Reverse transcriptase encoded by a retrotransposon from the trypanosomatid Crithidia fasciculata.
The long interspersed nuclear element (LINE)-like elements are a distinct family of eukaryotic transposons that contain a long open reading frame with limited sequence homology to retroviral reverse transcriptases. Unlike many retrotransposons, they lack long terminal repeats. The mechanism by which LINE-like elements move within the genomes of their hosts remains speculative. We have used an unusual approach to express and detect enzymatic activities associated with Crithidia retrotransposable element 1 (CRE1), a site-specific LINE-like element found in the insect trypanosomatid Crithidia fasciculata. A chimeric gene fusing the yeast retrotransposon Ty1 and the CRE1 open reading frame is constructed and then overexpressed in yeast. Fusion proteins are packaged into virus-like particles, which can be partially purified and directly analyzed for enzymatic activity. Here we demonstrate that CRE1 encodes an RNA-directed DNA polymerase. These data provide direct biochemical evidence that this widely distributed class of retrotransposons encodes reverse transcriptase and sets the stage for a detailed understanding of the mechanisms involved in LINE-like element transposition.
Gabriel
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A
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Boeke
J D
JD
D000818:Animals; D001483:Base Sequence; D016831:Crithidia fasciculata / Q000201:enzymology* / Q000235:genetics; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements; D005796:Genes; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013329:Structure-Activity Relationship
D004251:DNA Transposable Elements; D009841:Oligonucleotides; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D012194:RNA-Directed DNA Polymerase