DNA RESEARCH 6, 247-253 (1999) Short Communication Evaluation of a cDNA Scanning Method Concerning the Fidelity and Efficiency of cDNA Selection Using the YAC CIC3B1-S Region of Arabidopsis thaliana Chromosome 5 Reiko MOTOHASHI, 1 - 2 Takuya I T O , 1 Motoaki SEKI, 1 Kazuya ICHIMURA, 1 Kazuko YAMAGUCHI-SHINOZAKI, 2 and Kazuo SHINOZAKI 1 '* Laboratory of Plant Molecular Biology, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan1 and Biological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-2 Oowashi, Tsukuba, Ibaraki 305-0074, Japan2 (Received 5 April 1999; revised 7 June 1999) Abstract We previously reported a cDNA selection method using DNA latex particles to identify expressed genes in specific regions of genomes and named this cDNA scanning method (Hayashida et al., 1995 Gene 155-161). We applied the cDNA scanning method to the YAC CIC3B1-S DNA on Arabidopsis thaliana chromosome 5, and constructed a region-specific sublibrary in which cDNAs for genes on the YAC CIC3B1- S DNA were concentrated. We isolated 545 cDNA clones from the sublibrary, and determined partial sequence of them to produce expressed sequence tags (ESTs) derived from the YAC region. In total, 74 nonredundant groups of cDNAs were obtained from 545 cDNA clones. Forty-seven percent of these EST clones had significant homology to functional proteins such as protein kinases, LON protease, nucleic acid binding protein and chloride channel protein. We compared the cDNA sequences isolated by the cDNA scanning method to the Arabidopsis genomic sequence corresponding to the YAC CIC3B1-S region, and found that 69% of the selected cDNAs are located in the region. We discuss the fidelity and efficiency of the cDNA scanning method for cloning region-specific cDNAs and its useful application in positional cloning. Key •words: Arabidopsis thaliana; cDNA scanning method; YAC 1. Introduction mination of the entire genome sequence. In Arabidopsis, expressed sequence tags (ESTs) 5 ' 6 and molecular mark- Recently, whole genomic sequences of various organ- e rs are rapidly accumulating and yeast artificial chro- isms including many bacteria, yeast (Saccharomyces mosome (YAC), 7 bacterial artificial chromosome (BAC) 8 cerevisiae) (http://genome-www.stanford.edu/Saccharo- a n c i p i p h a ge libraries 9 are completely equipped. More- myces/), 1 and nematode (Caenorhabditis elegans) (http: o v e r, Arabidopsis genome sequencing will be finished by //www.sanger.ac.uk/worm/C.elegans.Home.html) 2 have the year 2001. Using these useful tools, many genes have been determined one after another. Now genomic been isolated by positional cloning, and their functions sequencing projects of several eukaryotes including have been analyzed. 10 " 12 However, main crops such as Arabidopsis thaliana (http://www.kazusa.or.jp/arabi/, maize and wheat have larger genomes and their genomic http://genome-www.stanford.edu/Arabidopsis/), 3 ' 4 rice, sequences are not easily determined. Moreover, mapping Drosophila melanogasta (http://fly2.berkeley.edu/), and m a n y EST clones on a genetic map is laborious and time human (http://gdbwww.gdb.org/) are in progress. Re- consuming. An efficient method for mapping expressed verse genetical approaches become more important for g e n e s o r c DNAs in specific genomic regions is necessary efficient functional analysis of each gene after the deter- t o identify candidate genes within specific regions that Communicated by Satoshi Tabata contain mutation loci in crops that have larger genomes. * To whom correspondence should be addressed. Tel. +81-298- We previously reported a cDNA selection method 36-4359, Fax. +81-298-36-9060, E-mail: sinozakiOrtc.riken. which we termed the cDNA scanning method. 13 - 14 The gojp , , , , cDNA scanning method is based on hybridization selec- t The nucleotide sequences reported in this paper are deposited -\rA/~i A/-I tl0n of in the DDBJ/EMBL/GenBank nucleotide sequence databases cDNAs using large fragments, Such as YAC, BAC with accession numbers AB018246, C99851-C99932. and PI clones, of genomic DNA that are covalently bound 248 cDNA Mapped on Arabidopsis YAC CIC3B1-S [Vol. 6, to latex particles. The hybridized cDNAs are eluted, 100 positive clones, major cDNA species were 3B1-43-1, amplified by PCR and cloned into AgtlO vector to con- 3B1-43-2, 3B1-45 and 3B1-81. For concentrating minor struct a cDNA sublibrary. The cDNA sublibrary was cDNA species, we removed major cDNAs from positive then screened with corresponding genomic DNA frag- clones by Southern hybridization using 3B1-43-1, 3B1- ments. The partial sequences of selected cDNAs provided 43-2, 3B1-45 and 3B1-81 as probes. Then, we obtained information about genes that are located on the genomic 545 positive cDNA clones for further sequence analysis. region. We think it important to evaluate the reliability of the2.2. Sequence analysis and characterization of selected cDNA scanning method for further application of this cDNA clones method. The 545 cDNAs selected from the YAC CIC3B1-S re- In this study, we applied this cDNA scanning method gion were sequenced from the 5' ends. Redundancy of to effectively concentrate cDNAs that are derived from these cDNA clones was examined by computer analy- a YAC clone CIC3B1-S Arabidopsis thaliana chromo- sis (Sequencher 3.0 software, Hitachi Software Japan). some 5, and determined their nucleotide sequences. Re- The clones with overlapping nucleotide sequences were cently, the nucleotide sequence of its corresponding ge- regarded as one group. Finally, we identified 74 non- nomic DNA has been reported. We then evaluated the redundant cDNAs by sequencing 545 clones derived from fidelity and efficiency of the cDNA scanning method to the CIC3B1-S region (Table 1). Based on an estimation isolate region-specific ESTs by comparing the ESTs with of one gene per 5 kb of Arabidopsis on average, these the corresponding Arabidopsis genomic sequence. groups cover approximately 69% of the existing genes on the CIC3B1-S region (Table 1). We confirmed that 2. Results screened cDNAs from the sublibrary were saturated. Se- quence homology of these cDNAs was examined with 2.1. Construction of a cDNA sublibrary of YAC the GenBank (release 102)/EMBL (release 51) database CIC3B1-S region in Arabidopsis using the FASTA program.16 Homology of the deduced To evaluate the fidelity and the efficiency of the cDNA amino acid sequences was examined with a nonredun- 17 scanning method, we used a YAC clone, CIC3B1-S, on dant protein database using the BLASTX program. Arabidopsis chromosome 5 as a model. The Arabidopsis PAM120 scores of > 80 of the result were considered CIC YAC library includes two CIC3B1 clones, CIC3B1-L to have significant homology. Based on these analyses, and CIC3B1-S. There are located on the long and short 89% of the clones were identical to registered sequences arm of Arabidopsis chromosome 5, respectively. The or ESTs of Arabidopsis, and 47% of them had significant CIC3B1-S clone has a length of 540 kb.15 The genomic homology to functional proteins from various organisms. sequence of the CIC3B1-S region was determined by the We isolated cDNAs encoding functional proteins such Cold Spring Harbor Laboratory group (WEB site). We as protein kinase (3B1-29, 60, 74), LON protease (3B1- compared cDNA sequences isolated by the cDNA scan- 61), nucleic acid binding protein (3B1-45) and chloride ning method with the genomic sequences and evaluated channel protein (3B1-81) (Table 2). Five cDNAs (3B1- the efficiency of the method in this study. 6, 3B1-26, 3B1-55-1, 3B1-58, 3B1-73) had no homology We used a PRL2 cDNA library derived from almost all to reported sequences in the public databases which in- organs of an Arabidopsis (Columbia ecotype). PCR prod- dicates that the cDNAs are derived from novel genes in ucts of DNA inserts derived from a cDNA library were the CIC3B1-S region. hybridized to the YAC CIC3B1-S genomic DNA cova- lently bound to latex particles. Nonhybridized DNA frag- 2.3. Comparison of nucleotide sequences of the identi- ments were washed away to concentrate cDNA fragments fied cDNA clones and genomic sequences derived from the genomic DNA. Then, we constructed a Recently, the nucleotide sequences of four BAC clones cDNA sublibrary using AgtlO vector, and screened pos- (F9D12, F21E10, F2P16, T21B4) within the CIC3B1-S itive clones by the YAC DNA as a probe. Among the region were determined. These BAC clones were assigned 200 clones in the sublibrary, 5-10 gave a positive sig- nal. The length of DNA inserts was about 300-2000 bp to the YAC map by hybridization by the Cold Spring (data not shown). We partially sequenced these cDNA Harbor Laboratory group (Web site). To estimate the clones from the 5' end with T7 primer. To select mi- fidelity of the cDNA scanning method for the selection nor species of cDNAs more effectively, major species of of expressed genes in a specific region, we compared the cDNAs were removed by hybridization. There was some nucleotide sequence of the identified cDNA clones and bias in the population of cDNAs in the sublibrary, which the BAC genomic sequences. We found that 58 out of might be due to differences in amplification by PCR, ef- 74 obtained cDNAs existed within the YAC CIC3B1-S ficiencies of cloning and a different population of cDNAs region (Fig. 1), which corresponds to 78% of the selected in the starting library. The first time we sequenced cDNAs. There are two BAC clones within the CIC3B1-S region of which sequence data have not been opened yet. No. 4] R. Motohashi et al. 249 T19G15 CIC2E3 3Bl-l,3Bl-5,3Bl-9,3Bl-10,3Bl-ll,3Bl-22 60.5 AF3 3Bl-33,3Bl-38,3Bl-39,3Bl-43-2,3Bl-44 F9D12 3Bl-45,3Bl-46-l,3Bl-51,3Bl-59,3Bl-67,3Bl-69 3B1 -70.3B 1 -72.3B 1 -79,3B 1 -81,3B 1 -82;3B 1 -83 3B1 -84.3B 1 -85.3B 1-86,3B 1-87-1,3B 1 -88 T27J6 Intend to sequence F21E10 3B1 -20,3B 1 -31,3B 1 -53,3B 1 -55-2 112 Kb 3B1-64.3B1-77 CIC3B1 (540 Kb) 06455 3B1-2,3B1-3,3B1-17,3B1-19,3B1-23,3B1-3O F2P16 3B1 -40.3B 1 -42,3B 1 -43-1,3B 1 -48,381 -52 110 Kb 3Bl-60,3Bl-61,3Bl-74,3Bl-75,3Bl-78 Tnll8 3Bl-18,3Bl-41,3Bl-46-2,3Bl-47 T21B4 3B1 -62,3B 1-65.3B 1 -66.3B 1-76 90 Kb 68.4 mi 125 F21A20 in Library or production F15A18 CIC12F8 Figure 1. Map positions of YAC CIC3B1-S and the EST clones on BAC clones, F9D12, T27J6, F21E10, F2P16, T21B4 and F21A20 are shown as thick bars. Representative RFLP markers hybridized to YAC and BAC are shown at the right with thin bars. Sequences of cDNAs on BAC F9D12, F21E10, F2P16, and T21B4 were submitted to GenBank and the production of a F21A20 library is still ongoing, and T27J6 sequencing has not yet begun. Table 1. Number of EST clones derived from the YAC CIC3B1-S region. YAC clone Sizea No. sequenced No. groups No. estimated % saturation0 (kb) clones existing genesb CIC3B1-S 540 545 74 108 69 a The length of CIC YAC DNAs is derived from Creusot et al.7 b The number of existing genes on each YAC DNA is based on an estimation of existence of one gene per 5 kb on average. c Percentage [(No. groups of sequenced clones)/(No. estimated existing genes) x 100]. Construction of C1C3B1-S region-specific cDNA sublibrary. Account that a region-specific cDNA sublibrary was constructed is given below. Detailed strategy of cDNA scanning were reported by Hayashida et al.13 and Seki et al.14 All the cDNA clones described were derived from a PRL2 cDNA library prepared from etiolated seedlings, roots, leaves, and flowering inflorescences of Arabidopsis thaliana (Columbia ecotype).5 cDNA inserts were amplified by PCR from the PRL2 cDNA library using pair of RV (5'-CAGGAAACAGCTATGAC-3') and M4 (5'-GTTTTCCCAGTCACGAC-3') primers. PCR conditions were as described by Seki et al.14 The PCR amplified cDNA was subjected to cDNA scanning as described by Hayashida et al.13 and Seki et al.14 The hybridized cDNAs were eluted from YAC DNA-latex particles. The eluted cDNAs were amplified by PCR using SP6 (5'-GTGAATTGAATTTAGGTGACA-3') and T7 (5'-AGGGAAAGCTGGTACGCCTG-3') primers. The region-specific cDNA sublibrary was cloned into the AgtlO vector with an EcoRI adapter. To isolate positive clones from the sublibrary, plaque hybridization was carried out as described by Sambrook et al.18 using the YAC CIC3B1-S DNA as a probe. These positive cDNA clones were selected and partially sequenced. YAC DNA templates were prepared and purified as described by Schmidt et al.15 250 cDNA Mapped on Arabidopsis YAC CIC3B1-S [Vol. 6, Table 2. Characterization of cDNAs that were selected with the YAC DNA clone, CIC3B1-S DNA-latex particles. Gene- accession genome Identical Related Encoded protein/Others features' High Organism number" sequence* registratJon" registration' score8 3B1-1 AB018246 AF077407 UDP-glucoronosyl AI00231 and UDP-glucosy( transferases AA713031 3B1-2 C99851 AF007270Di19mRNA 3B1-3 C99852 AF007270Di19mRNA AA66O487* 3B1-4 C99853 L46542' 3B1-5 C99854 AF077407 Z714S0.T44764 CLC-d chloride channel protein AA395428 3B1-6 C99855 3B1-7 C99856 W43671 3B1-9 C99868 AF077407 T22151,H37204,F14367, AA394350 3B1-10 C998S9 AF077407 AI100231 3B1-11 C99860 AF077407 AI10O231 3B1-12 C99861 AC004138 Z12017 predicted protein 115 Caenorhabd'nis elegans 3B1-13 C99862 AC005309 T04053 U93215 glutaredoxin isolog 339 Alabidopsis thaliana 3B1-14 C99863 AL031135 T45397,R30127,N97034, H3744,N37952,T44489, U26945.U26944 637 Alabidopsis thaliana R65483,R29982,T75992etc. R90440,N65251,N38143 etc. senescence-associated protein mRNA 3B1-15 C99864 AL022224 Z12017 predicted protein 105 Caenorhabditis elegans 3B1-17 C99866 AF007270 N37645.T13646 DNA polymerase III, alpha chain 3B1-18 C99867 AB008271 T04322,AA7126O4,N65O0O AA712370.T46317,T42562etc. 3B1-19 C99868 AF007270 234597.AA395358 T43703.W43384 serine hydroxymethyltransferase 3B1-20 C99869 AF058914 F13579.H36310 R30103.H36309 X89430 methyl CpG binding protein2 88 Homo sapiens 3B1-21 C99870 T44812 3B1-22 C99871 AF077407 UDP-glucoronosyl R84019 H36572.230909 and UDP-oJucosyl transferases 3B1-23 C99872 AF007270 Di19mRNA 3B1-24 C99873 AB015478 AA824726 X72617 mandelonitrile lyase 251 Prunus serotina 3B1-25 C99874 AC003680 F153O6.F153O5 024913* U96915sin3 associated 229 Homo sapiens pclypeptide p18(SAP18) 3B1-26 C99875 3B1-29 C99876 AF000147 AI0S5635" AF000147 UMP/CMP kinase 505 Alabidopsis thaliana U32330* (pyr6) mRNA 3B1-30 C99877 AF007270 AL021960BACF7J7 219 Alabidopsis thaliana predicted protein 3B1-31 C99878 AF058914 AF067773 aminoacyl-tRNA synthetases 3B1-33 C99879 AF077407 N38486.T45918.T42069, cDNAT75896,N37736,T42069 N37736.T75896 3B1-38 C99880 AF077407 UDP-glucoronosyl AA713031 and UDP-glucosyl transferases 3B1-39 C99881 AF077407 3B1-40 C99882 AF007270 DNA polymerase III, alpha chain 3B1-41 C99883 AF007271 contains weak to the SAPB protein 3B1-42 C99884 AF007270Di19mRNA X7B584.T04522.Z33919 etc. 3B1-43-1 C99885 AF007270 the SPOU family of rRNA methylases 3B1-43-2 C99886 AF077407 UDP-glucoronosyl AI10O231 X59814.T45310.T13741 X90959 Cor47 gene; dehydrin 292 Alabidopsis thaliana and UDP-qlucosyl transferases R29775,N37217,N38174 etc. ABA responsive gene 3B1-44 C99887 AF077407 cDNA N37539,R65355,AA394350, N65299.N65011 etc. N37424,Z34546,T88487 etc. 3B1-45 C99888 AF077407 H35953 L0729r,AF04557r, L07291 Alfalfa nucleic acid 927 Medicago saliva AF047428 - ,AC005312 binding protein (alfin-1) 3B1-46-1 C99889 AF077407 £ cofi cation R30560.F19910 transport protein ChaC 3B1-47 C99890 AF007271 MADS domain 3B1-48 C99891 AF007270Di19mRNA AA660487",X78584,T20623 etc. 3B1-49 C99892 AA042407,AA585949,AI099883, S47408 gfycine-rich protein 75 Alabidopsis thaliana W43529,N95880,AA597537 (atGRP-2 clone) 3B1-51 C99894 AF077407 nucleic acid binding protein Alfin-1 3B1-52 C99895 AF007270 Z25859-,Z25863-^2860", Z25859 826 Flaveria pringtei M87649',M87650* qlycine hydroxymethyltransferase 3B1-53 C99896 AF058914 Vigna radiata pectinacetylesterase precursor 3B1-55-1 C99898 3B1-55-2 C99899 AF058914 AF067773 aminoacyl-tRNA synthetase 3B1-58 C99902 3B1-59 C99903 AF077407 phosphoenolpyruvate N37505 synthase (ppsA) 3B1-60 C99904 X68525.X75432, X75431 AJ0O0732 ASKalpha gene 1329 Alabidopsis thaliana 3B1-61 C99905 AF007270 similar to the U88087,N38095,R65124 U85495* U88087 LON protease 794 Alabidopsis thaliana peptidase family S16 homolog mRNA 3B1-62 C99906 AF007271 contains weak to the F19867.F19751 No. 4] R. Motohashi et al. 251 Table 2. Continued. SAPB protein 3B1-63 C99907 AP000002 109 Pyrococcus horikoshii Pyrococcus horikoshii qenomic DNA 3B1-64 C99908 AF058914 aminoacyl-tRNA AF067773 synthetase 3B1-65 C99909 AF007271 T75860 sodium/hydrogen exchanger 3B1-66 C99910 AF007271 AA395155 3B1-67 C99911 AF077407 chaperonin containing N37479.AA712752.Z34940, TCP-1 complex gamma chain Z17442.Z17433 etc. 3B1-69 C99913 AF077407 UDP-glucoronosyl AA713031.Z29206 AI100231.AI100139 and UDP-glucosyl transfemses 3B1-70 C99914 AF077407 UDP-glucoronosyl R84019 Z30909,H36572,T22664, and UDP-qlucosyl transferases T42399.F14132 3B1-72 C99916 AF077407 H37204.AA394321 .H76508 AA394350,N65299.R65355, AF077407 BAC F9D12 844 Alabidopsis thaliana N37424.N65011 ,N37539 etc. predicted protein 3B1-73 C99917 3B1-74 C99918 AF007270 myosin heavy chain AJ000732 ASKalpha qene 3B1-75 C99919 AF007270 Di19 mRNA AA660487 3B1-76 C99920 AF007271 3B1-77 C99921 AF058914 AF067773 aminoacyl-tRNA synthetase 3B1-78 C99922 AF007270 MIPP proteins 3B1-79 C99923 AF077407 3B1-81 C99925 AF077407 Z71450.AA395428 T44764 Z71450 CLC-d chloride channel protein 307 Alabidopsis thaliana 3B1-82 C99926 AF077407 R65355,AA394350,R84203, N97138,H37204,H76508, cDNA T75896,N37736,T42069 T14127,T88487,N37424 N65299.N65011.H76607 etc. 3B1-83 C99927 AF077407 H35993.N37639 N37424.T14127, N65299, cDNA T75896,N37736,T42069 AA597333 AA597772,N65011.AA394321 etc. 3B1-84 C99928 AF077407 R65355.N37424.T14127, T88487,N37539,N65299, cONA T75896,N37736,T42069 AA394350 AA394321 ,H76508,H35993 etc. 3B1-85 C99929 AF077407 N38486,T42069,T45918, N97138,T22151,F14367, cDNA T75896,N37736,T42069 N37736,AA394350,T75896 H37204,R65355,R84203 etc. 3B1-86 C99930 AF077407 N37424,R65355,T88487, N65299.N65011 .H76508, cDNA T75896,N37736,T42069 T14127,H35993,AA394350 etc. AA394321 .N37539.AA597772 etc. 3B1-87-1 C99931 AF077407 H76508.AA394321, AA394350,N65299,R65355 AF077407 BAC F9D12 902 Alabidopsis thaliana H37204.H76607 N37424.N65011 .T88487 etc. predicted protein 3B1-88 C99932 AF077407 H37204 T22151,F14367,AA394350, AF077407 BAC F9D12 758 Alabidopsis thaliana N97138,N38486,T45918 etc. predicted protein a Genes are named tentatively according to the name of the YAC clone. b Accession numbers of the EST clones identified in this study. c Genomic sequence indicates the accession numbers of the genomic sequences that are identical to the selected cDNAs. d Identical registration indicates the accession numbers of the registered ESTs and genes of A. thaliana having more than 90% identity in 50 or more bp overlap with the selected cDNAs. e Related registration indicates the accession numbers of the registered ESTs and genes having less than 90% identity in 50-bp overlap with the selected cDNAs. Asterisk indicates the source of ESTs and genes except for A. thaliana. f Encoded protein/Others features indicates the accession numbers of the gene products having the highest similarity score (indicated in next column) and other species features of selected clones. g Source of the protein exhibiting the highest similarity score. Sequencing analysis of selected cDNA clones The selected cDNA was amplified by PCR using FW (5'-GCTGGGTAGTCC- CCACCTTT-3') and +80 (5'-CAGTTTTTCTTGTGAAGATTGGGGG-3') primers. The amplified cDNA inserts were sub- jected to sequence using T7 primer. Overlapping of the nucleotide sequences of the clones was examined by Sequencher 3.0 software. If all sequences are determined in the YAC CIC3B1-S showed that obtained cDNAs cover 56% of the predicted region, the number of selected cDNAs in this region will existing genes on this region (Table 3). Compared with increase by more than 58 cDNAs. percentage of (No. groups of sequenced clones)/(No. es- The length of lined up 3 BAC clones (F21E10, F2P16, timated existing genes) on Table 1, this percentage are T21B4) within the CIC3B1-S region is about 300 kb. To less than 69%, but shown similar value. estimate the efficiency of the cDNA scanning method, we The cDNA scanning method is a reliable technique for compared the nucleotide sequence of the identified cDNA cloning region-specific cDNAs. Moreover, we obtained clones and the genomic sequences of 3 BAC clones (Ta- new three cDNAs (3B1-20, 3B1-66, 3B1-76) by the cDNA ble 3). Fifteen expressed genes out of 23 predicted genes scanning method in addition to the genes identified by from computer analysis using the program Genefinder computer analysis. Using the cDNA scanning method (P. Green and L. Hillier) in BAC F2P16, 8 out of 16 in we can obtain expressed genes which are not identified BAC T21B4, and 6 out of 13 in BAC F21E10 could be by computer analysis. isolated using the cDNA scanning method. This result In conclusion, we estimated the fidelity and efficiency 252 cDNA Mapped on Arabidopsis YAC CIC3B1-S [Vol. 6, Table 3. Number of EST clones derived from line up 3 BAC clones. BAC clone Sizea No. estimated No. predicted No. groups % saturation01 (kb) existing genesb genes0 F2P16 110 22 23 15 65 F21B4 90 18 16 8 50 F21E10 112 22 13 6 46 Total 312 62 52 29 56 a The length of BAC DNAs is derived from Washington University Genome sequencing Center. b The number of existing genes on each BAC DNA is based on an estimation of existence of one gene per 5 kb on average. 0 The number of predicted genes from computer analysis using the program Genefinder by Cold Spring Harbor laboratory group. d Percentage of [(No. groups of sequenced clones)/(No. predicted genes) x 100]. of the cDNA scanning method using the Arabidopsis sequencing of cDNAs from Arbidopsis thaliana, Plant J., CIC3B1-S YAC region as a model, and showed that the 4, 1051-1061. cDNA scanning method is a useful method to efficiently 7. Creusot, F., Fouilloux, E., Dron, M. et al. 1995a, The identify expressed genes in specific regions of the genome. 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