Plant homologue of flap endonuclease-1: molecular cloning, characterization, and evidence of expression in meristematic tissues.
Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. We isolated a 1.4 kb cDNA from rice (Oryza sativa), termed OsFEN-1, encoding a protein which shows homology with the eukaryotic FEN-1 proteins. OsFEN-1 protein was overexpressed in Escherichia coli and purified to near homogeneity. DNA cleavage analysis using different branched DNA structures indicated that OsFEN-1 protein possesses both 5'-flap endonuclease and 5' to 3' double-stranded DNA exonuclease activities. OsFEN-1 protein incises a 5'-flap and 5'-pseudo Y structure one base 3' of the branched point in the duplex region. The enzymatic properties indicated that we succeeded in obtaining the gene and the protein of a plant counterpart of FEN-1. OsFEN-1 transcripts were expressed strongly in proliferating tissues such as root tips and young leaves that contain root apical meristem and marginal meristem, respectively. No expression was detected in mature leaves although the leaves were exposed to UV. We analyzed the spatial distribution pattern of OsFEN-1 transcripts by in situ hybridization. In the shoot apex, OsFEN-1 mRNA was abundant in the shoot apical meristem, tiller bud, leaf primordia, ligule primordia and marginal meristem of young leaves. In the roots, the transcript accumulated to high levels in the root apical meristem. Our results indicate that OsFEN-1 is expressed in tissues rich in proliferating cells, and its expression may be required for cell growth and organ formation.
Kimura
S
S
Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Chiba-ken, Japan.
Ueda
T
T
Hatanaka
M
M
Takenouchi
M
M
Hashimoto
J
J
Sakaguchi
K
K
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D045585:Flap Endonucleases; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017403:In Situ Hybridization; D018519:Meristem / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D018520:Plant Shoots / Q000201:enzymology / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D018076:DNA, Complementary; D018744:DNA, Plant; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases