Sl. Martin et al., FUNCTIONAL REVERSE TRANSCRIPTASES ENCODED BY A-TYPE MOUSE LINE-1 - DEFINING THE MINIMAL DOMAIN BY DELETION ANALYSIS, Gene, 215(1), 1998, pp. 69-75
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.