土田 修一

The Japanese murrelet (Synthliboramphus wumizusume) is an endangered small seabird species in Japan. Molecular sexing using PCR targeting of the gene encoding chromodomain helicase DNA-binding protein 1(CHD1) has been used for sex identification. Specifically, PCR using any of three commonly used primer sets (CHD1F/1R, 2550F/2718R and P2/P8) has permitted sexing in many bird species. CHD1F/1R and 2550F/2718R permitted molecular sexing in Japanese murrelet; however, P2/P8 did not permit. To generate a primer pair that permits efficient molecular sexing in this species, a new primer set, CHD1F1/1R1, was prepared to permit amplification of smaller products from degraded DNA samples. The electrophoretic patterns of PCR products amplified with the new primer set were easily classified as female or male. Additionally, the PCR product indicated the presence of a polymorphism in the fragment from chromosome W. The PCR fragments of long-type (WL) and short-type (WS) polymorphisms were observed only in females. When the distribution of the CHD1 gene on chromosome W of 61 female Japanese murrelet on Biroujima Island in Miyazaki Prefecture, WL and WS were observed in 90.2% and 9.8%. The DNA polymorphism is derived from the number of copies of a 32-bp-repeat unit, with WL and WS corresponding to two and one 32-bp-repeats, respectively.

OBJECTIVES: Following the massive earthquake that struck eastern Japan on March 11, 2011, a large amount of radioactive material was released into the environment from the damaged reactor of the Fukushima Daiichi Nuclear Power Plant (FDNPP). After the FDNPP accident, radiocaesium was first detected in muscle samples from wild Japanese monkeys exposed to radioactive materials, and haematologic effects, changes in head size, and delayed body weight gain were also reported, but little is known about the distribution of 137Cs in the organs and tissues of wild Japanese monkeys. RESULTS: We detected the 137Cs in various organ and tissue samples of 10 wild Japanese monkeys inhabiting the forested areas of Fukushima City that were captured between July and August 2012. Among muscle, brain, heart, kidney, liver, lung, and spleen, muscle exhibited the highest and the brain the lowest 137Cs concentration. The concentration (mean ± SD) of 137Cs in muscle, brain, heart, kidney, liver, lung, and spleen was 77 ± 66, 26 ± 22, 41 ± 35, 49 ± 41, 41 ± 38, 53 ± 41, and 53 ± 51 Bq/kg, respectively. These results can help us understand the biological effects of long-term internal radiation exposure in non-human primates.

Cat's AB blood group system (blood types A, B, and AB) is of major importance in feline transfusion medicine. Type A and type B antigens are Neu5Gc and Neu5Ac, respectively, and the enzyme CMAH participating in the synthesis of Neu5Gc from Neu5Ac is associated with this cat blood group system. Rare type AB erythrocytes express both Neu5Gc and Neu5Ac. Cat serum contains naturally occurring antibodies against antigens occurring in the other blood types. To understand the molecular genetic basis of this blood group system, we investigated the distribution of AB blood group antigens, CMAH gene structure, mutation, diplotypes, and haplotypes of the cat CMAH genes. Blood-typing revealed that 734 of the cats analyzed type A (95.1%), 38 cats were type B (4.9%), and none were type AB. A family of three Ragdoll cats including two type AB cats and one type A was also used in this study. CMAH sequence analyses showed that the CMAH protein was generated from two mRNA isoforms differing in exon 1. Analyses of the nucleotide sequences of the 16 exons including the coding region of CMAH examined in the 34 type B cats and in the family of type AB cats carried the CMAH variants, and revealed multiple novel diplotypes comprising several polymorphisms. Haplotype inference, which was focused on non-synonymous SNPs revealed that eight haplotypes carried one to four mutations in CMAH, and all cats with type B (n = 34) and AB (n = 2) blood carried two alleles derived from the mutated CMAH gene. These results suggested that double haploids selected from multiple recessive alleles in the cat CMAH loci were highly associated with the expression of the Neu5Ac on erythrocyte membrane in types B and AB of the feline AB blood group system.

Telomere shortening occurs when cells divide, both in vitro and in vivo. On the other hand, telomerase is able to maintain telomere length in cells by adding TTAGGG repeats to the ends of telomeres. However, the interrelationships existing among telomere length, telomerase activity and growth in vertebrates remain to be clarified. In the present study we measured telomere length (terminal restriction fragment length), telomerase activity and body growth of Oryzias latipes from the embryo stage until senescence. During the rapid growth stage (age 0-7 months), telomeres shortened in parallel with decreasing telomerase activity. Then, during adolescence (age 7 months - 1 year), telomeres lengthened quickly as growth slowed and telomerase activity increased. In the adult stage (age 1-4 years) characterized by little growth, telomerase activity decreased gradually and telomeres shortened. Our data indicate that telomere attrition and restoration are linked to growth and telomerase activity, and suggest that critical loss of telomere homeostasis is associated with mortality in this animal.

In April 2012 we carried out a 1-year hematological study on a population of wild Japanese monkeys inhabiting the forest area of Fukushima City. This area is located 70 km from the Fukushima Daiichi Nuclear Power Plant (NPP), which released a large amount of radioactive material into the environment following the Great East Japan Earthquake of 2011. For comparison, we examined monkeys inhabiting the Shimokita Peninsula in Aomori Prefecture, located approximately 400 km from the NPP. Total muscle cesium concentration in Fukushima monkeys was in the range of 78-1778 Bq/kg, whereas the level of cesium was below the detection limit in all Shimokita monkeys. Compared with Shimokita monkeys, Fukushima monkeys had significantly low white and red blood cell counts, hemoglobin, and hematocrit, and the white blood cell count in immature monkeys showed a significant negative correlation with muscle cesium concentration. These results suggest that the exposure to some form of radioactive material contributed to hematological changes in Fukushima monkeys.

【目的】ヒトにおけるShort tandem repeat（STR）多型は、親子鑑定・個体識別・人類遺伝学的解析など幅広く利用されている。さらに近年、性染色体特異的なSTRであるX‐STR、Y‐STRが開発され法科学的解析に重要な手法となっている。一方、動物においても、STR多型は個体識別・血統登録・品種分化などの手段として研究されているが、性染色体STRマーカーの開発はごく一部の種に留まっている。ニホンザルにおけるY-STR多型の開発は、ニホンザルの父系解析を可能とし、新たな集団遺伝学的知見が期待されることから、今回&nbsp;ニホンザルにおけるY染色体特異的STR座位の同定を試みたので報告する。<br>【材料および方法】日本獣医生命科学大学野生動物教育研究機構と福島市および新福島農業協同組合の研究協力協定に基づき得られたニホンザル標本より抽出したゲノムDNAを試料とした。Y-STR座位の同定は、82種類のヒトDYS座位を基にあらかじめPCR法を実施し、ニホンザル雄特異的な候補座位と判定した12遺伝子座について、1）塩基配列の解析、2）ニホンザル雄ゲノム特異的STRマーカーの再構築、3）雄20例を用いた多型性解析により実施した。<br>【結果および考察】これまでに解析した82種類のヒトDYS座位のうち、ニホンザルにおいて雄特異的バンドが増幅された5座位および雌雄で増幅バンドの移動度が異なる7座位の計12座位についてダイレクトシークエンス法により塩基配列を決定した結果、10座位で、(TC)(TATC)、GTT、TAGAなどの雄特異的STR配列が認められた。そこで、雄特異的STR遺伝子座（Y-STR）10座位の塩基配列をリファレンスとした。リファレンス配列では、STR配列を含む領域（199bpから380bp）においてヒトゲノム配列との相同性は65％から87％と座位間で差が認められた。さらに、ニホンザルの雄ゲノムに特異的でかつヒトゲノムでバンドが増幅しない、ニホンザルY-STRマーカーの設計を試みた結果、10種類のうち7種類の座位でニホンザルY-STRの作成が可能であった。なお、20個体（福島県ニホンザル）を最初のスクリーニングパネルとし開発した10種のニホンザルY-STRマーカーで多型性の有無について解析を進めており、現在少なくとも5座位（最大対立遺伝子数、5）で多型性を見出している。以上、今回ニホンザルにおけるY染色体特異的STR座位の同定は、父系マーカーを利用した新たなニホンザル集団の遺伝子構成の解明に繋がるものと考える。

The chemokine (C-X-C) receptor 1 (CXCR1) expressed on the neutrophil surfaces interacts primarily with interleukin-8 (IL-8) and has an important role in immune response. Two interesting single nucleotide polymorphisms (SNPs), SNP CXCR1+777G>C and SNP CXCR1-1768T>A, that exhibit an association with subclinical mastitis and milk quality in dairy cattle, respectively, have been reported in the bovine CXCR1 gene. The aim of this study was to demonstrate the presence of the two SNPs in the CXCR1 gene of Japanese Black cattle and examine the association between the SNPs and clinical diseases including intestinal and respiratory diseases in calves. Genotyping of the SNPs in healthy Japanese Black cattle showed that the SNPs were also present in Japanese Black cattle with gene frequencies of 0.37 and 0.15 for the C-type allele in SNP CXCR1+777 and for the A-type allele in SNP CXCR1-1768, respectively. Statistical analysis of the genotype distribution of the SNPs in the bovine CXCR1 gene in healthy and clinical intestinal or respiratory diseased Japanese Black cattle indicated no significant association of the SNPs with clinical diseases in the calves. However, a significant correlation of the number of A alleles in SNP CXCR1-1768 with white blood cell (WBC) and platelet counts was found in the disease group. It is possible that the SNP in the bovine CXCR1 gene plays a role in modulating the hematological profile of WBC and platelet counts.<br>

Previous studies of telomeres and telomerase have focused mostly on mammals, and data for other vertebrates are limited. We analyzed both telomere length (terminal restriction fragment length) and telomerase activity in a small freshwater teleost fish, the medaka (Oryzias latipes), and found that the telomeres shorten during ageing despite the fact that a considerable amount of telomerase activity is ubiquitously detectable throughout the life of the fish. Since the telomere attrition rate during development was greater than that in adulthood, telomere length is inversely correlated with the increase in body length. The difference in telomere length among medaka individuals was similar to that in humans, and the individual specific differences were evident even at the earliest embryonic stage. Telomerase activity was ubiquitously detectable not only in the body of the embryo but also in the systemic organs of mature individuals throughout their entire life span. These data suggest that telomere attrition during ageing in medaka, which is similar to that in humans, may be a major factor determining their mortality, and that telomere maintenance through strong telomerase activity may be required for the characteristic lifelong continuous growth of this fish.

Using a signal sequence-trap we identified a human gene encoding a polypeptide of 99 amino acids with a putative signal sequence. The gene was identical to keratinocyte differentiation-associated protein (Kdap), which was reported previously by Oomizu et al (Gene 256: 19-27, 2000) to be expressed in embryonal rat epidermis at the mRNA level. In humans, we found Kdap mRNA expression to be restricted to epithelial tissue at high levels. The 12.5 kDa protein was detected in culture supernatant of keratinocytes and those transfected adenovirally with the Kdap gene. In normal skin, Kdap protein was found exclusively within lamellar granules of granular keratinocytes and in the intercellular space of the stratum corneum. By contrast, in lesional skin of patients with psoriasis, Kdap was expressed more widely throughout suprabasal keratinocytes. When induced to differentiate in vitro, keratinocytes showed marked upregulation of Kdap mRNA expression similar to that of involucrin mRNA, but with differing kinetics. Finally, a spliced variant of Kdap mRNA was generated by alternative splicing mechanisms. Our studies indicate that human Kdap resembles rat Kdap with respect to tissue and cell expression at the mRNA level and that Kdap is a low-molecular-weight protein secreted by keratinocytes. Thus Kdap may serve as a soluble regulator of keratinocyte differentiation.

Microsatellite polymorphism due to differences in CT dinucleotide repeats was demonstrated in intron 14 of the canine BRCA1 gene. Genotype analysis of 103 unrelated dogs from 30 different breeds detected the presence of five alleles, including 10 of the expected 15 genotypes. Gene frequencies were biased and all alleles with the exception of one were below 0.1. This polymorphism, which occurs at the intron of canine BRCA1 should prove to be a useful marker fur detecting the loss of heterozygosity (LOH). One of the more notable findings of the present study was the detection of homozygotes of rare alleles. This finding identified an accumulation of rare alleles in specific canine breeds and demonstrated the usefulness of this characteristic for the biological study of dog evolution.

A cDNA coding for Feline liver xanthine dehydrogenase(XDH, EC1.1.1.204) was amplified by RT-PCR and cloned for determining the sequence:. The clones contained an open reading frame of 4002 base pairs encoding 1333 amino acid residues. The calculated molecular weight and isoelectric point were approximately 146 kDa and 7.0. Comparison of the deduced amino acid sequences indicated remarkable high homology, i.e., the amino acid residues of feline XDH shared approximately 90%, 87%, 87% and 86% identity with those of human, bovine, rat and mouse, respectively. The amino acid sequences of two putative iron-sulfur centers, one NAD binding site and one molybdenum binding site were well conserved among mammalian animals.

Transferrin (TF) polymorphism, one of the most useful genetic markers, have been studied extensively. TF*Dchi allele is widely distributed both among east Asian populations and American Indian populations. The TFDchi peptide was characterized by replacement of His by Arg at position 300 by amino acid sequencing. In the present study, one base substitution at the 956th nucleotide from the first nucleotide in the starting codon that induced His300Arg exchange was confirmed by direct DNA sequencing. The genotyping method used to detect the TF*Dchi allele involved the use of PCR-RFLP and restriction enzyme Acc II. Analysis of the 1765th nucleotide, which determines the common TF alleles, TF*C1 and TF*C2, in TF*Dchi cDNA indicated that the TF*Dchi allele is derived from the TF*C1 allele.

左眼の前房内に多数発生した浮遊性虹彩毛様体嚢腫によって視覚障害を示した雌, 8歳齢, 25.3.kgのラブラドール・レトリバーに対して, 眼内手術用の粘弾性物質を用いて前房内から浮遊性の虹彩毛様体嚢腫を押し出す方法によって摘出術を行ったところ, 針を付けた注射器で穿刺し一つ一つ吸引する方法よりもより簡便に嚢腫を摘出することができた。

膀胱癌と診断された雌のシェットランドシープドッグ, 9歳に対して膀胱全摘出を実施し, 尿路変更術として尿管尿道吻合術を行った。術後の経過は比較的良好で術後1ヵ月目頃からある程度の蓄尿が可能になった。この蓄尿は尿道が代償性に拡大したことによるもので興味深いものであった。

Transferrin(TF) polymorphism is one of the most useful markers in human genetics and forensic science. Three common phenotypes, TF Cl, TF C2-1 and TF C2, are determined by two autosomal codominant alleles, TF*Cl and TF*C2. We have analyzed the TF polymorphism at the nucleic acid level. The molecular basis for the TF polymorphism is determined by a single base pair mutation(C 1765 T) that results in a Pro570Ser amino acid substitution. TF*Dchi allele is a variant which is a marker of Mongoloid population. The His300Arg amino acid substitution in TF Dchi variant has been determined by amino acid sequencing. The molecular analysis that showed an Ato-G base exchange at position 956 in TF*Dchi allele confirmed the amino acid substitution in TF Dchi variant and indicated that TF*Dchi allele was derived from TF*C1 allele.

A genetic polymorphism of a human platelet polypeptide with a molecular weight of 28 kD detected by two-dimensional electrophoresis was investigated in family and population studies, and cell distribution. The 28-kD polypeptide showed autosomal codominant inheritance of two alleles. The gene frequencies of the two alleles were 0.925 and 0.075, respectively. The 28-kD polypeptide was observed in lymphocytes, neutrophils, eosinophils and monocytes, in addition to platelets. This polypeptide showed good reproducibility in electrophoresis, and appears to be useful as a genetic marker of the human genome in gene mapping and pedigree analysis.