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					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.             CIC library: a large insert YAC library for genome map-
The cDNA scanning method seems to be useful for the                     ping in Arabidopsis thaliana, Plant J., 8, 768-770.
selection of cDNAs in specific regions of larger genomes           8.   Chio, S. D., Creelman, R., Mullet, J., and Wing, R.
                                                                        A. 1995, Construction and characterization of a bacte-
that are not easily sequenced, such as maize and wheat,
                                                                        rial artificial chromosome library of Arabidopsis thaliana,
and a powerful method to identify candidate genes in                     Weeds World, 2(1), 17-20.
positional cloning of mutant genes.                                9.   Liu, Y.-G., Mitsukawa, N., Vazquez-Tello, A., and
   Acknowledgments: We thank Ms. H. Kanahara and                        Whittier, R. F. 1995, Generation of a high-quality PI
Ms. I. Furukawa for their skillful technical assistance.                library of Arabidopsis suitable for chromosome walking,
The PRL2 cDNA library and the CIC YAC library were                      Plant J., 7, 351-358.
kindly provided by Ohio State University's Arabidopsis            10.   Hardtke, C. S. and Berleth, T. 1998, The Arabidopsis
Biological Resource Center. This work was supported by                  gene MONOPTEROS encodes a transcription factor me-
a grant for Genome Research from RIKEN, and in part                     diating embryo axis formation and vascular development,
by a program for promotion of Basic Research Actives for                EMBO J., 17, 1405-1411.
Innovative Biosciences, the Special Coordination Fund of          11.   Moussian, B., Schoof, H., Haecker, A., Jurgens, G., and
                                                                        Laux, T. 1998, Role of the ZWILLE gene in the regula-
Science and Culture to K.S.T.I, and M.S. were supported
                                                                        tion of central shoot meristem cell fate during Arabidopsis
by a postdoctoral fellowship from Special Postdoctoral                  embryogenesis, EMBO J., 17, 1799-1809.
Researchers Program of RIKEN.                                     12.   Finkelstein, R. R., Wang, M. L., Lynch, T. J., Rao, S.,
                                                                        and Goodman, H. M. 1998, The Arabidopsis abscisic acid
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