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A cell line (PL38PB) was established from blood samples of a 6-month-old pig that was diagnosed with lymphoma with CD5 expression. Histopathological examination revealed neoplastic lesions in the spleen, liver and lymph nodes. Tumor cells were immunohistochemically positive for CD20 and immunoglobulin heavy chains (μ, γ and α). Membranous CD5 and cytoplasmic Immunoglobulin M (IgM), ​Immunoglobulin G (IgG) and ​Immunoglobulin A (IgA) were detected in PL38PB cells by flow cytometry. In addition, the cytoplasm of PL38PB cells were positive for IgM, IgG and IgA by immunofluorescent. However, no Ig secretion was detected in culture supernatant by Ouchterlony gel diffusion method. Results suggest that PL38PB cells express three Ig isotypes that are produced but not secreted.

For broad detection of pestivirus A (bovine viral diarrhea virus 1: BVDV1) and pestivirus B (BVDV2) by a reverse transcription loop-mediated isothermal amplification (RT-LAMP) test, the P25 primer set was designed using nucleotide sequences of 5’-UTR region of 1454 BVDVs. The base coverage of each primer against diverse BVDVs were more than 99% in each base position. The one step LAMP test with the P25 primer set could detect both BVDV1 (TK) and BVDV2 (KZ), but did not amplify 5 other bovine viruses. Detection limit of the LAMP test was 103 copies of synthesized DNAs, and 10−3 and 10−4 dilutions of viral RNAs of TK and KZ strains, respectively, whereas that with current Aebischer’s primer set was 10−2 dilution and negative of these RNAs, respectively. All of the 63 viral RNA samples of persistently infected (PI) cattle, consisting of the 1a (12), 1b (31), 1c (11), and 2a (9) subgenotypes, were broadly detected with the P25, while only 65% of them were positive with Aebischer’s primer set. The validation study showed that the RT-LAMP test with the P25 had 100% sensitivity and 100% specificity against that with updated Vilcek’s PCR primers. Also, by using the P26 primer set which contained 3 species-specific primers, all 63 RNA samples were clearly distinguished from BVDV1 or BVDV2 by the typing RT-LAMP test. These results indicate that the one step RT-LAMP test using P25 or P26 primer sets would be useful for broad detection and rapid differentiation of BVDV1 and BVDV2.

Approximately 20 major-to-ultratrace elements in each organ (mantle, muscle, liver, kidney) of a giant clam, Tridacna crocea, and 16 elements in symbiotic zooxanthellae were determined by ICP-AES, ICP-MS, and CHN coder. The biogeochemical properties of T. crocea were then investigated using multi-element profiling analyses. The values for most elements were the highest in the kidney among all organs. However, the concentrations of Mn, Co, Ni, Zn, Se, Ag, Cd, and Pb in the mantle were higher than those in the muscle and liver. Therefore, these results indicate that most trace elements taken in by T. crocea through zooxanthellae may finally accumulate in the kidney. Moreover, 14 trace elements in zooxanthellae were present in the concentration range from 181 µg g−1 for Zn to 0.58 µg g−1 for U based on dry weight after isolating zooxanthellae from the mantle. The concentrations of some trace metals in zooxanthellae were relatively higher than those in other organs except for the kidney. These results suggest...

Loop-mediated isothermal amplification (LAMP) is a potential screening test for avian influenza (AI), but its narrow detection spectrum limits its applications. To improve this narrow detection spectrum, 3 types of primers were compared for detection of diverse H5 subtype hemagglutinin (HA) genes. Four and 6 genes, of 10 genetically different H5 HA genes tested, were detected with S primers specific for A/duck/Tsukuba/9/2005 (H5N2) and with M primers (which contained mixed bases), respectively. In contrast, all 10 HA genes became positive with population primers (P primers) (a mixture of primers designed for each subpopulation of 2,202 HA genes). Our study indicated that the P primers for the forward inner primer (FIP) and backward inner primer (BIP) sites were essential for exhaustive detection, whereas those for the F3, forward loop (FL), backward loop (BL), and B3 sites were exchangeable with M primers. A base mismatch experiment demonstrated that HA genes with ≤2 base mismatches per primer site and ≤10 base mismatches per HA gene were amplifiable. Reverse transcription-LAMP was broadly reactive, specific for H5 subtype HA genes, and applicable to field samples, with the sensitivity of real-time PCR. The in silico analysis suggested that most H5 HA genes (2,586 positive genes/2,588 genes tested) registered in the GenBank database might be amplifiable. These results indicate that the use of subpopulation primers in LAMP allows exhaustive detection of diverse HA genes and H5 LAMP can be used as a reliable AI screening test in general diagnostic laboratories.

Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock-out and knock-in animals except for mice up to now. Very recently, a method of genome editing using zinc-finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene-targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT-K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT-K75 cells. In both cell lines, results from Cel-I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome-modified pigs.

PCBs are persistent environmental agents that induce multiple impairments in living beings. In this study we used a transgenic mouse model (Muta(TM) Mouse), carrying bacterial lacZ genes for mutation assays and for assessment of the genotoxic effect of PCB126 on fetal mice. Mothers of experimental groups were subjected to a single oral dose of PCB126 (125, 250 and 500 microg/kg) on the 10th day of pregnancy, respectively. Fetuses were autopsied on the 18th day of gestation. Cleft palate was observed in 2 out of 11 fetuses from 3 litters in 500 microg/kg treated group. Other external malformations were not observed. The DNA mutation frequencies (MF) of fetuses in each group were 1.15 +/- 0.24 x 10(-5), 0.90 +/- 0.20 x 10(-5) and 1.08 +/- 0.24 x 10(-5) in fetuses of 125, 250 and 500 microg/kg treated groups, respectively. The MF of controls was 0.81 +/- 0.22 x 10(-5). There were no significant differences among the groups. However, the MF of each treated group was a little highter than that of control group. Possible relationships between PCB and its mutagenic effects in the offspring of mice are discussed.

Bifidobacterium属は,高いプロバイオティクス効果および細菌の分類学のモデルケースとして興味深い状況にある。(独)理化学研究所バイオリソースセンター微生物材料開発室(JCM)に保存されているBifidobacterium属およびその近縁種で,ヒトを分解源とする基準株を検索したところ,9菌種(菌株)が候補としてあげられ,その全ゲノム解析を開始した。その菌種は,Table 1に示したとおりであるが,B. bifidum, B. breveおよびB. catenulatumの3菌種は全ゲノム配列を決定し,B. dentium, P. denticolens, S. inopinataおよびG. vaginalisの3菌種は,コンティグが15前後になっており,B. longumおよびb. scarcoviiについては,ショットガンライブラリの作製を完了した。各菌株について引き続きシークエンス作業を進め,全ゲノム解析後は,大規模なBifidobacterium属の比較ゲノム解析を行う予定である。, It is interested in Genus Bifidobacterium shows the probiotic effects and as the model case of taxonomy. The nine species of Bifidobacterium and the closest related species of the type strains isolated from human origin were obtained from the Microbe Division, Japan Collection of Microorganisms (JCM), RIKEN BioResource Center. We have started sequencing of the complete genome of nine species. We have done the complete genome sequencing of B. bifidum, B. breve and B. catenulatum. The contigs of Parascardovia denticolens, Scardovia inopinata and Gardnerella vaginalis is less than fifteen. The draft sequences of B. dentium genomes have been obtained, and the shotgun library of the B. scardovii and B. longum genomes were prepared. We are going to widely comparative genomics in the Family Bifidobacteriaceae, when all the genomes will be finished.

ホテイアオイを用いた水圏のファイトレメディエーション技術を開発するために,無菌ホテイアオイの培養に関する基礎研究を行った。まず,野外に於けるホテイアオイの湿重量変化と気温との関係を調べたところ,半月の平均気温が26℃の時,1日の湿重量が最大2.5%増加することがわかった。従って,無菌ホテイアオイをグロースキャビネット内で培養する際には,平均気温を26℃に設定することが望ましいと考えられた。また,大きな無菌ホテイアオイ個体を得るためには,通気性を良くするために培養器の開口部をアルミホイルで覆うのではなく,滅菌シートを利用した方が良いことがわかった。更に,無菌ホテイアオイを培養している培地の元素分析を行ったところ,一般的な植物組織培養に用いられるMS培地のFe,P及びCu濃度を高めた方が良いことが示唆された。, To develop phytoremediation technology for hydrosphere, we decided to utilize water hyacinth and performed the basic research for the aseptic culture. First, when the relations to the fresh weight change of water hyacinth and the temperature in the field were examined, it was understood that fresh weight has increased by 2.5 % per day at 26℃ of field temperature. Therefore, it was thought that the culture temperature for aseptic water hyacinth in the growth cabinet was higher than 26℃. Moreover, to obtain a big aseptic water hyacinth it was understood that it should be use the sterilization seat to improve ventilation. In addition, when elements concentration of the cultured medium was analyzed, it was suggested that the medium for water hyacinth should be modified the Fe, P, and Cu concentrations of the MS medium.

我々は,Lactobacillus reuteri JCM 1112^T, Lactobacillus fermentum IFO 3956, Lactococcus garvieae(ブリに対する毒性株), Lactococcus garvieae(ブリに対する無毒株)およびLactobacillus rhamnosusの全ゲノム解析を完了した。そして,Bifidobacterium breve, Bifidobacterium bifidumおよびBifidobacterium catenulatumについては,全ゲノム配列を決定中である。プロバイオティクスとは,"宿主の健康に対して貢献する生きた微生物"と定義付けられている。本プロジェクトでは,L.reuteriのプロバイオティクス効果を分子レベルで解明することを目的としている。そこで,分類学的にL.reuteriと近縁であるにもかかわらず,プロバイオティクス効果のほとんど認められないL.fermentumについても全ゲノム解析を行った。両菌種の比較ゲノム解析の結果,L.reuteriのゲノムには,抗菌物質であるロイテリン合成系にかかわる遺伝子の48kbの"genomicisland"を有しており,哺乳動物の腸内フローラバランスに良い影響を及ぼしていると推察された。我々は,glycerol dehydratase subunit motif(PFAM PFO2286-PFO2288)として,L.reuteriゲノムに3つの遺伝子(LR1633-LR1635)を検出し,gupCDE(glycerol utilizing enzymes)と命名した。本研究では,そのgupCDE遺伝子破壊候補株(L.reuteri Δ gupCDE)を得て,PCR解析やglycerol dehydratase酵素活性などから,L.reuteri Δ gupCDEを認めた。今後,親株(L.reuteriJCM1112^T株)とL.reuteri Δ gupCDEのそれぞれノトバイオート(モノアイソレート)マウスを解析して,プロバイオティクス効果を明らかにしていく予定である。, We determined the complete genome sequences of Lactobacillus reuteri JCM 1112^T, Lactobacillus fermentum IFO3956, Lactococcus garvieae (a toxic strain for yellowtail), Lactococcus garvieae (a non-toxic strain for yellowtail) and Lactobacillus rhamnosus.The genome analysis of Bifidobacterium breve, Bifidobacterium bifidum and Bifidobacterium catenulatum is on going. Probiotics are live microorganisms that confer health benefits on the host. The probiotic L. reuteri and non-probiotic L.fermentum are phylogenetically closest to L.reuteri in the Lactobacillus. To elucidate the molecular basis on the probiotic property exhibited by L.reuteri, comparative analysis of both genomes showed that L.reuteri harbored the unique 48kb genomic island containing the genes necessary for the biosynthesis of a reactive 3-hydroxypropionaldehyde (reuterin), which may influence the microflora balance in the gut. We identified three genes (LR1633-LR1635) with a glycerol dehydratase subunit motif (PFAM PF02286-PF02288) in the L.reuteri genome. These genes are designated gupCDE (glycerol utilizing enzymes) in this study. The gupCDE knock-out L.reuteri (L.reuteri Δ gupCDE) was obtained, and the reuterin synthesis will be conducted by glycerol dehydratase in the glycerol metabolism in vivo by the gnotobiotic mice mono-associated with L.reuteri and L.reuteri Δ gupCDE.

Gardnerella vaginalis strain JCM 11026 T was isolated from vaginal tracts of women. Here, we report the complete genome sequence of this organism.

We investigated the membrane transport of Na and K ions in red blood cells (RBCs) of northern fur seal (Callorhinus ursinus) by measurement of unidirectional fluxes. Like red blood cells of other carnivores, those of northern fur seal contain high Na and low K concentrations, which result from the lack of Na–K ATPase activity on their membranes. In physiological conditions, activities of bumetanide-sensitive Na, K–Cl cotransport and amiloride-sensitive Na/H exchange were measured. K–Cl cotransport and Na–Cl cotransport were not detected. Hypertonicity activated only Na/H exchange. We further examined the ion transport systems for regulatory volume increase (RVI) in red blood cells. In the hyperosmotic condition, shrunken RBCs restored their original cell volume in Na medium but not in Na-free medium, and this restoration with Na medium was inhibited by amiloride. From these results, it is suggested that RVI in northern fur seal RBCs are performed by amiloride-sensitive Na/H exchanger but not Na, K–Cl cotransporter.

プロバイオティクス乳酸菌としてLactobacillus reunteri (2, 039, 414 bp),そしてその知見のほとんどみられないLactobacillus fermentum(2, 098, 685 bp)の全ゲノム配列を決定した。両菌種は絶対ヘテロ発酵型の乳酸菌としては初めての全ゲノム配列決定となった。Lactobacillus属には,他にホモ発酵型と条件的ヘテロ発酵型の乳酸菌があるが,上記両菌種では,解糖(EMP)経路では必須の6-phosphofructokinase遺伝子の欠損が明らかとなり,そのためペントース・リン酸経路しか機能しなかった。各ORF内で3つ目の塩基の置換が非常に多く,そのことが遺伝子の機能は変わらず,G+C含量に10%もの違いが生じることが認められた。L. reuteriのゲノム中で多くのmobile elementsを発見したが,それはLactobacillus属が環境への適合性を高くし,生存性を有利にするのに貢献していることが示唆された。, We present the complete genome sequences of two lactic acid bacteria (LAB), probiotic Lactobacillus reuteri (2, 039, 414 bp) and non-probiotic Lactobacillus fermentum (2, 098, 685 bp), which share obligately the heterofermentative property in glucose metabolism and have a phylogenetically closest relation. The two genomes are compared with each other and with other lactobacilli exhibiting facultatively heterofermentative and obligately homofermentative properties. A striking common feature of the two lactobacilli is a lack of a gene encoding 6-phosphofructokinase essential for the Embden-Meyerhof-Pamas (EMP) pathway, so that the pentose phosphate pathway is utilized in them. Furthermore, numerous mobile elements in L. reuteri is found in the genomes. These data imply that a genetic plasticity-environment relationship may account for the diversity between these lactobacilli.

ホテイアオイを用いた水圏のファイトレメディエーション技術を開発するために,ホテイアオイの無菌化とホテイアオイで有効なプロモータの検討を行った。その結果,ホテイアオイの種子を滅菌し,外皮の一部を削除することで無菌化と発芽を行うことができた。また,高等植物において外来遺伝子を発現させるためによく使用されるCaMV35Sプロモータは,ホテイアオイでも利用できることがわかった。, To develop phytoremediation technology for hydrosphere, we decided to utilize water hyacinth. In this study, sterilization of water hyacinth as base technique for transgenic plant and search of a promoter to foreign-gene expression were performed. For sterilization of water hyacinth, the seed was a good material. However, even if the sterilized seed planted on the culture medium, budding was not observed. Then, when the seed which deleted a part of outer cover was planted, budding was observed. Furthermore, CaMV35S promoter, general promoter for higher plant, can use also for water hyacinth.

This study assessed a technique for the isolation and differentiation of dog erythroid progenitors. Mononuclear cells from peripheral blood were cultured in a two-phase liquid culture system. The colony burst was confirmed after the first-phase culture by using a semi-solid culture containing methylcellulose. By immunofluorescent observation or reverse transcriptase-polymerase chain reaction analysis, the cells which proliferated during the second-phase culture were found to express glycophorin, globin, and glutamate/aspartate transporter, which are markers of dog erythrocytes. This system is beneficial for the analysis of dog erythrocytes as a reasonable amount of well-matured cells (0.5×106/ml) are obtainable.

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.

K-Cl cotransport plays a crucial role in regulatory volume decrease of erythrocytes. K-Cl cotransport activities in dog erythrocytes with an inherited high Na-K pump activity (HK) and normal erythrocytes (LK) were compared. Nitrite (NO(2)) stimulated K-Cl cotransport activity in HK cells around 14-fold at 2.4 mM, and it also increased the Km value of this cotransporter. Real-time PCR and western blot analysis revealed that K-Cl cotransporter 1 was dominant, and that the quantity of K-Cl cotransporter 1 protein was comparable between HK and LK erythrocytes. These results suggest that the difference in cotransport activity was not caused by the amount of K-Cl cotransport protein but by a difference in the regulation system, which is susceptible to oxidant.

The rabbit is considered to be a valuable laboratory animal. We compared glycerol, lactamide, acetamide, and dimethylsulfoxide (DMSO) as cryoprotectants in egg-yolk diluent of ejaculated Japanese white rabbit spermatozoa for improvement of sperm cryopreservation methods. Rabbit semen was frozen with 1.0 M glycerol, lactamide, acetamide, or DMSO in plastic straws. Forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rate of forward progressive motile spermatozoa in lactamide (37.8 +/- 3.0%) was significantly (P

The present study investigated the effect of separation of spermatozoa by sonication or Piezo-pulse on in vitro development of oocytes injected with sperm heads in the rat. We also examined development to term of rat oocytes injected with sperm heads. Rat frozen-thawed spermatozoa were separated into heads and tails by sonication for 10 sec or Piezo-pulse in KRB medium, and each treated sperm head was injected into an ooplasm. The oocytes were observed for formation of two pronuclei and development to 2-cell embryos. The percentages of formation of two pronuclei and development to the 2-cell stage did not significantly (P>0.05) differ between the two groups. Oocytes injected with sonicated sperm heads that reached the pronuclear stage at 10 h after injection of sperm heads were transferred into 7 recipients. Five recipients became pregnant, and 8 living pups were obtained. The results indicate that rat oocytes injected with sonicated sperm heads can develop to term in vivo. Furthermore, no differ...

L. reuteriは, glycerol dehydratase (GD; EC 4.2.1.30)によってグリセロールからロイテリンを産生している。本菌株のグリセロール代謝は,グリセロールをGDによってロイテリンに変換し,1,3-propanediol dehydrogenase(EC 1.1.1.202)によって1,3-プロパンジオールに還元する還元反応系と,グリセロールをglycerol dehydrogenase(EC 1.1.1.6)によってジヒドロキシアセトンに変換する酸化反応系の2つの酸化還元反応系で構成されていると考えられる。従来,L. reuteriはGDによってグリセロールからロイテリンを産生していると報告されてきたが,ゲノム解析の結果から,GDではなくdiol dehydratase(DD; EC 4.2.1.28)に近い遺伝子構成であると言える。この遺伝子構造はdehydratase遺伝子を有する他のlactobacilliにおいても共通にみられた。特に, L. reuteriにおいて特徴的だったのは,L. collinoidesとL. brevisでは保存されているNADH: flavin oxidoreductase/NADH oxidase familyのドメイン構造を有する構造遺伝子が欠損している点であった。また,ロイテリン産生を考える上で重要な遺伝子であるglycerol dehydrogenaseと1,3-propanediol dehydrogenaseは, L. reuteriではpdu clusterとは異なるゲノム上の離れた位置に存在していた。つまり,dehydrataseとは別の制御系によって調節されている1,3-propanediol dehydrogenaseが,環境要因を感知し転写が抑制され,その結果,L. reuteriにおいてロイテリンが多量に産生されるものと考えられる。L. reuteriのpdu cluster下流でアデノシルコバラミン(AdoCbl)生合成系を検出した。L. brevisからは,AdoCbl生合成に関与する遺伝子群を検出することはできなかった。すなわち,L. reuteri以外の乳酸菌でAdoCbl生合成は確認されていないことから,他のlactobacilliでは,細胞外のコバラミンが存在している場合のみGDが機能するのに対し,L. reuteriは細胞外にコバラミンが存在しない場合でも,AdoCblを生合成し,ロイテリン産生が可能であると考えられる。以上,L. reuteriのpdu clusterの遺伝子構造,dehydrataseの特徴およびAdoCbl生合成系の存在が,L. reuteriのみでロイテリンが多量に産生される原因であり,L. reuteriの示す高いプロパイオテイクス効果の分子機構を解明する上で重要な知見である。, We attempted to determine the complete genome sequences of Lactobacillus reuteri JCM1112 (type strain) and Lactobacillus fermentum IFO3956. Production of antibacterial substances and adhesion factors for attachment to human intestinal cells largely influences contributed to the probiotic effects of lactic acid bacteria on the physiology. L. reuteri is known to produce a non-peptidic antibacterial substance, reuterin. Different from unlike bacteriocins, reuterin shows has an the antimicrobial activity against not only Grampositive bacteria but also Gram-negative bacteria, yeasts, fungi and protozoans. An operon in L. reuteri JCM1112 was found to encode the reuterin production system, whereas no orthologous counterpart was found in L. fermentum IFO3956. We analyzed a group set of genes involved in the reuterin synthesis in L. reuteri JCM1112, and found that the pdu cluster contained a class of genes responsible for polyhedral organelle formation, which were not present in the dha regulon, but lacked structural genes with the domain structure common to the NADH: flavin oxidoreductase/NADH oxidase family. In other bacteria that possess the dha regulon, the expressions of glycerol dehydratase and 1,3-propanediol dehydrogenase are subjected to synchronous transcriptional regulation to prevent the excessive production of cytotoxic reuterin. L. reuteri was shown to have a different regulatory system. Glycerol dehydrogenase conserved in L. reuteri might contribute to the maintenance of the intracellular oxidation-reduction balance when 1,3-propanediol dehydrogenase is being expressed. The pdu cluster of L. reuteri contained the structural genes homologous to propanol dehydrogenase (pduQ), propionaldehyde dehydrogenase (pduP) and propionate kinase (PduW) of S. typhimurium. This indicates that L. reuteri possesses an oxidation pathway to produce 3-hydroxypropionic acid from glycerol as well as a reduction pathway from glycerol to 1,3-propanediol via reuterin. A group of structural genes highly homologous to the genes involved in the biosynthesis of adenosylcobalamin (AdoCbl), a coenzyme of dehydratase was found in the downstream of the pdu cluster of L. reuteri. This is the first discovery for of the genes for the presence of AdoCbl biosynthesis in lactic acid bacteria. The hemABCL, cobACD and cysG genes were all found within the cob/cbi cluster in L. reuteri, while they are distantly located at different genomic loci in other bacterial species. The G+C content of the cob/cbi cluster, as well as the pdu cluster, was markedly lower than that of the other regions, which suggests that the gene cluster might have been acquired from other organisms through horizontal transmission. Thus, L. reuteri is able to transcribe almost all enzymes essential for the biosynthesis of AdoCbl from L-glutamate by a single transcription unit, indicating suggesting that L. reuteri could may produce AdoCbl more efficiently than any other bacteria. Our results also suggest that AdoCbl produced by L. reuteri might act may serve as a source of vitamin B_ in the intestine of humans incapable of synthesizing the substance. These genetic characteristics are specific only to L. reuteri in Lactobacillus, and are the causes for the production of reuterin in large quantities. In addition, it is considered that the efficient energy-production system and the inhibitory activity on the growth of other bacteria are importantly required for the contribution of probiotic lactic acid bacteria should contribute to the survivability in mammalian intestines, which is an important requirement for probiotic lactic acid bacteria.

The levels of verotoxin-1 and verotoxin-2 released by verotoxigenic Escherichia coli O157:H7 treated in vitro with sodium nitrite, sodium chloride and several antibiotics were evaluated. Of the three strains of E. coli O157:H7 used in this study, two strains produced both verotoxin-1 and verotoxin-2, and one strain produced only verotoxin-2. Treatment of E. coli O157:H7 with sodium nitrite (6000 mg/l, minimum inhibitory concentration) did not increase the levels of verotoxin-1 and verotoxin-2 compared with a treatment by sodium chloride or antibiotics. When the electron paramagnetic resonance spectrum of sodium nitrite-treated bacterial cells was examined at 77 K to clarify the mechanism for the anti-bacterial activity of nitric oxide derived from sodium nitrite, electron paramagnetic resonance signals with g-values of 2.035 and 2.010 were observed. These were identified as being derived from iron-nitric oxide complexes. It appears that the dinitrosyl iron complexes in the E. coli O157:H7 cells were generated from the reaction of iron-sulfur proteins (enzymes) with nitric oxide formed by the reduction of sodium nitrite. The amount of ATP was decreased by the presence of sodium nitrite in the cell suspension. These findings indicate that nitric oxide derived from sodium nitrite penetrated the cells and inactivated enzymes related to the respiratory chain.

Enterococcus faecalis TH10 is a lactic acid bacterial strain isolated from the Malaysian traditional fermented food, “tempeh”, and has antibacterial activity against various pathogens. To identify the antibacterial substance, the butanol extract of the culture supernatant of E. faecalis TH10 was fractionated by HPLC equipped with a reversed-phase partition column, and the elutes were subjected to antibacterial assay. As the activity was observed in a fraction eluted by ca 80% methanol, the fraction was analyzed by gas chromatography/mass spectrometry and 3-phenyllactic acid was identified as the major compound. Fractionation with an optical isomer separation column showed that the preparation contained D-and L-forms of 3-phenyllactic acid at a ratio of 2: 1. Authentic 3-phenyllactic acid showed antibacterial activity against various bacteria such as Staphylococcus aureus and Escherichia coil. These results suggest the possibility that 3-phenyllactic acid is a biopreservative.

亜硝酸ナトリウム(NaNO_2),塩化ナトリウム(NaCI)あるいは数種類の抗生物質で処理したベロトキシン産生大腸菌(VTEC)O157:H7の3株から,ベロトキシン(VT)1型と2型の放出量を定量した。VTEC O157:H7のうち,2株がVT1型とVT2型の両者を産出し,1株がVT2型のみ産出した。VTEC O157:H7をNaNO_2(最少発育阻止濃度である6,000mg/L)で処理したがVT1型およびVT2型の放出量は増加しなかった。NaNO_2由来の一酸化窒素(NO)の抗菌メカニズムを明らかにするために,NaNO_2で処理したVTEC O157:H7の細胞を77Kでの電子常磁性共鳴吸収(EPR)法に供した。その結果,g値2.035と2.010のEPRシグナルを検出し,細胞内に鉄硫黄タンパク質とNOが反応して形成されたジニトロシル鉄硫黄錯体が存在した。またATPの合成も阻害されていた。このことから,NaNO_2出来のNOは細胞内に入り,呼吸鎖に関与する酵素を不活化したものと考えられた。, The levels of verotoxin-1 and verotoxin-2 released from verotoxigenic Escherichia coli O157:H7 treated in vitro with sodium nitrite, sodium chloride and several antibiotics were estimated. Of the three strains of E. coli O157:H7 used in this study, two strains produced both verotoxin-1 and verotoxin-2 and one strain produced only verotoxin-2. Treatment of E. coli O157:H7 with sodium nitrite (6,000 mg/L, a minimum inhibitory concentration) did not increase the levels of verotoxin-1 and verotoxin-2 compared with treatment by sodium chloride or antibiotics. When the electron paramanetic resonance spectrum of sodium nitrite-treated bacterial cells was examined at 77K to clarify the mechanism of the anti-bacterial activity of nitric oxide derived from sodium nitrite, electron paramagnetic resonance signals with g-values of 2.035 and 2.010 were observed. These were identified as being derived from iron-nitric oxide complexes. It appeared that the dinitrosyl iron complexes in the E. coli O157:H7 cells were generated through reaction of iron-sulfur proteins (enzymes) with nitric oxide formed by the reduction of sodium nitrite. These findings indicate that nitric oxide derived from sodium nitrite penetrates the cells and inactivates enzymes related to the respiratory chain.

L.fermentumとL.reuteriは,分類学上の生理生化学的諸性状が一致することから同一菌種とされていたが,L.reuteriが独立する分類がなされた。分類学上の諸性状が一致するにもかかわらず,プロバイオティクス効果の知見はL.renteriに集中している。そこで,プロバイオティクス効果と遺伝的な背景を考察するために,両菌種の全ゲノム塩基配列を決定し比較ゲノムによる両菌種の遺伝子な特徴づけを行った。パルスフィールドゲル電気泳動法により両菌株のゲノムサイズを推定した。プラスミドは両菌株共に存在しなかった。染色体DNAの平均2.0kbと約10kb断片のゲノムライブラリーを構築した。各クローンに対してDYEnamic ET Terminator(Amersham Biosciences社)によるシークエンス反応を行い,キャピラリーDNAシークエンサーで解析した。得られたデータは,Phred/Phrapによるアッセンブルと,アノテーションはGenome Gambler(MKI社)により行った。ゲノムサイズは両菌株とも約1.8Mbでほぼ同様であったが,両菌株のゲノムの制限酵素地図的にはかなり異なっていた。ORFの数はそれぞれ1,600ぐらいで,輸送,エネルギー代謝に分類される遺伝子が主であった。両菌株よりβ-グルクロニダーゼ遺伝子は見つからなかった。ロイテリン(抗菌性物質)産生系は,L.reuteriにおいてオペロンとしてコードされていたが,L.fermentumには存在しなかった。プロバイオティクス効果においてヒト腸管付着因子の存在は重要であり,L.reuteriでいくつかの細胞付着性遺伝子が確認された。, L. fermentum and L. reuteri were separated from L. fermentum by polyacrylamide gel electrophoresis but not physicochemical phenotypes in 1980. On the other hand, L. fermentum is generally isolated from plant origin fermented foods and silage, whereas L. reuteri is typically isolated from mammalian intestinal tract and feces. Probiotics have recently been the focus of intense lactic acid bacterial researches interest. Although L. fermentum is phenotypically similar to L. reuteri, many research data of probiotics concentrate on L. reuteri not but on L. fermentum. The present studies are aimed at sequencing complete genomes of L. fermentum and L. reuteri, and analyzing those comparative genomes. We have sequenced the complete genomes of these species, and each genome contains approximate 1.8-Mb. The circular chromosomes of L. fermentum and L. reuteri have the average G+C contents of 52% and 39%, respectively. Five 23S rDNAs and six 23S rDNAs exist in the chromosomes of L. fermentum and L. reuteri, respectively. The numbers of insertion sequence (IS) elements and transposons (Tn) of L. fermentum are more than those of L. reuteri. L. reuteri possesses the structural genes determining reuterin (3-hydroxypropionaldehyde), an antimicrobial substance, production, but L. Fermentum does not possess this operon.

Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock-out and knock-in animals except for mice up to now. Very recently, a method of genome editing using zinc-finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene-targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT-K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT-K75 cells. In both cell lines, results from Cel-I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome-modified pigs.

我々は， ヒト消化管からの分離株であるL. rhamnosus ATCC 53103 株（別名としてGG 株）のゲノムを配列決定した。ATCC 53103 株は，最もプロバイオティクス効果の研究されている菌株の1 つである。本菌株のゲノムサイズは，3.0 Mb の染色体を有し，2,836 のタンパク質をコードしていた。糖代謝・アミノ酸代謝とその輸送に関係するタンパク質の他に，注目に値する防衛機構に関する遺伝子を多く保有していた。比較のゲノム分析で，広範囲なシンテニーがL. rhamnosus ATCC 53103 株とその近縁種であるL. casei ATCC 334 株（チーズ分離株）の間でみられた。しかし，L. rhamnosus ゲノムには多数の挿入，すなわち，PTS-タイプ輸送機システム，配糖体ヒドロラーゼと転写制御因子のために遺伝子から成る遺伝子群を含み，腸環境への潜在的適合性を反映していた。ATCC 53103 株には多くの細胞表面付着タンパク質が予測された。さらに，そのゲノムは，付着性に対して機能的なSpaCBA 線毛をコード化していた。L. rhamnosus ゲノムのこれらの特徴は，消化管生存に関与して，腸の粘膜と微生物叢との相互作用に貢献していると推察された。, We sequenced and analyzed the genome of Lactobacillus rhamnosus ATCC 53103 (also known as L. rhamnosus GG), a human isolate, which is one of the most studied probiotic strains. The organism harbored a 3.0-Mb chromosome encoding 2,836 protein-coding genes, and contained a remarkable number of proteins involved in carbohydrate and amino acid metabolism and transport, and defense mechanisms compared with other sequenced intestinal lactobacilli. Comparative genome analysis revealed that extensive synteny was found between L. rhamnosus ATCC 53103 and its closely related L. casei ATCC 334 (a cheese isolate), but the L. rhamnosus genome contains numerous insertions, which reflect potential adaptation to the gut environment, including six carbohydrate utilization gene clusters that consist of the genes for PTS-type transporter system, glycoside hydrolase, and transcriptional regulator. Genome analysis also predicted many cell surface adherence proteins, and its genome encodes functional SpaCBA pili shed on the importance of adhesion to mucus during colonization of the human. Collectively, these features in the L. rhamnosus genome are likely to contribute to the organisms’ gastric survival and promote interactions with the intestinal mucosa and microbiota.

ビフィズス菌（新菌種候補を含む）とその近縁種の10菌種の全ゲノム解析を完了し，ゲノムサイズは2.0～3.2Mbの範囲であった。各菌種のシンテニーの比較では，16S rRNA遺伝子配列による系統樹の近縁関係のとおり，B. breve と B. longum および B. catenulatum と B. adolescentis の組み合せでシンテニーはほぼ同じであった。それ以外の Bifidobacterium 属間では，近縁種のゲノム比較でよく見られるX（エックス）の形になっており，ori-ter軸を対称にした組み換えが複数回あったと考えられる。, We have sequenced seven species (including new species nov.) of bifidobacteria and the related three species. Their genomic size were the range of 2.0 to 3.2 Mb. We checked the location of the homologous genes in the chromosomes of ten species to compare the genomic structure of the species. According to the synteny of all the species chromosomes, the Bifidobacterium breve and B. catenulatum genes that showed high homologies to those of B. longum and B. adolescentis were picked up and aligned in the chromosomes or both bacteria, respectively. It was similar to show the phylogenetic relationships between the genomes of sequenced ten species, inferred from 16S ribosomal RNA gene sequences. The majority of the homologous genes in the other species of bifidobacteria appear to be located in versely in the related species of bifidobacteria.It is speculated that the genome backbones of the species are related to some extent but are still distinct.

ホテイアオイを用いた水圏のファイトレメディエーション技術を開発するために,ホテイアオイを利用することにした。本研究では,遺伝子導入のための材料とするために,ホテイアオイの外植片及び種子からのカルスの誘導を試みた。その結果,サイトカイニンとしてベンジルアデニンを,オーキシンとして2,4-Dを用いたところ,種子からカルスを得ることができたが,外植片からは得られなかった。しかしながら,この得られたカルスはその後増殖しなかったことから,更に検討する必要があることがわかった。, To develop phytoremediation technology for hydrosphere, we decided to utilize water hyacinth. In this study, we tried induction of callus from explants and the seeds of water hyathince for the material of gene transfer. The results were that the callus from seeds was obtained using BA as cytokinin and 2, 4-D as auxin, and not obtained from the explants. However, since the obtained callus did not grow, it turned out that it is necessary to examine a culture medium which changed the kind of plant hormone or these concentration.

A novel method to determine the content of 5-methylcytosine in DNA precisely, sensitively and simply has been developed using liquid chromatography (LC) with fluorescence detection. DNA was mildly digested by cooperation of nuclease P1 and DNase I, then the generated 2′-deoxynucleoside 5′-monophosphates (dNmp) were specifically derivatized fluorometrically at the position of the phosphoric acid moiety with 5-dimethylaminonaphthalene-1-[ N -(2-aminoethyl)]sulfonamide (dansylEDA), followed by the separation of fluorescent dansylEDA derivatives of dNmp on the reversed-phase partition column. The method was more than 10 times as sensitive as the conventional ultraviolet detection method.

We designed three hairpin ribozymes to cleave Escherichia coli beta-glucuronidase (GUS) mRNA and tested those activities in vitro. One of the ribozymes was designed to form 9 base pairs in total with the target GUS mRNA, and the other two ribozymes had longer substrate binding sites. All ribozymes cleaved the model substrate (100 bases long) at the predicted target site. Two ribozymes containing longer substrate binding sites cleaved the substrate much more efficiently than the other ribozyme containing shorter substrate binding site. Also, the ribozymes with long substrate binding sites had high activity against the full-length GUS mRNA (1.9 kilobases) and maintained the activity even at a low temperature, 26 degrees C, a general growth condition of plant cells. Effects of the substrate binding site length of the ribozyme on cleavage activity are discussed.

To detect the predominant lactobacilli in the intestinal flora of healthy thoroughbreds, we isolated lactobacilli from the feces of nine thoroughbreds (five males and four females; 0-15-year-old). The isolated lactobacilli comprise 17 species (37 strains), and they were classified into five groups: Lactobacillus salivarius (6 species), L. reuteri (6 species), Lactobacillus delbrueckii (3 species), L. buchneri (1 species) and L. vitulinus (1 species). On the basis of 16S rRNA gene sequences, we identified 3 other phylogenetic relatives belonging to the genus Lactobacillus. These results suggest that the intestinal flora of thoroughbreds may comprise many species of the genus Lactobacillus. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the 340-bp fragments of the 16S rRNA genes from the same nine fecal samples showed that L. hayakitensis, L. equigenerosi and L. equi are contained in all the samples, suggesting that these species are predominant lactobacilli in the intestinal flora of thoroughbreds.

The white-rot fungus T. versicolor UAMH 8272 produced two groups of laccases, each of which included several isoforms showing different isoelectric points (pI). Group 1 and group 2 laccases, respectively, displayed higher pI 5–6 and lower pI 3–4. Of the four cloned full-length laccase cDNAs, Lac 1 and Lac 4 were expressed in the heterologous protein expression system using Aspergillus oryzae. The measured pI of each Lac 1 and Lac 4 expressed in A. oryzae was lower than that of pI predicted from the amino acid composition. With this regard, isoelectric focusing of Lac 1 showed the presence of multiple protein bands in the 3.0–4.0 pI range, although the predicted pI value of Lac 1 was 4.7. Similarly, Lac 4 exhibited a pI value which was lower than that predicted (3.6 vs. 4.3, respectively). In all tested hydroxyPCBs, higher chlorinated hydroxyPCBs were less susceptible to in vitro degradation by laccase than lower chlorinated hydroxyPCBs. Although Lac 4 showed a generally higher activity than Lac 1, the two laccases were characterized by quite different substrate specificity toward two hydroxy-tetrachlorobiphenyl congeners. Two metabolites were obtained from the metabolism of hydroxy-pentachlorobiphenyl: a ten chlorine-substituted dimer with a C–O bond, and one with a C–C bond.

The behavior of heavy metals in wood during its carbonization process was examined. Cadmium in wood samples was found to be volatile when the samples were carbonized at 600 degrees C or higher, which demonstrated that removal of cadmium was feasible. Meanwhile, lead was found to be barely volatile even if the wood samples were carbonized at 1,000 degrees C or higher, which demonstrated that lead was difficult to remove and recover. The possibility of removing/recovering lead contained in wood by energization was then examined. By examining the concentration of sulfuric acid used as an electrolyte as well as load voltage, approximately 10% of lead was found to be recoverable.

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112(T) and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112(T) has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B(12)). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with (13)C(3)-glycerol demonstrated that L. reuteri JCM 1112(T) could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112(T) to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.

The rabbit is considered to be a valuable laboratory animal. We compared 2% acetamide and glycerol as cryoprotectants in egg-yolk diluent for ejaculated Japanese white rabbit spermatozoa to improve sperm cryopreservation methods. Fertility through artificial insemination, forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rates of forward progressive motility and plasma membrane integrity of the spermatozoa frozen with acetamide (27.1 +/- 8.3% and 24.5 +/- 6.5%) were significantly (P0.05) higher than those of the spermatozoa frozen with glycerol (16.3 +/- 10.9% and 14.3 +/- 7.6%). Though there was no significant difference in the kindling rates, the litter size of females inseminated with spermatozoa frozen with acetamide (6.0 +/- 1.1) were significantly (P0.05) higher than those of spermatozoa frozen with glycerol (3.0 +/- 0.4). The results indicate that 2% acetamide has a higher cryoprotective effect than 2% glycerol for sperm cryopreservation in the Japanese white rabbit.

The transport system responsible for glutamine, alanine and glutamate in MOLT4 human T4 leukemia cell line were characterized. Kinetic studies of sodium-dependent glutamine and alanine transport exhibited a single saturable high-affinity carrier with a Michaelis constant of 152 +/- 26 microm and 203 +/- 36 microm and a maximal transport velocity of 960 +/- 165 and 1096 +/- 208 nmol/10(9)cells/min, respectively. Glutamate uptake was less than one-tenth of glutamine and alanine, and linearly increased with glutamate concentration which was mediated by diffusion. 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), known as anion channel blockers, inhibited the sodium-dependent glutamine and alanine transport by 40% at 10 microm. Cellular contents of these amino acids in MOLT4 cells revealed glutamate to be the highest among them despite low glutamate influx. A glutamine metabolism study using whole cells indicated this high conversion rate from glutamine to glutamate, but no conversion to another amino acid. Based on these results, the high glutamate concentration in MOLT4 was speculated to be synthesized from transported glutamine by active glutaminase.

We examined motility, plasma membrane integrity, and binding capacity to homologous zona pellucidae (ZP) of frozen/thawed epididymal cat sperm as a model species for endangered felines. Epididymal spermatozoa from 20 domestic cats were frozen with freezing egg-yolk extender containing 3.0% glycerol in 0.25-ml straws. Post-thaw motility and plasma membrane integrity of the frozen/thawed spermatozoa were 31.8 +/- 2.4% and 32.2 +/- 4.2%, respectively. The frozen/thawed spermatozoa were co-cultured with frozen/thawed immature homologous oocytes with intact ZP for 3 h to examine their ability to bind to the ZP. Sixteen of the 20 frozen/thawed sperm samples demonstrated the ability to bind to ZP. These results indicated that the freezing system for epididymal sperm used in the present study gives appropriate information for banking the genetic resources of wild felid species.

Treatment with excessive amounts of Vitamin A during maternity induces fetal malformations. However, it is unclear whether these malformations are due to gene mutations or not. Using transgenic mice (containing lacZ gene showing beta-galactosidase enzymatic activity), we planned to observe whether gene mutations occur in the fetal tissues after treatment during maternity with Vitamin A (retinol palmitate). On the 11th day of pregnancy, mothers were given 30 mg (group 2), 150 mg (group 3) and 300 mg (group 4) of Vitamin A/kg body weight orally. Fetuses obtained on the 18th day of gestation showed malformations, such as cleft palate, origodactyly, brachydactyly and ectromeria. Most notably, cleft palate occurred dose dependently. The incidental rates were 100% in group 4, 58% in group 3 and 6% in group 2. The number of dead and absorbed fetuses also increased dose dependently with the treatments. DNA (integrated vectors containing lacZ genes) extracted from each fetus showed Vitamin A-induced lacZ mutations, especially in the malformed fetuses. The mutation frequencies were 4.99x10(-5) in group 4, 5.28x10(-5) in group 3 and 4.26x10(-5) in group 2. The frequencies of group 3 were significantly higher (p

A novel PCR assay was developed in order to examine the prevalence of Haemobartonella felis (H. felis) in Japanese domestic cats and which was able to differentiate of the Ohio strain and the California strain of H. felis. Blood samples from a total of 21 cats suspected of having haemobartonellosis were examined employing a novel PCR assay and demonstrated positive results in 18 cats which was confirmed by cytological examination of blood smears. Four out of 18 positive cats (22%) were infected with the California strain, whilst the other 12 cats (67%) were infected with the Ohio strain and two animals (11%) were infected with both strains. As most of the cats with moderate to severe anemia were infected with the Ohio strain, it is suggested that the most prevalent strain of H. felis in Japanese domestic cats might be the Ohio strain. In the present study, it was thought that molecular detection and characterization of H. felis may provide valuable information regarding the severity and prognosis of this illness.

A simple tubing stent was attempted to test a model for sutureless gastrointestinal anastomosis in 6 male rats at the age of 15 weeks. In the 3rd and 4th weeks after the operation, X-ray examination demonstrated that the gastrointestinal passage in the anastomotic site was quite satisfactory. There was no incidence of anastomotic leakage. In the 6th week after the operation, there were no macroscopic or microscopic ruptures, nor were there any obstructions at the anastomotic site. This simple sutureless method was effective at preparing anastomosis in the gastrointestinal tract in the rat and could be applied to other small experimental animals.

紫外線は，地球環境に於いて重要な生態学的因子である。この紫外線は，波長別にUV-C，UV-B及びUV-Aに分類されている。この中で特にUV-Bは生物に対する影響が強く，オゾン層の破壊に伴って照射量が増大すると予想されている。UV-Bの照射量の測定や生物への影響をモニタリングすることは重要であると考えられる。このようなことから，本研究課題では，UV-Bの生物影響をモニタリングするために，UV-Bの作用によって引き起こされるピリミジンダイマーの形成に注目し，このピリミジンダイマーの形成による転写の阻害を調べることができる遺伝子の構築を行うことにした。なお，本研究は重要な事項を含むため，この報告書に於いて詳細は記載しない。, Ultraviolet (UV) radiation is a significant ecological factor in the earth environment. This UV radiation is classified according to the wavelength at UV-C, UV-B, and UV-A. In particular, UV-B has strong influence on a living thing and it is expected that the amount of radiation increase with destruction of an ozone layer. Therefore, It is thought important to carry out the monitoring and measurement of the amount of UV-B radiation and the influence on a living thing. In this study, I note that formation of the pyrimidine dimer caused by the action of UV-B, DNA mutation detectable gene for ultraviolet radiation was constructed to monitoring influence of the living thing on UV-B. In addition, since this research includes important matters, it is not indicated for details in this report.