# Distributions of two recently inserted long interspersed elements of the L1 repetitive family at the Alb and βh3 loci in wild mice populations

Michel Bellis, Véronique Jubier-Maurin, Barbara Dod, Flavie Vanlerberghe, Anne-Marie Laurent, Christiane Senglat, Francois Bonhomme, Gérard Roizès

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## To cite this version:

Michel Bellis, Véronique Jubier-Maurin, Barbara Dod, Flavie Vanlerberghe, Anne-Marie Laurent, et al. Distributions of two recently inserted long interspersed elements of the L1 repetitive family at the Alb and βh3 loci in wild mice populations. Molecular Biology and Evolution, 1987, 10.1093/oxford-journals.molbev.a040449. hal-02175157

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# Distributions of Two Recently Inserted Long Interspersed Elements of the L1 Repetitive Family at the Alb and βh3 Loci in Wild Mice Populations¹

Michel Bellis,* Véronique Jubier-Maurin,* Barbara Dod,*

Flavie Vanlerberghe,† Anne-Marie Laurent,* Christiane Senglat,*

François Bonhomme,† and Gérard Roizès*

*Centre National de la Recherche Scientifique, Institut de Biologie, and †Institut des Sciences de l'Evolution, Université de Montpellier

The presence of the L1 sequences, L1Md4 next to the pseudogene βh3 and I12 found in the twelfth intron of the albumin gene, in certain strains of laboratory mice but not of others has led to the suggestion that these sequences were recent insertions into the *Mus musculus* genome. To be sure that they are really recent insertions and not relics of an ancestral chromosome, we investigated the presence or absence of these sequences in populations of wild mice belonging to the semispecies *M. m. domesticus* and *M. m. musculus* as well as in other species of the genus *Mus* and in related murids. The sequence I12 in the albumin gene was found in 34% of the chromosomes of the wild mice belonging to *M. m. domesticus* and to a lesser extent (6%) in *M. m. musculus*. Of 114 *M. m. domesticus* chromosomes, L1Md4 was found in only nine, seven of which came from the same locality. Its presence was associated with the haplotype HbbP, which is relatively rare in European populations of *M. musculus*. Since there was no evidence for the presence of these two L1 sequences in more distantly related species, we conclude that they are recent insertions in the *M. musculus* genome.

## Introduction

The structure of the L1 sequences—sometimes referred to as MIF1 (Brown and Dover 1981)—in primates and rodents had led to the suggestion that they could have been generated by RNA intermediates by means of a retroposon-like mechanism (Voliva et al. 1984a). A “retroposon” insertion of an L1 element has been suggested to have occurred in the cellular oncogene c-myc in canine transmissible venereal tumor (Katzir et al. 1985). There is, however, no direct experimental evidence for retroposition of L1 elements in primates or rodents. It has also been suggested that L1 elements may be transposable. However, the covalently closed, circular, extra-chromosomal DNAs containing L1 sequences and found in mouse thymocytes (Eujimoto et al. 1985), Hela cells (Jones and Potter 1985), and human fibroblasts (Riabowol et al. 1985) are most likely deletion products.

Whatever mechanism is responsible for their appearance, there is evidence for the recent insertion of L1 elements in *Mus musculus* at three different loci (Voliva et al. 1985).

1. Key words: long interspersed elements, L1 repeats, wild mice.

Address for correspondence and reprints: Dr. Gérard Roizès, CNRS (LP 8402-CRBM)-INSERM U249, Institut de Biologie, boulevard Henri IV, 34060 Montpellier Cedex, France.

Mol. Biol. Evol. 4(4):351–363. 1987.

© 1987 by The University of Chicago. All rights reserved.

0737-4038/87/0404-0003$02.00
al. 1984b; Burton et al. 1985; Jubier-Maurin et al. 1985). This suggestion, however, is based on the comparison of laboratory strains whose phylogenetic relations are not always clear (Bishop et al. 1985; Bonhomme 1986). Since the fixation of a new variant is generally a slow process, one cannot be certain that such a variant represents the maintenance of an ancestral polymorphism rather than a recent insertion. This problem can be approached by means of population studies coupled with evolutionary comparisons in related species, an approach that should allow one to conclude when such an element was inserted and, eventually, where it was introduced. They can also show whether there is linkage disequilibrium, which may be of biological significance, with other markers.

We have started to use this approach in our laboratory to study a number of orthologous elements of the L1 family, and the results provide further evidence that the two elements found in intron 12 of the mouse albumin gene (Kioussis et al. 1981; Cooper et al. 1984) and near the βh3 mouse globin pseudogene (Hutchison et al. 1984) are recent insertions in the *M. musculus* genome.

## Material and Methods

### Animals

The genus *Mus* (fig. 1) presents an array of related species at every stage of differentiation, among which the house mouse complex of species is the best known. This complex displays taxonomic units that show a complete panel of biosystematic

<figure><img src="image_3.png" /></figure>

FIG. 1.—A schematic phenogram showing the probable phylogenetic relationships between the species cited (Bonhomme 1986): 1, *Mus musculus domesticus* (*Mus* 1); 2, *M. m. musculus* (*Mus* 2A); 2C, *M. m. castaneus* (*Mus* 2C); 2B, *M. m. molossinus* (*Mus* 2B), which is a hybrid taxa between *Mus* 2A and *Mus* 2C; 5, *M. m. bactrianus* (*Mus* 5); 3, *M. spretus* (*Mus* 3); 4B, *M. spicilegus*, also known as *M. hortulanus* (*M. hortulanus*); 4A, *M. spretoïdes* (formerly known as *spicilegus*) southern form, which is sometimes referred to as *M. abotti* (*Mus* 4A); cervicolor, *M. cervicolor cervicolor*; pahari, *M. (Coelomis) pahari*; setulosus, *M. (Nannomys) setulosus*; and minutoïdes, *M. (Nannomys) minutoïdes*. The last three are now recognized as belonging to different—though closely related—murid genera (Bonhomme 1986). For convenience—and since, in certain cases, the taxonomy is still ambiguous—the biochemical groups proposed by Bonhomme et al. (1984) are also given. ↕ Indicates points where alternative branching order is possible. Dashed lines indicate the hybrid status of *M. m. molossinus*.
interactions, ranging from true sympatric species to parapatric semispecies interacting along a hybrid zone (Bonhomme 1986).

The probable phylogenetic relationships of the taxa referred to in the present paper are given in figure 1. The wild mice studied were caught in the contact zone between *M. m. domesticus* and *M. m. musculus* in Bulgaria and Denmark (see fig. 2). The classical inbred strains BALB/c, C57BP6, and DBA12 and a number of wind-derived laboratory lines established from animals of known geographical origin in our mouse colony were also used.

## DNA Samples

Total DNA was prepared from individual livers according to the method of Jahn et al. (1980). The cDNA clone pMSA433, covering exons 6–13 (fig. 3a) of the mouse albumin gene was provided by Dr. J. Sala-Trépat, and the insert was purified. The probe used to detect L1Md4 was a *Hind*III-*Eco*RI *β*h3-specific fragment extracted from the CE 17.6 phage provided by Dr. S. Martin (Hutchinson et al. 1984).

## DNA Transfer and Hybridization

DNA restriction fragments were transferred from agarose gels to diazobenzyl-oxymethyl paper (Southern 1975; Christophe et al. 1982) and then hybridized to $^{32}$P-labeled probes according to the method described by Feinberg and Vogelstein (1980). Hybridization was performed at 42 C for 16 h in 50% (v/v) formamide, 5 × SSPE (1 × SSPE = 0.15 M NaCl, 10 mM $NaH_2PO_4$, 1 mM ethylenediaminetetraacetate [EDTA]), 5 × Denhardt's solution (1 × Denhardt's solution = 0.02 mg Ficoll/100 ml, 0.02 mg polyvinyl pyrrolidone/100 ml, 0.02 mg bovine serum albumin/100 ml), 0.5% sodium dodecyl sulfate (SDS), and 100 µg salmon sperm DNA/ml. The filters were then washed three times in 2 × SSC (1 × SSC = 0.15 M NaCl, 0.015 M sodium

<figure><img src="image_4.png" /><figcaption>FIG. 2.—Maps of the region in Denmark (a) and Bulgaria (b) where the contact zone between *Mus* 1 and *Mus* 2A is located. The contact zone is indicated by the dashed line. The localities indicated in tables 1 and 2 are shown, except for Sofia and Sadovo, which fall outside the area represented on the map.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>FIG. 3.—a, Restriction map of the *Mus* 1 albumin gene with (+) and without (−) the L1Md element in the twelfth intron. □ Indicates the L1Md element. b, The presence or absence of this L1Md element detected after analysis of the restriction fragments obtained with *Sac*I, *Bam*HI, or *Pvu*II is indicated below each species. s and m indicate *M. (Nannomys) setulosus* and *M. (Nannomys) minutoïdes*, respectively. Only the restriction patterns that correspond to those present in *Mus* 1 were considered. These results were obtained by hybridizing Southern blots of *Bam*HI-*Hind*III-, or *Pst*I-digested DNA from the different species with the pMSA 433 cDNA probe indicated in panel a. n = No. of positive chromosomes/total no. of chromosomes studied.</figcaption></figure>

citrate, pH 7), 0.1% SDS, and two times in 0.2 × SSC, 0.1% SDS at 65 C. Filters were exposed at −70 C using Kodak XAR films and intensifying screens. Restriction-fragment-length polymorphisms (RFLPs) allowed us to distinguish between heterozygous and homozygous animals for the loci studied.

## Results

### The L1M Element of Intron 12 of the Serum Albumin Gene is a Recent Insertion in *Mus musculus* Populations

Four L1M elements are located in the albumin alpha-fetoprotein gene complex of BALB/c (Kioussis et al. 1981; Cooper et al. 1984). Elsewhere (Jubier-Maurin et al. 1985) we showed that the element present in intron 12 of the serum albumin gene was missing in the majority of the wild mice of *M. m. domesticus* (*Mus* 1) origin that we studied. For the region covering intron 12 of the albumin gene, the restriction map for *Bam*HI, *Sac*I, and *Pvu*II is shown in figure 3a. It can be seen that, for this region, the restriction-fragment pattern obtained with these enzymes depends on the absence or presence of the L1 sequence: when it is present one observes an extra band of 0.9 kb with *Bam*HI and two bands—one of 3.9 and one of 4.1 kb (instead of a single 7-kb band)—with *Sac*I; With *Pvu*II, which does not have a site in this particular L1 sequence, a fragment of ~8 kb instead of 7 kb is obtained when it is present. We used these RFLPs to detect the presence or absence of the L1 sequence in different species of the genus *Mus*.
In the present study, 41 (36%) of 114 mouse chromosomes of *M. m. domesticus* (*Mus* 1) origin had this element (table 1). This element was also found in 10 (6%) of the 164 *M. m. musculus* (*Mus* 2A) chromosomes studied (table 2). However, since *Mus* 1 and *Mus* 2A are two parapatric semispecies that come into contact and hybridize along a narrow zone crossing Europe from Denmark to Bulgaria (fig. 2) (Bonhomme et al. 1983; Boursot et al. 1984), it is difficult to decide whether these *Mus* 2A individuals received this element by means of introgression from *Mus* 1 individuals or acquired it by means of some other route. The proportion of *Mus* 1 mice that possess the element varies considerably, both from region to region and from one locality to another within a given region (table 1); for instance, in Bulgaria the frequency is ~14%, whereas in Denmark it is nearly 76%. If one averages the results that were obtained for the 18 individuals from other parts of the world (Algeria, Oceania, and Italy, where it is present; Spain, France, Israel, Egypt, United States, Morocco, and Tunisia, where it is absent), one obtains the value of ~16%. Since we were not able to test enough *Mus* 2A individuals from regions where one can be sure that introgression does not occur, one cannot exclude the possibility that the element is also polymorphic in this semispecies.

<table><caption>**Table 1**

Presence of an L1 Element in Intron 12 of the Albumin Gene and near the βh3-Globin Pseudogene of Various Mice belonging to the Semispecies *Mus* 1</caption><thead><tr><th rowspan="2">ORIGIN OF <i>Mus</i> 1<br>CHROMOSOMES</th><th colspan="2">NO. OF CHROMOSOMES<br>STUDIED/NO. OF CHROMOSOMES<br>POSITIVE</th></tr><tr><th>L1 Element<br>in I12</th><th>L1md4</th></tr></thead><tbody><tr><td><strong>Bulgaria:</strong></td><td></td><td></td></tr><tr><td>Vlas</td><td>16/3</td><td>18/7</td></tr><tr><td>Karnobat</td><td>4/0</td><td>4/1</td></tr><tr><td>Deskotna</td><td>2/0</td><td>2/0</td></tr><tr><td>Iabeltchevo</td><td>10/2</td><td>8/0</td></tr><tr><td>Chivarovo</td><td>2/0</td><td>2/0</td></tr><tr><td>Goritsa</td><td>4/0</td><td>6/0</td></tr><tr><td>Orizare</td><td>12/0</td><td>12/0</td></tr><tr><td>Bratovo</td><td>6/3</td><td>8/0</td></tr><tr><td>Sadovo</td><td>2/0</td><td>2/0</td></tr><tr><td><strong>Total</strong></td><td>58/8 (13.8%)</td><td>62/8 (12.9%)</td></tr><tr><td><strong>Denmark</strong></td><td></td><td></td></tr><tr><td>Odis</td><td>12/11</td><td>12/0</td></tr><tr><td>Egtved</td><td>22/15</td><td>22/0</td></tr><tr><td><strong>Total</strong></td><td>34/26 (76.5%)</td><td>34/0 (0%)</td></tr><tr><td><strong>Other</strong></td><td>18/3 (16.7%)</td><td>16/0 (0%)</td></tr><tr><td><strong>Total no. of strains</strong></td><td>4/4 (100%)</td><td>2/1 (50%)</td></tr></tbody></table>

NOTE.—Since many of the individuals studied were heterozygous for loci considered, the results are given as the number of chromosomes rather than the number of individuals. When only one band was detected, the animal was considered to be homozygous and two chromosomes were counted. In the case of inbred strains, only one chromosome was counted for each individual.
Representatives of other species were screened for the presence of the L1 element in intron 12 (results are given in fig. 3b); it was not detected in the 20 chromosomes coming from *Mus* 4A, *Mus* 4B 3, and the distantly related *M* (Nannomys) minutoides and *M*. (N.) setulosus, all of which diverged prior to the differentiation of the *M. musculus* complex of species (fig. 1). This result is a further indication that the L1 element of intron 12 of the albumin gene is a recent insertion in *M. musculus*; a species that probably diverged from the other species of the genus *Mus* >1 Mybp.

## The L1Md4 Element Adjacent to the Globin Pseudogene Bh3 Is a Recent Insertion in an *M. m. domesticus* Population

Voliva et al. (1984b) showed that the L1Md4 element near the globin pseudogene βh3 in BALB/c mice is absent from C57 mice and suggested that it is a recent insertion. We have investigated the presence or absence of L1Md4 in mice populations of different species.

For BALB/c and C57, the physical maps of the βh3 locus, showing the sites of EcoRI, BamHI, HindIII, and PstI (fig. 4), were reconstituted using the data in the literature (Jahn et al. 1980; Leder et al. 1980; Weaver et al. 1981; Hutchison et al. 1984; Voliva et al. 1984a, 1984b) and our own data. The PstI sites are not polymorphic, so a single band of 9.6 kb is obtained when L1Md4 is present and a 7.5-kb band is obtained when it is absent. Using this enzyme, we tested >100 wild mice, some wild-derived laboratory lines, and a few classical laboratory strains belonging to *Mus* 1 and *Mus* 2A. The results are summarized in tables 1 and 2. The RFLPs found for these individuals when BamHI and HindIII were used were compatible with the results obtained when PstI was used (figs. 4, 5).

The most important feature of this analysis is that only seven *Mus* 1 chromosomes from Vlas, Bulgaria, and only one from Karnobat, Bulgaria, were shown to possess L1Md4. Since the BALB/c mouse has the Hbb^d haplotype, we were interested to know

<figure><img src="image_6.png" /></figure>

FIG. 4.—Physical maps of the βh3 region of the C57 and BALB/c haplotypes: EcoRI (■), BamHI (●), HindIII (○), and PstI (▼) are indicated. The probe used was the HindIII/EcoRI fragment (■■), a gift of Dr. S. Martin. The sites shown by an arrow are those that are polymorphic in wild animals. The fragment indicated by the dashed lines is obtained in mice that do not have the L1 element but possess the polymorphic BamHI site.
<table><caption>**Table 2**

Presence of an L1 Element in Intron 12 of the Albumin Gene and near the βh3-Globin Pseudogene of Various Mice belonging to the Semispecies *Mus* 2A</caption><thead><tr><th rowspan="2">ORIGIN OF <i>Mus</i> 2A<br>CHROMOSOMES</th><th colspan="2">NO. OF CHROMOSOMES STUDIED/<br>NO. OF CHROMOSOMES POSITIVE</th></tr><tr><th>L1 Element in I12</th><th>L1Md4</th></tr></thead><tbody><tr><td><strong>Bulgaria:</strong></td><td></td><td></td></tr><tr><td>Sofia</td><td>12/2</td><td>12/0</td></tr><tr><td>Bilka</td><td>4/0</td><td>4/0</td></tr><tr><td>Dobromir</td><td>4/0</td><td>4/0</td></tr><tr><td>Komounari</td><td>4/0</td><td>4/0</td></tr><tr><td>Partizani</td><td>12/1</td><td>6/0</td></tr><tr><td>Bania</td><td>12/0</td><td>12/0</td></tr><tr><td>Kranevo</td><td>18/1</td><td>18/0</td></tr><tr><td>Sokolovo</td><td>32/0</td><td>32/0</td></tr><tr><td>Baltchik</td><td>6/2</td><td>6/0</td></tr><tr><td>Kavarna</td><td>4/0</td><td>4/0</td></tr><tr><td>Iavlata</td><td>6/0</td><td>6/0</td></tr><tr><td><strong>Total</strong></td><td>114/6 (5.3%)</td><td>108/0 (0%)</td></tr><tr><td><strong>Denmark:</strong></td><td></td><td></td></tr><tr><td>Losning</td><td>10/2</td><td>10/0</td></tr><tr><td>Hov</td><td>22/0</td><td>22/0</td></tr><tr><td><strong>Total</strong></td><td>32/2 (6.3%)</td><td>32/0 (0%)</td></tr><tr><td>Other</td><td>18/2 (11.1%)</td><td>18/0 (0%)</td></tr></tbody></table>

whether L1Md4 is always associated with this haplotype. This is not the case, for (1) none of the Hbb<sup>d</sup> haplotypes that we looked at were L1Md4 positive and (2) the L1Md4 sequences that we detected in the wild-mice populations of Vlas and Karnobat were all probably associated with the Hbb<sup>p</sup> haplotype (table 3).

A further indication that L1Md4 is a recent introduction in a few localized populations of *Mus* 1 is its absence from all the representatives of the other species in which the *Pst*1 sites defining the region have been conserved (*Mus* species 2A, 2B, 2G 4A, 4B, 3, *cervicolor*, and (*Coelomis*) *pahari*) (fig. 6).

## Parsimony Analysis of the R Region of 12 Published L1Md DNA Sequences

Voliva et al. (1984b) have argued that L1Md4 is a recent insertion because the poly A run found at the 3' end is almost pure. Unfortunately, the published sequence of the element for intron 12 does not include its 3' end (Cooper et al. 1984). However, phylogenetic analysis of the shared variant nucleotides of the R region of 12 published L1Md sequences shows that the two recently introduced elements belong to the same subfamily. This is illustrated in figure 7, which shows the most parsimonious unrooted tree obtained when a 184-nucleotide region is considered. Although, for practical reasons, all 400 nucleotides corresponding to the known I12 sequence were not treated together, similar subfamily patterns were found in the other parts of this region (not shown). Note that L1Md4 branches closest to I12. The representation used also shows that, in the majority of sites considered, the same mutation has occurred more than
<figure><img src="image_7.png" /><figcaption>FIG. 5.—Southern blots of *Pst*I- (a), *Bam*HI- (b), and *Hind*III- (c) cleaved DNA hybridized with the βh3 probe shown in fig. 4; genomic DNAs of BALB/c, C57, and wild *Mus* 1 animals (indicated by their extraction number) were treated as indicated in Material and Methods.</figcaption></figure>

once. This homoplasy is further evidence that the sequences have not evolved independently and strongly suggests that multiple short conversion events have played a role in the concerted evolution of this sequence family (Jubier-Maurin et al. 1989; Rogers 1985).

## Discussion

The presence or absence of two L1 elements—one in intron 12 of the mouse albumin gene and one next to the βh3 mouse globin pseudogene—has been investigated in wild populations of *Mus mus domesticus* and *M. m. musculus* and in representatives of other species of the genus *Mus*. The results provide further evidence that both elements were recently inserted into the *M. musculus* genome.

The fact that organisms as divergent as primates, rodents, and marsupials have a roughly equal number of L1 copies (Burton et al. 1985) suggests that this family was already highly repetitive in its common ancestor. One might expect, therefore, a relatively low insertion rate of new L1 copies. This, however, does not appear to be the case—if the data on those L1 elements for which the localization in the mouse genome
<table><caption>**Table 3**
Relation between the Presence (+) or Absence (-) of L1Md4 and the Hbb Haplotypes in *Mus* 1 and *Mus* 2A in the Present Study</caption><thead><tr><th rowspan="2">ORIGIN AND STATUS OF L1Md4</th><th rowspan="2">Hbb HAPLOTYPE</th><th colspan="2">SPECIES</th></tr><tr><th>Mus 1</th><th>Mus 2A</th></tr></thead><tbody><tr><td>Bulgaria:</td><td></td><td></td><td></td></tr><tr><td>-</td><td>s</td><td>28</td><td>52</td></tr><tr><td>-</td><td>d</td><td>2</td><td>20</td></tr><tr><td>-</td><td>p</td><td>3</td><td>28</td></tr><tr><td>-</td><td>ND</td><td>2</td><td>0</td></tr><tr><td>+</td><td>p</td><td>3</td><td>0</td></tr><tr><td>Genotype<sup>a</sup> +/-</td><td>p/s</td><td>4</td><td>0</td></tr><tr><td>Denmark:</td><td></td><td></td><td></td></tr><tr><td>-</td><td>s</td><td>25</td><td>14</td></tr><tr><td>-</td><td>d</td><td>7</td><td>24</td></tr></tbody></table>

is known is in any way typical (at least three recent insertions of the 13 L1 elements are known to be present in the albumin alpha-fetoprotein and β-globin gene complexes). We need more data on different orthologous elements before we can decide whether the insertion of new L1M copies is a process that adds and fixes them in the genome at regular intervals or whether the high reiteration frequency of the L1 family results

<figure><img src="image_8.png" /><figcaption>**FIG. 6.**—Southern blots of *Pst*I-cleaved DNA hybridized with the βh3 probe shown in fig. 4; genomic DNAs of BALB/c, C57, and *Mus* species 2A, 2B, 2C, 4A, 4B, 3, *cervicolor cervicolor*, and (*Coelomis*) *pahari* were treated as indicated in Material and Methods.</figcaption></figure>

<footnote><sup>a</sup> The phases of these four individuals could not be determined. ND = Not done.</footnote>
<figure><img src="image_9.png" /></figure>

FIG. 7.—a, Diagram showing the position in the L1Md sequence of the R region. The 184-nucleotide sequence is situated 80 nucleotides 3' of the *Bam*HI site. b, An unrooted tree for 12 R elements (Gebhard et al. 1982; Gebhard and Zachau 1983; Wilson and Storb 1983; Cooper et al. 1984; Voliva et al. 1984b) obtained by analyzing the nucleotide sequence of the region defined in panel a by means of the DNA PARS program from the PHYLIP package (provided by J. Felsenstein) for DNA. This program only considers shared variant nucleotides. ●—Indicates an event that occurs more than once in the tree; ○—indicates an event that occurs only once and is therefore compatible with the tree (shared-derived; synapomorphies); and +—indicates events that occur only once and that were not taken into account when constructing the tree (uniquely-derived; autapomorphies).

from a limited number of amplification and dispersal steps, (i.e., is a saltatory phenomenon). Under the former hypothesis, the similar number of L1 elements found in distantly related species could be explained by (1) L1's intrinsic property to duplicate itself by retroposition and (2) the capacity of the genome to accept and fix these duplications in mammalian populations. The recent evidence that the open reading frames present in this sequence might code for a reverse transcriptase such as protein supports this idea (Hattori et al. 1986; Loeb et al. 1986).

The phylogenetic analysis of the available R sequences indicates that L1Md4 and L12 are closer to each other than to the rest of the R sequences analyzed. Since the R region, which is located 3' of the open reading frames found in the consensus L1Md sequence, is less conserved than the other regions (Jubier-Maurin et al. 1985), such a close relationship is significant. It would be of interest to compare them with L1Md9, which is also present in the mouse β-globin gene complex and is thought to be a recent insertion (Burton et al. 1985). If L1Md9 turns out to be of the same type, one would have to consider even more seriously the suggestion made by Martin et al. (1984)—i.e., that the newly generated L1 copies originate from a limited number of progenitor elements that are conserved for functional reasons.

If retroposition is to play a role in the observed concerted evolution of the L1 family, at least some of the new inserted copies must become fixed in the population.
Fixation can occur as the result of either genetic drift or selective pressure. One would expect this to be a long process, especially if it occurs in large populations as the result of genetic drift. This idea is supported by the fact that the recent insertions L1Md4 and I12 are not yet fixed in the *musculus* genome, whereas at least one of the old L1 sequences in the intergenic region of the albumin alpha-fetoprotein locus, a sequence that was found to be present in all the *Mus* species tested, has become fixed (results not shown). Genetic drift and founder effects that occurred during the migration of these mice throughout Europe a few thousand years ago could explain the differential occurrence of I12 found in the Bulgarian (14%) and Danish (76%) *Mus* 1 populations. Since the few L1Md4 sequences that we detected in the wild-mice populations were most probably associated with the haplotype p, it is possible that the eventual fixation of this particular copy will depend on the factors determining the diffusion of this haplotype within the *Mus* 1 populations. The fact that HbbP is not found in Denmark might suggest that it and L1Md4 have not yet reached this region. This haplotype is common in wild populations of Asian mice (Miyashita et al. 1985) but until now has only been detected in *M. m. domesticus* in Southern Europe (Italy, Greece, and Bulgaria; C. Montgelard and J. Britton-Davidian, personal communication)). It has also been found in Israeli *M. m. domesticus* populations (Ritte and Neufeld 1982) and in *M. m. bactrianus* populations in Iran (Miyashita et al. 1985).

Although genetic drift would appear to be an adequate explanation for the diffusion of a new copy in a given population, one cannot exclude the possibility that direct or indirect selective pressures play a role in at least some cases. For instance, another explanation for absence of the HbbP haplotype in northern Europe is that it is selected against. Furthermore, Burton et al. (1985) have shown that the intergenic region between the Hbb-b1 and Hbb-b2 adult globin genes has undergone rearrangements involving L1 sequences. Although these are different in the HbbS and HbbD haplotypes, the distance between the two genes is maintained. This suggests that the whole region might be under selective pressure to preserve the distances between the coding genes in the locus. Again, population studies of these loci would shed light on the possible biological significance or consequences of such phenomena.

Although there is now considerable evidence that both conversion (Jubier-Maurin et al. 1985; Rogers 1985) and retroposition play a role in the process of molecular drive leading to the concerted evolution of the L1 family (Dover 1982, 1986), only when we have a more precise idea about the rate of insertion and fixation of new copies can the relative importance of these two mechanisms be estimated.

Since BALB/c (which is L1Md4 positive) and C57 (which is L1Md4 negative) are often considered as archetypes of the HbbD and HbbS haplotypes, respectively, one might have expected L1Md4 to be present in all genomes with the HbbD haplotype. Not only is this not the case, but none of the 53 HbbD genomes in wild mice that we tested were L1Md positive. Further population studies will show whether both HbbD/L1Md4-positive and HbbD/L1Md4-positive haplotypes exist in wild mice or whether the BALB/c haplotype is the result of a recombination between an HbbP/L1Md4-positive chromosome and an HbbD/L1Md4-negative chromosome that occurred during the formation of the strain. This, however, would be very surprising, since the HbbP haplotype only occurs in one (Av/SsJ) of the numerous inbred lines created during the first half of the present century.

## Acknowledgments

This work was supported by grants from CNRS, INSERM, and the Association pour la Recherche contre le Cancer (ARC). V.J.M. was supported by ARC and M.B.
and F.V. were supported by the Ministère de la Recherche et de la Technologie (MRT). We wish to thank S. Guerassimov and J. T. Nielsen for their help in obtaining the Bulgarian and Danish mice, J. L. Nahon for his technical advice on the preparation of diazobenzyloxymethyl paper, and A. Orth for her dedicated work in establishing the wild-derived lines.

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RICHARD B. FLAVELL, reviewing editor

Received June 15, 1986; revision received December 12, 1986.