Cloning, expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea.
We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial cDNA clones were isolated which were truncated at the 5' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topoisomerase purified to homogeneity. The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg2+. In the presence of Mg2+ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei.
Reddy
M K
MK
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
Nair
S
S
Tewari
K K
KK
D000595:Amino Acid Sequence; D002413:Cations, Divalent; D002467:Cell Nucleus / Q000201:enzymology; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant / Q000235:genetics*; D008274:Magnesium; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D002413:Cations, Divalent; D018076:DNA, Complementary; D018744:DNA, Plant; D004278:DNA, Superhelical; D011993:Recombinant Fusion Proteins; D004264:DNA Topoisomerases, Type I; D008274:Magnesium