Efficient amplification of Drosophila simulans copia directed by high-level reverse transcriptase activity associated with copia virus-like particles.
The number of retrotransposon copia per genome in Drosophila melanogaster cultured cells is two to three times higher than that in D. melanogaster embryo cells. Here, we have found that the genome of the related species, Drosophila simulans, contains in cultured cells more efficiently amplified copia DNA (approximately ten fold). Furthermore, we analyzed copia virus-like particles (VLPs) prepared from D. melanogaster and D. simulans cultured cells, which contain copia RNA and reverse transcriptase (RT) activity, and thus, play a major role in copia replication. The RT activity associated with the D. simulans VLPs was 25 times higher than that associated with the D. melanogaster VLPs. Taken together with the fact that copia is believed to transpose through an RNA intermediate, these results suggest that the amplification of copia DNA should relate to copia RNA-mediated transposition, and the higher RT activity associated with the D. simulans VLPs would lead to the efficient amplification of copia DNA. In a comparison between D. melanogaster and D. simulans copia nucleotide (nt) sequences, five nt substitutions, which cause the respective amino acid changes, were found in the copia RT-coding region. Polymerase chain reaction direct sequencing showed that these five substitutions are the vast majority in each Drosophila species. The substitutions, therefore, may be responsible for the high level of the RT activity associated with the D. simulans VLPs.
Yoshioka
K
K
Laboratory of Molecular Biology, School of Hygienic Sciences, Kitasato University, Kanagawa, Japan.
Kanda
H
H
Takamatsu
N
N
Togashi
S
S
Kondo
S
S
Miyake
T
T
Sakaki
Y
Y
Shiba
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D004330:Drosophila / Q000201:enzymology / Q000235:genetics* / Q000382:microbiology*; D004331:Drosophila melanogaster / Q000235:genetics / Q000382:microbiology; D004625:Embryo, Nonmammalian; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D015183:Restriction Mapping; D012190:Retroviridae / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification
D004251:DNA Transposable Elements; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D004247:DNA; D012194:RNA-Directed DNA Polymerase