Cloning and characterization of the gene encoding a mitochondrially localized DNA topoisomerase II in Dictyostelium discoideum. Western blot analysis.
We cloned a gene (topA) encoding DNA topoisomerase II from Dictyostelium discoideum nuclear DNA using oligo probes corresponding to the consensus amino acid sequences found in the gene in other eukaryotes. The gene encoding a predicted polypeptide of 1282 amino acids with M(r) of about 146 kDa, is a single copy that is expressed as a polyadenylated 4.5 kb RNA. The predicted amino acid sequence shares similarity with those of other eukaryotes with identity between 32 and 46%. The protein is 260-300 amino acids shorter in the C-terminal region and 50-80 longer in the N-terminal region than those of other eukaryotes. In TopA of D. discoideum, the N-terminal region with stretches of charged and hydrophilic amino acids is predicted to fold into an amphiphilic alpha-helix which is characteristic of a mitochondrial targeting signal presequence. Four independent polyclonal antibodies against bacterially expressed GST fusion proteins containing four portions of the polypeptide detected a single band on Western blots at about 135 kDa. Western blots analysis of subcellular fractions revealed that this protein is localized in mitochondria. The protein and the mRNA are present in growth phase and during development, although levels of both declined as development proceeded.
Komori
K
K
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
Kuroe
K
K
Yanagisawa
K
K
Tanaka
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004023:Dictyostelium / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D015183:Restriction Mapping
D004247:DNA; D004250:DNA Topoisomerases, Type II