{"_buckets": {"deposit": "d7d21d94-940e-405b-acdf-1a6d4bbd76a3"}, "_deposit": {"created_by": 1, "id": "880", "owners": [1], "pid": {"revision_id": 0, "type": "depid", "value": "880"}, "status": "published"}, "_oai": {"id": "oai:ir.soken.ac.jp:00000880", "sets": ["20"]}, "author_link": ["9811", "9812", "9810"], "item_1_biblio_info_21": {"attribute_name": "書誌情報（ソート用）", "attribute_value_mlt": [{"bibliographicIssueDates": {"bibliographicIssueDate": "1992-03-16", "bibliographicIssueDateType": "Issued"}, "bibliographic_titles": [{}]}]}, "item_1_creator_2": {"attribute_name": "著者名", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "小林, 麻己人"}], "nameIdentifiers": [{"nameIdentifier": "9810", "nameIdentifierScheme": "WEKO"}]}]}, "item_1_creator_3": {"attribute_name": "フリガナ", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "コバヤシ, マコト"}], "nameIdentifiers": [{"nameIdentifier": "9811", "nameIdentifierScheme": "WEKO"}]}]}, "item_1_date_granted_11": {"attribute_name": "学位授与年月日", "attribute_value_mlt": [{"subitem_dategranted": "1992-03-16"}]}, "item_1_degree_grantor_5": {"attribute_name": "学位授与機関", "attribute_value_mlt": [{"subitem_degreegrantor": [{"subitem_degreegrantor_name": "総合研究大学院大学"}]}]}, "item_1_degree_name_6": {"attribute_name": "学位名", "attribute_value_mlt": [{"subitem_degreename": "博士（理学）"}]}, "item_1_description_1": {"attribute_name": "ID", "attribute_value_mlt": [{"subitem_description": "1992019", "subitem_description_type": "Other"}]}, "item_1_description_12": {"attribute_name": "要旨", "attribute_value_mlt": [{"subitem_description": "　　In prokaryotes, the control of transcription initiation is a\u003cbr /\u003e key step in the regulation of gene expression. In order to\u003cbr /\u003e reveal the mechanism how the order of transcription is\u003cbr /\u003e determined among thousands of genes in a cell, it is important\u003cbr /\u003e to understand the intrinsic promoter strength for individual\u003cbr /\u003e genes (the term \"promoter strength\" refers to the relative rate\u003cbr /\u003e of synthesis of full length RNA product from a given promoter).\u003cbr /\u003e The level of transcription initiation is basically determined by\u003cbr /\u003e the sequence of the promoter, the start signal of RNA synthesis.\u003cbr /\u003e DNA sequence analyses of a wide variety of prokaryotic promoters\u003cbr /\u003e have indicated that promoters for the major form of \u003ci\u003eEscherichia\u003c/i\u003e\u003cbr /\u003e \u003ci\u003ecoli\u003c/i\u003e RNA polymerase (Eσ70) are composed of two conserved\u003cbr /\u003e hexanucleotide sequences, TATAAT and TTGACA, which are located\u003cbr /\u003e at 10 and 35 base-pairs, respectively, upstream of the\u003cbr /\u003e transcription initiation site, although a considerable variation\u003cbr /\u003e exists in the promoter sequence between genes within the same\u003cbr /\u003e organism. From thermodynamic and kinetic studies, these two\u003cbr /\u003e sequences are believed to determine the affinity to RNA\u003cbr /\u003e polymerase and the rate of DNA opening, altogether affecting the\u003cbr /\u003e promoter strength. However, little is known about the role of\u003cbr /\u003e individual bases within these two regions with respect to RNA\u003cbr /\u003e polymerase binding and DNA opening. In this study, I carried out\u003cbr /\u003e a systematic analysis of the relationship between the promoter\u003cbr /\u003e sequence and the promoter strength (Kobayashi, M. \u003ci\u003eet al.\u003c/i\u003e (1990)\u003cbr /\u003e Nucleic Acids Res., \u003cb\u003e18,\u003c/b\u003e 7367-7372).\u003cbr /\u003e　　A set of 18 variant ,lacUV5 promoters was constructed, each\u003cbr /\u003e carrying a single base substitution within the promoter -35\u003cbr /\u003e region (nucleotide positions from -36 to -31 relative to the\u003cbr /\u003e transcription start site). Using truncated DNA fragments\u003cbr /\u003e carrying these variant promoters and purified Escherichia coli\u003cbr /\u003e RNA polymerase holoenzyme(Eσ7O),  the in vitro mixed\u003cbr/\u003e transcription assays were performed to determine two parameters\u003cbr /\u003e governing the promoter strength, \u003ci\u003ei. e.,\u003c/i\u003e the binding affinity to\u003cbr /\u003e RNA polymerase (parameter I) and the rate of open complex\u003cbr /\u003e formation (parameter II).\u003cbr /\u003e　　Parameter I was affected to various extents, while parameter\u003cbr /\u003e II was mostly decreased except for two variant promoters, 34G\u003cbr /\u003e and 33G (the variant promoters were named according to the\u003cbr /\u003e position and base species of substitution). The 34G has a\u003cbr /\u003e sequence of TTGACA, which is completely identical with the\u003cbr /\u003e consensus sequence. The degree of change in parameter I mainly\u003cbr /\u003e depends on the position of base substitution. Base substitutions\u003cbr /\u003e at position -31 gave only a little effect; substitutions of C at\u003cbr /\u003e position -32 to any other base caused significant reduction;\u003cbr /\u003e base substitutions at position -35 also led to reduction,\u003cbr /\u003e although the effects were somewhat smaller than those of -32\u003cbr /\u003e base substitutions; the effects of base substitutions at\u003cbr /\u003e position -33, -34 and -36 were variable depending on the base\u003cbr /\u003e introduced. Among all possible sequences, TTGACA should be the\u003cbr /\u003e strongest promoter in terms of parameter I. The rate of open\u003cbr /\u003e complex formation (parameter II) was slower for most variant\u003cbr /\u003e promoters than for the reference promoter, except for the 34G\u003cbr /\u003e (consensus) and the 33G promoters. Again the promoter with the\u003cbr /\u003e consensus TTGACA sequence was the strongest.\u003cbr /\u003e　　In order to confirm these results, I next performed an\u003cbr /\u003e abortive initiation assay, in which the formation of initial\u003cbr /\u003e oligonucleotides is measured. The reaction conditions of the\u003cbr /\u003e abortive initiation assay were made identical to those of the\u003cbr /\u003e mixed transcription assay, except that ApA was added as a\u003cbr /\u003e primer, and ATP, GTP and CTP were omitted (and thus [α-32P]UTP\u003cbr /\u003e was a sole substrate). The final level indicates the binding\u003cbr /\u003e affinity to RNA polymerase (parameter I\u0027), while the reciprocal\u003cbr /\u003e of the time required for reaching plateau level represents the\u003cbr /\u003e rate of open complex formation (parameter II\u0027). The pattern of\u003cbr /\u003e the promoter strength determined by the abortive initiation\u003cbr /\u003e assay was essentially the same as that for the mixed\u003cbr /\u003e transcription assay. The degree of change in parameter I\u0027 is due\u003cbr /\u003e to both the position and species of base substitution. However,\u003cbr /\u003e all variant promoters except for 34G, displayed lower values of\u003cbr /\u003e parameter II\u0027 than the reference promoter. In the case of\u003cbr /\u003e parameter II\u0027, TTGACA was the only exception that was stronger\u003cbr /\u003e than the reference promoter, but all other base substitutions\u003cbr /\u003e resulted in marked reduction to less than half the level of the\u003cbr /\u003e reference promoter. The alteration pattern of both parameter I\u0027\u003cbr /\u003e and II\u0027, measured by the abortive initiation assay, was\u003cbr /\u003e essentially identical with that of parameter I and II determined\u003cbr /\u003e by the in vitro mixed transcription assay.\u003cbr /\u003e　　As an attempt to compare the promoter strength of the\u003cbr /\u003e  synthetic promoters measured by two in vitro assays with \u003ci\u003ein vivo\u003c/i\u003e\u003cbr /\u003e  activities, I performed β-galactosidase assay using variant\u003cbr /\u003e  lacUV5 promoter collections fused to the lacZ structural gene.\u003cbr /\u003e  The DNA fragments containing variant lacUV5 promoters were\u003cbr /\u003e  inserted between the inducible ara promoter and the lacZ,\u003cbr /\u003e  structural gene of plasmid vector pMS4342. I examined six\u003cbr /\u003e  variant promoters, which all showed unique promoter strength\u003cbr /\u003e  patterns in vitro. The promoter strength \u003ci\u003ein vivo\u003c/i\u003e was determined\u003cbr /\u003e  simply by monitoring β-galactosidase activity in the absence of\u003cbr /\u003e  arabinose. The promoter 34G was as strong as the reference, and\u003cbr /\u003e  the promoters 33G and 31T were intermediate while the others\u003cbr /\u003e  were weak (less than 25%) . When compared with the results of two\u003cbr /\u003e  in vitro transcription assays, the promoter strength \u003ci\u003ein vivo\u003c/i\u003e is\u003cbr /\u003e  in good agreement with parameter I measured by the productive\u003cbr /\u003e  initiation assay. The consensus sequence (34G) again exhibited\u003cbr /\u003e  the highest activity.\u003cbr /\u003e　　The following conclusions were drawn from the data presented:\u003cbr /\u003e  (1) Alteration in the promoter strength of variant promoters is\u003cbr /\u003e  dependent on both the position and base species of\u003cbr /\u003e  substitutions; (2) the consensus sequence (TTGACA) exhibits the\u003cbr /\u003e  highest values for both parameters; (3) base substitutions at\u003cbr /\u003e  nucleotide position -34 cause marked effect on both parameters;\u003cbr /\u003e  (4) cytosine at nucleotide position -32 cannot be replaced with\u003cbr /\u003e  other nucleotides without significant reduction of the promoter\u003cbr /\u003e  strength; (5) base substitution at nucleotide position -31\u003cbr /\u003e  exerts only a little effect on parameter I; (6) the promoter\u003cbr /\u003e  strength in vivo is in good agreement in parameter I of \u003ci\u003ein vitro\u003c/i\u003e\u003cbr /\u003e  promoter strength; and (7) the consensus sequence (TTGACA)\u003cbr /\u003e  exhibits the highest activity \u003ci\u003ein vivo\u003c/i\u003e as well as \u003ci\u003ein vitro.\u003c/i\u003e\u003cbr /\u003e　　This type of experiments has been ｄone as a collaboration\u003cbr /\u003e  research for the analysis of sequence-strength relationship of\u003cbr /\u003e  the promoter -10 region.", "subitem_description_type": "Other"}]}, "item_1_description_18": {"attribute_name": "フォーマット", "attribute_value_mlt": [{"subitem_description": "application/pdf", "subitem_description_type": "Other"}]}, "item_1_description_7": {"attribute_name": "学位記番号", "attribute_value_mlt": [{"subitem_description": "総研大甲第19号", "subitem_description_type": "Other"}]}, "item_1_select_14": {"attribute_name": "所蔵", "attribute_value_mlt": [{"subitem_select_item": "有"}]}, "item_1_select_8": {"attribute_name": "研究科", "attribute_value_mlt": [{"subitem_select_item": "生命科学研究科"}]}, "item_1_select_9": {"attribute_name": "専攻", "attribute_value_mlt": [{"subitem_select_item": "18 遺伝学専攻"}]}, "item_1_text_10": {"attribute_name": "学位授与年度", "attribute_value_mlt": [{"subitem_text_value": "1991"}]}, "item_1_text_20": {"attribute_name": "業務メモ", "attribute_value_mlt": [{"subitem_text_value": "（2018年2月19日）本籍など個人情報の記載がある旧要旨・審査要旨を個人情報のない新しいものに差し替えた。承諾書等未確認。要確認該当項目修正のこと。"}]}, "item_creator": {"attribute_name": "著者", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "KOBAYASHI, Makoto", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "9812", "nameIdentifierScheme": "WEKO"}]}]}, "item_files": {"attribute_name": "ファイル情報", "attribute_type": "file", "attribute_value_mlt": [{"accessrole": "open_date", "date": [{"dateType": "Available", "dateValue": "2016-02-17"}], "displaytype": "simple", "download_preview_message": "", "file_order": 0, "filename": "甲19_要旨.pdf", "filesize": [{"value": "349.9 kB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 349900.0, "url": {"label": "要旨・審査要旨 / Abstract, Screening Result", "url": "https://ir.soken.ac.jp/record/880/files/甲19_要旨.pdf"}, "version_id": "ad49a3da-07ec-4062-bfb4-dff7c0d7d087"}, {"accessrole": "open_date", "date": [{"dateType": "Available", "dateValue": "2016-02-17"}], "displaytype": "simple", "download_preview_message": "", "file_order": 1, "filename": "甲19_本文.pdf", "filesize": [{"value": "1.4 MB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 1400000.0, "url": {"label": "本文", "url": "https://ir.soken.ac.jp/record/880/files/甲19_本文.pdf"}, "version_id": "2d38d9c4-3a23-4d22-a08f-85dc77aff719"}]}, "item_language": {"attribute_name": "言語", "attribute_value_mlt": [{"subitem_language": "eng"}]}, "item_resource_type": {"attribute_name": "資源タイプ", "attribute_value_mlt": [{"resourcetype": "thesis", "resourceuri": "http://purl.org/coar/resource_type/c_46ec"}]}, "item_title": "大腸菌プロモーターの配列と強度の関連性", "item_titles": {"attribute_name": "タイトル", "attribute_value_mlt": [{"subitem_title": "大腸菌プロモーターの配列と強度の関連性"}, {"subitem_title": "Sequence-strength Relationship of theEscherichia coli Promoter", "subitem_title_language": "en"}]}, "item_type_id": "1", "owner": "1", "path": ["20"], "permalink_uri": "https://ir.soken.ac.jp/records/880", "pubdate": {"attribute_name": "公開日", "attribute_value": "2010-02-22"}, "publish_date": "2010-02-22", "publish_status": "0", "recid": "880", "relation": {}, "relation_version_is_last": true, "title": ["大腸菌プロモーターの配列と強度の関連性"], "weko_shared_id": 1}