Functional reverse transcriptases encoded by A-type mouse LINE-1: defining the minimal domain by deletion analysis.
Long interspersed elements, or LINEs, are retrotransposons that move via an RNA intermediate. In mice, one polymorphic variant of L1 has amplified relatively recently, giving rise to the A-type subfamily in species belonging to the genus and subgenus Mus. Retrotransposition of LINE-1 (L1) requires the function of the L1-encoded reverse transcriptase that is produced from open reading frame 2 (ORF2). Here, we employ a convenient yeast genetic assay to determine the reverse transcriptase activity of the ORF2 obtained from three A-type L1 elements: one, a cDNA from the RNA in ribonucleoprotein particles; another with a purported inactivating mutation; and the third, a hypothetical ancestral construct. Because there are no examples of A-type elements that have transposed recently to inactivate a gene, this assay is the first step towards demonstrating the functional capability of mouse A-type LINE-1 elements. One of the three elements was believed to have been inactivated during evolution by the substitution of leucine for a highly conserved phenylalanine or tryptophan residue among known reverse transcriptases. This mutation did not inactivate the L1 reverse transcriptase in the yeast assay; thus, all three of the elements tested encoded reverse transcriptase activity. We further examined the minimal reverse transcriptase domain within ORF2 by creating a series of deletions. The results demonstrate that removal of the L1 endonuclease domain from the N-terminal region of ORF2 does not affect reverse transcriptase activity as determined by this assay, and that approximately half of the ORF2 coding sequence from mouse A-type L1 elements is required for functional reverse transcriptase.
University of Colorado School of Medicine, Department of Cellular, Structural Biology, Program in Molecular Biology, 4200 E. Ninth Avenue, Box B111, Denver, CO 80262, USA. martins@essex.uchsc.edu
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Martin
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Li
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Epperson
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Lieberman
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D051379:Mice; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D018626:Retroelements / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D014162:Transfection / Q000235:genetics
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D018626:Retroelements; D012194:RNA-Directed DNA Polymerase