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Kafer, Chris,Thornburg, Robert W. 한국식물학회 2000 Journal of Plant Biology Vol.43 No.3
Two Expressed Sequence Tagged (EST) clones were identified from the Arabidopsis database as encoding putative cytidine deaminases. Sequence analysis determined that the two clones overlapped and encoded a single cDNA. This cytidine deaminase corresponds to the Arabidopsis thaliana gene, cda1. The deduced amino acid sequence was more closely related to prokaryotic cytidine deaminases than to eukaryotic enzymes. The cDNA shares 44% amino acid identity with the Escherichia coli cytidine deaminase but only 26 and 27% identity with human and yeast enzymes. A unique zinc-binding domain of the E coli enzyme forms the active site. A similar putative zinc-binding domain was identified in the Arabidopsis enzyme based upon primary sequence similarities. These similarities permitted us to model the active site of the Arabidopsis enzyme upon that of the E. coli enzyme. In this model, the active site zinc is coordinated by His^73, Cys^103, Cys^107, and an active site hydroxyl. Additional residues that participate in catalysis, Asn^64, Glu^66, Ala^78, Glu^79, and Pro^102, are conserved between the Arabidopsis and E. coli enzymes suggesting that the Arabidopsis enzyme has a catalytic mechanism similar to the E. coli enzyme. The two overlapping ESTs were used to prepare a single, full-length clone corresponding to the A thaliana cda1 cDNA. This cDNA was subcloned into pProExHtb and expressed as a fusion protein with an N-terminal His_6 tag. Following purification on a Ni-NTA-Agarose column, the protein was analyzed for its kinetic properties. The enzyme utilizes both cytidine (K_m= 226μM) and 2'-deoxycytidine (K_m=49μM) as substrates. The enzyme was unable to deaminate cytosine, CMP or dCMP.
Kafer, Chris,Thornburg, Robert 한국식물학회 2002 Journal of Plant Biology Vol.45 No.3
We have cloned the Arabidopsis thaliana uridine 5'-monophosphate synthase (UMP synthase) locus and characterized transcript levels of this gene as well as the transcript encoding a novel F-box protein found upstream of UMP synthase, Skp1-interacting partner 5 (SKIP5). The 3' end of the SKIP5 gene is 405 bp from the 5' end of the UMP synthase gene. To determine whether these proximate genes are coordinately or independently regulated, we have used an RT-PCR method to quantitate the transcript levels of each gene relative to 18S ribosomal RNA. Previous work has demonstrated an up-regulation of the UMP synthase gene in tobacco callus in the presence of compounds such as 5-fluoro-orotate and aminopterin that induce thymine starvation. Here, we present results showing that both the UMP synthase and SKIP5 genes are coordinately up-regulated in the presence of fluoroorotic acid.