Identification of the reverse transcriptase encoded by the Mauriceville and Varkud mitochondrial plasmids of Neurospora.
The Mauriceville and Varkud mitochondrial plasmids of Neurospora are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) that have characteristics of mtDNA introns and retroid elements. The plasmids contain a single long open reading frame (710 amino acids), whose amino-terminal half has structural similarity to reverse transcriptases. Using antibodies against synthetic peptides and trpE fusion proteins, we detected an 81-kDa protein encoded by this open reading frame in mitochondrial preparations from the plasmid-containing strains. This 81-kDa protein cosegregates with reverse transcriptase activity in sexual crosses and comigrates with reverse transcriptase activity in sodium dodecyl sulfate-polyacrylamide gels, where it can be assayed after renaturation of the protein. In glycerol gradients under nondenaturing conditions, the reverse transcriptase activity sediments at approximately 145 kDa, close to the value expected for a dimer of the 81-kDa protein. The 81-kDa protein represents most of the 710-amino acid open reading frame, but may be missing some amino acids at the amino terminus. The regions upstream and downstream of the putative reverse transcriptase domain lack sequences characteristic of gag, protease, RNase H, or integrase domains found in other retroid elements. The plasmid-encoded 81-kDa protein seems to be a novel type of reverse transcriptase that may provide insight into the evolution of these enzymes.
Department of Molecular Genetics, Ohio State University, Columbus 43210.
M T
MT
Kuiper
J R
JR
Sabourin
A M
AM
Lambowitz
D003001:Cloning, Molecular; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism*; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology / Q000235:genetics*; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012261:Ribonucleoproteins / Q000235:genetics / Q000302:isolation & purification; D013045:Species Specificity
D004272:DNA, Mitochondrial; D012261:Ribonucleoproteins; D012194:RNA-Directed DNA Polymerase