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服部, 憲尚
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金沢大学医学部医学科
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久慈, 一英
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矢倉, 公隆
概要:
本研究は、植物の特定DNA配列がゲノム内でどのような分布をしているかを明らかにする事を目的とし、ソラマメ由来の高度反復配列であるBamHlファミリ-(250bp及び1500bp配列)あるいはタバコゲノムより単離した自己複製能をもった配列(A
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RS)をプロ-ブとしたサザン・ハイブリダイズ法及びin situハイブリダイズ法による解析を行い以下のような結果を得た。 1.ソラマメ(vicia faba)BamHl ファミリ-の250bpと1500bpの相同配列が、カラスノエンドウ(Vicia angustifolia)およびスズメノエンドウ(Vicia hirsuta)のソラマメ属の野生種にも分裂散型反復配列として存在することが明かとなり、さらに、250bp配列と1500bp配列は古くは互いに隣接した形でのみゲノム内に存在したいたものが、進化の過程で分離しながら別のファミリ-を形成するようになったということが示唆された。またカラスノエンドウ染色体における1500bp相同配列の分布に注目すると、ほとんどの場合、長腕あるいは短腕のテロルメもしくは動原体付近に見出され、長腕の中間の位置には存在しないか、存在していてもわずかであることが分かった。1500bp配列が全ての染色体の全長にわたって存在しているソラマメゲノムにおける分布様式とこの結果を対比させて考えると、この配列が進化と共に分散化が強まっていったことが示唆される。2.タバコ ARSと相同な配列はタバコ以外のいくつかの植物種にも存在することが分かった。その中で、ソラマメについてin situ ハイブリダイゼイションを行ったところ、明確な結果は未だ得られていないが、傾向として、銀粒子が各染色体に存在することを示唆するパタ-ンが多数得られた。今後、さらに比活性の高いプロ-ブを得るか、ARSと相同な配列をソラマメから単離しされをプロ-ブに用いることにより確かなデ-タを得る必要があろう。 The purpose of this research is to clarify distributional patterns of specific DNA sequences in plant genomes. The following results were obtained by Southern blot hybridization and in situ hybridization utilizing highly dispersed repeated sequences of Vicia faba BamHl families and a cloned autonomously replicating sequence (ARS) from tobacco nuclear genome as probes. 1. Sequences which have homology to 250bp and 1500bp BamHl family sequences of V. faba were also present as dispersed repeated sequences in genomes of two wild Vicia species, V. angustifolia and V. hirsuta. In addition, it was suggested that all ancester sequences of the 250bp and 1500bp BamHl families were arranged adjacently in the ancient time and some of them were split to generate other sequence families such as 250bp and 1500bp families in the process of evolution. In situ hybridization of the 1500bp sequence to 6pairs of subtelocentric chromosomes of V. angustifolia revealed that this sequence were localized in telomeric regions of short or long arms, or in the region near centromeres of each chromosome but not in the middle regions of long arms of any chromosomes. From the fact that the 1500bp sequence distributes diffusely throughout all the chromosomes of V. faba, it was also suggested that the extent of dispersion of this sequence was increased with the evolution of Vicia genome. 2. By Southern blot hybridization analysis it was found that sequences homologous to tobcco ARS were present in several other plant genomes. Although clear patterns were not obtained yet in situ hybrization using sequence homology to tobacco ARS as a probe showed that silver grains were seemed to be distributed every chromosomes of V. faba. In order to obtain fine results, it is necessary to use probe with higher specific activity or to clone ARS-homologows sequences from V. fava genome and use the probes.<br />The purpose of this research is to clarify distributional patterns of specific DNA sequences in plant genomes. The following results were obtained by Southern blot hybridization and in situ hybridization utilizing highly dispersed repeated sequences of Vicia faba BamHl families and a cloned autonomously replicating sequence (ARS) from tobacco nuclear genome as probes.1. Sequences which have homology to 250bp and 1500bp BamHl family sequences of V. faba were also present as dispersed repeated sequences in genomes of two wild Vicia species, V. angustifolia and V. hirsuta. In addition, it was suggested that all ancester sequences of the 250bp and 1500bp BamHl families were arranged adjacently in the ancient time and some of them were split to generate other sequence families such as 250bp and 1500bp families in the process of evolution. In situ hybridization of the 1500bp sequence to 6pairs of subtelocentric chromosomes of V. angustifolia revealed that this sequence were localized in telomeric regions of short or long arms, or in the region near centromeres of each chromosome but not in the middle regions of long arms of any chromosomes. From the fact that the 1500bp sequence distributes diffusely throughout all the chromosomes of V. faba, it was also suggested that the extent of dispersion of this sequence was increased with the evolution of Vicia genome.2. By Southern blot hybridization analysis it was found that sequences homologous to tobcco ARS were present in several other plant genomes. Although clear patterns were not obtained yet in situ hybrization using sequence homology to tobacco ARS as a probe showed that silver grains were seemed to be distributed every chromosomes of V. faba. In order to obtain fine results, it is necessary to use probe with higher specific activity or to clone ARS-homologows sequences from V. fava genome and use the probes.<br />研究期間(年度):1989-1990, 研究課題/領域番号:01540562<br />出典：「植物ゲノムにおける特定DNA配列の分布」研究成果報告書 課題番号01540562 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）) 本文データは著者版報告書より作成
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水上, 勇治 ; Mizukami, Yuji
概要:
今回の研究において私達は種々疾患の甲状腺組織を用いノーザンブロット法,in situ hybridization法および,モノクローナル抗体TSH受容体を用いた免疫組織化学的染色法を行いTSH受容体遺伝子のmRNAレベルと蛋白レベルにおける
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発現状態を検索し,種々の甲状腺疾患における甲状腺濾胞細胞でのTSH受容体発現と濾胞細胞の機能さらに甲状腺ホルモン全体におよぼす影響を検討した.A.材料.1996年から1997年にかけて金沢大学第2外科にて手術で得られた甲状腺組織を対象とした.組織は切除後直ちに凍結して-80℃に保存された.B.プローブと抗体.TSH受容体遺伝子の検索に用いたプローブはTSH receptor cDNA vectorである。免疫組織化学染色に用いた抗体はマウス抗TSH受容抗体であり,これらはParis South大学Dr.E.Milgromより供与された.C.結果1.種々の疾患の人甲状腺組織を用い、TSH受容体遺伝子のmRNA発現、蛋白質発現をnothernblot法,in situ hybridization法,免疫組織化学法用い検討した.2.これら3種の方法を用いたTSH受容体発現は各疾患別にみてほぼ一致して結果が得られた.3.バセドー病甲状腺ではmRNAレベル,蛋白レベル共に発現が高度に増加し,バセドー病甲状腺におけるTSH受容体のup-regulation機構が作動していることが推測された.4.非機能性の甲状腺腺腫,乳頭癌においてもTSH受容体はmRNAレベル,蛋白レベルで発現が増加していた.5.未分化癌のTSH受容体はmRNAレベル,蛋白レベル共に発現が認められなかった.<br />In the present study, we investigated TSH-receptor expression in hyman thyroids using immunohistochemistry, in situ hybridization and northern blot. The relationship between the TSH-receptor expression and the thyroid function were examined.A.Materials and MethoedsThe surgically resected thyroid tissues of various thyroid diseases were used in this study. The thyroid specimens were immediately frozed at -80C and stored. The TSH-receptor cDNA vector and the anti-human mouse monoclomnal antibody against TSH-receptor protein were used. These were kindly supplied by Dr.E.Milgrom (paris South University, France).B.ResultsThe obtained results were as follows :1. The immunohistochemical, in situ hybridization and northern blot study showed the same results in the expression of TSH-receptor in human thyroids of each case. examined.2. In Graves' thyroids, the elevated TSH-receotor expression was observed in both of protein level and mRNA level. This finding supported the theory that the up-regulation mechanism of TSH-receptor is one of the major causes of Graves' hyperthyroidism.3. In the thyroid adenomas and the carcinomas which are functionally inactive, the elevated TSH-receptor expression was observed in the protein level and mRNA level.4. In the undifferentiated thyroid carcinomas, The TSH-receptor expression was not detected in the protein level and mRNA level.<br />研究課題/領域番号:08670196, 研究期間(年度):1996-1997<br />出典：「人TSH受容体発現のmRNAおよび蛋白レベルでの形態学的ならびに生化学的研究」研究成果報告書 課題番号08670196(KAKEN：科学研究費助成事業データベース（国立情報学研究所）) 本文データは著者版報告書より作成
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