A novel subfamily of LINE-derived elements in mice
Warren D. Flood,
Igor B. Rogozin,
Division of Animal Science, University of New England, Armidale 2351, NSW, Australia
Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk 90, Russia
Received: 11 February 1998 / Accepted: 4 August 1998
Abstract. Hybrid sequences have been described previously that
consist of a 5Ј region homologous to ORF2 of LINEs and a 3Ј end
that shares homology with a sequence located in the first intron of
immunoglobulin. The present investigation has revealed 14
new sequences from seven murine species, that show high homol-
ogy to those observed earlier. Database search has found several
new homologous hybrid sequences including one located in the
mouse T-cell receptor (Tcra) locus. Several interesting features of
this sequence include identical 15-bp flanking short direct repeats
as well as poly-A signal and A-rich sequence at the 3Ј end. We
have classified this set of sequences as LINE-derived elements
(LDEs), which constitute a newly observed subfamily. Compara-
tive analysis of these sequences suggests that a single recombina-
tion event was responsible for the production of an LDE progeni-
tor. The phylogenetic tree shows a number of elements that pre-
existed in the common ancestor of murine species and displays
different evolutionary rates. The time of LDE origin is estimated at
approximately 10–15 MYA.
The role of recombination events in the evolution of mobile ge-
netic elements in mammals has been studied previously. Observa-
tions have shown the existence of L1-Alu-associated sequences in
primate genomes (Miyake et al. 1983; Fujita et al. 1987), and study
of L1 elements in slow lory and galago has provided evidence that
ORF1 has at least two distinct progenitors (Stanhope et al. 1993).
In addition, Adey and associates (1994) demonstrated in rodents
that L1 has acquired novel promoter sequences over time from
non-L1 sources. Taken together, this suggests that changes of L1
and perhaps other types of mobile elements and genes arise from
recombination events and are subsequently utilized in evolution.
In a previous investigation, we found two sequences that re-
sulted from recombination between an L1 element and a se-
quence found within the first intron of the immunoglobulin C
gene (Filippov et al. 1992). This recombination event occurred in
ORF2 of L1, which encodes reverse transcriptase, an essential
enzyme in L1 transposition (Hattori et al. 1986, Fanning and
Starting from this point, we were able to construct PCR prim-
ers to study the genome of Mus musculus domesticus as well as
several other closely related murine species. Database search was
also used to find additional sequences.
This study had three major objectives. (1) To find other copies
of the L1 hybrid sequences in the murine genomes and to examine
whether they constitute an L1-derived sub-family. (2) To under-
stand whether a single recombination event was responsible for the
generation of progenitor sequence. (3) To reconstruct phylogeny of
the studied sequences.
Materials and methods
Samples, PCR amplification, cloning, and sequencing.
species studied include Mus musculus domesticus, M. m. musculus, M.
bactrianus, M. spicilegus, M. cervicolor, M. macedonicus, and M. spretus.
DNA samples from all species except Mus. m. domesticus were kindly
provided by B. Kunze and H. Winking from Medizinische Universita¨t
PCR was used to amplify sequences of interest. For this purpose we
selected a 135-bp region that covers the point of recombination between L1
and the sequence found in the first intron of the C
This region displays a relatively high level of conservation and provides a
good foundation on which to target primers. Primers were designed as a
consensus of known L1 and C
-like sequences. PL2 (5Ј-GAATTCC-
ATCAGACCTTCA-3Ј) is specific to the L1 homologous region, while
CE2 (5Ј-AAATTGTGACAAGTTCCT-3Ј) targets the C
gion. PCR reactions consisted of 30 cycles of denaturation at 94°C for 30
s, annealing at 58°C for 10 s, and extension at 72°C for 80 s. A final cycle
of 72°C for around 5 min was also performed. PCR reactions were well
reproducible, and controls were used to rule out contamination.
PCR products were ligated into pBluescript II KS+ phagemid (Strata-
gene) with the TA cloning strategy (Marchuk et al. 1991) and were trans-
formed into CaCl
-induced DH5␣ competent cells. Prior to sequencing,
clones were recovered through a plasmid mini prep procedure (Birmboim
and Dolly 1979). Clones were then sequenced in both directions by the
standard dideoxy method (Sanger et al. 1977).
All sequences obtained were submitted to GeneBank. Accessions num-
bers are U75333–U75343.
Database search was performed by using the follow-
ing computer programs: BLASTN (Altschul et al. 1990) and BLASTX
(Gish and States 1993) for comparison of a sequence vs. nonredundant
nucleotide and protein sequence databases (NR, supported by the National
Center for Biotechnology Information).
The phylogenetic tree was constructed by use of Jukes-Cantor cor-
rected distances and the neighbor-joining algorithm of Saitou and Nei
(1987). We used the MEGA package (Kumar et al. 1993) for all calcula-
Results and Discussion
LINE-derived elements (LDE). Figure 1 provides a schematic
view of the sequences studied in this investigation. It was shown
in the previous work (Filippov et al. 1992) that two sequences
found in Mus musculus domesticus on Chromosome (Chr) 17,
D17Leh80 (referred to further as tu80, in order to indicate that we
dealt with a part of D17Leh80) and D17Nov1 (referred to further
as nov1), resulted from a recombination event between a LINE
(L1-A2) and a sequence located within the first intron of the C
immunoglobulin gene. A database search revealed two additional
homologous hybrid sequences located on the complementary
strand of the mouse T-cell receptor alpha (Tcra) gene (Wilson et
al. 1992) on Chr 14 and in trypsinogene locus within T-cell re-
ceptor beta (Tcrb) gene cluster (Rowen et al. 1997) on Chr 6. The
first sequence will be referred to as tcra and the second as tcrb.
Correspondence to: A. Ruvinsky
Mammalian Genome 9, 881–885 (1998).
© Springer-Verlag New York Inc. 1998