Isolation and characterization of a gene encoding DNA topoisomerase I in Drosophila melanogaster.
We synthesized a DNA probe specific for the gene encoding eucaryotic DNA topoisomerase I by the polymerase chain reaction. The sequences of the primers for this reaction were deduced from the regions with extensive homology among the enzymes from the fission and budding yeasts, and the human. From the clones isolated by screening a Drosophila cDNA library with this DNA probe, two cDNA clones of 3.8 and 5.2 kb were characterized and completely sequenced. Both cDNA sequences contain an identical open reading frame for 972 amino acid residues. The 3.8 kb messenger RNA is likely generated by using a polyadenylation site 5' upstream to that used in generating the 5.2 kb mRNA. The predicted amino acid sequence shows that a segment of 420 amino acid residues at the amino terminus is hydrophilic, similar to the amino terminal 200 residues in the yeast and human enzymes. Furthermore, the Drosophila enzyme is unique in that the amino terminal 200 residues are enriched in serine and histidine residues; most of them are present in clusters. The rest of the Drosophila sequence is highly homologous to those from yeast and human enzymes. The evolutionarily conserved residues are identified and are likely the critical elements for the structure and function of this enzyme. A plasmid vector containing the cloned cDNA was constructed for the expression of Drosophila protein in Escherichia coli. The enzymatic and immunochemical analysis of the polypeptide produced in this heterologous expression system demonstrated that the expressed protein shares similar enzymatic properties and antigenic epitopes with DNA topoisomerase I purified from Drosophila embryos or tissue culture cells, thus establishing the bacterial expression system being useful for the future structure/function analysis of the Drosophila enzyme.
Hsieh
T S
TS
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710.
Brown
S D
SD
Huang
P
P
Fostel
J
J
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015342:DNA Probes; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000196:embryology / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D015342:DNA Probes; D004264:DNA Topoisomerases, Type I