A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud

AbstractMammal–fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements

マイクロプレート オ モチイタ オス シバヤギ ケッショウ チュウ 5αジヒドロテストステロン ノ コウソ メンエキ ソクテイ

雄シバヤギ血漿中5α-ジヒドロテストステロン(5α-DHT)に関する酵素免疫測定法(EIA)について検討した。抗血清は抗5α-DHT-11α-Succinate-BSAを,酵素標識ホルモンには5α-DHT-11α-Succinate-peroxidase(5α-DHT-HRP)を用いた。抗血清は100,000倍に希釈・使用が可能であった。また,抗血清にはテストステロンが30%交叉反応するため,Bond Elut CN-Uを用いたカラムクロマトグラフィーによる精製を行い,被検血漿中の5α-DHTとテストステロンを分離・測定した。その結果,酢酸エチル : ベンゼン=2 : 98の展開液を流したところ,1.00-4.25mlの範囲に5α-DHTが溶出された。添加回収試験において添加量0.1-1.0pgの各濃度での回収率は,平均100.45%±2.13となった。再現性試験における頸静脈血および精巣静脈血の測定内変動係数(n=6)は各々6.38%および5.94%となり,測定間変動係数は8.36%ならびに11.53%となった。以上の結果から,雄シバヤギの血漿中5α-DHT濃度を,本法を用いて測定することが可能であることを明らかにした。Enzymeimmunoassay (EIA) for 5α-dihydrotestosterone (5α-DHT) in male Shiba-goat blood plasma was examined. The antiserum used 5α-DHT-11α-succinate-peroxidase (5α-DHT-HRP) for the enzyme labeling hormone in respect of 5α-DHT-11α-succinate-BSA. The use was possible for the antiserum at 100,000 times. The 5α-DHT in plasma was purified and separated from the testosterone by column chromatography using Bond Elut CN-U, since antiserum for 5α-DHT had cross reaction (30%) to the testosterone. The 5α-DHT was washed away by the development liquid of ethyl acetate: benzene=2 : 98 and was collected in the range between 1.00 and 4.25ml. Recovery rates of 5α-DHT each concentration of the addition 0.1～1ng to Shiba-goat plasma were 100.45%±2.13 of the averages. Inter-assay coefficient of variation (C.V.) became respectively 6.38% and 5.94% in jugular and testicular vein blood,while for intra-assay, they became 8.36% and 11.53%. It was possible to analyse 5α-DHT concentration in blood plasma of the male Shiba-goat from the above result using this method

マイクロプレート オ モチイタ オス シバヤギ ケッショウ チュウ テストステロン ノ コウソ メンエキ ソクテイ ホウ ノ ケントウ

Testosterone-3(E)-carboxymethyloxime-BSAを抗原として作成した抗体,ならびに酵素標識ホルモンとしてTestosterone-3(E)-carboxymethyloxime-peroxidaseを使用し,雄シバヤギにおける血漿中テストステロン濃度の酵素免疫測定法(EIA)を検討した。本実験では高い測定感度が得られる二抗体法を用い,作成した抗血清は,350,000倍に希釈しても使用可能な高い力価を有していた。血漿に10～100pgのテストステロンを添加した添加回収試験では,回収率が平均102.8±2.8%となった。測定内変動係数(N=6)は雄シバヤギ頸静脈血漿で5.08%,精索静脈血漿では7.32%,測定間変動係数(N=6)は,頸静脈血漿で5.74%,精索静脈血漿で6.13%であった。以上の結果から,本方法によって雄シバヤギ血漿中テストステロンの測定が可能となった。Enzymeimmunoassay (EIA) of testosterone was examined in which an individual antibody, and testosterone-peroxidase-conjugate were used for that, was measured in male Shiba-goat plasma. An anti-testosterone-3(E)-carboxymethyloxime-BSA antibody was used as an anti-serum, and testosterone-3(E)-carboxymethyloxime-peroxidase was used as a steroid-enzyme conjugate. The anti-serum was diluted by using 2nd antibody method which could get high measurement sensitivity of 350,000 times. Recovery rates of testosterone for each concentration with the addition of 10～100pg to Shiba-goat plasma were 102.8±2.8% of the averages. Inter-assay coefficient of variation (C.V.) for testosterone level from jugular and testicular vein blood plasma samples were 5.08% and 7.32% respectively, as for intra-assay, they became 5.74% and 6.13%. These results suggest that the EIA method is extremely suitable to measure testosterone concentration in blood plasma of the male Shiba-goat

PROTEINS IN EMBRYONIC BOVINE ENAMEL

The fractionation and the amino acid composition of the organic matrix of embryonic bovine enamel was studied. EDTA- and water-soluble fraction dissolved in phosphate buffer showed a temperature-dependent properties; at low temperatures it formed a clear solution, but at room temperature heavy precipitations occurred. This phenomenon was reversible. EDTA- and water-soluble fraction prepared in mild conditions was first chromatographed on Sephadex G-25 column. Four fractions were obtained. The main fraction comprised nearly half of the material and its molecular weight was found by gel filtration to be greater than 3,500. The temperature-dependent phenomenon above stated was observed only in the main fraction. The result of the fractionation with Sephadex G-100 revealed that a portion of the main fraction had molecular weight greater than 100,000. The results of the amino acid analysis showed that the EDTA- and water-soluble fraction was a high proline component. On the other hand, the EDTA insoluble fraction was a high proline-glutamic acid-histidine component. The EDTA-insoluble fraction was extracted with organic solvents and water. About 73% was solubilized, and the amino acid composition of the extracted residue relatively changed from the mother fraction; the residual protein contained only small amount of histidine. These results were discussed with reference to enamel maturation