Single embryo culture is useful for assessing the developmental competence of an embryo in detail. Recently, a device made of poly(dimethylsiloxane) (PDMS), which is biocompatible and nontoxic, has been widely used for culture various types of cells. However, PDMS plates are porous, causing the serious osmolality increment of the medium (over 600 mOsm/kg from Day 4 to Day 7). Here, we report that curing the PDMS under low pressure (LP-PDMS) greatly reduced the porosity, resulting in a constant osmolality of the medium. The blastocyst rate of single bovine embryos cultured with LP-PDMS microwell (MW) plates was the same as that of group-cultured embryos (25 embryos/50 mu l droplet; control, P>0.05). These results indicate that MWs on a plate made of PDMS cured under low pressure can be successfully used for individual embryo culture.

Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.

Aims: To understand the mechanisms by which cattle circulate and accumulate vitamin E, we cloned the cDNA for bovine α-tocopherol transfer protein (α-TTP) and examined its expression in different tissues. Methodology: A full length α-TTP cDNA was amplified from bovine liver by RT-PCR. Poly (A)+ RNA obtained from bovine tissues was subjected to RT-PCR analysis to examine α-TTP mRNA expression. Western blot analysis was performed using polyclonal antibody raised against an oligopeptide derived from bovine α-TTP to examine α-TTP protein expression in various bovine tissues. The localization of α-TTP in bovine lung tissue was examined by immunostaining with antibovine α-TTP polyclonal antibody. Results: The open reading frame consists of 846 nucleotides encoding 282 amino acids with 98% and 83% identities to sheep and rat orthologs, respectively. Bovine α-TTP mRNA and protein were expressed most strongly in liver and lung, whereas expression of α-TTP mRNA and protein are reported to be very weak or absent in human and rodent lungs. In the lung, immunostaining suggested that α-TTP is expressed specifically in alveolar walls, which consists of alveolar cells, epithelial cells of small bronchi, and endothelial cells of pulmonary blood vessels. Conclusion: These results suggest that, in the lung, α-TTP is involved in supplying vitamin E to alveolar surfactant in order to protect the lung tissue from oxidative stress, and that this role may be more important in bovines than in other mammals.

Early assessment of carcass traits and meat quality characteristics during fattening is expected to improve the efficiency of beef cattle production. Yet, there have been few reports on developing an assessment index for feeding condition of Japanese Black cattle. In this study, to identify protein biomarkers for predicting carcass traits and meat quality characteristics of Japanese Black cattle by proteomics, we first compared protein expression profiles of perirenal white adipose tissue between low and high merit steers in five carcass traits (carcass weight, rib eye area, rib thickness, subcutaneous fat thickness, and beef marbling standard [BMS] number). We then investigated the involvement of the lower- or higher-expressed proteins in metabolic pathways and evaluated the expression of vimentin proteins by western blot analysis in individual steers. We identified 45 proteins which were significantly expressed in low or high merit groups of five carcass traits. Coordinate involvement of some identified proteins in glycolysis/gluconeogenesis and citrate cycle pathways was found in high merit groups of carcass weight, subcutaneous fat thickness and BMS number. The expression of vimentin protein, which was significantly expressed in the high BMS number group, was confirmed in individual steers. Taken together, these results suggest the possibility of these identified proteins as protein biomarker candidates for predicting carcass traits and meat quality characteristics during fattening of Japanese Black cattle.

Development of assisted reproductive technologies is necessary to obtain fertilized oocytes in a subfertile transgenic mouse strain. Here, we showed the application of laser-assisted drilling of the zona pellucida to in vitro fertilization of cryopreserved mouse oocytes with sperm from subfertile transgenic mice (C57BL/6N-Tg(UCP/FAD2)U8 strain). After cryopreservation by vitrification, the recovery and survival rates of the zona-drilled mouse oocytes were 97% (97/100) and 94% (91/97), respectively. In vitro fertilization of the cryopreserved zona-drilled mouse oocytes with sperm from the subfertile transgenic mice was greatly facilitated (60%, 55/91) compared to that of the cryopreserved zona-intact mouse oocytes (11%, 81/768). In vitro fertilized embryos that developed to the 2-cell stage were again cryopreserved by vitrification, and after warming they were transferred into recipient females. Subsequently, six viable offspring were delivered, and all were confirmed to be transgenic mice. These results indicate that laser-assisted zona drilling of oocytes combined with cryopreservation by vitrification may be a useful approach for large-scale production of in vitro fertilized embryos for managing transgenic mouse strains with reproductive disabilities such as subfertile sperm.

Linoleic acid (18:2n-6) and a-linolenic acid (18:3n-3) are polyunsaturated fatty acids that are essential for mammalian nutrition, because mammals lack the desaturases required for synthesis of Delta12 (n-6) and n-3 fatty acids. Many plants can synthesize these fatty acids and, therefore, to examine the effects of a plant desaturase in mammals, we generated transgenic pigs that carried the fatty acid desaturation 2 gene for a Delta12 fatty acid desaturase from spinach. Levels of linoleic acid (18:2n-6) in adipocytes that had differentiated in vitro from cells derived from the transgenic pigs were approximate to10 times higher than those from wild-type pigs. In addition, the white adipose tissue of transgenic pigs contained approximate to20% more linoleic acid (18:2n-6) than that of wild-type pigs. These results demonstrate the functional expression of a plant gene for a fatty acid desaturase in mammals, opening up the possibility of modifying the fatty acid composition of products from domestic animals by transgenic technology, using plant genes for fatty acid desaturases.

P-glycoprotein functions as an ATP-dependent efflux pump that extrudes cytotoxic drugs out of the cells before the drugs reach their intracellular targets, so conferring multidrug resistance on the cancer cells. P-glycoprotein transports many structurally dissimilar cytotoxic drugs that act on different intracellular targets. In the cystic fibrosis transmembrane conductance regulator (CFTR), which acts as a chloride channel and belongs to the ATP binding cassette (ABC) superfamily of transporters like P-glycoprotein, amino acid substitutions in TMI and TM6 modulate the anion selectivity or ion conductance of the ion channel. We examined if some amino acids in TM1 of P-glycoprotein are involved in the substrate recognition, as in CFTR. Our data suggest that three amino acids His(61), Gly(64), and Leu(65) in TM1 would make a part of the substrate binding pocket, and that these amino acid residues may be involved in deciding the range of most suitable molecular sizes of the substrates.

To identify the roles of the two nucleotide-binding folds (NBFs) in the function of human P-glycoprotein, a multidrug transporter, we mutated the key lysine residues to methionines and the cysteine residues to alanines in the Walker A (W-A) motifs (the core consensus sequence) in the NBFs. We examined the effects of these mutations on N-ethylmaleimide (NEM) and ATP binding, as well as on the vanadate-induced nucleotide trapping with 8-azido-[alpha-P-32]ATP, Mutation of the W-A lysine or NEM binding cysteine in either of the NBFs blocked vanadate-induced nucleotide trapping of P-glycoprotein. These results suggest that if one NBF is non-functional, there is no ATP hydrolysis even if the other functional NBF contains a bound nucleotide, further indicating the strong cooperation between the two NBFs of P-glycoprotein. However, we found that the effect of NEM modification at one NBF on ATP binding ar the other NBF was not equivalent, suggesting a non-equivalency of the role of the two NBFs in P-glycoprotein function. (C) 1998 Elsevier Science B.V. All rights reserved.

Recently, we showed that the amino acid at position 61 in TM1 of human P-glycoprotein is important in deciding the substrate specificity of this protein. In this work, we investigated whether the amino acids other than His(61) in TM1 of P-glycoprotein are also essential in the function of this protein, Nine amino acids residues, from Ala(57) to Leu(65) in TM1, were independently substituted to Arg, and analyzed the drug resistance of cells stably expressing each of these mutant P-glycoproteins, The mutant P-glycoproteins Ile(60) --> Arg, His(61) --> Arg, Ala(63) --> Arg, Gly(64) --> Arg, and Leu(65) --> Arg were normally processed and expressed in the plasma membrane, Substrate specificities of mutant P-glycoproteins Gly(64) --> Arg and Leu(65) --> Arg were quite different from that of the wild type, and similar to that of the His(61) --> Arg mutant, while the Ile(60) --> Arg and Ala(63) --> Arg mutant P-glycoproteins showed similar substrate specificities to that of the wild-type P-glycoprotein, suggesting that not only the amino acid residue at position 61 but also those at position 64 and 65 are also important in deciding the substrate specificity of P-glycoprotein. These three amino acids His(61), Gly(64), and Leu(65) would form a compact region on an alpha-helix arrangement of TM1, These results suggest that a region consisting of His(61), Gly(64), and Leu(65) in TM1 would participate in the formation of the recognition site for substrates of P-glycoprotein. (C) 1997 Federation of European Biochemical Societies.

In CFTR, a member of the ABC superfamily and a chloride channel, amino acid substitutions in its transmembrane domains 1 and 6 (TM1, TM6) have been reported to modulate the anion selectivity or ion conductance of the ion channel. In P-glycoprotein, no amino acid substitution in TM1, but some in TM6, have been reported to modify the substrate specificity of this protein. In this work, we demonstrated the involvement of His(61), which is in the middle of the predicted TM1, in the function of P-glycoprotein. His(61) was replaced by all other amino acid residues, and each of the mutant cDNAs was introduced into drug-sensitive human carcinoma cells, KB3-1. The drug-resistance profile of cells stably expressing each mutated P-glycoprotein was investigated by comparing their relative resistance to vinblastine, colchicine, VP16, and adriamycin. The resistance to vinblastine was increased by replacing His(61) by amino acids with smaller side chains, while it was lowered by replacing by amino acids with bulkier side chains. The reverse effect was observed for resistance to colchicine and VP16. The resistance to adriamycin was increased by replacing by amino acids with bulkier side chains except Lys or Arg, which have a basic side chain. We also showed that the replacement of His(61) by Phe and Lys greatly impaired the efflux of calcein AM, while the replacement had no effect on the efflux of rhodamine 123. These results suggest that an amino acid residue at position 61 in TM1 is important in deciding the substrate specificity of P-glycoprotein.

We review how P-glycoprotein recognizes a wide variety of compounds and how it carries its substrates across membranes. Amino acid substitutions that affect the substrate specificity of P-glycoprotein have been found scattered throughout the molecule. In particular, some amino acid residues in the putative transmembrane domain (TM)1 together with TM5-6 and TM11-12 may help to govern substrate specificity. The features that substrates for P-glycoprotein share are also discussed. The amphipathy of a substrate may decide whether the substrate can be intercalated into the lipid bilayer of the membrane. In addition, only certain molecular volumes and tertiary structures may mab it possible for the substrate to fit into the substrate-binding site(s) of P-glycoprotein.

Multidrug resistance-associated protein (MRP), a member of the ABC superfamily transporters, functions as an ATP-dependent efflux pump that extrudes cytotoxic drugs from the cells. Although glutathione has been considered to play an important role in the function of MRP, there is no convincing evidence that glutathione directly interacts with MRP. Here we demonstrate that vanadate-induced trapping of 8-azido-ATP in MRP was stimulated in the presence of glutathione, oxidized glutathione and the anti-cancer drugs VP-16 and vincristine. MRP in membrane from a human MRP cDNA transformant was specifically photolabeled with 8-azido-[alpha-P-32]ATP by the vanadate-trapping technique. Vanadate and Mg2+ were required for trapping of nucleotides, and vanadate trapping of nucleotides was inhibited by excess ADP as well as ATP. These results suggest that a stable inhibitory complex MRP . MgADP . Vi, an analog of the MRP . MgADP . Pi transition state complex, is formed in the presence of vanadate, Glutathione as well as anticancer drugs would directly interact with MRP, and stimulate the formation of the transition state of the ATPase reaction of MRP.

The human MDR1 gene encodes the multidrug transporter P-glycoprotein (Pgp). Although the MDR2/Pgp shares about 80% identity at the amino acid level with the MDR1/Pgp, the MDR2/Pgp cannot act as a multidrug transporter. We examined the drug sensitivity of Saccharomyces cerevisiae expressing either the human MDR1/Pgp or MDR2/Pgp. The human MDR1/Pgp conferred about 4-fold resistance to aureobasidin A, a cyclic depsipeptide antifungal antibiotic, on the drug-sensitive yeast strains. Interestingly the human MDR2/Pgp also conferred about 2.5-fold resistance to aureobasidin A. The resistance to aureobasidin A conferred by the MDR2/Pgp as well as by the MDR1/Pgp was overcome by vinblastine, verapamil, and cyclosporin A, depending on their concentrations, but not by colchicine. Aureobasidin A probably interacts directly with Pgps, because it overcame multidrug resistance of human cells and inhibited azidopine photoaffinity labeling of MDR1/Pgp in human cell membranes. These results suggest the possibility that the human MDR1 and MDR2/Pgps have conserved domain(s) for drug recognition.

The initiation of replication from oriV(RSF1010), the replication origin of the broad host-range plasmid RSF1010, depends on RepA (helicase), RepB' (primase), and RepC (intiator protein), encoded by RSF1010 itself, while this initiation event in E. coli is independent of dnaA, dnaB, dnaC, and dnaG [Scherzinger et al, (1984) Proc. Nat], Acad. Sci. USA 81, 654-658; Scholz et al, (1985) in: Plasmids in Bacteria, pp, 243-259, Plenum, New York; Haring and Scherzinger (1989) in: Promiscuous Plasmids of Gram-negative Bacteria, pp, 95-124, Academic Press, London; Scherzinger et al, (1991) Nucl, Acids Res, 19, 1203-1211], We showed in this work that a newly constructed origin consisting of an oriV(RSF1010) and a DnaA protein binding site, the dnaA box, inserted near oriV(RSF1010) (oriV(RSF1010)-dnaA box) could function without RepB' primase, but required RepA and RepC, This oriV(RSF1010)-dnaA box could not replicate in a dnaA46 strain in which only RepA and RepC were supplied, even at a permissive temperature, These results indicate that an inserted dnaA box can functionally substitute for the RSF1010-specific ssi signals, the RepB' dependent priming signals in OriV(RSF1010), and can direct a priming pathway different from the RSF1010-specific one, but related to DnaA protein.

The broad host range plasmid RSF1010 requires for its replication in Escherichia coli three plasmid-encoded proteins and specific nucleotide sequences ssiA, ssiB, and iterons in the oriV(RSF1010). In Pseudomonas aeruginosa, a recombinant mini-RSF1010 plasmid lacking ssiB lost its replication ability, but a miniplasmid lacking ssiA or carrying a primosome assembly site in place of ssiA could replicate. Moreover, ssiA, as a sole ssi signal, in the orientation that ssiB had originally taken was sufficient for replication of the miniplasmid. These results indicated that only one RSF1010-specific ssi signal in the orientation that ssiB takes in wild-type oriV(RSF1010) was essential for replication of RSF1010. Replication of the miniplasmids was dependent on the three plasmid-encoded proteins, RepA, B', and C, as in E. coli. (C) 1994 Academic Press, Inc.

The broad host-range plasmid RSF1010 contains two oppositely oriented priming signals, ssiA and ssiB, for DNA synthesis dependent on the origin of vegetative DNA replication (oriV). If either ssiA or ssiB was deleted or inverted, the RSF1010 miniplasmids containing engineered oriVs were maintained at low copy numbers, replicated abnormally as dimers, and accumulated specific single strands in the Escherichia coli strain supplying the three RSF1010-encoded RepA, RepB', and RepC proteins. Interestingly, an additional intracellular supply of the Sog primase (the sog gene product of plasmid CoIIb-P9) reversed the replication deficiency of these miniplasmids with respect to all three aspects described above. These were also true f or the RSF1 01 0 miniplasmids in which either ssiA or ssiB was replaced by the primosome assembly site (PAS) or by the G4-type ssi signal (G site). Furthermore, comparative analysis of the functional contribution of the two oppositely oriented ssi signals to the DNA replication of RSF1010 showed that, irrespective of their types, ssi signals conducting the initiation of DNA chain elongation away from the iterons were functionally more important than ones in the inverted orientation. We consider that this functional difference reflects the inherent properties of the initiation mechanism of RSF1010 DNA replication.

The two single-strand DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010) of the broad host-range plasmid RSF1010 contain proposed stem-loop structures. Nine single base-change mutations in the stem of the ssiA structure, each of which destroyed a relevant base pairing, damaged the ssiA activity. A second single-base change was introduced into each of the nine ssiA mutants in such a way that the base pairing was restored. Only three out of nine second base changes that restored the base pairing restored the ssiA activity up to the wild-type level. Thus, the three are intramolecular suppressors. The results strongly suggested that, in the area of the stem of ssiA where the suppressor mutations fell, base pairing was the most important structural parameter for the ssiA activity. By contrast, it is most probable that, in the other part of the stem of ssiA, both base-pairing and the intrinsic base sequence were the major determinants of the ssiA activity.

To analyze the RSF1010-specific priming mechanism, a library of randomly mutagenized ssiA sequences was constructed by chemical synthesis using mixed nucleotide phosphoramidites. Synthetic ssiA sequences with the single base-substitutions were assayed for the SSI activity in E. coli JM109 expressing RepB' primase. It was demonstrated that the activity of ssiA was damaged markedly by single base-substitutions within the possible stem-loop structure and its 3'-flanking region. It is conceivable that these domains are critical in recognition and primer synthesis by RepB' primase.

α-tocopherol transfer protein(α-TTP)は、ビタミンEの一つであるα-tocopherol(α-toc)に特異的に結合する細胞質局在タンパク質である。ヒト、マウス、ラットでは、α-TTPは肝臓で最も強く発現して、食餌中より摂取されたビタミンEからα-tocを選抜し、体中に循環させることに機能する。本研究では、動物におけるビタミンEの供給源となる植物を主食とする草食動物のウシよりα-TTPのcDNAを単離し、その発現部位について検討を行った。また、α-TTPをHEK293細胞に発現させ、その機能の検討を行った。

ウシより新規に単離されたMRP1とヒトのMRP1とのアミノ酸レベルでの同一性は９１％あるのにもかかわらず、両者の基質特異性は大きく異なることが明らかにされている。このようなウシMRP1とヒトMRP1の基質特異性の違いは、両者のアミノ酸の違いによって引き起こされると予想される。そこで、ウシMRP1のヒトMRP1とアミノ酸の種類が異なる部位に変異を導入することで数種類の変異型ウシMRP1cDNAを作製した。この変異型ウシMRP1cDNAをCMV（Cytomegalovirus）プロモーターの下流に連結し、さらにその下流にIRES（internal ribosome entry site）とG418耐性遺伝子を連結して、MRP1とG418耐性遺伝子をbicistronicに培養細胞内で発現するためのベクターを構築した。このように構築した発現ベクターをヒト由来の培養細胞へ導入し、変異型ウシMRP1発現細胞を得た。得られた変異型ウシMRP１発現細胞と、野生型ウシMRP１発現細胞の抗ガン剤に対する耐性度を測定、比較することを通じ、変異したアミノ酸がMRP１の基質認識

MRP1（ABCC1）は、17個の膜貫通へリックスと2個のATP結合ドメインをもつ膜タンパク質である。MRP1は、ガン細胞の細胞膜に発現すると、様々な抗ガン剤を細胞内から外へ排出することにより、細胞を複数の抗ガン剤に対して耐性化する。このタンパク質の大きな特徴は、様々な抗ガン剤をはじめ、重金属等の多様な物質を基質として認識して細胞内から外へ輸送できることである。しかし、MRP1による多様な基質の認識・輸送機構は明らかにされていない。我々は、ウシとヒトのMRP1のアミノ酸配列、及び基質特異性の違いを利用して、MRP1の基質の認識や輸送に関与するアミノ酸部位を同定しようと試みた。

マウス胚性幹細胞におけるABCトランスポーター・Bcrp1 mRNAアイソフォームAの転写調節領域の解析

ウシ肝臓よりα-TTPの全長のcDNAを単離した。単離したcDNAの塩基配列から、ウシα-TTPは282アミノ酸から構成されると予想された。ウシのα-TTPはラット、マウス、ヒトのα-TTPとアミノ酸レベルでそれぞれ83%、83%、88%の同一性を有していることがわかった。ウシにおけるα-TTPの組織ごとの発現量を検討した結果、マウスとは異なり、肝臓以外の組織においてもα-TTPが相当量発現していることが明らかになった。またウシでは、肝臓におけるα-tocopherolの蓄積量が、他の組織と比較して特異的に多くないことを示唆する結果を得た。以上の結果から、ウシにおけるα-TTPの機能は、マウスやヒトとは異なることが示唆された。

これまでに我々は、抗がん剤輸送タンパク質ABCC1 (MRP1)のN末端側領域に含まれるウシ由来ABCC1の98番目のMet(Met98)をArgに、または309番目のArg(Arg309)をTrpに置換すると、基質特異性が大きく変化することを明らかにしてきた。そこで、これらの部位がABCC1の基質輸送にどのように関与するのかを検討するために、それぞれ部位を、側鎖の電荷の異なる他のアミノ酸に置換した変異体の活性を調べた。その結果、ウシABCC1のMet98をAspやLysに置換した変異体、およびArg309を、GluやPheに置換した変異体は、野生型とほぼ同様の耐性度のパターンを示した。以上の結果より、これらの部位におけるABCC1と基質との相互作用には、アミノ酸の側鎖の電荷よりも、側鎖の形状が重要であることが示唆された。

ABCC1は、17個の膜貫通へリックスと2個のATP結合ドメインをもつ膜タンパク質である。ABCC1は、ガン細胞の細胞膜に発現すると、様々な抗ガン剤を細胞内から外へ排出することにより、細胞を複数の抗ガン剤に対して耐性化する。このタンパク質の大きな特徴は、様々な抗ガン剤をはじめ、重金属等の多様な有害物質を基質として認識して細胞内から外へ輸送できることである。しかし、ABCC1による多様な基質の認識・輸送機構は明らかにされていない。我々は、ウシとヒトのABCC1のアミノ酸配列、及び基質特異性の違いを利用して、ABCC1の基質の認識や輸送に関与するアミノ酸部位を同定しようと試みた。

マウスES細胞の未分化維持機構においてABCトランスポーターBcrp1が与える影響について解析した。

ウシα-TTP mRNAは肝臓で強く発現している一方で、肺においても発現していることがわかった。ウシ肝臓より、ウシα-TTP cDNA（2740bp）を単離した。ウシα-TTPは282アミノ酸から構成され、ラット、ヒト、マウスのα-TTPとそれぞれ83％、88％、83％の同一性を有していた。

MRP1(ABCC1)は、ATPの加水分解のエネルギー利用して抗がん剤等の有害物質を細胞内から外へ輸送するトランスポータータンパク質である。ウシ、およびヒトMRP1の機能とアミノ酸配列の差異を利用して、MRP1の基質認識に関与するアミノ酸を同定しようと試みた。その結果、ウシMRP1のN末端側に存在する膜貫通ドメイン(MSD0)に存在する98番目のMetをArg、またはLysに置換すると、MRP1のドキソルビシンに対する耐性度が特異的に上昇することがわかった。また、MSD0に続く細胞内ループドメイン(L0)に存在する309番目のArgをTrpに置換した場合も、ドキソルビシンに対する耐性度が上昇することが判明した。

マウスＥＳ細胞分化誘導過程におけるにABCトランスポーター Bcrp1 アイソフォームの発現様式およびRNAiによるアイソフォームAノックダウンES細胞樹立について試みた。

マウスＥＳ細胞の分化誘導過程におけるにABCトランスポーター Bcrp1 アイソフォームの発現様式について解析した。

ウシMRP1cDNAに部位特異的変異を導入した5種類の変異体のcDNAを作製し、ヒト培養細胞KB3-1およびHEK293に導入、発現させた。変異体発現細胞の数種類の薬剤に対する耐性度を調べた。その結果、ドキソルビシンおよびVP16に対して耐性度の変化した変異体があることがわかった。

抗がん剤を生産する放線菌のゲノムDNA上より新規のABCタンパク質を含む約9kbpの断片を単離し、その全長の塩基配列を決定した結果、単離した断片上には、6つのORFが存在すると推定された。そのうちの4つは、ABCタンパク質に特有のATP結合ドメインサブユニットを含む、何らかの物質輸送タンパク質複合体を形成していると予測されました。また、残りの2つのORFは、細菌や植物において細胞内シグナル伝達を行っているtwo-component systemを構成するタンパク質をコードしていると推定された。

ウシより単離したMRP1の基質特異性を調べたところ、ウシMRP1とヒトMRP1は、アミノ酸レベルでの同一性が約90％もあるにもかかわらず、両者の基質特異性は明確に異なることがわかった。この結果は、ウシMRP1とヒトMRP1との間で異なっている全体の10％ほどアミノ酸のいずれかが、MRP1による基質の認識および輸送に直接関与しているアミノ酸であることを示唆している。そこで、両者の間で異なっているアミノ酸を分子生物学的手法によって別のアミノ酸に改変した変異MRP1を作製し、その変異体の基質特異性などの活性を調べた。

ウシMRP1cDNAに部位特異的変異を導入し、4種類の変異体のcDNAを作製した 。これらの変異体および野生型のウシMRP1をヒト培養細胞KB3-1に導入し、G418で選抜して得られた細胞について、4種類の薬剤に対する耐性度を調べた。その結果、ウシMRP1の1088番目の変異体が導入された細胞では、耐性度のパターンが変化していることがわかった。

抗がん剤アクチノマイシンＤを産生する放線菌ゲノムDNAより、新規のABCタンパク質をコードすると予想される遺伝子の部分断片を単離した 。単離した遺伝子断片のうちの1種類には、４つのORFが存在すると推定された。４つのORFは、ATP結合領域のみからなるABCタンパク質、2つの膜結合タンパク質、輸送基質結合タンパク質をそれぞれコードしていると推定された。

RT‐PCR法によって、マウスの肝臓からビタミンEの代謝に関与すると予測されるタンパク質をコードするcDNAを得た。この遺伝子の機能解析を培養細胞を用いて行った。

ウシMRP1cDNAに部位特異的変異を導入し、4種類の変異体のcDNAを作製した 。これらの変異体および野生型のウシMRP1をヒト培養細胞KB3-1に導入し、G418で選抜して得られた細胞について、4種類の薬剤に対する耐性度を調べた。

放線菌Stretomyces sp. NRRL11395は、抗がん剤アクチノマイシンＤを生産する能力をもつ微生物である。 この微生物は、細胞内で生産した転写阻害活性をもつ抗がん剤アクチノマイシンＤをそのまま細胞内に蓄積すると自身を害するので、それを細胞外に輸送する機構を有すると予想される。この放線菌より、自らの生産するアクチノマイシンＤを細胞外へ排出するのに関わるP-糖タンパク質に類似のABCタンパク質遺伝子の単離を行った。

抗がん剤アクチノマイシンＤを産生する放線菌Streptomyces sp. NRRL11395のゲノムDNAより、新規のABCタンパク質をコードすると予想される遺伝子の部分断片を2種類を単離した

脂肪酸不飽和化酵素FAD2を過剰発現するトランスジェニックマウスの表現型に対する遺伝子相互作用の解明を目的として、マイクロアレイを用いた網羅的遺伝子発現解析を行なった。

このタンパク質は細胞膜に局在する膜タンパク質であり、構造に類似性のない様々な種類の抗がん剤を、細胞内から外へ排出することによって、がん細胞を多剤耐性化する。MRP1タンパク質の基質認識に関与するドメインを決定することを通じ、MRP1の基質認識および輸送機構を明らかにしようと考えた。

トランスジェニックマウスにおける導入遺伝子の染色体上挿入位置を、Inverse PCRを用いて同定した。

Multidrug resistance protein 1 (MRP1), a member of the ATP-binding cassette (ABC) family of membrane transport proteins, functions as an energy-dependent efflux pump that extrudes many kinds of xenobiotics out of cells. In order to identify the domains involving recognition and transport of substrates of MRP1, we replace several non-conserved amino acids in bovine MRP1 by corresponding ones of the human ortholog by site-directed mutagenesis, and examine whether the amino acid substitutions alter the substrate specificity of bovine MRP1.

ダチョウの成長ホルモン（GH）遺伝子は5つのエキソンから構成されていた。そのプロモーター領域には、ほ乳類においてGHの脳下垂体特異的な転写を誘導する転写因子Pit1の結合配列が3カ所見いだされた。

第93回本大会でオレイン酸をリノール酸に不飽和化するホウレンソウ由来ω6（Δ12）脂肪酸不飽和化酵素fad2 cDNAの上流に脂肪組織特異的プロモーターaP2遺伝子の転写調節領域をつなげた融合遺伝子を導入したトランスジェニックマウス（A系統）の作製について報告した。本実験では、脂肪組織でリノール酸合成能を獲得したトランスジェニックマウスに関して生理学的解析を行った。

本実験では、エネルギー代謝に着目してTgマウスにおける不飽和化酵素遺伝子の機能的発現による影響についてより詳細な分子的機構を明らかにすること目的に、エネルギー代謝に関係するタンパク質uncoupling protein（脱共役タンパク質、UCP）の発現量をNorthern blot及びWestern blot法により検討した。

本実験では、第91回本大会ですでに報告したfad2遺伝子を構成的に発現させたトランスジェニックマウス（B系統）について、より詳細な表現型を明らかにすることを目的に生理学的検討を加えたので報告した。

MRP1 は抗がん剤などの有害物質排出タンパク質である。 ウシ MRP1 とヒト MRP1 のアミノ酸配列、 及び基質特異性の比較から、 MRP1 の分子内において有害物質の認識・輸送に重要な機能を果たす部位が示唆された。 （英文）

我々が単離したウシ MRP1 と、 ヒトより単離された MRP1 を抗がん剤に感受性の高いヒト由来の培養細胞で発現させ、 それぞれが細胞に付与する抗がん剤に対する耐性度を比較し、 ウシ MRP1 の機能の特色を調べた。

乳牛の乳腺より新たにウシ MRP1 の全長 （1530 アミノ酸） を含む cDNA を単離した。 ウシ MRP1 をヒト由来の培養細胞で発現させ、 その機能解析を行ったところ、 その基質特異性はヒト MRP1 の特異性とは大きく異なることがわかった。

MRP1 は､ ガン細胞の抗ガン剤耐性に関与するポンプタンパク質である｡ マウスを用いて乳腺に発現する MRP1 の機能を調べた結果､ MRP1 が有害物質の母体から乳汁への移行阻止に機能することが示された｡

授乳中のウシ (ホルスタイン) の乳腺よりウシ MRP1 の全長 (1530 アミノ酸) を含む約 6.2 kb の cDNA を単離した｡ ウシ MRP1 をヒト由来の培養細胞で発現させると､ 細胞は抗ガン剤耐性を獲得した｡