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In order to satisfy and accelerate such expectation, so called “Millennium Project ” has been established by the Japanese government in collaboration with four ministries and an agency with a budget of 20 billion dollars in total by the year of 2005. Considering significance of genome information derived from human cDNA, a new project, supported by the supplementary budget, "Full-length human cDNA Sequencing" has started. This project aimed at not only determining about 10,000 independent full-length or long human cDNA clones isolated at Japanese research institutes, but also analyzing partially their functions. The project formation including member companies and collaborating research institutes which also supply clones to be analyzed are summarized in the figure 1. The member companies were supplied with clones from Institute of Medical Science, The University of Tokyo (IMS) and Helix Research Institute Inc. (Helix), and these teams determined entire base sequence from the libraries of full-length human cDNA prepared by oligo- capping method developed by Maruyama & Sugano. A team of Kazusa DNA Research Institute (Kazusa) determined partial sequence of mostly 4-5kbp-long cDNAs, and also determined entire sequence of some of their selected clones by using shotgun-cloning method. Sequence data obtained were analyzed by homology search, and translated into amino acid coding sequences where applicable, and then analyzed protein functions using prediction programs such as SOSui. A large comprehensive cDNA database was constructed to analyze full-length cDNA sequences for functional annotation. Concerning analysis of gene functions, expression profile was quantitatively examined by ATAC-PCR method developed by Kato, and database was constructed. In order to construct integrated gene expression profile, we also established technologies to manufacture cDNA microarray for high throughput assay. It is important to analyze relationship between disease and its relevant genes, and we tried in silico chromosonal mapping implementing genetic information database. Moreover, as a follow-up of this project, we have organized a consortium to utilize the outcome of the project, aiming at protection of intellectual property of cDNA information obtained, such as for example, through patent application with some functional analysis. In this Project, 9,123 clones of full-length human cNDA sequence were determined and 2,000 partial sequence data were also determined. The results of sequence analysis by member companies were summarized in Table 1. Total 7,604 clones of full-length sequencing data were disclosed to the consortium members during a period from October, 1999 to April, 2000. Full- length cDNA sequences of 2,212 human genes were published in the DNA Data Bank of Japan (DDBJ; an international database located in the National Institute of Genetics) on February 22nd, 2000, which may contribute to the progress of international genome research. -3- MITI / Agency of Industrial Science and Technology NEDO Technology Research Association for Biotechnology Committee Full-length cDNA Sequence and Long cDNA Sequence and Functional Analysis Functional Analysis Institute of Medical Science. University of Tokyo Helix Research Institute. Kazusa DNA Research Institute • Kyowa Hakko Kogyo Co., Ltd. • Otsuka Pharmaceutical Co., Ltd. • Hitachi, Ltd. •Aisin Cosmos R&D Co., Ltd. • Mitsui Knowledge Industry Co., Ltd. • Hitachi Science Systems, Ltd. • Nisshinbo Industries, Inc. •UNITECH. Co., Ltd. ■Daiichi Pharmaceutical Co., Ltd. I (Joint Research) •Takara Shuzo Co., Ltd. • Graduate School of biological • Hitachi Software Engineering Sciences, Nara Institute of Science and Technology Figure 1. Full-length human cDNA Sequencing Project < Project Formation) Table 1. Results of cDNA Sequencing of member companies Total : 9071 clones Long cDNA Full-length cDNA : 8951 : 1 20 Team Kazusa DNA Helix Research Inst : Inst. Medical Science : Research 5001 3950 Inst. : 1 20 Kyowa Otsuka Hitachi Member Takara Nisshinbo Hakko Pharmac Fujiya Hitachi Science Aisin Cosmos Companies Shuzo Industries Kogyo eutical Systems H7$t 1928 965 1057 2501 500 1500 500 120 -4 2. %%%# (*). xm&m (*) ZL--T*y* (*) % (*) BSSMtar fob: h cDNAY/A (#) B3[^x>XyXfAX (*). $£i£ (#) - =lX^EX9F%3r yy yy ^--7T y y x& m2 / w y y n -5- 3. flF3e»BteJ:Vfia m 9 E I* s i 5! 1 1 1 . Ttfe* c DNAE?IJS:$St>**€^-togtl(D tto#pisro 2.9 e.±M c DNAE^iJSrSSUiSe^-roS^ro 3. DNAE^IJ^S (#F-S 4. DNABaM*g&m#Gfm4W rL-—x y y# 5. cDNAT-?-^-^m% 6.4*cDNA ro$XSS?lJft$»V*«gE-6- c DNAr—9 —/<—H (# 0 VzSfEBfr 7. A# c DN 8. FcDNAy/Aj|f|f 0 9. ^cDNA©tWfe cDNA#*& SfiiSW MDNA^^nTKfl 1 0. DNA-v^DTWdttSiie^-tSilgro 0iz:yy 1 1. ## c DN A(Dmm@E9iJ%S&«:#### 1 2 . ftE C D N A l 3. ft-&pS#WF9E su / n — 6 — SB 1 * J$*roe$ 1. 1 1. fc hy/AWty^- (f 0f?e0r!S >>-f A-^WSe® ^(*#+577,677? n-yo^-y ^7“ cDNA»5'**ga?ii^fc t e, &- 3- K-f 5 "TBEttOiSiV'cDNA? D — y 5. ilft Sn/c-S(7)*ffl®exicSI LT, £ b i i t)K, Zhb 6. ^*SiM 5r^£LfccDNA?D-yJ81 L, (%# L t. Full-length cDNA sequencing and functional prediction of gene products. 1. As the candidates for analysis of the full-insertion sequences of human cDNA clones, 8,143 clones were selected by analyzing their S'-terminal sequences with bioinformatics approaches from 77,677 clones of the cDNA collections in Institute of Medical Sciences, University of Tokyo, which were collected from cDNA libraries constructed by the oligo-capping method. 2. Among the selected clones, 4,957 kinds of cDNA clones were distributed to the sequencing teams, Fujiya Bioscience Institute, Otsuka pharmaceutical, and Kyowa Hakko Kogyo. 3. After confirming the identity of the terminal sequence of each clone with that used for the clone selection, the full-insertion sequences of 1,928 clones were determined. 4. Homology analysis, prediction and evaluation of the coding sequences, evaluation of the full-rates, and prediction of signal sequences and transmembrane domains were performed in 3,950 clones. 5. Further analyses in their expression profiles and functions were performed in a part of the clones. 6. These cDNA clones was collected and stored in containers for future studies. -7- 1 . 2 mmmm, ie #¥#%#, ies7ji, j^ws, mmm&Qm&znft h h^±#cDNA7< 7 7 U — b7jM $ tlfc^^:5cDNA7 □ — > 0 £t£SE7J £ 77< 7 —7 j: D^^b/co biSlTH,f^>tl7°7X$HgiJt{ligI (Genesis Workstation) \Z £ 0 7° 7 X ^ HDNA^iHl, '> — 7 X/XKS * tA, ¥ (CPE-ABltt®! 07 ^ t: 7 U “ MDNA '>— 7X/f“ (ABI3700) CT« E$E7J£#tSbfco ftEte, EteEE26.5%, ###E f*J&3.0%, IE^/MltoEl7.8%> IES¥JI§3.2%, E^$bE¥^® 8.0%, #38.8%7^6o ¥ cDN AE 6 M 7 E b 7 2kbp <£ 0 l,926bp7& o fco 70 7 t>807 ^ / ^b(70ORF#b^l/2 t 0^1±#:068%7$) o 7. BMJT'te. ¥^fl shies/mi, s^mm¥#m, mm, m, jes/ess EE(DJE^S< , -en^'ni,327bp, l,481bp, l,560bp, l,559bp, 2,059bp, 2,187bp, 2,355bp tt£~D T^&o 5'-###R#%0#$ ^¥^349bp, 3'-###R#%0#$ ^¥^805bp7$)O fc0 ORF0¥*9E6te798bp (S>£ 7 > A° 7 RS2667 ^ / ^g)7$>6. ±###7H, GenBank, UniGene, GENSEQ& ESt7—7 A —X thy b b/clSc^^#:(7)E72%tcE ^7^0 ftTz b, b 0±BB#7j:##8 0Bfl7£7 7—7& ^#:(D^46%(Cf8^T^>o 75%tegenomicE7Jfi¥0 &0, /^TSDNAE^ l^# ##(X7 b <5 KIAA7 D — >43 fg # tE ft] 0 unknown-cDNAE 7J 7 $> <5 „ b 4l b 0 E 7J tiESTth M#(b7:g;^####M%$4l7b^b7 3->7&6. B D 025%^S^Jf7S 0#, f-7^-Xt:Si$tl/;^D“>l:i^t?)o h hESTx-7 te$tiEl687fr (1999.12.17$^) Ii$n7^7 b hSfK705^67S7E^7;5 bSt)tL7V^o 70<£7&¥7££:3iiJ*i^0ESTb t: y hb^t^0^l>§b(hit b07 -8- Full-length cDNA sequencing and functional prediction of gene products Otsuka Pharmaceuticals sequenced full-length cDNAs developed by Institute of Medical Science in The University of Tokyo. We used primer-walking method for sequencing. We extracted plasmid DNAs and purify after sequencing reactions by TECAN Genesis Workstation and sequenced them by PE-ABI 3700 Autosequencer. Clones are derived from adipose tissue (26.5%), umbilical vein (3.0%), proximal urinary tubule (2.6%), small intestine mucosa (17.8%), small intestine (3.2%), cardiac coronary artery (8.0%) and lung (38.8%). Average length of cDNA is 1,926 bp. 68 % of the clones have more than 80 amino acid. The length of cDNA of each tissues are adipose tissue (1,506 bp), umbilical vein (2,059 bp), proximal urinary tubule (1,559 bp), small intestine mucosa (2,355 bp), small intestine (1,327 bp), cardiac coronary artery (1,481 bp) and lung (2,187 bp). The length of 5 -UTR (untranslated region) is 349 bp, that of 3 -UTR is 805 bp and ORE is 798 bp long on the average. Homology searches were done against nucleotide databases, such as GenBank, UniGene, GENSEQ. 72% of clones show homology with EST, however, few of them have annotation about their function. One quarter of the clones matched registered clones after end-sequence selection. In summary, about 30% of clones have no homology with ESTs. Conversely, 19% of clones match patent database and half of them are not registered in public database. -9- 1 . 3 *1*1,057* D->^gH^lJ^^^#tb^o 7 U->(7)|*Iim> katoIIlEJI£fi*(kat)4107 □ - >, (kaia)9* □ ->, 1ESAMJ&ti* (coif) 148* U, W M±&;Mfi;fc(hrc) 253* □->> SM¥MMEti;fc(cas)827 □->, fifSSJ9&6 3fc(hsi)l55 7 D — >, In stl,057* □ —7T&-3 fc = 1:1 uit 96 a y°u- httso^-Sv-a.T;v&> oiAGENkitm^^^!t#(#Mu^m, wmatm, # %m&fe®Mn,05i\zm Lrcz-t-v bsue l,000£r* 1J y bfdo ^SE^J BB^iJx-vfe>yj < gB?iJ£j$IS£-ti"SS/^pJIE&ATGCV 7 h yxy^s/su mmit, mmt, Mmmtttm^tz. y- yfi^^L/to c(Dcan, am ® 7 n - > 0#mDNAmm, 7-7x77^^, y-^yyt>yu>y, ammm# gB^j(D##^6^^mmgB^A#mm^%(D&mm(7)mm^mfb^gi6a, 1,057® ©7 □ 6##l,798,208%&(DgByj^##^^L^o ^ nB M 1,000® tl *f L T 105.7%© ±B(D&oi-^ssn/tcDNAgBym> r#%%k ScDNAyy ^ mfumn >v-yyAmi:M^2:Ra cat: 1^3 >v-y yAmm^gByjm^, gByj^^y-A^^T 5,i53> 2,129(2000^ 2^AB^&)T$,*. i>#DNAy-^/X>71:T^MT6C(!:l:j;3T, 51^ E^lJ^^y-A^#:T2,212, ^340 (2000^3 ^ 23 B T $> Z>» -10- Full-length cDNA sequencing A total of 1057 clones newly screened by eliminating the repetition of the same clone as previously known genes were subjected to sequence analysis. The new clones contained 410 clones derived from katoIII cell (kat), 9 clones from normal small-intestinal mucosal cells (kaia), 148 from normal colorectal cells (coif), 253 from epithelial cells in the renal cortex (hrc), 82 from coronary arterial smooth muscular cells (cas) and 155 from testicular cells (hsi). To prepare the template DNA for sequencing, we compared the efficacy between Fujiya manual method using a 96-well plate and QIAGEN kit method. The latter was found more appropriate in respect of convenience although both methods were available for preparation of template DNA suitable for Sanger's reaction from quantitative and qualitative aspects and data of high quality were obtainable. The number of clones of which sequences had been determined reached 1,057, which was beyond the initial goal number of 1,000. The max and min lengths of those sequences were 4,690 bp and 346 bp, respectively and the mean was 1680. Determination of the sequences for 998 of them were made only by primer walking method, whereas that for 59 clones was done by the method in combination with random method such as shotgun or transposon insertion. Assembly of the sequence data was made using ATGC software, which allows to rapidly and accurately combine with the sequences and the sequencing became high-speed, convenient and highly accurate. By starting unidirectional walking from both ends strand-independently, it became possible to design a primer with ease and few artificial mistakes, and manage the project, indicating that the walking strategy was effective to control the status of clones and reduce the burden to construct and edit the consensus sequence assembly. Shotgun and transposon insertion techniques were introduced since those were able to read regions that were difficult to read because they contained some repeated sequences of which primer-designing was impossible or sequences which terminate the Sanger's reaction. Transposon insertion was mainly used to cover the primer walking method for 59 clones and shotgun method was further applied for 4 of them. For the unreadable region because of a long poly A stretch, five different primers, (T)19 (V/A/C/G/T) were additionally used to make complete determination of both strands with accuracy. Speed-up for the respective steps of large-scaled sequence analysis including purification of template DNA, sequencing reaction, data assembling and accurate construction of final sequences were achieved. Thus, base sequences for 1,798,208 bp in total from 1,057 clones were finally determined. This corresponds to the achievement ratio of 105.7% for 1000 as the goal of clone number to determine. The outcomes of cDNA sequences analyzed as mentioned above were disclosed to an utilization consortium, leading to an establishment of intellectual assets. The total number of sequences disclosed to the consortium was 5,153 for all sequence analysis groups and 2,129 for group IMS alone (at the end of Feb. 2000). Thereafter, an international contribution was made through opening the outcomes into a public DNA data bank. The number of data opened to DDBJ was 2,212 for all groups and 340 of them were analyzed by Fujiya ( on March 23, 2000). 11- 1 . 4 5S±ScDNAE5iJi*5g£tfae?g#)CD«»ll6^;!->7.xAroM5S sosui&i*© #rL©$@&#A. raSScDNACSLfclBff ->XtA4, #»©CPU &iS*LfciStt|g7 ‘j;l7-XXT--y a >£, !/--> a A AvXAAti: *95000* ©cDNAEf>J£-ffiLTl~ 2 0 -cmmzMVr-tz>z.trt»smT°$>v, lt-st*5±e>c 4^WJ. WfcSrSIc^MSftfcEfiJx-X©IR9& J£bJI6T*-5„ *«*0X37^> LTA#©T-X©4= A^6##©&V— Tl>5. &^##©f-X^-Xlc79bT#M###&e^o)hlK*&#E*3 C£K j: 9 , Orthlogitfs^ ££>*■ 9 < #ST#5<, cDNAEfJ £iM IC*:* L fc © 5 . yy AEfl- ESTEfJ. 75 y SEMS^t X y?:#*bT!#9#lt. X37©S^l: -12- Development of functional analysis system of gene products for Full-length cDNA sequencing In this research, the system which automatically did a suitable analysis for FLcDNA was constructed by using suitable relational database (Sybase) for the handling of an efficient workstation equipped with two or more CPU and a large amount of data including a new technique such as SOSui in addition to a traditional technique like a homology and a motif search, etc. This system can analyze the FLcDNA sequences of about 5000 at high speed within one or two days. It also be possible to have a look at an analysis result by extracting necessary information. Moreover, not only the public data base such as GenBank, SWISS-PROT, PIR, etc. can be automatically imported, but also the newly developed analysis method can be added to the system very easy. This system can be divided roughly into two parts, i.e., core program and the interface program. The a core program where various function prediction programs are executed, a huge input FLcDNA sequences, and the results of function prediction are efficiently stored, retrieved, and displayed. (1) Core program The core program is roughly divided into "Nucleic acid sequence analysis part" and "Amino acid sequence analysis part". Each analysis program is automatically executed, and the result is stored on a relational database (Sybase). In the N.A. part, the following programs can be automatically executed, i.e, a homology search program (BLAST2), a detection tool of the repeats, the function analysis tool of protein translation area (detection of ORF, Testcode, homology search against EST data base). In the A.A. part, translation program (Translate) and other verious analytical programs (SOSui, Motifs, ProfileScan, CoilScan, HTHScan, SPScan) were implemented. (2) Interface program The interface was designed to handle a large amount data with high-speed process.The user gets on this system taking the FLcDNA sequences in the lump by specifying a storage place through the network. As for sequences, it can be handled after dividing them into two or more groups, and the addition of other sequences is also possible at any time. The user only selects sequences and the analytical programs, and all processing will be automatically done. Each parameter of an analysis program can be arbitrarily determined. The score and the character string of an analysis results become a key to add the examination from various aspects to an analytical result. The Ortholog relationships can be displayed comprehensibly by the result of homology searching against the data base of various species (human, mouse, worm, E.coli, etc.). All the results were displayed linear in order to overview the whole results together including homology search against the public data base of genomes and the EST, and other amino acid analysis programs. Moreover, the result of the amino acid analysis to the protein translation region displays the part to which the function and the structure was identified after the translated area is displayed linear to another kind. In addition, three reading frames can be translated into the amino acid, the result of the homology search executed for the known amino acid be arranged, display, and the frame shift may be detected. The progress condition of an analysis each program can be confirmed at any time as a system management tool by the job monitor. 13- 1 . 5 *###TmcDNAT-f r*l8»t hcDNATVFL7"D->';c7 8rS^S*cDNA7 o->®±#E9i|A%^e*fi6. ctuiimcE^jyt $2*1 Site ^dtSBgSfi&ff 7lc$,/tO. ■5-ffl*a^D7T'f U & 8«riHTofEaisss5*siLt, 7-+f> • 7'n-y -# WT-&-5S* #J$lcS*58#PiH, RNAS43 *1*3 x K0,'S^B*yn-7x7 h® ±3 tC#- ATAC-PCR (Adaptor Tagged Competitive PCR) B. ^'MOit > ~f f^ip S iaSST^S!# **$*fcU £6£«ft«*¥K*¥®OTStitW± (Cj;oTIS%SnS:g*tt6Cit$#?T$.%„ *E%TB, ATAC-PCR®>\< X *- 7' 7 h- ft'>XxA®«ISSrff FL7/n-7x7 h B1W91-ATE?iJ9i$2 fl7c 7 n- >® %8 fillg*iRWL, *(®#¥lfrffi©cDNAx-7*-X*#fi£-f 3 d tSSSi Lfc„ A -f X ;lr - 7r 7 h • yXfifflliCtt, 96* to^-yfcfxU-Sr&ofcBiii DNA Analyzer*#* U +F > 7P;U® M *96*e?¥fi-PfT X 5 * 7 CSC# L ft. ATAC-PCR*ffl®Sfi^#SS9*,7'f 7 —StoEffV 7 h^f-^fflSV 7 h& 7S1V7 h©E%t>ff-ofc» 2©iBS, 6B86Mlcd3B4«®e®ievt%ayn7 7'f * *ig$fc0l00»fi^lC7iVrTS**5^ 2*lB, / -If >7’n 7 h &*tt#LTK + (g®*m#e* *£, ATAC-PCRi /-+f >7'D 7 ht© tkKStBtrfrofc *2 3, ATAC-PCR® fi A MICE A 'Stlfc. %$*$»!£**$*®/ts6c atac-pcr®S«t6](Ditlsi1® 6*B<$*l*i§E#zp* L/t#IS®S6ff, (D <*»©iMR»*©«ffl, ©8J$W®1f >:/$«S®&M. ® v tv --> g > • *-7 ’*^s£L, 2 *1: *oT##%©A *7 BfJofc. B?W® 7 D-/l;7UTIi, E^H*$®ESi:* 6ICATAC-PCRC =k5SI6*ff ®- *#%1=&©TB. 1739afiT®#J6^6941*e*ICO®T, 7B«Po1®fga^n 7 7 -i *%-/0 7 7<;l/d3*aE^#*A^5®0RF*#m*®##*@*: ^5., «i6»«McDNAx-7*-X*##bfco C®###®A®T-7*-XB#a me?®####t:&/co, ii#i:*oTt)*»T#(8T*9, 4-s®8gJ!i»^n •5. -14- Development of cDNA database for functional analysis In the 'Applied technological development of strategic Human cDNA' FL Project by IMSUT(Institute of Medical Science, The University of Tokyo) Team nucleotide sequence of newly identified Full-length cDNA clones were determined. As a functional analysis of novel genes, expression profiling is extremely important. Although Northern Blotting Method is the most reliable and common method to study gene expression profiling between tissue, the method is difficult to be applied in the case of this project which studies lots of genes, because it takes a lot of time and cost. ATAC-PCR (Adaptor Tagged Competitive PCR) developed in Japan by Dr. Kikuya Kato (Nara Institute of Science and Technology) only need little sample, is highly quantitative and sensitive, and this method result in high repetition occurrences. The research constructed the ATAC-PCR High-Through Put System, and FL Project IMSUT Team collected information on the novel clones, and developed a database of functional analysis. In order to construct High-Through Put System, UNITECH introduced high-speed DNA Analyzer with 96 capillary and made it possible for each unit with 96 gene to handle all samples. Also developments of gene specific primer planning software, data processing software and data control software were made for ATAC-PCR. As a result, we made it possible to fix quantity of 100 gene per day as highly reliable expression profile between 6 tissue. This is by far, reliable processing ability as compared to Northern Blot Method. The comparison experimentation was made between ATAC-PCR and Northern Blot Method and the result showed that ATAC-PCR has greater detect capability to Northern Blot Method. Expression level of newly identified gene is generally low and in order to find fix quantity on a stable basis, ATAC-PCR was technologically developed. They are; l.to measure gene of the original tissue as an internal standard, 2.use of multiple restriction enzyme, 3.examine the concentration of each sample at the measurement, and these studies resulted to improve the frequency in acquiring samples. Also the use of three-step concentration from original gene of internal standard enabled to make calibration curve, and to get high standard data which brought the highly reliable fix quantity samples. The clones distributed from the IMSUT were studied with ATAC-PCR, and for this particular research, 941 genes out of 1739 were used between 7 tissue to get appearance information. The ORF estimation from nucleotide sequence, and ATAC-PCR analysis result were put together as cDNA database for functional analysis. This exceedingly reliable database will be useful for the development of functional analysis of new genes in the medical industry. -15- 1 - 6 ±#cDNA(DmmaE9]&2&#:t###ecDNAf-f-/<-XS/%:rA(D4#m *5*£ttEl2Stt3r Ltz.rn.fci-(Dmmmwit z && ;m LT. ^ y * y t h^scDNA^ 04t>fxy^^fAX, 0 t&y LT. 5,001^ n-ycDS ®EyiJ£fc£Lfc0 0 £ gift iff T? ft. r <7)\H2,50\? L/c0 $ fc, ±%#A&cDNAT —^ A#cDNA(D##^T y T —y 3 y&iftfifrSt-fT 5 ft«S:M|gi"5 r £ ^ J; >9 . Lfc£gcDNAK?iJ£te*A (1) ^ficDNA(7)BflyiJ^:S ##gi^7 y “DNA'>—yyy —<7)^At-«t o , y 7 yyy-^-Eii-^oiigo, sayij^r -Exy/HcSequencher&mV'^yy^ i& S E yiJ ^ i£ £ LTE ffit/co mm&rn. y &~y (2) ±m#^C^cDNAy—y ^^7" A(D## 1) ±m#y-y -<-;%##?)im#mife=i Hyiyits#!©)!#, RTfMVri&miz.m^'it&mmM&ft V\ r cb^^T^^y^yA^^I^L/Co ^*DB3£^<7) £«><£> g i&T y y — y 3 yy*y a £ lx n, DDBJ^o^ftcDNAy — y iftfflor-y — ## y y. y A ^ L T ^ orft yy — y 3 yyyy an y —y^< —7.y — y (-&<(<##Tyy-y3yy%yA, ^y^xyyyy-yayy^yA^M^L, y 2) it, ±m^y k°yyyy y-y a yy^yA(D^m(-, 3) 5,000 y o — yw^fcDNAC^t^t^f-^ 7, t LX. _hEStify —y £: E^#J -16- Sequence analysis of full-length cDNA clones and construction of a large systematic cDNA database The purpose of the project is to construct intellectual bases in Japan by analyzing gene functions based on the sequences of full-length cDNA clones which were isolated at Helix Research Institute (HRI). The project planned to determine the sequences of 5001 clones at Hitachi Ltd., TaKaRa Shuzo Co. Ltd., Hitachi Science systems Co. Ltd. and Nisshinbo Co. Ltd. Hitachi Ltd. analyzed 2501 clones, and also we constructed a database system to analyze full- length cDNA sequences for functional annotation. (1) Sequence analysis of full-length cDNA clones High throughput analysis system was established by introducing some instruments that is Hydra micro-dispensers, Gene Amp PCR system and multi-capillary DNA sequencer. Primer walking method was used for the analysis. Primers were synthesized based on the selected sequences from sequencing results with a software of Oligo, and data were assembled with a software of Sequencher. We reported the sequencing data of 2501 clones after solving several problems resulted from sequences and clones. (2) Construction of a large systematic cDNA database 1) We developed large-scale database techniques including a data pre-processing system, a registration system to public databases and an integrated analysis and display system. We are able to confirm sequencing accuracy, estimate a start codon and edit sequences based on the analysis data using a data pre-processing system. For the purpose of registration to public databases, we constructed a system, which helps registering full-length cDNA sequences to DDBJ as auto-annotation system. ORF annotation system, a functional annotation system based on the database search and a mapping annotation system were integrated systematically. 2) We constructed a system to identify disease-related genes and polymorphic region based on the mapping annotation system described above. This enabled us to pick up the sequences of the related genes and information. 3) An integrated analysis and display system was constructed to analyze the sequences of 5001 full-length cDNA clones. -17- 1 . 7 ±#cDNA(DS*aE9i&2(D#9E ax ^ v y ? T. ^ y (4^ n->/) ^#mi-6^^L-C^-C##Lybo a#&y7 7-7(D#@3: o tz\fi) tz Wc L o *#%e@L-C. -fy^-r X 5Ktt£0)#tt:ll£|l£®tfc-f-5 — ? ^ y \/ 's Jf&ffli & x $r)VT y y-f-S L Sequences analysis of full-length cDNA clones Our objective in this project is that both strand sequences of 500 full-length cDNAs, which were established by the Helix Research Institute, are analyzed by using the primer-walking method. These cDNA clones are originated from the libraries of the established cell lines and human organ tissues such as NT2-cell, ovary, placcenta and thyroid. The sequences of all but 36 clones have been read without troubles. The templates of the thirty six clones have obstacles such as lots of false-primming sites, secondary-structure formation, impurity and long repeat sequence. We managed to read the sequences by long time-consuming tasks in which lots of primers were designed or subclones were made by the shotgun method. The difficult sequence intends to need lots of primes. The ratio of designed primer number to the useful one for each clone let us know that NT2RM4, NT2RP3 and THTROl libraries may have difficult sequence clones. From these results we established the accurate sequences of all 500 full-length cDNAs and also improved our sequencing ability. —18 — 1 . 8 ±ficDNA®t6SE9J3fc$ *e>ti>3 3Ef5rJS#itfSDNAl^;|/T-8SilWT£-5=k5 ffiox, bh8e^c>f£$BE?iHiim, e« >X6b*®|S]±S0-5„ M #: 69 C It 10,000bp/day T'&-z> fcMtirffeJ] & 35,000bp $ T Bl £ _h If •5. ^S6cDNA©#*E?JftSiaHTro=t x icffofco 56f, -\'J y?XiMi'5>SS bfcMftJ: D ->-7->X£jS©«ffli&.|>X‘7X3 FDNA£M8bfe. 7#C, -/XX5 KdnaCX □ —- >X,snTtA57c3::6cDNA©5fcSt6SE?|J6 8t6b, # BflfcSSE fij&mcxxf • if/$bzt. cfi&miATtab*i=*;sbZ:#*Em=# v-«K4BlECbfc. «*», HPLC£ffl b/t #87)1-2-$T$> 0 , |s|#l:#.» ©XX'l'7-$:#84 5©tt8©TMT, 6'7ltffiAsW'oTb£. 4©7bA, 77-f 7-0»BAiy--)->X»fff©*acai.; tai^MT^fcOT, d; 0 ffiflE7)^ffl®Zj: X x< 7-#8%&*ab7b. ttt08S. *f7bC#Stbrcffig*-h U -y yT0*SJtt C± 0 , 20*©:/7f 7-£30»T«B-r3 st/SHiJIIC&ofc. ciiia$E*S©6f8©S $T$ 0 , #$69%%*Efil©##C*#<##b^. se-c, =ko#e7)^oamt:%*EM&*)g-#-&-x< , pcrki $, y;pE68-^'>--y > X*#CO©T©#M&lf oTh. —-St, y-'r>X'rJH:-l-*A75 h'5: #9of &CCj; 0. i«GC®*©E5iJ£J; D»EBC&Jt-f-5 S $7c, X-X-> XKJStotH X ;l/�mS7&U7< V-9--x;l/tt-C©->-X>XSS$fi:'5 AtCiD. ##%### UAEm©##ca#69&#b 7b. SBC, #$©Au#*E9'j©*#tg Nb/t. $7c, SffcCABI 3104-7 27 D-v-dr >+)—£* A b 7b S tea D, #C/\- FffiT ©###*7)17 "j -fbtzMfS' D T& < , 4 CfflV^fcLI-CORttSas 4a-'ABia8©8-'> —X >-9--©#tt&S^bTfRE4ff 7 CtCJ; 0 , «xnc«ff^feC*i:C7aiP7-5St*i -19- otz. a wft b O^rt^tTo fcJ&H. ft) 10,000bp/day "C - >r y X il& A £ 35,000 bp/daytc $ "C 31 #-t If 5 Z i: & t±j^/i0 t LX. 0 b Sequence analysis of full-length cDNA clones The molecular biology, which has been drastically advanced in recent years, has opened the way to explain the mechanisms of many biological events at DNA level. Thus, information of human DNA sequence seems to have a potential to create novel businesses, such as developing new medication and gene therapy. Therefore, it is necessary to promote characteristic analysis related human DNA to lead the potential market. To address this request, the strategic analysis of “complete ” length cDNA, directly linked to the functional studies of proteins, has started as a national project. In this project, we Nisshinbo planed to sequence 500 complete-length cDNA clones, constructed and retained by Helix institute. In parallel with this analysis, we also had a plan to enhance overall sequence capacity up to 35,000 bp/day, 3.5-fold increase from the beginning. The complete-length cDNA sequences have been analyzed as follows. First, we prepared plasmid DNA that contains complete-length cDNA insert as a template for sequencing analysis. Next, we determined terminal sequences of the insert (first run). The sequence data from the far end of the first run could be used to prepare the primer for the second run into the region of insert. In this way, the new primers were designed and prepared around 100-bp from far end, and applied for the analysis of subsequent sequences. This process, called primer walking, can be repeated many times to sequence extensive tracts of insert cDNA. Finally, the whole sequence of complete-length cDNA was determined by assembling all fragmental sequences that were obtained by the primer walking analysis. As a result, we Nisshinbo successfully finished to sequence all 500 clones within the period of this project. We established the new simplified purification system of primers, since it requires considerable time and complicated steps using conventional HPLC method when deal with a number of primers. Thus, construction of new system was an important factor for rapid sequencing. The new system using a simple cartridge enabled the short-time purification. With this process, 20 primers were purified within 30 minutes. It allowed 6 times speed up and significantly easy treatment, and enabled effective sequencing analysis. To determine the sequences more precisely and speedy, we have been made improvements in PCR condition, gel composition, and sequencing condition. For examples, -20- formamide addition into gel has brought better results for GC-rich region, and non-cycling reaction named isothermal method was effective in sequencing long polyA region. On the other hand, introduction of new ABI 310 capillary sequencer has increased sequencing capacity, moreover, taking advantages of individual sequencers was useful in accurate determination of difficult region to sequence. As a consequence, we successfully improved the sequencing efficiency from 10,000 bp/day at initial to 35,000 bp/day after the conditions optimized. In conclusion, Nisshinbo has successfully determined all 500 complete-length cDNA clones and improved on overall sequencing ability to 3.5-fold increase from the beginning. It means that all of our purposes for this project have been accomplished, and our approach to this project can be achieved because we produced the expected results. -21- 1 . 9 £6cDNA0fflSR9J9fc$> cDNA^$g#$lO##. &tfDNA"7-f £ □ 7 flFll t$«r ¥^10¥SNEDO^3^:SlE^STfTtin/r hcDNAy/Afbm##M5Ej ^ U rf^-f v 7° & £ < — X U 7 n — > ££ STc^ScDNACDSSEyj&^/^EE&E^L IH%-t-7T$>5 = QSEja (#) A u '7 7 XE^lE^T5,000{@ 7h. ®/^100%(C#^%(AgBW7 PCR^^#(7)4%#^# L < M^h(D (±fo(D6%& T> E2l Hi % ME FbI ft ^ 5,oooSE cd cdna Sequence analysis of full-length cDNA clones , Establishment for stock and delivery system, and DNA microarray process technology The main theme of operational technology development of human cDNA genome application, which is financialy supported by NEDO grant 1998, is sequence determination of full length cDNA made by oligo cap cloning method. TAKARA Shuzo Co.,Ltd proceeded with three project, namely. ©Sequencing analysis for 1,500 full length cDNA clones from 5,000 cDNA discovered by Helix Institute. ©Establishment for stock and delivery system for 5,000 cDNA clones discovered by Helix Institute. ©Amplification and purification of PCR fragment from cDNA for making DNA array,with the corporation of Hitachi Software Co.,Ltd. The result is as follow ® & ©are completely finished and ©is finished at 94% . The reason why ©is not completely finished is that 6% of all clones has very poor yield and we could not make all the PCR product in the period of this project. -22- 1.10 cDNA77 7 □ 7 U7 Ic«t5 HSV7 h»i7i>y-7U *E5ETtt. 'x'J y 7XE%0rl:T#6n^h hcDNA^'f 75'J C'fi'?., 5S±S*s £fceStl7c5,000H©cDNA7 D — 5cSficDNA"7'f >D7 L7 (EfiK&WtoilliE i, -?7 » nr I/'HC j:%%S)Rt1r*?iro#iERy:seES BlSi: LTV>5.. CCTil. i6 s.R&DNA-r'f zui’V'isen. satt©ff«. &tf»e y^BSStirtoHiESfTofc, DNA7 7 ^ □TK ff J5$ftW©tlu ltt&Zc D5,0007.#-y H6i$S£Lfc7l/7©ff®©£ettti:, 320~3507 l/7/fl tz. $/t. f#^b^7k7©7%@M^#%#ie (ttffl-iJI67V"fZfffi£7l/'f) tt¥i$ 93% tti-Dtz (lli)©fFST48ttfF/$) . btt 6 =fc 0 , 19 —&an*^3>fi$gtt©i6HDNA 7 7 7 □ 7 1-7 DNA@$ffl3- MrilUfcXrH K# 7 X £5#E<£ffl V. LfciBS, ^ V -L- U 7>n- hySeL-TM8tt©A(xca^*UML^. #5%j; 0«i@bZ:MfW]©a%:6 77 >n7H JttBlA'f 7*V 77-fer-Zg >L7cSS %, 7 1/41 3#S©fii$©7 —y-y hDNASSSfflViT/N'f 7U 77 if— -> g > lfc£8S, iSi$© SSg&EBSSJlto LZt. 2wg, 5Kg*©mRNA^6i@SLyc7 —7 y h DNAiSESrtil ©T A7 7 U 77-b'—-> g >£fi7A 7 —y y hDNAi§f$6JIBIC#il&mRNAiitti;2«g£fi7b *5fi75:Mf£;B#M£#ISbfc„ A<7vy -23- Development of gene expression analysis technology for the use of DNA microarray This project uses fully sequenced five thousands (5,000) of full length Human cDNA clones among the cDNA libraries obtained at the Helix Research Institute and targets to establish technologies and techniques to produce DNA microarray for the use and verification for gene expression analysis of those cDNA clones. For those objectives, we have studied conditions, stability, and reproducibility to produce a high quality DNA microarray, and proved its effectiveness for the gene expression analysis. 1. Establishment of a DNA microarray production technique A quantity productivity of a DNA microarray with five thousands (5,000) DNA clones on one microarray was three hundreds twenty (320) to three hundreds fifty (350) per month. And a ratio of a usable microarray to defective one (yield rate) was average 93% (forty eight (48) microarraies can be produced by one (1) production operation). A fundamental technology for quantity production of the homogeneous and highly reproducible DNA microarray has been established. (1) The poly-L-lysine coating was found out to be the most stable and reproducible method after the comparison of five different kind of DNA stabilization coating method. (2) The DNA microarray lasts for a little less than two months after the production, according to a series of hybridization experiments at a different date(s) from the production. (3) The most suitable composition of the target DNA solution was found out by comparing the three DNA solutions at different kind of composition. (4) The most suitable amount of mRNA was confirmed to be two (2) fig, after comparing the hybridization experiments with a target DNA prepared from two (2) and five (5) fig mRNA, respectively. (5) The most suitable hybridization time was confirmed by comparing hybridization results at different time period. (6) The most suitable hybridization washing condition was confirmed by comparing different kind of washing conditions. 2. Evaluation of stability and reproducibility Randomly selected DNA microarray was applied for competitive hybridization at the same experimental condition, and dispersion of the results was evaluated. The results have confirmed that the DNA microarray production and the efficiency of the hybridization show the high level of stability and reproducibility of the DNA microarray. 3. Verification for the use of gene expression analysis Two different kind of target Human DNA (Liver and Brain) was applied for competitive hybridization against the produced DNA microarray, and the measurement of the amount of transcription of each gene was attempt. As a result, a gene(s) expressed only in the brain, in liver, or in both was detected to prove that the DNA microarray can detect the variation of the gene expression with high reproducibility. -24 1.11 6EcDNA<0i££E$im$&tX8te«*Tlc|!|-f fc5££tt7-f • 3X^EXE%9r ^ LtlI^cDNA04J1:\ t M>S(7)SmcDNAc7)j^SgdyiJ(7)^:S^ff ofCo £ /c, cDNA S L^o SEcDNAOj^SSByiJ^^^X^ft, t h t>E(7)SEcDNAy d —y (4—5kb) 2000#(DM ^(D^E^^&^SrfTofc, r (D^^SaM(Dtf^^^Jffl It, (D##y d - y 120#(DilglJ^^^ L/^o P-yl-OV'T, y a y hyfy?£lc«t *9 ^SSEyiJcD^^^rff V^x 120#(7)j^SEyiJ^:^^^T L/c0 E^T-^ld:, Long length cDNA sequencing and development of new technologies for functional analysis In the analysis for genome, it is important to analyze cDNA as information for the investigation of gene function. We have achieved the sequencing of long length cDNA of human spleen that Kazusa DNA Research Institute possesses. We have also studied the technology of targeted end-labeling by using three stranded DNA formation and detection of fluorescent signals, as the basic technology necessary for making the DNA chip of long length cDNA. We accomplished the sequencing of both 5’ and 3' end sequence of human spleen long length cDNA that has 4-5kb length. Utilizing this information of the partial sequence, we selected 120 clones of the possible new gene by comparison to several data bases. Then, we completed the whole sequence of each clone by the shotgun method. These informations of the whole sequence of long length cDNA are useful as the new data base for the analysis of gene function. Using the end labeling method for DNA with three strand DNA formation directed by recA protein, we studied the targeted end labeling technology of long length DNA and the fixation technology of the end labeled DNA on a solid surface. We could get the high efficiency of the end labeling through the optimization of the reaction to introduce efficiently an amino base into the -25- one end of a long length DNA strand. The optimization of the fixation reaction was also carried out to fix efficiently the end labeled DNA on the surface of a slide glass with a covalent bond. As a result of using new technology contained the end labeling and the fixation by the covalent bond, we could obtain the same detection capacity as that of PCR method. In the study of the detection of fluorescent signals on a solid surface, we constructed the trial detector using cooled CCD camera and macro irradiation system. This trial detector possesses the basic performance. -26- i.i2 aacDNA »efme? «*M#e>nntf. 7«&©#»d e&s tJBtJtis. b holes -f-j'^-XOMIM»^6$«lcE-fal*S$5ISm-rc«. *EflS®fi¥69#HT$.a» e#:A-> KIWS^WfflT'^a. fit, E?iHi$e*IC3 >ba-5'l: j: «$r*65ln SilicoSS&ftT -y b > 7*©#ft £&W L ft. *£#:*&+»* 1C it. MBitfi o, bioscouT-esmufc L T, UniGene £ GeneMap©* IR&K^ftLfcSa^J Mi T y y*#©T-^<-%&## L^b. *iCr-?M«fil:*#it sain Silico*6#:t y fcf > d/SrHISttif S £ itfffijfefc. me? a;bAic». y y adnah ©d- >y;b e^f njxt &&. a€tt5$S© Development functional analysis of long length cDNA In case we search for relationship between gene and disease, genetic information is very important. If we can get chromosomal band location, then we can get the information about human disease information from OMIM. We build the integrated genetic information database by bioSCOUT. UniGene has chromosomal band location and sequence data. GeneMap has physical mapping information but does not have sequence data. We need the sequence data when we try In Silico Mapping. So we extracted the sequence data from the integrated genetic information database. We built two kinds of databases. One database is the sequence database including UniGene and GeneMap. Another database is the integrated map information database. We established In Silico chromosomal mapping system. When we get evidence for relationship between gene and disorder, we need genomic DNA samples. We collected the 20 samples of 5 families, which we got informed consent. We extracted genomic DNA from 20 samples. -27- i 3 Horn###.?)##. ^#$m ^ m-o fL. |gP^fb(D#^(caj^LT^igM#(/)#%# SrfT o tz-o *9 t. t tbfco j£-?u'y F L, &W^(D%}&}MiM1t&\xl 5 tz. &6(-8^ 27 0 3 v y ”— v TA^iiifco 5Fj5£l2¥2^ 22 0 , 2212i@(7)^^ScDNA^ cr - > ^ $ gd £'J t# $ £ S^X - 9 "<“ * "C^)6 0 *DNAir-f/<>f (DDBJ) (ddkMLfco To promote the project, the technology committee was established. The committee consists of seven members. Director of the committee is assistant professor Sugano, Institute of Medical Science, University of Tokyo. During the fiscal year, the technology committee meetings of all 12 companies were held twice, usual technology committee meetings were held five times, and sub-committee meetings were held twelve times to mission complete. Collaborative research agreements were made between RAB (Research Association for Biotechnology) and Institute of Medical Science, University of Tokyo, Helix Research Institute, Inc., Kazusa DNA Research Institute, and Graduate School of biological Sciences, Nara Institute of Science and Technology. R&D information was collected by attending scientific meetings and international symposiums, and technical information was exchanged. Member of the consortium was 33 at the end of March, 2000. The information collected by those attendees and the project results was summarized. A consortium was organized to utilize results of this project on 27th August, 1999, as intellectual property rights such as patents will be protected. Full-length cDNA clones of 2212 human genes were prepared and the base sequences were determined. These sequences were published in the DNA Data Bank of Japan (DDBJ; an international database located in the National Institute of Genetics) on February 22, 2000. -28- i.i4 tStolBSlStto 3CmE2l0T T194-8533 3C3fCfWEBmM0T3-6-6 T 771-0192 SSW@Srfl)Hl*|IITj)ll*aSf 463-10 W^F-E T 257-0031 #$)IIWSSr1?Eg228 siSSgR T164-8555 %RS43EKS41$f2-7-14 3-3. —y /? 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Maruyama & Sugano, Gene 138 171-174 1994. (2) • Dynaclust. (^'4 ~Y ^ M>) • BLAST ver.2.0 Altschul, S.F., Lipman, D.J. et.al. Nucleic Acids Res. 25:3389-3402. 1997. (http://www.ncbi.nlm.nih.gov/BLAST/ ) • NetStart A.G.Pederson & H.Nielsen. ISMB 5 226-233 1997. • TestCode J.W.Fickett Nucleic Acids Res. 10 5301-5318 1982. • MasudaScore Y.Iida & T.Masuda Nucreic Acids Res. 24 3313-3316 1996. • TranStart. E, MS • RepeatMasker. Phil Green (http://www.genome.washington.edu/UWGC/analysistools/phrap.htm ) • Cross match. Phil Green (http://www.genome.washington.edu/UWGC/analysistools/phrap.htm ) • Phred/Phrap/Consed. Phil Green (http://www.genome.washington.edu/UWGC/analysistools/phrap.htm ) • Autofinish. Phil Green (http://www.genome.washington.edu/UWGC/analysistools/phrap.htm ) • Sequencher ver.3.0. (Gene Codes Inc.) • Hybsimulator ver.3.0. (AGTC Inc.) • Amplify. Bill Engels (rM^n>-y • BLASTN. (GCG Package ver.10.) • BLASTP. (GCG Package ver.10.) • GENCORE FrameSearch (Compugen GENCORE Frame N2P ver. 4.5) • SPCIAS. /NK /StR (X,^$DNA#^Bf) • SOSui. (^#%±#) • Signal?. Nielsen et al. (t>-7“ f %#^:) (3) * • GenBank (http://www.ncbi.nlm.nih.gov/Genbank ) • GenPept (http://www.ncbi.nlm.nih.gov/ ) • UniGene Gregory D. 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(aa) S(bp) flBto (ACXX) 255 1,506 156 (61) 230 (hu v) 29 2,059 24 (83) 342 /H#%# (KAIA) 171 2,355 130 (76) 345 25 1,559 21 (84) 276 (REC) svimm^mm (cas ) 77 1,481 65 (85) 284 lE^/hE (HIS) 30 1,327 20 (65) 207 B (LNG) 373 2,187 235 (63) 238 1#+ 960 1,926 651 (68) 267 £/c. ORFfc btltz.653 ? n — >(D^X\ 5'-#%^##(D# $ #349bp, 3’-#SE1W ^c(D# ^ (±805 bp-efco;fc 0 ORF(D5F^ESti798bp (#^^ EA 266T ^ / #) X&'itz. o ORF^ £ ftfc ^ p t. ORF/cDNA(%)^iSt:|£t^(7)'e(j:Jt^^ p — yZ,S%(D^A# He j:6mRNA(Df-xrX(D ORF^-a Ay-ev^ 6 . i&VMi, * y^° y £ fi&T L t> V' x. ft i' z £ *^ p v" —)W#f ESTt"— y /< —x o tzQ £6, NONSTx — £ t y h (D|^46%(e ti^t^>0 Z <7) 9 h , 75%(t genomicE^lJ 6 %k (D ; No-hit, 101 -800 (D b 7(Z^#:^b-Cl^V^t(D^^35%, #^P^—(D&6t(7b ^V^(2, EST#(D DNA^rb ^ — g[ L6 & (DZ,S46%. ^ ^ (21^ —B: L^: (7)(219%(7) 183 ^ P — y "C, ^(DcHeilNONSTT ——^ — as y. (7)^.7?^ < GENESEQ(D 2 9 ^ 7. ^(DiF — £> b <5 o Zb2 "Cb, O y^-(D$)6 y ^ -69 H r^r tn n '> CO beta-glucanase , CO acety lglutamate kinase, '> h 'y 3 y /< :& CO glucosyl transferase, t h CO O-linked GlcNAC trnasferase , ^ ^ CO phospholipase C#co k h 5 ^ # x. 6 fl <5 t h #f , ^7’> COphospahatidylinositol triphosphate binding protein, ## CO GTP binding protein , # # CO iron-transport protein , t h CO Ca-dependent phospholipid binding protein , y y h COurea transporter# (O h y V y zK — $ w-JHqp'S ^wmm?. mmmm, #co#m6#m* o V h^RT-PCR(CZ FISH, i&lMt, RH y t° > ? {Z. £ % %k&fc{\L #C0^^, ]) 'S \f -r > y ?'s ;< y %(Dm% t £3?5t£<0|pl£, V , KO-^yy^m k F##(7)9 fimfrb$u&t 3. 4 -^T, (ccv^T, ^ ^/ 3. 5 simxtt 1) Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A, Sugano S;Genc 1997 Oct 24;200( 1-2): 149-56;Construction and characterization of a full length-enriched and a 5’-end-enriched cDNA library 2) Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ;J Mol Biol 1990 Oct 5;21 5(3):403-10;Basic local alignment search tool. -70 *4* 5±ficDNAEM3fc$ 4.1 MS «m Ic 9t$ L £ £ L ZtS& :»ia®«ti»y/A«%#»T»555£»cDNASFOTF%5£ 6 CffLB8i •§£®4:ftS*#ICfflSSeL =k7 6t»tt. yVA®«|gBy-®#Bg^f6fflS*M%®fci6®ttMS$6BC*y-5r t^Biet" 4. 2 tmatx*» *11 -y k>y&icj:5cDNA7'f y? u -a. 0f« y y ;ex m ^scfif e. c -71- 1057 5-f75U-SiJ ±icy^-f 7—^^% 998 ...... -...... £fr 480 kat (Katoin tans) 410 /mm 7.2 kaia 9 2.61 coif 148 ±lC7>^A;i 59 hrc (K$K±6IBga) 253 5>^A;$¥i6|E«$ 5.36 cas (a$!iE¥;ft®s$oea) 82 hsi (fit*) GC ## 50.4% 155 w-m&mm 1680 ...... —...... 1798208 a it 1057 -72- 96, 373XL, $;tHDNA Analyzer 3700H=koT##fL, 7X7X$r#/c. ^(:77< 7 —j;oT7 D-7^^ 0 1##&&#/:(% ATGC7-7X7X7 vt77U 7 7 h 7X7 (7 7 h7x7IM)&mWTE?iJ^-7 7 vi:777J77a37-t:77-X7-7X7X(Ai##, y7<7-^fi&l:T #Am(7,8)aw-D^7>yA&^#mL^. 4. 3 eaa##a (1) mmi£%mmMk(D%m 7°7x^ MDNAmmm^LT^zirn^-3.7;i/& (El) £ rfcE ±TM#(:f7vx lE^I^E^^n^SSISc^itgeL/t(E2) 0 E243qiagen _0^cd ^:7^ QIAGEN kit(D@# (REAL: R E A L. prep96 kit, Turbo: QIAprep96 Turbo kit, Ultra: QIAwell96 Ultra kit) £ , +G50ti96A7°l/- M±4£T'0Sephadex-G5OX t:>7 L/c C £&. +ExSAP^Exonuclease I&U^X hf 6 ^Alkaline PhosphataseMS L ft Z. £. . 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AtVB#8lj7,7'f 7 —SgLft427 □ —MICKl LTt2, A©¥#S6X8, #XS**136T $> o ft. 4.4 SSS ¥#l< >D— f-:S2kb8#©cDNA7 n —MSAMtc^rSEJiiln^ttS'f 5ft*l:ii, 3AE ?iJ$ftg-r5.fcii)®->-7x>->>7*ffl«SDNAe, 6 • Sfi»f*fft5CitfegT»4. 962t¥l/- K±8ICT7)l/* U SDSft £(t7 79 X 5 b*#ES®ffi%$7tttSXCJ:0. -A-fttMS. • MS*St^of-?7'jt/9'JCI ff#&;&t£;ift©i*,S»©BBAE?iJx-7©#f#£nJ(it;:l.ft. $ft, «#bJj|6ftM-7l> '>>7*7'>>®®^W->»J:Dfl,«fe6.n-5»ffoI|g^*X«#:ictMt'T, -f >D— MW *#^6iwsi$6iLic*7#x®ie*i=]®*^roX7'f7-se$M66-r5.#iii8esffli"5.r LftU, y>7Xtt®*CT^:ftE?iJ&ft^lffi AESJ ® AS £ # 0 7 M 7'A C ¥ x -6 M a y f-AMft®*»i, #S^#@2^©X*^&Ct:< Wft6#$ljT&S. -«tLT06C. 7 0->Colfl506(^7 7-&*5018$S) SrXEtCMS T-y>7'Alctt®-®o/tE®tt¥lR 0 8866.6® ASS:¥Uc»X h 5 >X# VXSXtttttE 0 M666® ASG@@A^±C&#@-6^*)6. £ft7 □ — MftDNA©## X*. X77— IS fflb>ft®fPLfflttfc. May f A >&® SWEATS&. Lft Uftft'6*#—MTSSflAfto ft —76~ 7>yA&aLTiifm#% &#k:kL ^#^T&3/Co -MaLTimvC, ^O->hrcO14960#MS?'J(21OO#&)&^fl@#t:#L T/\—7°o^ ^M^o —>^i99#&(D@g^J^io[g#0)g^4i'5##[ #%Mmaa?'j&#LT&o, #$m^UA X h (T)l9(V/A/C/G/T) 6Wo/:5##(D#gijy7 HcDNA^YX x V —^ □ — >!!£: UTltsf 105?E1(D h hcDNAX n —>&± 3'%^UAXM/'yf-^#W^^^GC#m^50.4%T& o/%. ^ ^:<(ht,39@79^n —>^|%#mRNA^A^LTW6W#B%^^#^^l^:o L#7bc gg^J^^^-A^#:T5153, A#%T2,129(2000^2^ABm%)T&6. W'&m, DDBji: ^^6^^^(2000^3^23B^&) H, ag^J^^f—A#:#:T2,212(^^%^^:&^340)T$)6. ^#^1, $3* 4^ vH>^^cDNA7< 6^^ 4. 5 (1) Paul, S. and Declan, B. (2000) Celera turns to public genome data to speed up endgame. Nature 403:119 (2) Maruyama, K. and Sugano, S. (1994) Oligo-capping: A simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138 : 171-174 (3) Suzuki, Y., Yoshimoto-Nakagawa, K., Maruyama, K., Suyama, A. and Sugano, S. (1997) Construction and characterization of a full length-enriched and a 5-end-enriched cDNA library. Gene 2QQ: 149-156 (4) #:@'ilRi#u #*^^(l999)#M§%t: hcDNAfB^K#T^%7°Oi/J:X h E: Y /- V —Vol.57,No.7,pp47-48 -78- (5) Deininger, P.L. (1983) Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem 129 : 216-223 (6) Yamazaki, M., Ono, A., Watanabe, K., Sasaki, K., Tashiro, H. and Nomura, T. (1995) Nucleotide sequence surrounding the locus marker D21S246 on human chromosome 21. DNA Res 2: 187-189 (?) Stellwagen, A.E. and Craig, N.L. (1997) Gain-of-function mutation in TnsC, an ATP-dependent transposition protein that activates the bacterial transposon Tn7. Genetics 145: 573-585 (8) Stellwagen, A.E. and Craig, N.L. (1997) Avoiding self: two Tn7-encoded proteins mediate target immunity in Tn7 transposition. 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SOSuiH £ 5 MS 6 SI wBIJ fciRsnfcra i3m±ommm^u t¥$['£tlfcb < 300 o 200 4 5 6 7 8 9 10 11 12 13 14 15 ^ "j Hi2:sosuin HTHScan CoilScan SPScan cDNA * 230 1001 763 #& SPScanT^£B£ tltc i/ ^ 7 H£J^T IZtjsT 0 0 5 1015 20 25 30 35 40 45 50 55 60 65 70 -95- 5. 5 1. Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402 2. Gribskov et al. (1988) CABIOS 4(1): 61-66 3. von Heijne, G. (1987) Sequence Analysis in Molecular Biology: Treasure Trove or Trivial Pursuit. 4. McGeoch, D. (1985) Virus Research 3:271-286 5. Claverie, J. M. and Audic S. (1996) CABIOS 12(5):431-439 6. Lupas, A. (1996) In Methods in Enzymology 226:513-525 7. Pickett, J. (1982) Nucleic Acids Res. 10(17):5303-5318 8. Mitaku, S. (1998) Bioinformatics 14(4):378-379 9. Wheeler, DL et al. (2000) Nucleic Acids Res. 28(1):15-18 10. Apweiler, R. et al. (2000) Nucleic Acids Res. 28(1):15-18 11. Bairoch, A. et al. (1999) Nucleic Acids Res. 27(1):215-219 12. Jurka, J (1998) Curr. Opin. Struct. 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Annotation Analysis Analyze Motif if-lAtl Add Prosite (PS) 08 Condition Motif }HIH mRAi F AM A H (Average hydrophobicity) Integrated Analysis P Value 1 E FO Clustering Result Quality P High (H) P Mic Very Low(VL) HTH A RAC FAMILY 1, F’ATTERN Bacterial regulRt: ry prot /yi) ZINC,FINGER C2H2; PATTERN Zinc finger, C2H2 type.doi ZINC.PROTEASE, PATTERN. Neutral zinc metallopeptidas-= 2FE2S_FERREDOXIN; PATTERN. 2Fe~2S ferredoxins, iron- Add Pfam (PF) DB Condition 4FE4S„FERREDOXIN; PATTERN. 4Fe~4S ferredoxins, iron- Domain |AhpC/TSA family AA_TRANSFER,CLASS.1; PATTERN Aminotransferases cl E Value [0 AA_TRANSFER,CLASS„2; PATTERN Aminotransferases cl _AddJ AA_TRANSFER_CLASS.5; PATTERN. Aminotransferases cl AA_TRNA_LIGASE.I, PATTERN. Aminoacyl-transfer RNA s AA_TRNA_LIG ASE.II.2, PATTERN. Ami noacyl-transfer RNA And / Or Condition No. A p No.B IF " A And B" Add | * A Or B* Add Integrated Analysis - Netscape EMEl Oz-OKF) mm Result of Integrated Analysis F DB Search & Sequence F Expression Profile r Mapping AA sequence code : a-bb / a~bbn a:0-0riginal / R-Revised(by "Check System") ,tf-7 bb:F0,F1 ,F2,R0,R1 ,R2(6 Frame by FrameMaker), FAn,RAn(ATGpr Forward/Reverse No.n),FX(Fixed) Seap^ffce RetrietW^Ad Score Sequence Motif Name ATGpr Psort Sosui ' Pros it.g Pfam 0-FA1 0-FA1I O-FO 0-F1 0-FA1 0-FA11 / 0-RA3 C-HEMBA1 000001 0-FA1 0-FX none 0-FA1 L pattern ^§2.599998%:nuclear 1 Domain > 0.810000 1 3.0RF7/f-y3>yXfA(l*lt5 130 (0i6). 1) ammaeux h 0 Swiss-prot 0 ft—V— K$?#T fit aIi L ft Swiss-prot 0 ft “ *7 — K1W ft -3 T > H ill ft -S Swiss-prot 0 X > h U — ft B# %0i#i^< >AftXft-^0AmcDNAE^J0X> h U-^-^^^ft^,o #Swss-prot0EC No.^?t/f fa^lftcEC No. ft Swiss-prot0 X > h U — ft## L, BiMO^lW >A^f- X 0A#cDNA@g7'J0X> h • Swiss-prot 0 ft S; ft ft X h ## m^L7cft#0ftftX Sft^^Swiss-prot0Xft h U-ft##L, B##0i%Wft ft A ftXft-ft 0£6cDNAE7lJ0Xft h U -ft-KSftft5o # Swiss-prot 0 ft fa^L/7cft%@ft#^0$)^) Swiss-prot 0 X ft h U — ft ## L» Bitift 0 ?6 ft ft 7N ft Xft^ft 0±#cDNA@g^J0Xft h U -ft^K^ftft6. #Owl0ftSftftX h ffltft f^^L^cft#0ftftX h ft^a tfOw!0Xft h U -ft##L, B##0i^VW ft/\ftXft “ ft 0 ft: HcDNAE7J <7)X > h #Owl0ft#@^B## j#%L/cft#@l:M#0&6Owl0Xft h U -ft##L, B##0^^ft ftAftXft- ft 0 ft 6 cDNAE 70 0 X ft h #mRNA(h(7)|p|—1±## fa£ LfcrnRHA^Xj-ft'S IrJ—ftftTX — M ft## L, # ^ T 6 ft >A^J“ X0ft6cDNAE7O 0x ft h u £o #mRNA^0^ft###T m^L^mRNA(:^ft-6^ft#0^ftft^T3- Mft##L, m^ft-^ftftAftX^- ft 0 ft 6cDNAE 70 0 X ft b U “ft-H^ftf £o ^-###^^ft### j: 0#6^6ft ft/\ftXx^ft0ft&cDNAE7iJ0Xft h U - 0-Kfti$cftU 2) M#k<;i/(7)m-# c e-mi, ^toi/-#m(7)^m#6a6miRi#(7)X3 7^e)#m$^^Mmix<;i/(7) mW1]fe\Z.-O^TmmtZ>o NCBI (National Center for Biotechnology Information) L /cblastxft fOffl ~T %> <, d ^ 0 o'^Oii'Tc ##ET0ftft ftAft%07a 7 Swiss-prot#0ft ftAftM0^-ft^- X (FASTAX;*- —VyH ^-0^^(hbT, ft^-xm0XfthU-##^. BM0fB4# -132- >gi|l 169338 |sp|P2191 2|DHSB_HUMAN SUCCINATE DEHYDROGENASE (UBIQUINONE) IRON-SULFUR PROTQN PRECURSOR (IP) (IP SUBUNIT OF COMPLEX II) Length = 280 Score = 581 bits (1481), Expect = e-166 Identities = 278/281 (98%). Positives = 7tK)/281 (98%) Query 134 MWVALSLRf^PATTLGGACLC^SRGAQTAAATAPRIKKFAlYRWDPDKAGDKPHMQT 313 MAAWALSLRRRLPATTLGGACLQAS^GAQTAAATAPRIKKFAIYRWDPDKAGDKPHMQT Subject: 1 MAAVMALSLRRRLPAXTLGGACLQASRGAQTAAATAPRIKKFAIYRWDPDKAGDKPHMQT 60 P-V alue = logl O(Expectfi) I dentity ex.) logl0(e-166) Decision Tree: id <= 53 : | id <= 47 : | | pval <= -41.4202 : | | | pval <= -44.6383 : c (15.0) | | | pval > -44.6383 : | | | | id <= 44 : c (4.0) | | | | id > 44 : b (2.0) | | pval > -41.4202 : | | | pval <= -0.0409586 : c & 14.0/5.0) | | | pval > -0.0409586 : 0i 8. y :iu x'a -134- 3) ^ScDNAE^L RtXSwiss-protOX> h U—+ —7— — OCD^ScDNABB^HC^ B LT, t^U'J — &f&0j^t#f>nfcSwiss-prot©X > h V-, m^U'OV, &£V^£(DX.> h U-^^'ti'^-7-^-7- H era CIM % y f L, lx/<;i/^4e(D4:-7 - El9(DS^'f# 6 4lS = T&:b*> , ^rficDNABB^J HE — 7— -5 o CD 7^T'cf-5o 7c H-E-S^TEEn- HB:te, zh^E IDh, IDm, IDK NID04P ^)VX^ U T lA 5 „ # £ IDh |1| —14 iW < > NID11 IrI —14 ^ 5) ^AE## ^AE##"C11, mRNABB^iJ B:(D^^t4^^1-^E3- F cDNABB £0 -135- / \SPX> MJ 4=©*- r ^ScDNA 82?l| K-1 1 K-2\ K-3 K-4 K-n C-XXX< POO; A B C-XXX2 C : D i C D C-XXX< J(B> : D D A C-XXX< , D A C-XXX5 D C B C C-XXX6 D D C-XXX7 B C [ C \ C-XXX8 D D C C-XXX9 B \ i D C-XXX1 0 \ / B c ■' • ' ** V' •: * *• * "■ " • • 0 1 9.ific DNAlB^lJi ^r— V~ KOH# Ic / Is >= 90% Tzt^ytyV ^ >= 300bp Ic / Is < 50% Identity > 95% NIDdH-'ttfcU X Ic : zq>tZ>-yX^. Is : Query R?lJ£Subjectfi£?lj0*sU fiO> K?'JH 0 2 0. IrI—I4S*^1"5>E=-- K 3’ 5 nasq query sbject 02 1. ^gcDNASB^J (hmRNA&U^ EST(CO^T nast NF Bn . . hpH NS $ t h_2 2_G (20) PF $th_21_G (5) 4° $th 11 G $th 12 G nasq (5) (20) El 2 2 — K -137- • OwiESt-^X bmtir • Swiss-prot 41—9 — K 4^-9- M«k^#1x^71/a^a. • Swiss-prot EC No.###E EC No.tmfa U'Ol%Mtir4kfrt'?Z>o • Swiss-prot • Swiss-protES "X* ^ X h E^x^X H2:M#1x<;i/$:##^EaTa. • OwlESx^X h###E E#x4^X • f7^JH' MB! hy h L^^^cDna SH^J cdx > ^Ta. 023^^f \z, OT^a. (1) ##C43WT#mL^^E(D#^ (2) ##(D*@m#Sa&^#cDNAE9J(DX> h U (3) ^ScDNAE^J(DX> h U — M T a Swiss-prot ^ 7c ^ / ^3 j: ^ Owl (D # # Swiss-prot (C D T$> a o >X •^srapRX ^x^tc^tta^^Dv—v >x<> f/J'tDy —th—f O h77pby h (7) X > h U — ## Acc (X/h'J — ^0 'J >Xf4)> EC No. 0%EC No.(h4^—9 — M(7)M#^#^(7) U > X #) > DeffinitioiK Keyword OK 4^—9 —K ^K±b v EC No.^3i:r;+“9- H £ >bl;, M#k^7l/A, B (ab), C (ABC) 1C 43 O'a ^ Swiss-protX > h U “-ffc £:I^ thTT a o $ 6 (C , <7) S tk 7^ 6 > *€“ u >XTa. • ^EDy-X-fO h'y9°b'y h<7779-fX> H43 j;^M#lx^7l/## lx ^\)l/ (R-revel) b, 4r4l$r et#^ a^:)6(7)^-@/i9X'—Xo -138- Owlicilf 0 t D $/ — th — f 7 h © x > h u — Accx EC No.x Definition> Keyword by'fhy h©7v-f/> m24lzmmr / x-va LTx +-y-Fl:fgLMftf pJ|gT$>^o C(D=k3(:x ^yXfAlc^^m @B?m-e - E^TS^ScDNAge^lJOlWISM^^ # L Xio (3) a. < >/\9XT#m$#l^cDNA(Z)±&d3 6^6. a-c^x mA -5yu y fy -a-ij, *ist ^Sd^Jlf > F 7-^7 chWX^o #&x European Bioinformatics Institute (EBl) Ttl te/W Z? U y H 7 — t) — £rx“7 ^ — L x J T^t)^x EBI^NCBI©X-7e##76 C 61: j;oTx ##%0±©%##/\< 7*U y H v-73-i-^SUT^^dbEST-^dbSTS0gB^J1WW5:5j<6b^ ^ $ 5KX ^nt> (026)0 ±sb©xo©u (0 25x 026)x < >/\7%©@B7J##^^a##a u >7^#^ c a^oj#c^6o < >A^7XtCd3V^TE#L^cDNAOBB^JtC^ST^^fe#:±(7Mir fl1W#(DS^^x ^C1:U >/7T6^m##om^^B%aT%0 #l:x ^@##6L/Tx #m%0^ ^^x m^TZ'f >/\7x©x-7£j# ###©^±^0-5o $ bl:x LT^Xx v bf >y$a6<>AC7X^:#cDNA6:^m##^^^ormCT6o 027CT v H>^##r©^66©x-7^U >^©##^^"7o C fl 6 ©##(:: j;oTx ^:#cDNA(:^T & < o X-lf-A ^(DfmV^btHZ^LTx Smt~5^ScDNA£x Ty ¥>?<&&$& tmMffimtt&lZ 028CTvbf>y##(D##^m:A7jim@(De!ie^fo C(Dm@^6genernap -139- ~>r 15) WB §iiaWP 'if >cn»-x Integrated Annotation Analysis / Modify Query cDNA-DB -9-K A^J Search System Menu ECNoA* W Select leeclem CSP : Series-prel) SP Keyword ifetottA* SP EC No. SP Ortansm Taxocu SP Text f Or R-Le«el » A B 7'M-r A^ AND/OR Lofk«l Owety ECNo 0 2 3.EtET/T-'>3 8tX£#cDNA (P30G41 i-23460 P44411 P5697S (PI 8476 P21762 P36700 006B*r\P3S704 P35705 P48822 P52552 P80002 P91883 0 04120 0 77834 PI 8476 P21762 P26830 P30041 P34227 P48537 i 02 4.mHZ7 /-T-v3 jr-9-nc#g L/"cEEttf-7'> -140- mm a £ile Edit yiew Qo Qpnmumcatw bate esse Mapping Analysis Select Sequence Pand is RHMap SG3 (cR) r RHMap GB4 (cR) DB Sdect P dbSTS P dbEST P GenBankEST f~ GenBank mRNA P GenBank genomic f~ GenBank unfinished genomic Blast ______E Value Range fo . (To Method Sdect Sequence by Name Sequence Name jc-HEHBAiOOOOOl I* Sdect Sequence by Chromosome Number and Distance Chromosome Number 121 (SG3:0-1630cR GB4:0-266.94cR) Distance from top (cR) |l0290 o Map li$t< dbSTS,dbEST ^ST,genomic ) mm Otoe No. Sequence Name tor SG3 1 C-HEMBAl 000001 21 44.3-49.9 1308 P0.7 genomic AF064859. 1 5 polymorphism 13_ 2 C-HEMBA1000001 21 34.7-35.4 888 F genomicAP000044. 1 5 polymorphism 1 3 C-HEMBAl 000001 "21 34.7-35.4 888 F genomic JC604 59.1 5 polymorphism 1 4 C-HEMBAl 000001 21 34.7-35.4 891 PO.O genomic AP000044.1 5 polymorphism 1 5 C-HEMBA1000001 21 34.7-35.4 891 PO.O genomic JC60459.1 5 polymorphism 1 lamtHeT «hw ' 112250 Bone dysplasia with medullary fibrosarcoma 147660 Interferon, alpha, deficiency i 238300 Hyperglytanerraa, nonkitotic, type I ; 247640 Leukemia, acute lymphoblastic > K^( 02 8 -143- /itilcDNA© >J X h ^i28^ C^to &±#cDNAt:#LTkL (028). b. iieOyXfAi^^T, £#cDNAgB?iJ #, A#cDNAE^(D^m##a^^^C(b^Te6. cn&$UmLT, XvtfXX#^ T#^yc^#cDNAE^(D^m##<&#**^ XXXA&im^L/:. 028(:i-(D#f f. ^#cDNAE^J(:*LT^m##m&^^T6. (4) iWa-T-f <-X(Z)## ^±E%L/tStff'>XT-A^E^UTfiJfflT#^'>X7LA^MBU^ (029). 03O(: 0RF7/f-y 3 >> f'"57/<-X'tt-f l"I^'< SlbT / 7 — XgX, Tvkf>^7/T-y3>(D"e^l^^U:^LT##^m:$:j@^L"r, 7fl6^± T^fef^ficDNAga^J^U X h oJt^T'fe^o (5) f^^s/x^Ac ±#cDNA###(:MT5 7/ 7-X a X&# cc-rn, £t\ #6^i^±#cDNAga^j(D#m^ ##f L/c. ^6gB^J#(D^$^, A U y X XEX □- >£j 4000@B^J(:3^T^A^$§^^[g3i(:^T. gB^Jiooo#*#^ 63000#*# (c*(c^#L (FL)= it 3& (D #) H , ^#^-X<-XT&6 UniGene (7) ^ # mRNA (UniGene) > S H 2 tl tz H X V ^DNAE^u^tDfiEcDNA (KIAA) <£> # £ £ 7 L . Z.(D£o\Z, * X U X x X h TS 6 nfc±#cDNAte> £ il £ T CD 2 £2" £ & X D X x X h T# 6*l7;:£#cDNA 7. 4 emaatf^&omaRB (1) $§# M5$ -144- frfejj&tLTURLrzo ^n->©n>^^^©rajs ^$>o^^x 2^^AT2298Xn->(D^ SE^IJ^S^^TL, 3^*IttSt)201^n->jiJPU, ^H-2499^7 D-yOllr^Tl fto b. ^S^iacDNAf“^^“XyXfA(Dii ® IT, "T —^IiJ&LIIvX'tAx B :> X 7 A x ###3 j; Uf$a;#(Z)Mi A X7A^^^Lyc« f“^iy®lyXfA(5;l t^x Hz/UMTa&mmaWx evx caawm%i/x^A&##BL;t. ffijT y f“'> a yyXfAiITti, DDBJ^O^HcDNAx — X SBffltiD'fr — XfEfi£5: ##7X7AALTx 0RF7/7-7g>7X7Ax 7-X<-X It — 7 (C S < SlbT yf —ya yyXtA, X y H > X 7 / 7 — v 3 yyXtA$:l^ U 7tl 6 (D^-o ###7 7X 7 A 1/yho © LTx ±iax v ^>^7/7-7a >XX7A(7)^m(:x ^m7-X<-X(D##$:m^^t)1t6 C6 la-at, ^Sx^iMyXrA^iiLfco ® 5,OOOX n-XD^r^cDNA^ttf £^£-x — x^ — X £ IT, _LEjW#r-T-X (2) ^#^lx ^^Utc^ScDNABB^iJ-tWIgAX LTx c M##x %#y/A##g;&x $6cm^Lyh7-x<-x7X7A^M%LTv^ W. ^ tl 'b o 7. 5 am## [1] Wadman, M., Human genome deadline cut by two years, Nature, 395, 207, 1998. [2] Maruyama, K., Sugano, S., Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides, Gene, 138, 171, 1994. [3] Ota, T., et al., "Full-Length cDNA Project toward a High Throughput Functional Analysis", Microbial & Comparative Genomics, 2, 204, 1997. 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