JP2005230001A - Protein translation controlling factor obtained by using single-stranded dna oligomer complementary to ribosome rna base sequence, initiation factor obtained by using the single-stranded dna oligomer complementary to the ribosome rna base sequence, and protein translation controlling method using the single-stranded dna oligomer complementary to the ribosome rna base sequence - Google Patents
Protein translation controlling factor obtained by using single-stranded dna oligomer complementary to ribosome rna base sequence, initiation factor obtained by using the single-stranded dna oligomer complementary to the ribosome rna base sequence, and protein translation controlling method using the single-stranded dna oligomer complementary to the ribosome rna base sequence Download PDFInfo
- Publication number
- JP2005230001A JP2005230001A JP2004180996A JP2004180996A JP2005230001A JP 2005230001 A JP2005230001 A JP 2005230001A JP 2004180996 A JP2004180996 A JP 2004180996A JP 2004180996 A JP2004180996 A JP 2004180996A JP 2005230001 A JP2005230001 A JP 2005230001A
- Authority
- JP
- Japan
- Prior art keywords
- stranded dna
- base sequence
- dna oligomer
- rna base
- protein translation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108020004414 DNA Proteins 0.000 title claims abstract description 41
- 230000014616 translation Effects 0.000 title claims abstract description 32
- 102000053602 DNA Human genes 0.000 title claims abstract description 24
- 108020004682 Single-Stranded DNA Proteins 0.000 title claims abstract description 24
- 102000005877 Peptide Initiation Factors Human genes 0.000 title claims abstract description 23
- 108010044843 Peptide Initiation Factors Proteins 0.000 title claims abstract description 23
- 230000000295 complement effect Effects 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 14
- 108020004418 ribosomal RNA Proteins 0.000 claims description 25
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 102000034356 gene-regulatory proteins Human genes 0.000 claims 1
- 108091006104 gene-regulatory proteins Proteins 0.000 claims 1
- 238000001243 protein synthesis Methods 0.000 abstract description 9
- 238000012258 culturing Methods 0.000 abstract description 2
- 239000003755 preservative agent Substances 0.000 abstract 1
- 230000002335 preservative effect Effects 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 30
- 108020004566 Transfer RNA Proteins 0.000 description 23
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 19
- 108020004999 messenger RNA Proteins 0.000 description 17
- 108020004465 16S ribosomal RNA Proteins 0.000 description 15
- 108020004463 18S ribosomal RNA Proteins 0.000 description 14
- 241000588724 Escherichia coli Species 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 230000002062 proliferating effect Effects 0.000 description 7
- 210000003705 ribosome Anatomy 0.000 description 7
- 230000010261 cell growth Effects 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical class O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- 108020005098 Anticodon Proteins 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 108020005075 5S Ribosomal RNA Proteins 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241000206602 Eukaryota Species 0.000 description 3
- 108091081024 Start codon Proteins 0.000 description 3
- 230000005757 colony formation Effects 0.000 description 3
- 239000003636 conditioned culture medium Substances 0.000 description 3
- 108091036078 conserved sequence Proteins 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 102000002278 Ribosomal Proteins Human genes 0.000 description 2
- 108010000605 Ribosomal Proteins Proteins 0.000 description 2
- 241000269368 Xenopus laevis Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940116977 epidermal growth factor Drugs 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 2
- -1 23S rRNA Proteins 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000013446 GTP Phosphohydrolases Human genes 0.000 description 1
- 108091006109 GTPases Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000221961 Neurospora crassa Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000003923 Protein Kinase C Human genes 0.000 description 1
- 108090000315 Protein Kinase C Proteins 0.000 description 1
- 241000588767 Proteus vulgaris Species 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 101150004071 SRR1 gene Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000007244 Zea mays Nutrition 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000001035 methylating effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000033667 organ regeneration Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 125000001151 peptidyl group Chemical group 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 229940007042 proteus vulgaris Drugs 0.000 description 1
- 230000015338 rRNA methylation Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
Images
Abstract
Description
本発明は細胞のたんぱく質翻訳調節因子及びイニシエーションファクター並びに細胞のたんぱく質翻訳調節方法に関するものである。 The present invention relates to a cellular protein translation regulatory factor and initiation factor, and a cellular protein translation regulatory method.
たんぱく質翻訳はリボソームの仲介によってメッセンジャーＲＮＡ（以下、「ｍＲＮＡ」とする。）と転移ＲＮＡ（以下、「ｔＲＮＡ」とする。）の相互作用により行われる。ｔＲＮＡに備わったアンチコドンは相補的にｍＲＮＡに結合するがリボソームにも相互的に作用する。リボソームのｔＲＮＡ結合部はＰ ｓｉｔｅ（ｐｅｐｔｉｄｙｌ ｔｒｓｎｓｆｅｒａｓｅ ｃｅｎｔｅｒ）と呼ばれ、高Ｍｇ2+濃度状態ではｍＲＮＡが存在しなくても結合が起こる。Ｐ ｓｉｔｅは１６ＳリボソームＲＮＡ（以下、「１６ＳｒＲＮＡ」という。）の特定の位置の塩基であろうと考えられている。カリフォルニア大学サンタクルーズのＮｏｌｌｅｒ ＨＦらのグループはリボソームのｓｍａｌｌ ｓｕｂｕｎｉｔの一部である１６ＳｒＲＮＡの９６６番、１４０１番のグアニンをメチル化することによってＰ ｓｉｔｅにおけるｍＲＮＡとｔＲＮＡの結合を阻害することを明らかにした。また、９２６番のグアニンをメチル化すると先程の高Ｍｇ2+濃度状態でのＰ ｓｉｔｅへのｔＲＮＡ結合を阻害するようになる。これらのリボソームＲＮＡ上の位置は古くから微生物に広く塩基配列が保存された領域、すなわちａｒｃｈａｅ ｂａｃｔｅｒｉａ（古細菌）から原核生物に至るまで１６ＳｒＲＮＡに４ヶ所認められ、約２０塩基長のオリゴマーとして存在する。これらの保存領域に挟まれた数百塩基長のＲＮＡ配列は、属あるいは種として分類学上規定される特異的な菌の集団におおむね一致して同じ配列がみられる。このＲＮＡ塩基配列は実際はＤＮＡとしてコードされている。ＤＮＡにコードされたＲＮＡ塩基配列はｒｒｎオペロンと呼ばれ、二つのプロモーターの後ろに１６ＳｒＲＮＡ、ｔＲＮＡ、２３ＳｒＲＮＡ、５ＳｒＲＮＡ、ｔＲＮＡの順にスペーサー領域を介して連結している。それぞれのｔＲＮＡは二つのｔＲＮＡＧｌｕの組み合わせか、あるいはｔＲＮＡｌｌｅとｔＲＮＡＡｌａの組み合わせで存在し、この２種類のｒｒｎオペロンが数個染色体上に存在する。リボソームＲＮＡの保存領域に関しては重要な機能を担う配列が保存されているとの認識が早くからみられ、リボソームＲＮＡ上の1本鎖ＲＮＡ構造にその機能が託されていると考え、その検索がアミノアシルｔＲＮＡの解析方法に則って行われるようになった。 Protein translation is performed by the interaction of messenger RNA (hereinafter referred to as “mRNA”) and transfer RNA (hereinafter referred to as “tRNA”) through ribosome mediation. The anticodon provided in tRNA binds complementarily to mRNA, but also interacts with ribosomes. The tRNA-binding portion of the ribosome is called P site (peptidyl tsnsferase center), and binding occurs even in the absence of mRNA in a high Mg 2+ concentration state. P site is considered to be a base at a specific position of 16S ribosomal RNA (hereinafter referred to as “16S rRNA”). The University of California, Santa Cruz, Noller HF, et al. Group found that it inhibits mRNA and tRNA binding in Psite by methylating 966 and 1401 guanines of 16S rRNA, which is part of the small subunit of the ribosome. did. In addition, methylation of 926th guanine inhibits tRNA binding to P site at the high Mg 2+ concentration state. The positions on these ribosomal RNAs have been recognized in four locations in 16S rRNA from ancient times to regions where base sequences are widely conserved in microorganisms, that is, from archeae bacteria to prokaryotes, and exist as oligomers of about 20 bases in length. . The RNA sequences with a length of several hundred bases sandwiched between these conserved regions are almost identical to the specific bacterial populations that are taxonomically defined as genera or species. This RNA base sequence is actually encoded as DNA. The RNA base sequence encoded by DNA is called rrn operon, and 16S rRNA, tRNA, 23S rRNA, 5S rRNA, and tRNA are linked in this order via spacer regions behind two promoters. Each tRNA exists as a combination of two tRNA Glu or a combination of tRNA lle and tRNA Ala , and these two types of rrn operons exist on several chromosomes. Regarding the conserved region of ribosomal RNA, it was recognized early that the sequences responsible for important functions were conserved, and the function was entrusted to the single-stranded RNA structure on ribosomal RNA. It came to be performed according to the analysis method of tRNA.
ドイツのマックスブランク研究所のＥｒｄｍａｎｎ ＶＡらのグループは配列が明らかなオリゴヌクレオチドを合成し、リボソームＲＮＡとの相補的結合能をみてリボソームＲＮＡの1本鎖構造上の位置と配列を決定している。すなわちＥ．ｃｏｌｉとＢ．ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓの５ＳｒＲＮＡ配列の二次構造にみられる１本鎖領域と２本鎖領域が存在することを推定しそれを決定することを行った。彼らはあらかじめ配列のわかっているオリゴマー（３、４塩基）を合成し、常温でハイブリダイズさせ、１本鎖状態を保持している位置と配列を特定している。５ＳｒＲＮＡの約１２０塩基配列の１０番、３０番、６０番、７０番、８５番、９５番の塩基位置に１本鎖領域が認められ、二次構造が提案されている。 The Erdmann VA group at the Max Blank Institute in Germany synthesizes oligonucleotides with known sequences and determines their position and sequence on the single-stranded structure of ribosomal RNA based on their complementary binding ability to ribosomal RNA. . That is, E.I. E. coli and B.I. We estimated and determined the presence of single- and double-stranded regions in the secondary structure of stearothermophilus 5S rRNA sequence. They synthesize oligomers (3 and 4 bases) whose sequences are known in advance and hybridize at room temperature to specify the position and sequence that maintain a single-stranded state. A single-stranded region is recognized at the base positions of the 10th, 30th, 60th, 70th, 85th, and 95th positions of about 120 base sequences of 5S rRNA, and a secondary structure has been proposed.
その後ブラウン大学のＧｅｒｂｉ ＳＡ、エジンバラ大学のＣｏｘ ＲＡのグループでは、真核細胞同士のリボソームＲＮＡの比較あるいは原核細胞との比較を行って機能部位の検索を行つている。真核生物としてＸｅｎｏｐｕｓ ｌａｅｖｉｓ、Ｎｅｕｒｏｓｐｏｒａ ｃｒａｓｓａ、Ｄｒｏｓｏｐhｉｌａ ｍｅｌａｎｏｇａｓｔｅｒ、原核生物としてＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉを用い２８Ｓ、２３Ｓ、１８Ｓ（真核生物）、１６Ｓ（原核生物）を対象にしている。ここでもリボソームＲＮＡの機能領域を明確にするため進化の過程で保存された領域に着目している。制限酵素で５０〜３００塩基毎にマッピングを行いそれについて異種同士から抽出したヌクレオチドをブローブにしてＤＮＡ−ＤＮＡあるいはＤＮＡ−ＲＮＡハイブリダイゼーションを行って異種間で共通の塩基配列すなわち保存された領域を特定する作業を行っている。その結果、（１）各種間に普遍的な保存領域が認められ、（２）その保存領域はリボソームＲＮＡのＤＮＡコード領域内に認められスベーサー領域にはほとんど認められない。（３）１８ＳｒＲＮＡあるいは１６ＳｒＲＮＡの３’末端側の保存領域は生物全般に広く認められ、ｍＲＮＡの結合域Ｓｈｉｎｅ ａｎｄ Ｄａｌｇａｒｎｏ ｂｏｘを強く推定している。（４）１６Ｓ、１８Ｓ内には他にもいくつかの保存域がみつけられている。（５）またｔＲＮＡの結合域とｒＲＮＡのメチル化についての相関についても予想を立てている。 Later, Gerbi SA from Brown University and Cox RA from Edinburgh University searched for functional sites by comparing ribosomal RNA between eukaryotic cells or prokaryotic cells. Xenopus laevis, Neurospora crassa, Drosophila melanogaster as eukaryotes, Escherichia coli as prokaryotes, and 28S, 23S, 18S (eukaryotes), 16S (prokaryotes) are targeted. Here, too, attention is paid to regions conserved during the evolution process in order to clarify the functional region of ribosomal RNA. A restriction enzyme is used for mapping every 50 to 300 bases, and nucleotides extracted from different species are probed for DNA-DNA or DNA-RNA hybridization to identify a common base sequence between different species, that is, a conserved region. I am doing work. As a result, (1) a universal conserved region is recognized among various types, and (2) the conserved region is found in the DNA coding region of ribosomal RNA, and is hardly found in the baser region. (3) A conserved region on the 3 'end side of 18S rRNA or 16S rRNA is widely recognized in all living organisms, and strongly suggests the binding region Shine and Dalgarno box of mRNA. (4) Several other storage areas are found in 16S and 18S. (5) We have also predicted the correlation between the tRNA binding region and rRNA methylation.
さらにＥ ｃｏｌｉ（１９７８）、Ｐｒｏｔｅｕｓ ｖｕｌｇａｒｉｓ（１９８１）の１６ＳｒＲＮＡ、Ｚｅａ ｍａｙｓの葉緑体ｒＲＮＡ（１９８０）、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅの１８ＳｒＲＮＡ（１９８０）、Ｘｅｎｏｐｕｓ ｌａｅｖｉｓの１８ＳｒＲＮＡ（１９８１）、ヒトとマウスのミトコンドリア１２ＳｒＲＮＡ（１９８０）、Ｓ．ｃｅｒｅｖｉｓｉａｅミトコンドリア１５ＳｒＲＮＡ（１９８０）の塩基配列がほぼ同時期に発表された。また、Ｅ．ｃｏｌｉの１６ＳｒＲＮＡの二次構造が５ヶ所から提出され細菌に普遍的な二次構造として認められた。これによってペプチド結合を触媒するリボソームの機能が保存配列に存在し、二次構造上に位置が推定されることが確実になってきた。このように１９８０年代初頭になされたリボソームＲＮＡの構造と機能の研究は冒頭に述べたＮｏｌｌｅｒ ＨＦらの成果に繋がっていくことになった。 Further, E coli (1978), Proteus vulgaris (1981) 16S rRNA, Zea mays chloroplast rRNA (1980), Saccharomyces cerevisiae 18S rRNA (1980), Xenopus laevis 18S rRNA (1981), human and mouse r 80 ), S. The base sequence of cerevisiae mitochondrial 15S rRNA (1980) was published at about the same time. In addition, E.I. The secondary structure of E. coli 16S rRNA was submitted from 5 sites and recognized as a universal secondary structure in bacteria. This has ensured that the function of the ribosome that catalyzes peptide bonds exists in the conserved sequence and that the position is presumed on the secondary structure. Thus, research on the structure and function of ribosomal RNA made in the early 1980s led to the results of Noller HF et al.
リボソームＲＮＡの保存塩基配列は１６ＳｒＲＮＡ、１８ＳｒＲＮＡの３’末端部にＳｈｉｎｅ ａｎｄ Ｄａｌｇａｒｎｏ領域があるが、他にもグアニンがメチル化することによって、ｍＲＮＡ、ｔＲＮＡの結合が阻害される領域として４ヶ所、その他に４ヶ所見出されている。１９８７年の時点で発明者Ｈｏｓｈｉｎａ Ｓらはかかる機能が明らかになる前にそのうちの２ヶ所を任意に用いることで本来無菌的状態を維持している生体部位に起こる細菌感染症を普遍的に検出し、その病原体を識別する方法を提案した（特許第３１３５９０９号）。１６ＳｒＲＮＡに存在する保存的配列は各々約２０塩基あり、生体内でもこれをプライマーとする複製酵素の伸展反応があるのだろうと推定される。そこでこの共通な塩基配列の中から二つを選んでプライマーとし、いわゆる遺伝子増幅を行えば、原因菌に特異的な配列を検出可能な量だけ得られることになるのである。 The conserved nucleotide sequence of ribosomal RNA is the Sine and Dalgarno region at the 3 'end of 16SrRNA and 18SrRNA, but there are four other regions where the binding of mRNA and tRNA is inhibited by methylation of guanine. 4 places have been found. As of 1987, the inventor Hoshina S and others universally detected bacterial infections occurring in living body parts that originally maintained aseptic conditions by arbitrarily using two of these functions before they became apparent. And proposed a method for identifying the pathogen (Japanese Patent No. 3135909). There are about 20 bases of conserved sequences present in 16S rRNA, and it is presumed that there is a replication reaction of replication enzymes using this as a primer in vivo. Therefore, by selecting two of these common base sequences as primers and performing so-called gene amplification, an amount specific to the causative bacterium can be detected.
さらに、細胞のたんぱく質翻訳調節因子としては明確になっていないグロースファクターやホルモンは転写調節を行っている。グロースファクターは、例えば細胞内のリン酸がたんぱく質の受容体を刺激しその刺激が次々と他のたんぱく質に伝達されるという信号伝達が生じ細胞の分裂が促進される。ホルモンは転写調節遺伝子に直接結合しその結果細胞の分裂が促進されることがある。 In addition, growth factors and hormones that are not clearly defined as cellular protein translational regulators regulate transcription. In the growth factor, for example, intracellular phosphate stimulates a protein receptor, and the stimulation is transmitted to other proteins one after another, and cell division is promoted. Hormones may bind directly to transcriptional regulatory genes and thus promote cell division.
次にイニシエーションファクターについて述べる。１６ＳｒＲＮＡや１８ＳｒＲＮＡの塩基配列の中には細菌、真菌、植物、動物など細胞で共通の配列がみられる。特に５３０ループ、９２６倍位、１４０１倍位は塩基配列の共通する領域で二次構造、立体構造も共通した性状を示している。これらの領域は１６ＳｒＲＮＡや１８ＳｒＲＮＡのＰ ｓｉｔｅ中に存在しておりｍＲＮＡのコドンをｔＲＮＡのアンチコドンが認識するデコーディング機能にかかわる部位である。即ちリボソームのｓｍａｌｌ ｓｕｂｕｎｉｔ（１６ＳｒＲＮＡ、１８ＳｒＲＮＡとリボソームたんぱく質の複合体）にｍＲＮＡとｔＲＮＡが会合する場所である。 Next, the initiation factor will be described. Among the base sequences of 16S rRNA and 18S rRNA, common sequences are found in cells such as bacteria, fungi, plants and animals. In particular, the 530 loop, the 926-fold position, and the 1401-fold position are the regions having the same base sequence and exhibit the same secondary structure and three-dimensional structure. These regions are present in the 16S rRNA and 18S rRNA P sites, and are sites related to the decoding function in which the anticodon of tRNA recognizes the codon of mRNA. That is, it is a place where mRNA and tRNA are associated with a ribosomal small subunit (complex of 16S rRNA, 18S rRNA and ribosomal protein).
たんぱく質翻訳の開始には、第１段階としてこのＰ ｓｉｔｅ部位にホルミル化されたメチオニン−ｔＲＮＡ（ｆＭｅｔ−ｔＲＮＡiMet：ＵＡＣ）とｍＲＮＡの転写開始コドン（ＡＵＧ）が相補的に結合したものが定着する。つぎにリボソームのｌａｒｇｅ ｓｕｂｕｎｉｔ（２３ＳｒＲＮＡ、２８ＳｒＲＮＡとリボソームたんぱく質の複合体）がかぶさっていわゆるリボソーム粒子が形成される。そして、ｓｍａｌｌ ｓｕｂｕｎｉｔとｌａｒｇｅ ｓｕｂｕｎｉｔが結合した段階でペプチド鎖伸長反応が始まる。さらにリボソーム、ｔＲＮＡ複合体のｍＲＮＡに対する１コドンの移動（トランスロケーション）が引き続いて起こる。このリボソームのｓｍａｌｌ ｓｕｂｕｎｉｔ中にある５３０ループ、９２６倍位、１４０１倍位は立体構造でその位置を決めると７０〜１００Åの間に入る。リボソーム上でのたんぱく質翻訳の第１段階ではＰ ｓｉｔｅにｆＭｅｔ−ｔＲＮＡiMetが結合する反応が起こる時、ｆＭｅｔ−ｔＲＮＡiMetがＡ ｓｉｔｅに誤って結合しなく、引き続きｍＲＮＡの開始コドンＡＵＧがｆＭｅｔ−ｔＲＮＡiMetのアンチコドンと結合する。この時、ｆＭｅｔ−ｔＲＮＡiMetとＰ ｓｉｔｅの結合の後につぎのｔＲＮＡが会合してこないことがみられる。さらにｌａｒｇｅ ｓｕｂｕｎｉｔが最後まで会合してこないことなど一連の阻害反応がみられ、この現象を担う因子が存在する。 At the start of protein translation, as a first step, a formylated methionine-tRNA (fMet-tRNAi Met : UAC) and a transcription start codon (AUG) of mRNA are fixedly bound at this P site site. . Next, ribosomal large subunits (23SrRNA, 28SrRNA and ribosomal protein complex) are covered to form so-called ribosomal particles. Then, the peptide chain elongation reaction starts at the stage where the small subunit and the large subunit are combined. Furthermore, 1-codon movement (translocation) with respect to the mRNA of the ribosome and tRNA complex occurs subsequently. The 530 loop, 926-fold, and 1401-fold in the small subunit of this ribosome fall within the range of 70 to 100 mm when the position is determined by the three-dimensional structure. When the reaction to be combined fMet-tRNAi Met to the P site occurs in the first stage of protein translation on the ribosome, fMet-tRNAi Met is not bound by mistake to the A site, it will continue to start codon AUG of the mRNA fMet-tRNAi Binds to Met anticodon. At this time, it can be seen that the next tRNA does not associate after the binding of fMet-tRNAi Met and Psite. Furthermore, a series of inhibitory reactions such as the fact that large subunits do not associate to the end are observed, and there are factors responsible for this phenomenon.
この現象のそれぞれを役割分担してたんぱく質翻訳を開始し、たんぱく質翻訳が順調に正確に進むための働きをしている因子をイニシエーションファクター（Ｉｎｉｔｉａｔｉｏｎ Ｆａｃｔｏｒ、ＩＦ）という。イニシエーションファクターには原核生物ではＩＦ１、ＩＦ２、ＩＦ３の３種類があり、このうちＩＦ２はＧＴＰを脱リン酸化するＧＴＰａｓｅである。また、ＩＦ２はたんぱく質翻訳の第１段階で働きｆＭｅｔ−ｔＲＮＡiMetがｍＲＮＡのＡＵＧに結合する際に関与する。真核生物ではイニシエーションファクターとしてｅＩＦ２がＩＦ２と同様の働きをしている。ＩＦ１はリボソームｓｍａｌｌ ｓｕｂｕｎｉｔに最初に結合してｆＭｅｔ−ｔＲＮＡiMetがＰ ｓｉｔｅに結合するための空間を提供し、ＩＦ３はｌａｒｇｅ ｓｕｂｕｎｉｔが結合するのを防ぐ「くさび」の役をしていると考えられるが、ＩＦ１とＩＦ３はまだ本体が明らかになっていない。
新規のたんぱく質翻訳調節因子及び新規のイニシエーションファクター並びに新規のたんぱく質翻訳調節方法を提供する。 Provided are a novel protein translational regulator and a novel initiation factor and a novel protein translational regulation method.
リボソームＲＮＡに存在する保存的配列を合成した１本鎖ＤＮＡオリゴマーを細胞内に取込ませると増殖性細胞がさらに増殖することが見出された。 It was found that proliferating cells proliferate further when single-stranded DNA oligomers synthesized with conserved sequences present in ribosomal RNA were incorporated into the cells.
本発明によれば、例えば、細胞を用いて有用たんぱく質を多量に生産できる。再生医療で組織細胞を増殖させる場合に上皮細胞増殖因子（Ｅｐｉｄｅｒｍａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ）と本発明であるオリゴマーＤＮＡを加えると細胞が良く増殖し、必要な臓器再生技術を早期に得られる。あるいは免疫細胞に本発明であるオリゴマーＤＮＡを与えれば、免疫細胞のたんばく質合成能力を制御でき、免疫機能をコントロールでき、免疫能を賦活できる。あるいは、がん細胞に本発明であるオリゴマーＤＮＡを与えると、がん細胞のたんばく質合成能力を制御でき、がん細胞を殺せる可能性がある。細菌に本発明であるオリゴマーＤＮＡを与えれば抗生物質のように使える可能性がある。細菌の１６ＳリボソームＲＮＡだけに存在し、ヒトや植物の１８ＳリボソームＲＮＡに無いオリゴマー配列が認められているので、識別して使うことができる。さらに、イニシエーションファクターとして使用することにより、より効果的に細胞のたんぱく質合成能力を制御できる。 According to the present invention, for example, useful proteins can be produced in large quantities using cells. When tissue cells are proliferated by regenerative medicine, epidermal growth factor and the oligomeric DNA of the present invention are added, so that the cells proliferate well and necessary organ regeneration techniques can be obtained at an early stage. Alternatively, when the oligomeric DNA of the present invention is given to immune cells, the protein synthesis ability of immune cells can be controlled, the immune function can be controlled, and the immune ability can be activated. Alternatively, when the oligomeric DNA of the present invention is given to a cancer cell, the protein synthesis ability of the cancer cell can be controlled and the cancer cell may be killed. If the oligomeric DNA of the present invention is given to bacteria, it may be used like an antibiotic. Since oligomer sequences that are present only in bacterial 16S ribosomal RNA and not in human or plant 18S ribosomal RNA are recognized, they can be identified and used. Furthermore, by using it as an initiation factor, the protein synthesis ability of cells can be controlled more effectively.
図１はＥ．ｃｏｌｉの１６ＳｒＲＮＡの塩基配列図である。表１に示すようにＥ．ｃｏｌｉのリボソームの保存領域のうち１６ＳＲＲＵ１（５’ＣＡＧＣＡＧＣＣＧＣＧＧＴＡＡＴＡＣ３’（配列番号１））と人の１８ＳｒＲＮＡとラットの１８ＳｒＲＮＡとの一致率は１００％であり、Ｅ．ｃｏｌｉのリボソームの保存領域のうち１６ＳＲＲＵ５（ＧＣＡＣＡＣＡＣＣＧＣＣＣＧＴ（配列番号３））と人の１８ＳｒＲＮＡとの一致率が９３％、ラットの１８ＳｒＲＮＡとの一致率が１００％であり、Ｅ．ｃｏｌｉリボソームの保存領域のうち１６ＳＲＲＵ２（ＣＣＧＴＣＡＡＴＴＣＣＴＴＴＧＡＧＴＴＴ（配列番号３））と人の１８ＳｒＲＮＡとラットの１８ＳｒＲＮＡとの一致率が９５％であるようにＥ．ｃｏｌｉや高等の哺乳類の間で進化の進み具合に差があるにも関わらず共通の遺伝子配列を持つものがある。
リボソーム塩基配列の保存領域はたんぱく質合成機能を行う場の領域とされ、リボソーム立体構造では１本鎖で存在することが徐々に明らかになってきた。ラット繊維芽細胞Ｒａｔ６の１８ＳｒＲＮＡ塩基配列に相補的なＤＮＡ塩基配列を３つ合成した。３つのＤＮＡオリゴマーは、ＳＲＲ１１（５’ＣＡＧＣＡＧＣＣＧＣＧＧＴＡＡＴＡＣ３’（配列番号１））、ＳＲＲ２２（ＣＣＧＴＣＡＡＴＴＣＣＴＴＴＧＡＧＴＴＴ（配列番号２））、ＳＲＲ５３（ＧＣＡＣＡＣＡＣＣＧＣＣＣＧＴ（配列番号３））である。 The conserved region of the ribosome base sequence is regarded as a region for performing protein synthesis, and it has gradually become clear that it exists as a single strand in the ribosome conformation. Three DNA base sequences complementary to the 18S rRNA base sequence of rat fibroblast Rat 6 were synthesized. The three DNA oligomers are SRR11 (5'CAGCAGCCGCCGTAATAC3 '(SEQ ID NO: 1)), SRR22 (CCGTCAATTCCCTTTGAGTTT (SEQ ID NO: 2)), SRR53 (GCACACACCGCCCGT (SEQ ID NO: 3)).
ラット線維芽細胞Ｒａｔ６を９６ウエル培養シャーレで上皮細胞増殖因子（以下、「ＥＧＦ」という。）加ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ）を入れて２週間培養した。その後たんぱく質合成機能領域のＤＮＡオリゴマーＳＲＲ１１とＳＲＲ２２の１０ｎｇ／ｍＬを上記ＥＧＦ（１０ｎｇ／ｍＬ）添加ＤＭＥＭに加えてさらに培養を３週間続けた。ＥＧＦを加えたがＤＮＡを加えていないＲａｔ６細胞を（あ）ケース、ＥＧＦを加えＤＮＡを加えたものを（い）ケースとする。これらの条件培地に馴化したＲａｔ６細胞を一般に使われる１０％子牛血清（ＣＳ）とＥＧＦ（１０ｎｇ／ｍＬ）を加えていない培地（以下、「Ａケース」という。）、一般に使われる１０％子牛血清（ＣＳ）を加えＥＧＦ（１０ｎｇ／ｍＬ）を加えていない培地（以下、「Ｂケース」という。）、ＤＮＡオリゴマーを除いたＥＧＦ（１０ｎｇ／ｍＬ）を加えた培地（以下、「Ｃケース」という。）に戻して培養を１週間から３週間行った。培地交換はその間３から４日ごとに適宜行った。その結果を表２に示す。
（あ）ケースで培養したＲａｔ６細胞をＡケースの培地に置き換えると１１個のコロニーを形成した。Ｂケースに置き換えた場合ではコロニー数は６１個に増加した。Ｃケースで培養を行うとコロニー数は１７０個であった。一方、（い）ケースで培養を行ったＲａｔ６細胞はＢケースに置き換えた場合コロニー数は７７個であった。Ｃケースではコロニー数は８３２個であった。
（あ）ケースで培養をし続けた場合に比べ、それにオリゴマーＤＮＡを添加して培養を続けた（い）ケースのＲａｔ６細胞では、Ｂケースの標準培地に戻したものではコロニー数はわずかに増加したが、Ｃケースに戻した場合ではコロニー数が強く増加した。また、ＳＲＲ１１の代わりにＳＲＲ５３を加えて同様の実験を試みたところ細胞増殖能が認められた。
(A) When the Rat6 cells cultured in the case were replaced with the medium in the A case, 11 colonies were formed. When replaced with B case, the number of colonies increased to 61. When culture was performed in C case, the number of colonies was 170. On the other hand, when Rat6 cells cultured in (i) case were replaced with B case, the number of colonies was 77. In C case, the number of colonies was 832.
(A) Compared to the case of continuing culture in the case, the number of colonies slightly increased in the case of Rat6 cells in which the oligomer DNA was added and continued in the case (i) in the case of returning to the standard medium of B case However, when it was returned to C case, the number of colonies increased strongly. Moreover, when SRR53 was added instead of SRR11 and the same experiment was tried, the cell growth ability was recognized.
以下参考に詳細な実験経過を記載する。第１日（１１月９日）Ｒａｔ６細胞を約２週間ＥＧＦで培養する。第１３日（１１月２２日）Ｒａｔ６細胞をＥＧＦ（１０ｎｇ／ｍＬ）のみとＥＧＦ（１０ｎｇ／ｍＬ）とオリゴＤＮＡ（ＳＲＲ１１＋ＳＲＲ２２）（１０ｎｇ／ｍＬ）を添加した条件培地に交換。Ｒａｔ６細胞は正常発育。第１８日（１１月２７日）ＥＧＦ（２０ｎｇ／ｍＬ）を増量。第２２日（１２月１日）ＥＧＦ（１０ｎｇ／ｍＬ）に減量。Ｒａｔ６細胞の発育は正常を維持している。第２６日（１２月５日）ＥＧＦ＋オリゴＤＮＡ（ＳＲＲ１１＋ＳＲＲ２２）を添加した培地でのＲａｔ６細胞の発育が増殖性に変換し、コロニー形成が見られる。ＥＧＦのみを添加した培地でのＲａｔ６細胞の発育が増殖性に変換し、コロニー形成が見られる。また、ＳＲＲ２２＋ＳＲＲ５３（ＧＣＡＣＡＣＡＣＣＧＣＣＣＧＴ）を添加した系でもＲａｔ６細胞の発育が増殖性を示した。特にＳＲＲ１１＋ＳＲＲ２２を添加した場合の細胞増殖能が最も強力であった。第２６日（１２月５日）９６ウェルプレートにそれらのコロニーを選び、細胞を１×１０５ｃｅｌｌ／ｗｅｌｌづつ軟寒天培地に植え継ぐ。Ａ：上層を０．３％寒天にＥＧＦのみ添加、Ｂ：上層を０．３％寒天にＥＧＦとオリゴＤＮＡ（ＳＲＲ１１、ＳＲＲ２２）を添加、下層はすべて０．５％寒天で何も添加せず。第３３日（１２月１２日）９６ウェルプレートにそれらのコロニーを選び、細胞を８×１０３／ｗｅｌｌづつ接種。条件培地は１０％ＣＳとＥＧＦ（１０ｎｇ／ｍＬ）をそれぞれ添加した培地に戻した。第３９日（１２月１８日）増殖性細胞に一般に見られるフォーカス形成とコロニー形成が見られた。第５４日（１月２日）結果観察した。 The detailed experimental process is described below for reference. Day 1 (November 9) Rat6 cells are cultured in EGF for about 2 weeks. Day 13 (November 22) Rat6 cells were replaced with conditioned medium supplemented with EGF (10 ng / mL) alone, EGF (10 ng / mL) and oligo DNA (SRR11 + SRR22) (10 ng / mL). Rat6 cells develop normally. Day 18 (November 27) EGF (20 ng / mL) increased. Day 22 (December 1) Reduced to EGF (10 ng / mL). Rat6 cell growth remains normal. Day 26 (December 5) Rat6 cell growth in a medium supplemented with EGF + oligo DNA (SRR11 + SRR22) is converted to proliferative and colony formation is observed. Rat6 cell growth in a medium supplemented with only EGF is converted to proliferative and colony formation is observed. In addition, the growth of Rat6 cells showed proliferative properties even in a system to which SRR22 + SRR53 (GCACACACCGCCCGT) was added. In particular, the cell growth ability when SRR11 + SRR22 was added was the strongest. Day 26 (December 5) Pick those colonies into 96-well plates and inoculate the cells on soft agar at 1 × 10 5 cells / well. A: Only EGF is added to 0.3% agar for the upper layer, B: EGF and oligo DNA (SRR11, SRR22) are added to 0.3% agar for the upper layer, and nothing is added to the lower layer with 0.5% agar. . On day 33 (December 12), select those colonies in 96-well plates and inoculate the cells with 8 × 10 3 / well. The conditioned medium was returned to a medium supplemented with 10% CS and EGF (10 ng / mL). On day 39 (December 18), focus formation and colony formation generally seen in proliferating cells were observed. On day 54 (January 2), the results were observed.
実験ではＤＮＡオリゴマーＳＲＲ２２を加えた場合にコロニー数が最も増加したことから、ＳＲＲ２２の塩基配列の位置がたんぱく質翻訳の主たる場を提供していると考えられる。リボソームＲＮＡの１本鎖領域で塩基配列が進化の過程で保存された部位に（ＳＲＲ２２にはＴが多く見られる）ｍＲＮＡのポリＡテールが結合する。そこで１本鎖ＤＮＡオリゴマーＳＲＲ２２をＤＮＡ合成して細胞に与えると、フリーのｍＲＮＡが結合しそれがリボソームＲＮＡのＳＲＲ２２部位に結合しやすくなる。ｍＲＮＡとｒＲＮＡの結合する速度を加速して触媒反応を行うと推測される。１本鎖ＤＮＡが化学物質としてたんぱく質合成やリボソームの働きについて触媒のように寄与していると推測される。本発明では１本鎖ＤＮＡオリゴマーによるたんぱく質翻訳調節因子を提供する。また、本発明では細胞に本発明である１本鎖ＤＮＡオリゴマーを細胞内に取込ませ細胞を培養したんぱく質翻訳調節する方法を提供する。 In the experiment, when the DNA oligomer SRR22 was added, the number of colonies increased most, so it is considered that the position of the base sequence of SRR22 provides the main field for protein translation. The poly A tail of mRNA binds to a site where the base sequence is conserved in the process of evolution in the single-stranded region of ribosomal RNA (SRR22 has many Ts). Therefore, when the single-stranded DNA oligomer SRR22 is synthesized and given to cells, free mRNA is bound and it is easy to bind to the SRR22 site of ribosomal RNA. It is presumed that the catalytic reaction is carried out by accelerating the binding speed of mRNA and rRNA. It is speculated that single-stranded DNA contributes like a catalyst to protein synthesis and ribosome function as a chemical substance. The present invention provides a protein translational regulator using a single-stranded DNA oligomer. The present invention also provides a method for regulating protein translation by culturing cells by incorporating the single-stranded DNA oligomer of the present invention into the cells.
さらに、イニシエーションファクターとしてのリボソームＲＮＡ塩基配列に相補的な一本鎖ＤＮＡオリゴマーについて述べる。イニシエーションファクターは、たんぱく質合成において、Ｐ ｓｉｔｅにｆＭｅｔ−ｔＲＮＡiMetが結合する反応が起こる時、ｆＭｅｔ−ｔＲＮＡiMetがＡ ｓｉｔｅに誤って結合しなく、引き続きｍＲＮＡの開始コドンＡＵＧがｆＭｅｔ−ｔＲＮＡiMetのアンチコドンと結合するなどたんぱく質翻訳の開始、たんぱく質翻訳が順調に正確に進むための働きをしている。そして、本発明によるリボソームＲＮＡ塩基配列に相補的な一本鎖ＤＮＡオリゴマーはイニシエーションファクターとして作用しｆＭｅｔ−ｔＲＮＡiMetをＰ ｓｉｔｅに誘引する働きなどたんぱく質翻訳の開始、たんぱく質翻訳が順調に正確に進むための働をするため急激なたんぱく質合成を促し、その結果急激な細胞の増殖に至ったと考えられる。本発明ではイニシエーションファクターとしてのリボソームＲＮＡ塩基配列に相補的な一本鎖ＤＮＡオリゴマーを提供する。 Furthermore, a single-stranded DNA oligomer complementary to the ribosomal RNA base sequence as an initiation factor is described. Initiation factor in protein synthesis, when the reaction occurs to bind the fMet-tRNAi Met to P site, fMet-tRNAi Met is not bound by mistake A site, subsequently initiation codon AUG of the mRNA is fMet-tRNAi Met anticodon It works for the start of protein translation such as binding to the protein, and for protein translation to proceed smoothly and accurately. Since the single-stranded DNA oligomer complementary to the ribosomal RNA base sequence according to the present invention acts as an initiation factor, the protein translation starts and protein translation proceeds smoothly and accurately, such as the function of attracting fMet-tRNAi Met to Psite. It is thought that rapid protein synthesis was promoted because of the action of this, resulting in rapid cell proliferation. The present invention provides a single-stranded DNA oligomer complementary to a ribosomal RNA base sequence as an initiation factor.
最後に、イニシエーションファクターの働きをもったリボソームＲＮＡ塩基配列に相補的な一本鎖ＤＮＡオリゴマーについて述べる。リボソームＲＮＡ塩基配列に相補的な一本鎖ＤＮＡオリゴマーには細胞を急激に増殖させる働きがあるがイニシエーションファクターそのものではない可能性が科学上ある。しかし、イニシエーションファクターと同様ないしイニシエーションファクターに匹敵する働きを有することから、本発明では、イニシエーションファクターの働きをもったリボソームＲＮＡ塩基配列に相補的な一本鎖ＤＮＡオリゴマーを提供する。 Finally, a single-stranded DNA oligomer complementary to the ribosomal RNA base sequence having the function of an initiation factor is described. There is a possibility that a single-stranded DNA oligomer complementary to a ribosomal RNA base sequence has a function of rapidly proliferating cells but is not an initiation factor itself. However, since it has a function similar to or comparable to the initiation factor, the present invention provides a single-stranded DNA oligomer complementary to the ribosomal RNA base sequence having the function of the initiation factor.
新たに細胞内でたんぱく質を多量に生産させることができ医学的実験や治験に貢献し、さらには細胞を増殖させたり増殖を制限させるなどすることで、がん細胞のたんぱく質合成能力を制御できる可能性があるなど新規の医薬品の開発に繋がる。 It is possible to newly produce a large amount of protein in the cell, contribute to medical experiments and clinical trials, and further control the ability of cancer cells to synthesize proteins by proliferating or limiting the growth of cells. This leads to the development of new medicines.
１ ＳＲＲ１
２ ＳＲＲ２
３ ＳＲＲ５
1 SRR1
2 SRR2
3 SRR5
Claims (6)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2004180996A JP2005230001A (en) | 2004-01-20 | 2004-06-18 | Protein translation controlling factor obtained by using single-stranded dna oligomer complementary to ribosome rna base sequence, initiation factor obtained by using the single-stranded dna oligomer complementary to the ribosome rna base sequence, and protein translation controlling method using the single-stranded dna oligomer complementary to the ribosome rna base sequence |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2004012053 | 2004-01-20 | ||
| JP2004180996A JP2005230001A (en) | 2004-01-20 | 2004-06-18 | Protein translation controlling factor obtained by using single-stranded dna oligomer complementary to ribosome rna base sequence, initiation factor obtained by using the single-stranded dna oligomer complementary to the ribosome rna base sequence, and protein translation controlling method using the single-stranded dna oligomer complementary to the ribosome rna base sequence |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2005230001A true JP2005230001A (en) | 2005-09-02 |
Family
ID=35013583
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2004180996A Pending JP2005230001A (en) | 2004-01-20 | 2004-06-18 | Protein translation controlling factor obtained by using single-stranded dna oligomer complementary to ribosome rna base sequence, initiation factor obtained by using the single-stranded dna oligomer complementary to the ribosome rna base sequence, and protein translation controlling method using the single-stranded dna oligomer complementary to the ribosome rna base sequence |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2005230001A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006311830A (en) * | 2005-05-09 | 2006-11-16 | Sadayori Hoshina | Regulatory factor of genetic information expression using single-stranded oligonucleotide complementary to ribosome rna base sequence, and method for regulating genetic information expression |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63289A (en) * | 1986-03-27 | 1988-01-05 | モンサント カンパニ− | Increase in protein production using novel liposome bonding area in bacteria |
| EP0502271A1 (en) * | 1989-04-17 | 1992-09-09 | The Standard Oil Company | 16s rRNA oligonucleotide probes for the identification of sulfate-reducing bacteria |
| JPH07289295A (en) * | 1994-04-28 | 1995-11-07 | Kirin Brewery Co Ltd | Method for detecting or identifying lactic acid bacteria |
| JPH10327859A (en) * | 1997-05-09 | 1998-12-15 | Hoechst Res & Technol Deutsche Gmbh & Co Kg | Control of translation start in eukaryotic cell |
| WO2003018750A2 (en) * | 2001-08-22 | 2003-03-06 | Isis Pharmaceuticals, Inc. | Molecular interaction sites of 23s ribosomal rna and methods of use |
| JP2003265199A (en) * | 2002-03-13 | 2003-09-24 | Ncimb Japan Co Ltd | Method for analyzing flora by using t-rflp |
-
2004
- 2004-06-18 JP JP2004180996A patent/JP2005230001A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63289A (en) * | 1986-03-27 | 1988-01-05 | モンサント カンパニ− | Increase in protein production using novel liposome bonding area in bacteria |
| EP0502271A1 (en) * | 1989-04-17 | 1992-09-09 | The Standard Oil Company | 16s rRNA oligonucleotide probes for the identification of sulfate-reducing bacteria |
| JPH07289295A (en) * | 1994-04-28 | 1995-11-07 | Kirin Brewery Co Ltd | Method for detecting or identifying lactic acid bacteria |
| JPH10327859A (en) * | 1997-05-09 | 1998-12-15 | Hoechst Res & Technol Deutsche Gmbh & Co Kg | Control of translation start in eukaryotic cell |
| WO2003018750A2 (en) * | 2001-08-22 | 2003-03-06 | Isis Pharmaceuticals, Inc. | Molecular interaction sites of 23s ribosomal rna and methods of use |
| JP2003265199A (en) * | 2002-03-13 | 2003-09-24 | Ncimb Japan Co Ltd | Method for analyzing flora by using t-rflp |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006311830A (en) * | 2005-05-09 | 2006-11-16 | Sadayori Hoshina | Regulatory factor of genetic information expression using single-stranded oligonucleotide complementary to ribosome rna base sequence, and method for regulating genetic information expression |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hajnsdorf et al. | Multiple activities of RNA-binding proteins S1 and Hfq | |
| Wood et al. | Nucleotide sequence of the Rickettsia prowazekii citrate synthase gene | |
| Burton et al. | Three divergent mitochondrial genomes from California populations of the copepod Tigriopus californicus | |
| WO2017043656A1 (en) | Method for converting genome sequence of gram-positive bacterium by specifically converting nucleic acid base of targeted dna sequence, and molecular complex used in same | |
| TWI893012B (en) | Eukaryotic semi-synthetic organisms | |
| Clifton et al. | Evolutionary repair reveals an unexpected role of the tRNA modification m1G37 in aminoacylation | |
| Lange et al. | Genome-wide analysis of growth phase-dependent translational and transcriptional regulation in halophilic archaea | |
| JP5867586B2 (en) | Hydrocarbon synthase gene and use thereof | |
| Stantial et al. | Transcription-coupled repair of UV damage in the halophilic archaea | |
| JP3972106B2 (en) | Genome library production method and genome library produced by the same method | |
| US20190218533A1 (en) | Genome-Scale Engineering of Cells with Single Nucleotide Precision | |
| Lax et al. | Symmetric adenine methylation is an essential DNA modification in the early-diverging fungus Rhizopus microsporus | |
| JP2005230001A (en) | Protein translation controlling factor obtained by using single-stranded dna oligomer complementary to ribosome rna base sequence, initiation factor obtained by using the single-stranded dna oligomer complementary to the ribosome rna base sequence, and protein translation controlling method using the single-stranded dna oligomer complementary to the ribosome rna base sequence | |
| Chen et al. | Zorbamycin has a different DNA sequence selectivity compared with bleomycin and analogues | |
| Gruegelsiepe et al. | Antisense inhibition of Escherichia coli RNase P RNA: mechanistic aspects | |
| JPWO2018174097A1 (en) | A method for converting a nucleic acid sequence of a cell, which specifically converts a nucleobase of DNA targeted by using an endogenous DNA modifying enzyme in a cell, and a molecular complex used therefor | |
| JP2022513599A (en) | Auxotrophy strain of Staphylococcus bacterium | |
| Ohyama et al. | Gene content, organization and molecular evolution of plant organellar genomes and sex chromosomes—insights from the case of the liverwort Marchantia polymorpha | |
| KR101406153B1 (en) | Method for synthesis of gene using serial oligonucleotide primers | |
| Alqahtani et al. | CRISPR-Cas: Effectors, mechanism, and classification | |
| Verma et al. | MoxT toxin of Bacillus anthracis exhibits sequence specific ribonuclease activity | |
| WO2002024950A2 (en) | Methods and means of rna analysis | |
| Schmidt et al. | Epigenetic control of tetrapyrrole biosynthesis by m4C DNA methylation in a cyanobacterium | |
| JP4793842B2 (en) | DNA amplification method using random RNA primer | |
| Xie et al. | Multi-copy single-stranded DNA in Escherichia coli |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20070228 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20091221 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20100217 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20100324 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20100623 |
|
| A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20100705 |
|
| A912 | Re-examination (zenchi) completed and case transferred to appeal board |
Free format text: JAPANESE INTERMEDIATE CODE: A912 Effective date: 20100903 |