Shared sequence variants of Mus spretus LINE-1 elements tracing dispersal to within the last 1 million years.

Abstract

LINE-1 repetitive sequences contain a record of an evolving population of transposons within the mammalian genome. Of the 100,000 copies of LINE-1 sequences per genome there are many shared sequence variants representing changes occurring within the propagating LINE-1 elements themselves, rather than changes that occur during retrotransposition or after an element inserts in the genome. These shared sequence variants define families of LINE-1 elements which have spread within specific periods of time. We have been interested in studying events in LINE-1 evolution since the speciation of Mus spretus and Mus domesticus approximately 3 million years (Myr) ago. To do this, we have collected LINE-1 sequences that have shared sequence variants specific to M. spretus. The sampled LINE-1 elements were sequenced at their extreme 3' ends, where the density of sequence variants is highest. The new sequences define six new M. spretus-specific sequence variants. Of these, we have found one that could be used to screen for LINE-1 elements arising in the last 1 Myr, which we argue is a critical sample for understanding the dynamics of LINE-1 propagation.

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Copyright 0 1994 by the Genetics Society of America
Shared Sequence V a r i a n t s of Mus spretus LINE1 Elements Tracing Dispersal to
Within the Last 1 Million Years
N. Carol Casavant and Stephen C. Hardies
Department of Biochemistry, University of Texas Health Science Center at Sun Antonio, Sun Antonio, Texas 78284-7760
Manuscript received August 13, 1993
Accepted for publication February 9, 1994
ABSTRACT
LINE-1 repetitive sequences contain a record of an evolving population of transposons within the
mammalian genome. Of the 100,000 copies of LINE-1 sequences per genome there are many shared
sequence variants representing changes occurring within the propagating LINE-1 elements themselves,
rather than changes that occur during retrotransposition or after an
shared sequence variants define families of LINE-1 elements which have spread within specific periods
of time. We have been interested in studying events in
and Mus domesticus approximately 3 million years (My) ago. To do this, we have
sequences that have shared sequence variants specific to M. spretus. The sampled LINE-1 elements were
sequenced at their extreme 3' ends, where the density of sequence variants is highest. The new sequences
define six new M. spretus-specific sequence variants. Of these, we have found one that could be used to
screen for LINE-1 elements arising in the last 1 MV, which we argue is a critical sample for understanding
-
the dynamics of LINE-1 propagation.
element inserts in the genome. These
LINE-1 evolution since the speciation of Mus spretus
collected LINE-1
L
the murine genome via transposition [for reviews see
SKOWRONSKI
and SINGER (1986), SCOTT et al. (1987),
EDGELL
et al. (1987), HUTCHISON
INGER et al. (1992)l. The LINE-1 elements are organized
into a small number of families in each murine species
(MARTIN et al. 1985; HARDIEs et al. 1986; RIKKE et al.
1991), with each family characterized by a particular set
of sequence variants. These shared sequence variants
are acquired by propagating elements that pass them
onto the future generations, whereas other substitutions
that are acquired in a pseudogene copy and are not
passed onto future generations are
ants. The shared sequence variants reflect the sequence
of propagating parental elements,
families represent the existence of more than one
propagating element. Analysis of the shared sequence
variants therefore yields information about the number
of propagating elements and how they are related one
to another.
Previous studies of shared sequence variants have in-
dicated that large families amplified at various points in
the past, sometimes splitting into two subfamilies, and
sometimes dying out as new families arose. For example,
comparisons of 5' ends of murine LINE-1s have indi-
cated that new families can arise (referred to as A, F, or
V type LINE-1) that have had their 5' control sequences
replaced (ADEY 1991; SCHICHMAN
et al. 1993;JUBIER"AURIN et al. 1992). Anciently derived
families have been described in mouse and rat (PASCALE
et al. 1993), and in Peromyscus (KASS et al. 1992). Split-
INE-1 elements are repetitive sequences that have
been dispersed to approximately 100,000 sites in
et al. (1989) and DEIN-
called "private" vari-
and the different
et al. 1992; SCHICHMAN
Genetics 137: 565-572 (June, 1994)
ting of LINE-1 into multiple families has also been de-
scribed in man (SCOTT et al. 1987; JURKA
comparisons paint a broad picture of the evolution of
LINE-1; however, the details of the process have been
more difficult to ascertain.
In contrast to the large families described above, DOM-
BROSKI et al. (1991) defined a family of only six human
LINE-1 elements that includes a currently active propa-
gating element as judged by having made a copy within
a currently living human. Although this family appar-
ently dates back 6 million years (Myr) , it has contributed
only slightly to the human LINE-1 total replicative ca-
pacity. The question becomes whether there are many
small replicating subfamilies within the umbrella of a
large amplifjmg family, or if these six members consti-
tute a nonrepresentative family.
To address the existence of small subfamilies in the
mouse, we will need to sequence a set of LINE-1 ele-
ments from the analogous time period for the mouse.
The time period in question has been ascertained for
the mouse from the analysis of substitution patterns in
LINE-1 coding sequence
(HARDIEs et al. 1986). This
work closely analyzed the pattern
LINE-1 sequences after they had become separated
from the major propagating lineage, which was called a
molecular driver. The result was that after separating
from the molecular driver, LINE-1 sequences appeared
to have had a period of about 1 Myr in their history
during which their coding sequences were still under
selective pressure. The sequence that was under selec-
tive pressure could not have been the element that was
actually sampled, because most of those elements are
1989). These
of substitution in

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566
N. C. Casavant and S. C. Hardies
presumed to be truncated. That means that most ele-
ments must have propagating parents in their histories
that were separate from the major propagating lineage
for about 1 Myr. Those parents only made small families,
because you never sample more than one progeny from
each parent. All of this means that small families are
active in the mouse for about 1 Myr before they either
die out or become big families. Therefore, if small sub-
families are to be observed while they are still active,
sampled elements must be less than 1 Myr old.
If small subfamilies of this kind are active today, they
should exist as the youngest components of larger fami-
lies that have spread successfully in the last few Myr. So,
to find 1-Myr-old subfamilies, we started by defining a
LINE-1 family that is both large and vigorously propa-
gating, and therefore would have a suitable number of
1-Myr-old members. A suitable family of about 9000
members has been recently identified in Mus spretus,
characterized by the presence of two specific sequence
variants named Ms496 and Ms416 (RIKKE et al. 1991;
RKKE
and HARDIES 1991). These shared sequence vari-
ants have been acquired and amplified in less than 3-5
Myr, the time since speciation between Mus domesticus
and M. spretus (BONHOMME
et al. 1984). In this study, we
have collected LINE-1 sequences from this family and
expanded the list of its characteristic sequence variants,
looking to define even younger subfamilies.
We searched for additional shared sequence variants
in the Ms496 family by a targeted polymerase chain re-
action (PCR) amplification and cloning strategy. To ob-
tain the maximum number of sequence differences
within a short segment of the LINE-1 element, we chose
to sequence the 3‘ most region (adjacent to the poly-
adenylation site) for two reasons: (1) this region is the
most populous among LINE-1 elements and (2) the 3‘-
untranslated region has acquired mutations within the
propagating elements at a faster rate (RIKKE et al. 1991).
In addition, this study extends the characterized region
within the M. spretus-specific population first described
by RIKKE
et al. (1991) by an additional 469 bases for a
total of over 900 bases characterized.
The majority of sequence variants observed were spe-
cific to M. spretus and were shared among the reported
members of the Ms496 family. The relative lack of un-
shared mutations was expected since there has been
little time after the dispersal of the individual LINE-1
elements for them to acquire private mutations. There-
fore, the majority of sequence differences observed
within the collected family members define the se-
quence of actively propagating LINE-1 elements. A total
of six new M. spretus-specific shared sequence variants
were identified and ordered within the Ms496 family.
The sequences analyzed to date do not yet tell us the
relationship between small and large families. To answer
that question, we need to further increase the sample of
sequences confined to the last 1 Myr. The current results
will enable us to obtain this sample, because they estab-
lish a shared sequence variant that is suitable to use as
a hybridization probe to screen for the required 1-Myr-
old LINE-1 elements.
MATERIALS AND METHODS
SSLl-PCR A mouse of the M. spretus Spain strain
SPRET/Ei was obtained from J. Nadeau (The Jackson Labo-
ratory, Bar Harbor, Maine). Mouse DNA was prepared as de-
scribed by RIKKE et al. (1991). M. spretus LINE-l elements
were amplified with oMS496HIII and cloned as described by
CASAVANT and HARDIES (1993). Those filters to be probed with
oMs6936 (CATTGCACACACTAG) or oSCHla (TCATTTA-
CATTTCCAATGCT) were prehybridized and hybridized as
described by RIKKE et al. (1991). Each filter was washed 3 times
for 20 min in 6 X SSC at 39” for oMs6936 and room tem-
perature for oSCHla. The J’-untranslated region probe (CASA-
VANT and HARDIES 1993) was nick translated, hybridized and
washed as described by WAHL et al. (1979). Filters were ex-
posed overnight on Kodak X-OMAT AR film.
DNA sequencing: The individual clones were sequenced
using the dideoxy chain termination method (SANGER
1977). Both strands of each reported sequence were com-
pleted using internal LINE-1 primers.
Construction of LINEl phylogenetic tree: Comparative se-
quence data from M. spretus and M.
used to construct the tree. All of the elements called M. do-
mesticus originate from inbred strains that are actually hybrids
between Mus musculus musculus and Mus musculus domes-
ticus (BONHOMME
and GUENET 1989). M. domesticus sequences
were LlMdA2 (LOEB et al. 1986), LlMdA13 (SHEHEE
1987), R5 (GEBHARD
et al. 1982), LlMdl and L1M4 (VOLIVA
al. 1983, with corrections from SHEHEE
LlMdl7 (SHEHEE
et al. 1989). The M. spretus sequences were
SPR48,49,68 and Dl3SHl (CASAVANT
SC2, SC3, SC4, SC5, SC6 and SC7 (this report). The tree was
constructed using the method of maximum parsimony (FITCH
1977). There were minimal discordancies; those causing un-
certainty in placement of branches are explicitly discussed in
the results.
The sequence of the LINE-1 ancestor at the time of the
M. spretus/M. domesticus speciation was estimated for display
purposes only. It is essentially a consensus after the removal of
species-specific variant bases.
et al.
domesticus elements were
et al.
et
et al. 1989), and
and HARDIES 1993), and
RESULTS
Collecting M. spretus LINEl elements: Members of
the Ms496 family in both M. spretus and in an inter-
specific hybrid mouse were recovered by subfamily spe-
cific LINE-1 PCR (SSL1-PCR)
1993). In this method, a PCR primer specific for the
defining Ms496 sequence variation in the family is
paired with the “bubble” anchor of RILEY et al. (1990)
located within the flanking DNA. This allowed collec-
tion of sequences from the 3’ most 489 bp of the ele-
ments, which is the richest region for informative se-
quence variants (RIKKE et al. 1991).
Previous experience with amplification between a
LINE-1 primer and the bubble anchor revealed a ten-
dency to preferentially amplify truncated LINE-1 ele-
ments (CASAVANT and HARDIES 1993). To seek out ele-
ments with intact 3‘ ends, a library of the amplified
(CASAVANT and W I E S

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LINE-1 Shared Sequence Variants
567
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FIGURE 1.-Comparison of
M. domesticus LINE-1 sequences. The coordi-
nates are based on LlMdA2 (LOEB et al. 1986).
The comparison extends from the base after
the 496 mutation at position 6874 to the poly-
adenylation site (AATAAA).
quence represents the LINE-1 sequence as it
would have looked at the time of speciation
between M. spretus and M. domesticus. Only
differences from the ancestor are shown for
the other sequences. Dashes were inserted to
maintain alignment where necessary. Spaces
indicate that sequence is not available. The se-
quences are entered in the phylogenetic order,
with R5 through SC6 representing elements
produced before speciation, LlMdA2 through
LlMd4 representing M. domesticus-specific el-
ements, and SPR68 through D13SH1 repre-
senting M. spretus-specific elements. The
boxes highlight candidates for M. spretus- and
M. domesticus-specific shared sequence vari-
ants. The double lined boxes enclose the M.
spretus variants, and the single lined boxes en-
close the M. domesticus variants. Of these, the
M. spretus variants at 7102 and 7224, and the
M. domesticus variants at 6954,6938, and 6955
are uncertain as to their species specificity.
Boxes were left open wherever other bases in-
terfered with their closure. The underlined se-
quence between 7326 and 7345 is the comple-
ment to oSCHlA.
M. spretus and
The ancestor se-

Page 4

568
N. C. Casavant and S. C. Hardies
products was probed with an oligonucleotide (oSCHla)
from the extreme 3' end of LINE-1 (position 7337-7356
in Figure 1). Post-hybridization washing was conducted
at low stringency so that mismatches could be tolerated.
Prior to probingwith oSCHla the library was hybridized
to a nick translated fragment containing the 3' end of
the LINE-1 element to determine which clones con-
tained LINE-1 sequence. The sequences of six of the
oSCHla positive LINE-1 elements (SC2-7) are shown in
Figure 1. Each contains a complete 3' end except SC5,
which has a poly(A) tail beginning 91 bp before the
usual polyadenylation site. SC5 passed the screen with
oSCHla because there is a second LINE-1 element with
an intact 3' end in the immediate flanking region (not
shown).
Defining M. sfwetusspecific variants: To detect M.
spretus-specific sequence variants, the sequences were
aligned with six representative LINE-1 elements from M.
domesticus. Three elements, LlMdAQ (LOEB et al. 1986),
LlMdA13 (SHEHEE et nl. 1987) and LlMd4 (Vol.nr~ pt nl.
1983), were previously described as having spread rela-
tively recently in M. domesticus. A fourth element,
LlMdl7 (SHEHEE
sequence compared well with the younger elements.
The remaining two, LlMdl (VOl.rV.4 pt nl. 1983) and R5
(GERHARD
et ai. 1982), are known to have arisen shortly
before the speciation between M. spretus and M. do-
mesticus. A phylogenetic tree was derived (Figure 2)
which placed the new M. sprptu.5 sequences within this
known framework. This tree served to segregate the el-
ements by common content of sequence variants.
SC2, SC3, SC4 and SC7 are organized as if descended
from a single evolving sequence, although we believe
that the evolving sequence actually represents a lineage
of ancestral propagating elements (HARDIES and
1990). They contain shared sequence differences as ex-
pected for members of the Ms496 family. Therefore,
these sequences were presumably primed specifically by
the oMS496 primer, and should be M. spretus-specific
inserts since the 496 mutation is known to have arisen
after speciation. Sequence variants shared by SC2, SC3,
SC4 and SC7 represent mutations spread during the
pansion of this family, and are candidates for M. spretus-
specific shared sequence variants. The M. spretus and
M. domesticus sequences in Figure 1 are arranged in the
same order as on the tree, and compared to the common
ancestral sequence computed for the M. spretus/M. do-
mesticus split. Candidates for sequence
M. spretus and M. domesticus-specific subfamilies have
been boxed in Figure 1 and the candidates for
spretus-specific variants have been tabulated in Table 1.
Four of the candidates (6936, 7265, 7285 and 7024)
should definitely be M. spretus-specific by the following
argument. They appear on the tree after SC2, which is
presumed to have carried the 496 mutation, which in
turn is known to have arisen after speciation. Further
pt nl. 1989), was chosen because its
RIKKE
ex-
variants defining
M.
i'B%
: : : : I
6953
6970
7037
7055
6888
69 64
7002
7049
7081
7131
7f71
7256
7285
7306
7347
r
1
FIGURE S.-Molecular
and M. sflrrtus LINE-] sequences. Dashed brackets indicate
the range of mcertainty in placing those sequences that are
truncated, and therefore missing some informative positions.
Branch lengths are not drawn to scale. Only shared sequence
variants are indicated. The stippled bar, representing the spe-
ciation between M. sflrrtus and M. domeslicus, has been ex-
tended to encompass shared sequence variants for which spe-
cies specificity and branch order is uncertain.
phylogenetic tree of M. domrslirus
verification is provided by three previously characterized
3' truncated members of the Ms496 family (mMsPCR48,
mMsPCR49 and mMsPCR68; CASAVANT
1993) which are indicated as SPR48, SPR49 and SPR68
on Figures 1 and 2. Only general positions on the tree
for these four element5 are shown because the infor-
mative positions 3' of 6987 have been deleted by trun-
cation. However, they clearly
6936. Therefore, by mapping above 6936, they confirm
this variant's sprptus-specific character. Also, since 6936
maps above 7024, 7024 must be M. spretus-specific.
The remaining four candidates (6920,7102,7146 and
7224) are shared by all four of SC2, SC3, SC4 and SC7.
These variants therefore, have arisen early in the ances-
tral lineage, and should be characteristic of most mem-
bers of the Ms496 family, as are the defining variants,
Ms496 and Ms416. Whether they came before or after
Ms496 can not be determined within this sample of se-
quences. Two of them (7102 and 7224) have the dis-
turbing property that they also appear in L1 Md4, but not
and HARDIES
arose after 6920 and before

Page 5

LINE-1 Shared Sequence Variants
569
TABLE 1
Candidates for M. sprettrs-specific variants
Base change
Positiona ancestor:variant
M. spretus clonesb
6920 C:T SC2, 3, 4, 7, SPR48, 49, 68
and D13SHl
6936
7024'
7102d2 e
7146
7224'
7265
7285-7306
T:C
T:C
T:C
GT
T:C
A:G
SC3, 4, 7, Dl3SHl
sc3,4
sc2, 3, 4, 7
sc2, 3, 4, 7
sc2, 3, 4, 7
sc3, 4, 7
sc3, 4,
7
22hp:DEL.
a The coordinants are based on LlMdA2 (LOEB et al. 1986).
The clones listed are those that inserted after speciation.
Clones SPR49 and Dl3SHl are truncated and therefore do not
contain sequence at this site or any other site 3'.
Clones SPR48 and SPR68 are truncated and therefore
contain sequence at this site or any other site 3'.
eM. spretus specificity of these positions is uncertain, because
LlMd4 also contains the putative M. spretus base: at 7102, 13 addi-
tional mouse sequences did not contain this variant; at 7224, of 17
additional M. domesticus LINE-1 elements, only LlMd2, an older
element, did contain this variant.
do not
other M. domesticus elements (Table 1). LlMd4 had
been assigned as one of the youngest M. domesticus el-
ements because it has no unshared mutations in a more
5' sequenced segment (MARTIN et al. 1985, HARDIES
1986); and it maps in Figure 2 as part of a M. domesticus-
specific family. However, the position of LlMd4 is based
on only three positions (6954, 6938 and 6955) that dis-
agree with 7102 and 7224. Because LlMd4 is young and
relatively free of unshared differences, it should not be
expected to be involved in a lot of coincidences, espe-
cially two aligning with the same M. spretus subfamily.
So we consider the placement of LlMd4, as well as the
character of all five variants in question to be unresolved
at present. This region of uncertainty is marked by the
wide stippled region in Figure 2.
We provisionally consider the
(6920 and 7146) to be M. spretus-specific, although con-
firmation is lacking. This makes a total of six M. spretus-
specific shared sequence variants that are characteristic
of the consecutive subfamilies and can be used to order
elements with respect to their age. Of these, Ms6936 and
Ms7024 are supported the best.
The assignment of variant 6936 as M. spretus-specific
conflicts with information derived from the remaining
two new sequences, SC5 and SC6. These two elements
fell on the tree together with older elements that in-
serted into the genome prior to speciation between M.
spretus and M. domesticus. They share sequence variants
with LlMdl and R5 at positions 6953,6970,7037,7055
and for SC5 at 7156. These are positions that are known
to have changed in the ancestral lineage before the spe-
ciation of M. spretus and M. domesticus, and before the
expansion of the Ms496 family. SC6
variants for this assignment: the M. spretus-specific bases
at 6920 and 6936, and the absence of the base charac-
et al.
last two candidates
has three discordant
teristic of older elements at 6913. This is too much to
explain by coincidence. SC6 could have arisen by in vivo
recombination, but there is also an obvious PCR artifact
consistent with this pattern. We believe that the oMS496
primer did prime and partially extend on a M. spretus-
specific element as intended, but the strand
completed before it dissociated. This incomplete strand
then primed on another LINE-1 element in a later cycle.
Thus SC6 is an artificial recombinant between a M.
spretus-specific LINE-1 and an older LINE-1 element.
Regardless of the etiology of SC6, we have treated it as
two separate entities (SC6 left and SC6 right) in our
analyses. In contrast, SC5 appears to truly be an older
element on which the oMS496 primer primed nonspe-
cifically. SC5 shares a single M. spretus-specific variant
at 6936, which we believe is a coincidence. Further in-
formation confirming that variant 6936 is truly M.
spretus-specific is presented below.
How to search for small subfamilies: We are seeking
a way to determine if the Ms496 family is
smaller concurrent subfamilies thus indicating indepen-
dent progenitors. Concurrent subfamilies indicative of
multiple independent progenitors are exemplified by
the state of the M. domesticus sequences in Figure 2. To
distinguish separate propagating elements, at least two
progeny from each parent must be sampled; that is,
clades must be observed in the data. For example LlMd4
and LlMdl7 share different variants (positions 6938
and 6955) than LlMdA2 and LlMdA13. This arrange-
ment indicat'es that the ancestor of LlMd4/LlMd17
and LlMdA2/LlMdA13 split into two separate propa-
gating elements whose copies form two separate
clades. This particular example is flawed, because in-
formation from the key variants is self-contradictory, as
discussed above. Nonetheless, this pattern of mutually
exclusive shared sequence variants is the key to identi-
fylng concurrent subfamilies indicative of independent
progenitors.
In contrast to the appearance of the M. domesticus
sequences, the M. spretus subfamilies appear to be con-
secutive rather than concurrent. For example, SC3/SC4
is a more recent subfamily of the larger group SC3/
SC4/SC7/SC'2. Since all four repeats could have been
descended from the same parent, SC3/SC4 is not a sepa-
rate clade. ThLerefore clades must be observed to estab
lish the existence of truly separate parents.
Note that the data do not exclude that SC3, SC4, SC7
and SC2 might be representative of separate clades. For
example, if additional LINE-1 elements were sampled
that shared a variant with SC2, but did not have variant
7024, then that would set SC2 off into a separate clade
requiring a separate propagating parent.
these elements may be representative of a separate
clade, however, due to the small sample size none was
observed. The sample size required to either visualize or
was not
split into
Any one of