of yet another independent domestication. The D2 haplotypes that distribute high frequency (almost 100%) throughout the Chinese breeds were dominant in Bhutan, Myanmar, and Vietnam. These results suggest an existence of human-mediated dispersal of domestic pigs from north to the south during the historical expansion of Sino-Tibetan and Tai peoples. The D3 haplotypes previously reported in north India were found in sympatric domestic and wild pigs in Bhutan. The D3 haplotype is an important proof of independent domestication event and/or great gene flow between wild and domestic pigs in the foot of Himalaya.

A genetic linkage map for Suncus murinus was previously constructed with 11 marker loci. In this study, we developed 172 new microsatellite and three RFLP markers, and re-constructed a new framework map by combining all markers. The new map comprises 42 markers that are distributed into 12 linkage groups, two of which are assigned to chromosomes, and spans 403.5 cM with an average inter-marker distance of 13.5 cM.

The musk shrew (Suncus murinus) is an insectivore species that inhabits tropical and subtropical Asia widely. To clarify the genetic relationship among wild musk shrew populations, we examined the electrophoretic variants of biparentally inherited genetic markers at 10 loci coding for eight blood proteins/enzymes in a total of 639 animals and compared the results obtained from the mitochondrial DNA data. The principal-component analysis performed using the allele frequency data revealed that the 17 populations could be divided into two major groups, a South Asian group and a Southeast Asian group that includes several island populations bound by Myanmar. The degrees of genetic divergence among populations were higher within the Southeast Asian group than within the South Asian group. This finding was incongruent with the mtDNA diversity. Analysis conducted at the individual level showed that a shrew from the central region in Myanmar that carries a South Asian type of mtDNA showed the electrophoretic variants specific to the Southeast Asian group, suggesting that this region is a contact zone between the two major groups.

The beta 3-adrenergic receptor (ADRB3) is a G protein-coupled receptor that is involved in regulating energy homeostasis. We have studied DNA sequences of porcine ADRB3 to find candidate genetic polymorphisms for economically important growth and performance traits in pigs. Five novel haplotypes derived from the three In/dels and 44 SNPs were identified among domestic pigs and wild boars. Three of them encode non-synonymous amino acid sequences by five missense polymorphisms and a frameshift by a thymine insertion. The amino acid polymorphic sites were distributed as follows: one substitution was in extracellular loop 1, three substitutions were in intracellular loop3 and one substitution and the deletion of two amino acids were at the carboxyl-terminal. There was no polymorphism in the transmembrane domains. In addition, we surveyed the allelic frequency of the thymine insertion that cause frameshift in South-east Asian local pigs, including some commercial breeds and wild boars. This thymine insertion was distributed widely in the domestic pigs and wild boars. The frequencies of this allele were relatively low in Western improved breeds, while they were very common in Asian breeds and wild boars in Asia. This result indicates that this insertion originally occurred in ancient Asian wild boars and then circulated among Asian domestic pigs. This allele also spread over Western breeds, probably through the introgression of Asian pigs into European stocks during the 18th and 19th centuries.

Previous QTL studies have identified 24 QTLs for body weight and growth from 3 to 10 weeks after birth in an intersubspecific backcross mouse population between C57BL/6J and wild Mus musculus castaneus that has 60% of the body size of C57BL/6J. The castaneus allele at the most potent QTL (Pbwg1) on proximal chromosome 2 retards growth. In this study we have developed a congenic strain with a 44.1-Mb interval containing the castaneus allele at Pbwg1 by recurrent backcrossing to C57BL/6J. The congenic mouse developed was characterized by significantly higher body weight gain between 1 and 3 weeks of age and lower weight of white fat pads at 10 weeks of age than C57BL/6J. However, no clear difference in body weight at 1-10 weeks of age was observed between congenic and C57BL/6J strains. QTL analysis with 269 F(2) mice between the two strains did not identify any QTLs for body weight at 1, 3, 6, and 10 weeks of age, but it discovered eight closely linked QTLs affecting body weight gain from 1 to 3 weeks of age, lean body weight, weight of white fat pads, and body length within the Pbwg1 region. The castaneus alleles at all fat pad QTLs reduced the phenotypes, whereas at the remaining growth and body composition QTLs, they increased the trait values. These results illustrate that Pbwg1, which initially appeared to be a single locus, was resolved into several loci with opposite effects on the composition traits of overall body weight. This gives a reason for the loss of the Pbwg1 effect found in the original backcross population.

In order to investigate the genetic diversity of the Bhutanese cattle, complete mitochondrial DNA displacement loop sequences from 30 Bhutanese cattle were determined and analyzed these in conjunction with previously published sequences. Sequence comparisons and phylogenetic analyses revealed the presence of B.taurus mitochondrial DNA haplotypes (13.3%), suggesting the genetic introgression of B.taurus genetic materials into Bhutanese population. The remaining B.indicus haplotypes indicated two clades of mitochondrial haplogroups, I1 and I2. The high frequency of I2 haplotype (46.2 %) was observed in Bhutanese cattle. Mean nucleotide divergence values were calculated within populations and Bhutanese population revealed higher value (0.75 %) than those of India (0.41 %) and China (0.19 %) . The results suggested the high genetic variability in Bhutanese cattle consisting of several haplogroups of mitochondrial DNA.

Objectives: This study aimed to develop a novel type 2 diabetes model designated the HND (Horio–Niki diabetic) mouse, by transferring diabetogenic genes from wild castaneus mice (Mus musculus castaneus) captured in the Philippines into laboratory mice (C57BL/6J:B6). Methods: Offspring from the cross between a wild male and a B6 female were backcrossed to the sire. One male backcross which exhibited fasting hyperglycemia was crossed with a B6 female to comprise the fundamental stock (F0). Thereafter, full-sib mating was performed, and mice with impaired glucose tolerance were selected and bred from the F2 generation. Characterization of the phenotype of HND mice and insulin release from their islets was evaluated with F12 generation males. Results: The male HND mice were lean, and spontaneously exhibited impaired glucose tolerance at a high incidence rate at 6 weeks of age. Their serum insulin levels in response to intraperitoneal glucose were markedly attenuated. However, glucose-induced insulin release from isolated HND islets was not affected. Notably, inhibition of glucose-induced insulin release by epinephrine was more pronounced in HND islets than in B6 islets. Moreover, in vivo treatment of HND mice with the α2-adrenergic receptor agonist clonidine resulted in marked hypoinsulinemic hyperglycemia. Conclusions: We suggest the HND mouse may be a distinctive and useful model for type 2 diabetes with impaired neural control of insulin secretion.

The FGS/Kist strain of mice, a new animal model for focal glomerulosclerosis (FGS) in humans, was previously established by recurrent selection for high proteinuria, which is a principal marker of FGS, from descendants of CBA/Nga and RFM/Nga strains. We performed a genome-wide scan for quantitative trait loci (QTLs) affecting proteinuria in a population of 356 backcross progeny derived from a cross between FGS/Kist and the standard normal strain, C57BL/6J. Five proteinuria QTLs (Ptnu1-5) were detected at the genome-wide 5% or less level. Ptnu1 and Ptnu2, located on Chromosomes (Chrs) 8 and 17, respectively, had main effects on proteinuria and also interacted epistatically with each other. However, Ptnu3 on Chr 9 and Ptnu4 and Ptnu5 both on Chr 15 had epistatic interaction effects only. Except for the epistatic interaction effect of Ptnu4 and Ptnu5, all alleles derived from FGS/Kist were responsible for the high proteinuria. These results indicated that the genetic control of proteinuria is complex and the identified QTLs may provide new insights into the pathogenesis of FGS in mice as well as in humans.

Avian circadian rhythms are regulated by a multiple oscillatory system consisting of the pineal, the suprachiasmatic nucleus (SCN) and the eye. In the present study, ontogeny of circadian clock in the pineal and the SCN of chick embryo was examined using Per2 expression as a marker. A daily rhythmicity of Per2 expression was first detectable at embryonic day (ED) 18 in the pineal and at ED 16 in the SCN under light-dark (LD) cycles. The amplitude of the rhythmicity increased during the development. In contrast, little expression was observed during the development in constant darkness. These results suggest that although circadian clock matures by the end of the embryonic life in chicken, LD cycles are required for the expression of the Per2.

Using nucleotide sequences of the mitochondrial DNA (mtDNA) cytochrome b and SRY genes, we examined the genetic status of two major groups of domestic cattle, the humpless taurine (Bos taurus) and humped zebu (B. indicus), using 10 cattle populations in Asia. Several sequence polymorphisms specific for each major group were found, although the frequency of these polymorphisms varied in each population. Six major mtDNA-SRY composite types were observed. The Mishima, Mongolian, Korean, Chinese Yellow and Sri Lanka cattle populations had a full match between the mtDNA and SRY sequences, specifically the taurine/taurine type or zebu/zebu type. A non-match type (zebu/taurine type) was found at a high frequency in the Bangladesh (83.4%) and Nepal populations (83.3%). Our results suggest that these non-match type populations developed from genetic hybridization of different strains. Also, the domestication history of modern Asian domestic cattle could be explained by male-mediated introgression. Additionally, our results suggest the occurrence of introgression of mtDNA from other Bibos or Poephagus species into native cattle populations. The existence of other mtDNA-SRY composite types, such as the Bali-zebu and yak-zebu types in Indonesia (85.7%) and Nepal (16.7%), respectively, suggests that genetic introgression also occurred from other genera into domestic cattle during the process of domestication.

Six flying fox species, genus Pteropus (four from the Philippines) were investigated using complete cytochrome b gene sequences (1140 bp) to infer their evolutionary relationships. The DNA sequences generated via polymerase chain reaction were analyzed using the neighbor-joining, parsimony, and maximum likelihood methods. We estimated that the first evolutionary event among these Pteropus species occurred approximately 13.90 ± 1.49 MYA. Within this short period of evolutionary time we further hypothesized that the ancestors of the flying foxes found in the Philippines experienced a subsequent diversification forming two clusters in the topology. The first cluster is composed of P. pumilus (Philippine endemic), P. speciosus (restricted in western Mindanao) with P. scapulatus, while the second one comprised P. vampyrus and P. dasymallus species based on the analysis from first and second codon positions. Consistently, all phylogenetic analyses divulged close association of P. dasymallus with P. vampyrus contradicting the previous report categorizing P. dasymallus under subniger species group with P. pumilus, P. speciosus, and P. hypomelanus. The Philippine endemic species (P. pumilus) is closely linked with P. speciosus. The representative samples of P. vampyrus showed a large genetic distance of 1.87%. The large genetic distance between P. dasymallus and P. hypomelanus, P. pumilus and P. speciosus denotes a distinct species group.

Complete cytochrome b gene sequences (1140 base pairs) in species of Megachiroptera were ascertained in order to deduce their phylogenetic relationships, using samples of Cynopterus brachyotis, Eonycteris spelaea, Ptenochirus jagori, Pteropus vampyrus, and Rousettus amplexicaudatuscollected from the islands of Luzon and Mindanao in the Philippines. Genetic divergence between samples of R. amplexicaudatus, E. spelaea, and C. brachyotis was very small. On the other hand, a large genetic distance was detected between species of Megachiroptera. The phylogenetic tree using neighbor-joining, parsimony, and maximum-likelihood methods generated similar topologies, reflecting the evolutionary associations among megachiropteran species. We estimated that Megachiroptera separated from Microchiroptera 50.2 million years ago (MYA), and split further approximately 32.4 MYA, forming three lineages: E. spelaea, R. amplexicaudatus, and P. vampyrus and the P. jagori - C. brachyotis cluster. The third lineage, composed of P. vampyrus and the P. jagori - C. brachyotis cluster, branched out 31.9 MYA. We hypothesize that R. amplexi- caudatus diverged from the three members of the subfamily Pteropodinae examined, and its phylogenetic relationship with E. spelaea remains unclear. Resume : Les sequences completes du gene cytochrome b (1140 paires de base) chez des megachiropteres ont ete etu

The direct R-banding fluorescence in situ hybridization (FISH) method was used to map 18S-28S ribosomal RNA genes and 10 human cDNA clones on the chromosomes of the musk shrew (Suncus murinus). The chromosomal locations of 18S-28S ribosomal RNA genes were examined in the five laboratory lines and wild animals captured in the Philippines and Vietnam, and the genes were found on chromosomes 5, 6, 9, and 13 with geographic variation. The comparative mapping of 10 cDNA clones of human chromosome 1 demonstrated that human chromosome 1 consisted of at least three segments homologous to Suncus chromosomes (chromosomes 7, 10, and 14). This approach with the direct R-banding FISH method is useful for constructing comparative maps between human and insectivore species and for explicating the process of chromosomal rearrangements during the evolution of mammals.

Forty-one cDNA clones of human functional genes were newly mapped to chromosomes of the musk shrew (Suncus murinus, Insectivora) by fluorescence in situ hybridization, and a comparative cytogenetic map of 51 genes, including 10 genes reported in our previous study, was constructed between human (HSA) and musk shrew (SMU) chromosomes. In this comparative map, the 51 genes localized to human autosomes, except HSA 8, 16, and 20, were mapped to 15 shrew autosomes, except SMU 4, 16, 17 and 18. Twelve conserved segments were identified between human and shrew chromosomes, and six segments among the musk shrew, human, and mouse. Our results defined the presence of at least one inversion and several interchromosomal rearrangements that occurred during evolution after the two species diverged from a common ancestor. Localization of three major histocompatibility complex (MHC) genes to shrew chromosome 3 suggested that the MHC genes of the musk shrew are located in a cluster on chromosome 3. The cytogenetic map constructed in this study is the first cytogenetic map with many functional genes in insectivore species. This approach provides clues for clarifying the chromosomal evolution in this order.

In order to construct a chicken (Gallus gallus) cytogenetic map, we isolated 134 genomic DNA clones as new cytogenetic markers from a chicken cosmid DNA library, and mapped these clones to chicken chromosomes by fluorescence in situ hybridization. Forty-five and 89 out of 134 clones were localized to macrochromosomes and microchromosomes, respectively. The 45 clones, which localized to chicken macrochromosomes (Chromosomes 1–8 and the Z chromosome) were used for comparative mapping of Japanese quail (Coturnix japonica). The chromosome locations of the DNA clones and their gene orders in Japanese quail were quite similar to those of chicken, while Japanese quail differed from chicken in chromosomes 1, 2, 4 and 8. We specified the breakpoints of pericentric inversions in chromosomes 1 and 2 by adding mapping data of 13 functional genes using chicken cDNA clones. The presence of a pericentric inversion was also confirmed in chromosome 8. We speculate that more than two rearrangements are contained in the centromeric region of chromosome 4. All 30 clones that mapped to chicken microchromosomes also localized to Japanese quail microchromosomes, suggesting that chromosome homology is highly conserved between chicken and Japanese quail and that few chromosome rearrangements occurred in the evolution of the two species.

We performed a genome-wide quantitative trait locus (QTL) analysis of body weight at 10 weeks of age in a population of 321 intercross offspring from SM/J and A/J mice, progenitor strains of SMXA recombinant inbred strains. Interval mapping revealed two significant QTLs, Bwq3 (body weight QTL3) and Bwq4, on Chromosomes (Chrs) 8 and 18 respectively, and five suggestive QTLs on Chrs 2, 6, 7, 15 and 19. Bwq3 and Bwq4 explained 6% of the phenotypic variance. The SM/J alleles at both QTLs increased body weight, though the SM/J mouse was smaller than the A/J mouse. On the other hand, four of the five suggestive QTLs detected had male-specific effects on body weight and the remainder was female-specific. These suggestive QTLs explained 5-6% of the phenotypic variance and all the SM/J alleles decreased body weight.

We constructed the comparative cytogenetic maps of X chromosomes in three rodent species, Indian spiny mouse (Mus platythrix), Syrian hamster and Chinese hamster, using 26 mouse cDNA clones. Twenty-six, 22 and 22 out of the 26 genes, which were mapped to human, mouse and rat X chromosomes in our previous study, were newly localized to X chromosomes of Indian spiny mouse, and Syrian and Chinese hamsters, respectively. The order of the genes aligned on the long arm of human X chromosome was highly conserved in rat and the three rodent species except mouse. The present results suggest a possibility that the rat X chromosome retains the ancestral form of the rodent X chromosomes.

We constructed comparative cytogenetic maps of the Chinese hamster to mouse, rat and human by fluorescence in-situ hybridization using 36 cDNA clones of mouse, rat, Syrian hamster, Chinese hamster and human functional genes. In this study, 30 out of the 36 genes were newly mapped to Chinese hamster chromosomes. The chromosomal homology of the Chinese hamster was identified and arranged in 19, 19 and 18 segments of conserved synteny in mouse, rat and human, respectively. Additionally, two of the 19 segments homologous to mouse chromosomes were initially identified in this study.

Summary We sequenced the 16S rRNA gene in mitochondrial DNA to characterize mithun located in Bhutan and to increase our understanding of its origin. We compared mithun with yak, European cattle, Bhutanese zebu and Indian zebu. Sequencing revealed low nucleotide diversity within the mithun population and their phylogenetic proximity to gaur. A close relationship between Bhutanese mithun and gaur was confirmed by an additional comparison with wild gaur specimens from three locations in Bhutan. Direct domestication of mithun from gaur was supported, while maternal contribution from the cattle lineage during domestication was not supported.

A genome-wide scan for quantitative trait loci (QTLs) controlling body weight at 10 weeks after birth was carried out in a population of 387 intersubspecific backcross mice derived from a cross between C57BL/6J inbred mice (Mus musculus domesticus) and wild mice (M. m. castaneus) captured in the Philippines, in order to discover novel QTLs from the wild mice that have about 60% lower body weight than C57BL/6J. By interval mapping, we detected four QTLs: a highly significant QTL on Chromosome (Chr) 2, which was common in both sexes; two significant QTLs on Chr 13, one male-specific and the other female-specific; and a suggestive male-specific QTL on X Chr. By composite interval mapping, we confirmed the presence of the three QTLs on Chrs 2 and 13, but not of the male-specific X-linked QTL. The composite interval mapping analysis newly identified three QTLs: a significant male-specific QTL on Chr 11 and two highly significant female-specific QTLs on Chrs 9 and X. Individual QTLs explained 3.8-11.6% of the phenotypic variance, and all the QTL alleles derived from the wild mice decreased body weight. A two-way analysis of variance revealed a significant epistatic interaction between the Chr 2 QTL and the background marker locus D12Mit4 on Chr 12 only in males. The interaction effect unexpectedly increased body weight. The chromosomal region containing the Chr 2 QTL did not coincide with those of growth or fatness QTLs mapped in previous studies. These results suggest that a population of wild mice may play an important role as new sources of valuable QTLs.

A novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica) was isolated from genomic DNA digested with restriction endonuclease, Bg/II. Sequence analysis of three different-size clones revealed the presence of a tandem array of a GC-rich 41 bp repeated element. This sequence was localized by fluorescence in situ hybridization (FISH) primarily to microchromosomes of Japanese quail (2n = 78); approximately 50 of the 66 microchromosomes showed positive signals, although hybridization signals were also detected on chromosomes 4 and W. This satellite DNA did not cross-hybridize with genomic DNA of chicken (Gallus gallus) and Chinese painted quail (Excalfactoria chinensis) under moderately stringent conditions, suggesting that this class of repetitive DNA sequences was species specific and fairly divergent in Galliformes species.

Hereditary, persistent, right oviduct manifested in an inbred line (PNP/DO line) from the Fayoumi breed of chickens was investigated for form of expression and mode of inheritance. Females in the PNP/DO line have varying lengths of elongated right oviducts, besides the normal left ovary, and oviducts that generally possess, irrespective of their total length, regions similar to those normally observed in a left oviduct. Observations of embryos indicated that the existence of right oviduct in this line could be attributed to the slow regression of right Mullerian duct during the embryonic stage, and left-right asymmetry in female genital system is intrinsic in this line. Intracrosses of the PNP/DO line produced 93% of female embryos with persistent right Mullerian ducts at the next generation, and reciprocal crosses of the PNP/DO line and control strains produced 5 and 30% of female embryos with persistent right Mullerian ducts in the F1 and N2 generations, respectively. Mating results suggested that this mutant trait is controlled by two pairs of autosomal recessive genes with major effects and numerous loci that have minor effects. Thus, expression of this trait is due to the interaction of major loci and the background genotype.

The karyotype of the tamaraw (Bubalus mindorensis, 2n = 46) was investigated by RBG-banding technique and compared with those of the river and the swamp cytotypes of domestic water buffalo (B. bubalis). The tamaraw karyotype consisted of 6 submetacentric and 16 acrocentric autosome pairs (NAA = 56), and X and Y chromosomes. The RBG-banded karyotype of the three taxa had a high degree of homology, and the tamaraw karyotype could be explained by a Robertsonian translocation between chromosomes 7 and 15 and by a telomere-centromere tandem fusion between chromosomes 4p and 12 of the standardized river buffalo cytotype (2n = 50, NAA = 58). The buffalo satellite I and II DNAs were localized to the centromeric regions of all the tamaraw chromosomes. The biarmed chromosome 2 of the tamaraw resulting from the fusion between chromosomes 7 and 15 of the standard contained much larger amounts of the satellite I DNA than the other biarmed chromosomes, suggesting that this chromosome was formed by a relatively recent Robertsonian fusion. The (TTAGGG)n telomeric sequence was specifically localized to the telomeric region of all the buffalo chromosomes. The 18S + 28S rDNA was localized to the telomeric regions of the chromosomes 5p, 7, 19, 21, and 22 of the tamaraw and of their homologous chromosomes in the river and swamp buffalo cytotypes.

We discovered two mouse strains (SMXA-5 and SMXA-9) with hyperinsulinemia among the substrains and progenitor strains (SM/J and A/J) of the SMXA recombinant inbred (RI) strains, and characterized the two strains at 20 weeks of age. SMXA-5 (mean +/- S.E.M: 9.6 +/- 1.7 ng/ml) and SMXA-9 (7.7 +/- 1.3 ng/ml) males had higher serum immunoreactive insulin levels than SM/J (1.4 +/- 0.3 ng/ml) and A/J (1.1 +/- 0.1 ng/ml) males in the nonfasting condition. The hypoglycemic response to insulin at 30 min after injection was significantly less in SMXA-5 males than in SM/J mice. Glucose tolerance test revealed that the incidence of impaired glucose tolerant males was 58% (11/19) in SMXA-5 and 42% (10/24) in SMXA-9 strains, but none in SM/J and A/J strains. SMXA-5 (209 +/- 29 mg/dl) and SMXA-9 (235 +/- 31 mg/dl) had higher serum triglyceride levels than SM/J (126 +/- 14 mg/dl) and A/J (89 +/- 5 mg/dl) males in the nonfasting condition. Histologic examination revealed enlarged islets in the pancreas of hyperinsulinemic SMXA-5 male mice. Moreover, SMXA-5 and SMXA-9 mice exhibited mild obesity. SMXA-5 and SMXA-9 males were therefore characterized by hyperinsulinemia, impaired glucose tolerance, hypertriglyceridemia and mild obesity which resembled some of the phenotypes of human Syndrome X, although both progenitor strains were normal so far as we examined. Since the RI strains are a powerful tool to facilitate polygenic-trait analysis, SMXA-5 and SMXA-9 mice will be useful materials to investigate the genetic basis of complex diseases, and are possible new metabolic models in relation to hyperinsulinemia.

In the SMXA recombinant inbred (RI) strains, we measured body weight, blood insulin and lipid (triglyceride, total cholesterol and phospholipid) levels in each strain. In the five traits, mean values of substrains varied remarkably and showed a continuous spectrum of distribution, suggesting control by multiple genes at distinct loci for each trait. We also screened for quantitative trait loci (QTLs) involved in the five traits. Suggestive QTLs for body weight (Chromosomes 1 and 6), insulin (Chromosomes 1, 3, 10 and 17), triglyceride (Chromosomes 4 and 11) and phospholipid (Chromosome 18) levels were detected. The SMXA RI strains are unique tools for analyzing genetic factors that influence body weight, blood insulin and lipids levels.

SM/J and A/J inbred strain of mice have different characteristics in circadian behaviors such as free-running period (τ), phase relationship (ψ) between light-dark cycles and activity rhythms, and amount of wheel-running activity. To determine the genes which affect these behaviors, a quantitative trait locus (QTL) analysis using SMXA recombinant inbred strains derived from SM/J and A/J mice was performed. Concerning τ, two regions on chromosomes (Chrs) 7 and 18 surpassed the genome-wide suggestive level. As for ψ, one suggestive QTL was detected on Chr 7. The QTLs which affect daily activity counts under light-dark cycles and constant darkness were mapped to the same chromosomal regions on Chrs 1 and 17, respectively. The provisional QTLs detected in the present study might be useful for understanding the complex mechanism regulating circadian behaviors.

The complete mitochondrial DNA (mtDNA) control region was cloned and sequenced in the musk shrew, Suncus murinus, Insectivora. The general aspect was similar to that found in other mammals. We have found in two locations of this region the presence of arrays of tandem repeats like those in other shrew species. One array was located in the left domain containing the termination-associated sequences (TAS) and the length of a copy was 77 bp. The other repeats were situated upstream from the recognition site for the end of H-strand replication in the right domain and were 20 bp long. The left halves of the control region containing the former repeats were sequenced and compared in several laboratory lines and wild animals from different localities, variations in copy number of repeated sequences were found both among individuals and within an individual. A comparative study of repeated sequences provides useful indication for the origin and evolution of tandem repeated sequences. Strand slippage and mispairing during replication of mtDNA with concerted manner is currently regarded as a dominant theory to account molecular mechanism for tandemly repeated sequences, and the pattern of sequence and length variation in our study supports this theory. Our results, however, suggest that the evolution of the repeated sequences containing the TAS in the musk shrew might go through the process of two steps; at the first step one complete repeated and several incomplete repeated sequences had reproduced in common ancestor of the shrew, and the second stage step-up of complete repeated sequences occurred with concerted evolution after differentiation into continental and insular groups.

We investigated an odontometrical difference in the mandibular molars (M1, M2, and M3) of two laboratory strains of the musk shrew (Suncus murinus) originating in Bangladesh (BAN strain) and Tokunoshima Island of Japan (TKU strain). We used skulls from two strains of shrews that were maintained under identical laboratory conditions. Mesiodistal and buccolingual crown diameters in the trigonid and talonid of the mandibular molars were measured with a measuring microscope, calibrated to 0.001 mm. The crown proportion was expressed by the crown indices calculated from the measurements. Size reduction was analyzed quantitatively according to the reduction index. All crown dimensions were significantly larger in BAN shrews than in TKU shrews (P < 0.01). Sexual differences were noted in the talonid dimensions, while interstrain differences were clearly evident in the trigonid dimensions. The crown indices in M1 showed the least interstrain difference of the three molars. The crown indices showed that TKU shrews had relatively larger buccolingual diameters and talonid diameters than BAN shrews, and the reduction indices showed that TKU shrews had relatively larger M2 and M3 than BAN shrews. To extract the variance components of tooth shape, a principal component analysis was performed after the variables were standardized. After Varimax rotation, each factor was interpreted. The first three factors accounted for 79.9% of all variances. The first component represented the mesiodistal crown proportion of the trigonid-to-talonid crown component. The second and third components represented the relative size of buccolingual diameters in the distal molars for M1. The principal component scores showed that TKU shrews had relatively larger talonids and distal molars than BAN shrews.

The EDS (early-onset diabetes in suncus) colony has been developed as a new closed breeding colony of the musk shrew (Suncus murinus, Insectivora) exhibiting a high incidence of spontaneous diabetes mellitus. We investigated the characteristic features of diabetic shrews in this colony. All diabetic shrews are characterized by glycosuria (Tes-tape value > or = 3+), hyperglycemia (23.3 +/- 0.8 mmol/l) and polyuria, and they were affected by the age of 3 months. Cumulative incidence (64.1% in males and 27.8% in females) was kept intact after the age of 3 months. The growth pattern of diabetic shrews was similar to that of non-diabetic shrews, and obesity was not consistent in diabetic shrews. The intraperioneal glucose tolerance test revealed both impaired glucose tolerance and impaired insulin secretion in diabetic shrews. Insulin sensitivity of diabetic shrews decreased in the intraperioneal insulin tolerance test. Neither severe hypertrophy nor lymphocytic infiltration was observed in pancreatic islets of diabetic shrews. These facts suggested that diabetic shrews in the EDS colony should be classified as early-onset non-insulin dependent diabetes mellitus (NIDDM) without obesity. Early-onset of severe hyperglycemia with impaired glucose tolerance is a distinctive character compared with other non-obese NIDDM models in rodents. We concluded that the diabetic shrews in the EDS colony are a new animal model of human NIDDM without obesity.

メコンデルタを中心にベトナム南部で多く飼育されている在来アヒルのCoにおいて,血液タンパク質多型を遺伝標識として,主に他のアジア産アヒルとの類縁関係を推測した.ベトナム南部のCan Thoを中心とした5地点で,全部で214羽のCoから採血した.水平式デンプンゲル電気泳動法ならびに水平式ポリアクリルアミドゲル電気泳動法により多型を分析した血液タンパク質は,これまでに多型が見いだされている次の10種類である.血漿エステラーゼ-1(Es-1),血球エステラーゼ-3(Es-3),血球エステラーゼ-4(Es-4),血球エステラーゼ-D2(Es-D2),血漿ポストアルブミン-1(Pa-1),血漿ポストアルブミン-4(Pa-4),血漿ポストアルブミン-1(Ptf-1),血漿ホスフォヘキソースイソメラーゼ(PHI),血球酸性ホスファタ-2(Acp-2),血漿ロイシンアミノペプチダーゼ-2(LAP-2).Coの場合,このうちEs-D2には多型が観察されなかった.Pa-4にはAバンドの陽極側に新たなバンドが観察され,これをバンドCと命名し,共優性遺伝子Pa-4Cにより支配されているものと仮定して扱った.Co全体でPa-4Cの頻度は低かった(0.091).Coにおいて,Acp-2BおよびLAP-2Aの頻度は他のアヒルに比べて高く,Es-1Aの頻度は低かった.集団間の遺伝的距離に基づいてUPG法による枝分かれ図を描いたところ,Coは日本,中国,台湾のアヒルで構成されるクラスターと遺伝的に遠く,またインドネシア在来アヒルならびにカーキキャンベルからなるクラスターとも比較的遠いことが示された.遺伝子頻度の分散共分散行列による主成分分析を行って,その第一から第三までの主成分得点に基づいて三次元の散布図を描いたところ,枝分かれ図と同様の結果が示された.Coは東北アジア産アヒルともインドネシア在来アヒルとも遺伝的に異なっており,それらとは異なるマガモ祖先を持っていることが示唆された.

The mutation designated "open eyelids at birth" arose spontaneously in the BAN strain of the musk shrew (Suncus murinus). This is the first report on this kind of mutation in the shrew. Mating experiments suggested that the mutant character is controlled by an autosomal recessive gene with about 43% penetrance. The symbol oeb(open eyelids at birth) was proposed for the mutant gene. Affected shrews were fully viable and fertile. Phenotypically, the affected shrews were characterized by one or both eyelids open at birth. By 9 days of age, the exposed and dried eyeballs usually dropped off and became missing or sometimes looked microphthalmic. Except for the eye abnormality, the affected shrews showed no obvious external abnormalities. Light-microscopically, the affected shrews with missing eyes or microphthalmia-like eyes did not have complete eye structures, and only remnants of some eye tissue were observed. In the phenotypically normal oeb/oeb shrews with closed eyelids at birth, no histological eye abnormalities were noted. Thus, the mutant character observed may simply be caused by a failure of the eyelids to close.

Immunodetection of methicillin-resistant Staphylococcus aureus (MRSA) by conventional methods employing mammalian immunoglobulins has unknown detection limits, and often yields false-positive results because of the presence of S. aureus protein A, which binds the Fc region of mammalian IgG. In this study, a new PBP2a-specific chicken IgY antibody was developed in inbred and conventional chickens, and used for the detection of MRSA using whole cell lysate samples. Our results showed that this chicken IgY antibody minimized the side effects of protein A. Moreover, enzyme-linked immunosorbent assay and immunochromatography systems were used with a monoclonal and polyclonal anti-PBP2a IgY antibody, clearly differentiating MRSA from methicillin-sensitive S. aureus and other methicillin-sensitive Staphylococcus spp. The detection limit of the immunochromatography was 10(8) colony-forming units; therefore, 1 colony on an agar plate was adequate to distinguish MRSA from non-MRSA. The specificity and sensitivity of this assay were almost similar to that of a commercially available latex agglutination test; however, the procedure used in this study was less complicated. The entire detection procedure, including sample preparation, takes only 20 min and does not require special equipment. Therefore, the use of this IgY antibody as a new tool for the detection of MRSA is highly recommended.

Supraependymal fibers of the house musk shrew (Suncus murinus) were examined by conventional scanning electron microscopy (SEM), backscattered electron (BSE) imaging of enzyme immunohistochemistry and by immunotransmission electron microscopy in the dorsal part of the third ventricular wall. In this region, ependymal cells were not so heavily ciliated and conventional SEM studies showed two main categories of supraependymal fibers. The first type consisted of long fibers fasciculated which were distributed over the ventricular surface between the anterior commissure and the subfornical organ. The second category was a thin fiber which was observed on the ependymal luminal surface. Some of these fibers had varicoses or terminal-like swellings. This type of supraependymal fiber seemed to originate in the first type of fiber bundles. To confirm the nature and the distribution of serotonin-immunoreactive supraependymal fibers, BSE imaging using immunohistochemical reactions was used. Serotonin-immunoreactive structures were shown as highlighted structures by means of a backscattered electron mode. These investigations revealed that the majority of both types of supraependymal fibers observed by conventional SEM contained serotonin. A moderate number of serotonergic supraependymal fibers was observed on the ventricular surface of the subfornical organ. Immunohistochemical studies using Vibratome sections of identical ventricular regions revealed the presence of serotonin-immunoreactive processes, with the use of light- and electron-microscopy. They were distributed in in the third ventricle just adjacent to the ependymal luminal surfaces. These fibers contained immunoreactive large cored vesicles and immunonegative small clear vesicles.

Measurement values of sixteen external characters and karyotypes were described for one female and five males of the adult captive tamaraws, Bubalus (B.) mindorensis endemic to the island of Mindoro. Principal component analyses among bubaline species, using nine or ten of the characters as variables, derived more than 95% of total variance on the first component axis, in which the tamaraw was located almost at the midpoint of two distant bubaline groups : the dwarf water buffaloes of Sulawesi (Anoa depressicornis and A. quarlesi) and the Asiatic water buffaloes (the swamp and the river types of Bubalus bubalis) . All the six tamaraws had 46 chromosomes in diploid form (2N) and 58 as the fundamental number (FN) including the sex chromosomes. In contrast, all the bubaline species of Syncerus, Bubalus and Anoa were reported to have FN=60 with exception of the swamp type of the Asiatic buffalo (FN=58 but 2N 48) . The X chromosome of the tamaraws was the largest acrocentric, and the Y was also acrocentric, probably among the smallest acrocentrics. The karyotype of the tamaraws differs from that of the swamp type buffalo both by one additional pair of metacentrics and the two decreased pairs of acrocentrics in the autosomes.