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 v ia an RNA intermediate. In mice, one polymorphic variant of L1 has amp lified relatively recently, giving rise to the A-type subfamily in spe cies belonging to the genus and subgenus Mus. Retrotransposition of LI NE-1 (L1) requires the function of the L1-encoded reverse transcriptas e 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 c DNA from the RNA in ribonucleoprotein particles; another with a purpor ted inactivating mutation; and the third, a hypothetical ancestral con struct. Because there are no examples of A-type elements that have tra nsposed recently to inactivate a gene, this assay is the first step to wards demonstrating the functional capability of mouse A-type LINE-1 e lements. One of the three elements was believed to have been inactivat ed during evolution by the substitution of leucine for a highly conser ved phenylalanine or tryptophan residue among known reverse transcript ases. This mutation did not inactivate the L1 reverse transcriptase in the yeast assay; thus, all three of the elements tested encoded rever se transcriptase activity. We further examined the minimal reverse tra nscriptase domain within ORF2 by creating a series of deletions. The r esults demonstrate that removal of the L1 endonuclease domain from the N-terminal region of ORF2 does not affect reverse transcriptase activ ity as determined by this assay, and that approximately half of the OR F2 coding sequence from mouse A-type L1 elements is required for funct ional reverse transcriptase. (C) 1998 Elsevier Science B.V. All rights reserved.