the mouse genome

Origins of the mouse

The precise origin of the mouse and human lineages has been the subject of recent debate. Palaeontological evidence has long indicated a great radiation of placental (eutherian) mammals about 65 million years ago (Myr) that filled the ecological space left by the extinction of the dinosaurs, and that gave rise to most of the eutherian orders²³. Molecular phylogenetic analyses indicate earlier divergence times of many of the mammalian clades. Some of these studies have suggested a very early date for the divergence of mouse from other mammals (100–130 Myr^{23, 24, 25}) but these estimates partially originate from the fast molecular clock in rodents (see below). Recent molecular studies that are less sensitive to the differences in evolutionary rates have suggested that the eutherian mammalian radiation took place throughout the Late Cretaceous period (65–100 Myr), but that rodents and primates actually represent relatively late-branching lineages^{26, 27}. In the analyses below, we use a divergence time for the human and mouse lineages of 75 Myr for the purpose of calculating evolutionary rates, although it is possible that the actual time may be as recent as 65 Myr.

Origins of mouse genetics

The origin of the mouse as the leading model system for biomedical research traces back to the start of human civilization, when mice became commensal with human settlements. Humans noticed spontaneously arising coat-colour mutants and recorded their observations for millennia (including ancient Chinese references to dominant-spotting, waltzing, albino and yellow mice). By the 1700s, mouse fanciers in Japan and China had domesticated many varieties as pets, and Europeans subsequently imported favourites and bred them to local mice (thereby creating progenitors of modern laboratory mice as hybrids among M. m. domesticus, M. m. musculus and other subspecies). In Victorian England, 'fancy' mice were prized and traded, and a National Mouse Club was founded in 1895 (refs 28, 29).

With the rediscovery of Mendel's laws of inheritance in 1900, pioneers of the new science of genetics (such as Cuenot, Castle and Little) were quick to recognize that the discontinuous variation of fancy mice was analogous to that of Mendel's peas, and they set out to test the new theories of inheritance in mice. Mating programmes were soon established to create inbred strains, resulting in many of the modern, well-known strains (including C57BL/6J)³⁰.

Genetic mapping in the mouse began with Haldane's report³¹ in 1915 of linkage between the pink-eye dilution and albino loci on the linkage group that was eventually assigned to mouse chromosome 7, just 2 years after the first report of genetic linkage in Drosophila. The genetic map grew slowly over the next 50 years as new loci and linkage groups were added—chromosome 7 grew to three loci by 1935 and eight by 1954. The accumulation of serological and enzyme polymorphisms from the 1960s to the early 1980s began to fill out the genome, with the map of chromosome 7 harbouring 45 loci by 1982 (refs 29, 31).

The real explosion, however, came with the development of recombinant DNA technology and the advent of DNA-sequence-based polymorphisms. Initially, this involved the detection of restriction-fragment length polymorphisms (RFLPs)³²; later, the emphasis shifted to the use of simple sequence length polymorphisms (SSLPs; also called microsatellites), which could be assayed easily by polymerase chain reaction (PCR)^{33, 34, 35, 36} and readily revealed polymorphisms between inbred laboratory strains.

Origins of mouse genomics

When the Human Genome Project (HGP) was launched in 1990, it included the mouse as one of its five central model organisms, and targeted the creation of genetic, physical and eventually sequence maps of the mouse genome.

By 1996, a dense genetic map with nearly 6,600 highly polymorphic SSLP markers ordered in a common cross had been developed³⁴, providing the standard tool for mouse genetics. Subsequent efforts filled out the map to over 12,000 polymorphic markers, although not all of these loci have been positioned precisely relative to one another. With these and other loci, Haldane's original two-marker linkage group on chromosome 7 had now swelled to about 2,250 loci.

Physical maps of the mouse genome also proceeded apace, using sequence-tagged sites (STS) together with radiation-hybrid panels^{37, 38} and yeast artificial chromosome (YAC) libraries to construct dense landmark maps³⁹. Together, the genetic and physical maps provide thousands of anchor points that can be used to tie clones or DNA sequences to specific locations in the mouse genome.

Other resources included large collections of expressed-sequence tags (EST)⁴⁰, a growing number of full-length complementary DNAs^{41, 42} and excellent bacterial artificial chromosome (BAC) libraries⁴³. The latter have been used for deriving large sets of BAC-end sequences³⁷ and, as part of this collaboration, to generate a fingerprint-based physical map⁴⁴. Furthermore, key mouse genome databases were developed at the Jackson (http://www.informatics.jax.org/), Harwell (http://www.har.mrc.ac.uk/) and RIKEN (http://genome.rtc.riken.go.jp/) laboratories to provide the community with access to this information.

With these resources, it became straightforward (but not always easy) to perform positional cloning of classic single-gene mutations for visible, behavioural, immunological and other phenotypes. Many of these mutations provide important models of human disease, sometimes recapitulating human phenotypes with uncanny accuracy. It also became possible for the first time to begin dissecting polygenic traits by genetic mapping of quantitative trait loci (QTL) for such traits.

Continuing advances fuelled a growing desire for a complete sequence of the mouse genome. The development of improved random mutagenesis protocols led to the establishment of large-scale screens to identify interesting new mutants, increasing the need for more rapid positional cloning strategies. QTL mapping experiments succeeded in localizing more than 1,000 loci affecting physiological traits, creating demand for efficient techniques capable of trawling through large genomic regions to find the underlying genes. Furthermore, the ability to perform directed mutagenesis of the mouse germ line through homologous recombination made it possible to manipulate any gene given its DNA sequence, placing an increasing premium on sequence information. In all of these cases, it was clear that genome sequence information could markedly accelerate progress.

Origin of the Mouse Genome Sequencing Consortium

With the sequencing of the human genome well underway by 1999, a concerted effort to sequence the entire mouse genome was organized by a Mouse Genome Sequencing Consortium (MGSC). The MGSC originally consisted of three large sequencing centres—the Whitehead/Massachusetts Institute of Technology (MIT) Center for Genome Research, the Washington University Genome Sequencing Center, and the Wellcome Trust Sanger Institute—together with an international database, Ensembl, a joint project between the European Bioinformatics Institute and the Sanger Institute.

In addition to the genome-wide efforts of the MGSC, other publicly funded groups have been contributing to the sequencing of the mouse genome in specific regions of biological interest. Together, the MGSC and these programmes have so far yielded clone-based draft sequence consisting of 1,859 Mb (74%, although there is redundancy) and finished sequence of 477 Mb (19%) of the mouse genome. Furthermore, Mural and colleagues⁴⁵ recently reported a draft sequence of mouse chromosome 16 containing 87 Mb (3.5%).

To analyse the data reported here, the MGSC was expanded to include the other publicly funded sequencing groups and a Mouse Genome Analysis Group consisting of scientists from 27 institutions in 6 countries.

Sequencing strategy

Sanger and co-workers developed the strategy of random shotgun sequencing in the early 1980s, and it has remained the mainstay of genome sequencing over the ensuing two decades. The approach involves producing random sequence 'reads', generating a preliminary assembly on the basis of sequence overlaps, and then performing directed sequencing to obtain a 'finished' sequence with gaps closed and ambiguities resolved⁴⁶. Ansorge and colleagues⁴⁷ extended the technique by the use of 'paired-end sequencing', in which sequencing is performed from both ends of a cloned insert to obtain linking information, which is then used in sequence assembly. More recently, Myers and co-workers⁴⁸, and others, have developed efficient algorithms for exploiting such linking information.

A principal issue in the sequencing of large, complex genomes has been whether to perform shotgun sequencing on the entire genome at once (whole-genome shotgun, WGS) or to first break the genome into overlapping large-insert clones and to perform shotgun sequencing on these intermediates (hierarchical shotgun)⁴⁶. The WGS technique has the advantage of simplicity and rapid early coverage; it readily works for simple genomes with few repeats, but there can be difficulties encountered with genomes that contain highly repetitive sequences (such as the human genome, which has near-perfect repeats spanning hundreds of kilobases). Hierarchical shotgun sequencing overcomes such difficulties by using local assembly, thus decreasing the number of repeat copies in each assembly and allowing comparison of large regions of overlaps between clones. Consequently, efforts to produce finished sequences of complex genomes have relied on either pure hierarchical shotgun sequencing (including those of Caenorhabditis elegans⁴⁹, Arabidopsis thaliana⁴⁹ and human¹) or a combination of WGS and hierarchical shotgun sequencing (including those of Drosophila melanogaster⁵⁰, human² and rice⁵¹).

The ultimate aim of the MGSC is to produce a finished, richly annotated sequence of the mouse genome to serve as a permanent reference for mammalian biology. In addition, we wished to produce a draft sequence as rapidly as possible to aid in the interpretation of the human genome sequence and to provide a useful intermediate resource to the research community. Accordingly, we adopted a hybrid strategy for sequencing the mouse genome. The strategy has four components: (1) production of a BAC-based physical map of the mouse genome by fingerprinting and sequencing the ends of clones of a BAC library⁴⁴; (2) WGS sequencing to approximately sevenfold coverage and assembly to generate an initial draft genome sequence; (3) hierarchical shotgun sequencing of BAC clones covering the mouse genome combined with the WGS data to create a hybrid WGS-BAC assembly; and (4) production of a finished sequence by using the BAC clones as a template for directed finishing. This mixed strategy was designed to exploit the simpler organizational aspects of WGS assemblies in the initial phase, while still culminating in the complete high-quality sequence afforded by clone-based maps.

We chose to sequence DNA from a single mouse strain, rather than from a mixture of strains⁴⁵, to generate a solid reference foundation, reasoning that polymorphic variation in other strains could be added subsequently (see below). After extensive consultation with the scientific community⁵², the B6 strain was selected because of its principal role in mouse genetics, including its well-characterized phenotype and role as the background strain on which many important mutations arose. We elected to sequence a female mouse to obtain equal coverage of chromosome X and autosomes. Chromosome Y was thus omitted, but this chromosome is highly repetitive (the human chromosome Y has multiple duplicated regions exceeding 100 kb in size with 99.9% sequence identity⁵³) and seemed an unwise target for the WGS approach. Instead, mouse chromosome Y is being sequenced by a purely clone-based (hierarchical shotgun) approach.

Sequencing and assembly

The genome assembly was based on a total of 41.4 million sequence reads derived from both ends of inserts (paired-end reads) of various clone types prepared from B6 female DNA. The inserts ranged in size from 2 to 200 kb (Table 1). The three large MGSC sequencing centres generated 40.4 million reads, and 0.6 million reads were generated at the University of Utah. In addition, we used 0.4 million reads from both ends of BAC inserts reported by The Institute for Genome Research⁵⁴.

Table 1: Distribution of sequence reads

Full table

A total of 33.6 million reads passed extensive checks for quality and source, of which 29.7 million were paired; that is, derived from opposite ends of the same clone (Table 1). The assembled reads represent approximately 7.7-fold sequence coverage of the euchromatic mouse genome (6.5-fold coverage in bases with a Phred quality score of >20)⁵⁵. Together, the clone inserts provide roughly 47-fold physical coverage of the genome.

The sequence reads, together with the pairing information, were used as input for two recently developed sequence-assembly programs, Arachne^{56, 57} and Phusion⁵⁸. No mapping information and no clone-based sequences were used in the WGS assembly, with the exception of a few reads (<0.1% of the total) derived from a handful of BACs, which were used as internal controls. The assembly programs were tested and compared on intermediate data sets over the course of the project and were thereby refined. The programs produced comparable outputs in the final assembly. The assembly generated by Arachne was chosen as the draft sequence described here because it yielded greater short-range and long-range continuity with comparable accuracy.

The assembly contains 224,713 sequence contigs, which are connected by at least two read-pair links into supercontigs (or scaffolds). There are a total of 7,418 supercontigs at least 2 kb in length, plus a further 37,125 smaller supercontigs representing <1% of the assembly. The contigs have an N50 length of 24.8 kb, whereas the supercontigs have an N50 length that is approximately 700-fold larger at 16.9 Mb (N50 length is the size x such that 50% of the assembly is in units of length at least x). In fact, most of the genome lies in supercontigs that are extremely large: the 200 largest supercontigs span more than 98% of the assembled sequence, of which 3% is within sequence gaps (Table 2).

Table 2: Basic statistics of the MGSCv3 assembly

Full table

Anchoring to chromosomes

We assigned as many supercontigs as possible to chromosomal locations in the proper order and orientation. Supercontigs were localized largely by sequence alignments with the extensively validated mouse genetic map³⁴, with some additional localization provided by the mouse radiation-hybrid map³⁷ and the BAC map⁴⁴. We found no evidence of incorrect global joins within the supercontigs (that is, multiple markers supporting two discordant locations within the genome), and thus were able to place them directly. Altogether, we placed 377 supercontigs, including all supercontigs >500 kb in length.

Once much of the sequence was anchored, it was possible to exploit additional read-pair and physical mapping information to obtain greater continuity (Table 2). For example, some adjacent supercontigs were connected by BAC-end (or other) links, satisfying appropriate length and orientation constraints, including single links. Furthermore, some adjacent extended supercontigs were connected by means of fingerprint contigs in the BAC-based physical map. These additional links were used to join sequences into ultracontigs. In the end, a total of 88 ultracontigs with an N50 length of 50.6 Mb (exclusive of gaps) contained 95.7% of the assembled sequence (Fig. 1). Continuity near telomeres tends to be lower, and two chromosomes (5 and X) have unusually large numbers of ultracontigs.

Figure 1: The mouse genome in 88 sequence-based ultracontigs.

The position and extent of the 88 ultracontigs of the MGSCv3 assembly are shown adjacent to ideograms of the mouse chromosomes. All mouse chromosomes are acrocentric, with the centromeric end at the top of each chromosome. The supercontigs of the sequence assembly were anchored to the mouse chromosomes using the MIT genetic map. Neighbouring supercontigs were linked together into ultracontigs using information from single BAC links and the fingerprint and radiation-hybrid maps, resulting in 88 ultracontigs containing 95% of the bases in the euchromatic genome.

High resolution image and legend (96K)

Proportion of genome contained in the assembly

This was assessed by comparison with publicly available finished genome sequence and mouse cDNA sequences. Of the 187 Mb of finished mouse sequence, 96% was contained in the anchored assembly. This finished sequence, however, is not a completely random cross-section of the genome (it has been cloned as BACs, finished, and in some cases selected on the basis of its gene content). Of 11,452 cDNA sequences from the curated RefSeq collection, 99.3% of the cDNAs could be aligned to the genome sequence (see Supplementary Information). These alignments contained 96.4% of the cDNA bases. Together, this indicates that the draft genome sequence includes approximately 96% of the euchromatic portion of the mouse genome, with about 95% anchored (Table 1).

Genome size

On the basis of the estimated sizes of the ultracontigs and gaps between them, the total length of the euchromatic mouse genome was estimated to be about 2.5 Gb (see Supplementary Information), or about 14% smaller than that of the euchromatic human genome (about 2.9 Gb) (Table 3). The ultracontigs include spanned gaps, whose lengths are estimated on the basis of paired-end reads and alignment against the human sequence (see below). To test the accuracy of the ultracontig lengths, we compared the actual length of 675 finished mouse BAC sequences (from the B6 strain) with the corresponding estimated length from the draft genome sequence. The ratio of estimated length to actual length had a median value of 0.9994, with 68% of cases falling within 0.99–1.01 and 84% of cases within 0.98–1.02.

Table 3: Mouse chromosome size estimates

Full table

Quality assessment at intermediate scale

Although no evidence of large-scale misassembly was found when anchoring the assembly onto the mouse chromosomes, we examined the assembly for smaller errors.

To assess the accuracy at an intermediate scale, we compared the positions of well-studied markers on the mouse genetic map and in the genome assembly (see Supplementary Information). Out of 2,605 genetic markers that were unambiguously mapped to the sequence assembly (BLAST match using 10^-100 or better as an E-value to a single location) we found 1.8% in which the chromosomal assignment in the genetic map conflicted with that in the sequence. This is well within the known range of erroneous assignments within the genetic map³⁴. We tested 11 such discrepant markers by re-mapping them in a mouse cross. In ten cases, the data showed that the previous genetic map assignment was erroneous and supported the position in the draft sequence. In one case, the data supported the previous genetic map assignment and contradicted the assembly. By studying the one erroneous case, we recognized that a single 36-kb segment had been erroneously merged into a sequence contig by means of a single overlap of two reads. We screened the entire assembly for similar instances, affecting regions of at least 20 kb. Only 17 additional cases were found, with a median size of the incorrectly merged segment of 34 kb. These are being corrected in the next release of the MGSC sequence. We are continuing to investigate instances involving smaller incorrectly merged segments.

We also found 19 instances (0.7%) of conflicts in local marker order between the genetic map and sequence assembly. A conflict was defined as any instance that would require changing more than a single genotype in the data underlying the genetic map to resolve. We studied ten cases by re-mapping the genetic markers, and eight were found to be due to errors in the genetic map. On the basis of this analysis, we estimate that chromosomal misassignment and local misordering affects <0.3% of the assembled sequence.

Quality assessment at fine scale

We also assessed fine-scale accuracy of the assembly by carefully aligning it to about 10 Mb of finished BAC-derived sequence from the B6 strain. This revealed a total of 39 discrepancies of 50 bp in length (median size of 320 bp), reflecting small misassemblies either in the draft sequence or the finished BAC sequences. These discrepancies typically occurred at the ends of contigs in the WGS assembly, indicating that they may represent the incorrect incorporation of a single terminal read.

At the single nucleotide level in the assembly, the observed discrepancy rates varied in a manner consistent with the quality scores assigned to the bases in the WGS assembly (see Supplementary Information). Overall, 96% of nucleotides in the assembly have Arachne quality scores 40, corresponding to a predicted error rate of 1 per 10,000 bases. Such bases had an observed discrepancy rate against finished sequence of 0.005%, or 5 errors per 100,000 bases.

Comparison with the draft sequence of chromosome 16

We also compared the sequence reported here to a draft sequence of mouse chromosome 16 recently published by Mural and co-workers⁴⁵. Because the latter was produced from strain 129 and other mouse strains, it is expected to differ slightly at the nucleotide level but should otherwise show good agreement. The sequences align well at large scales (hundreds of kilobases), although the assembly by Mural and co-workers contains less total sequence (87 compared with 91 Mb) and includes a region of approximately 300 kb that we place on chromosome X. There were differences at intermediate scales, with our draft sequence showing better agreement with finished BAC-derived sequences (approximately fourfold fewer discrepancies of length 500 bp; 20 compared with 5 in about 2.8 Mb of finished sequence). These could not be explained by strain differences, as similar results were seen with finished sequence from the B6 and 129 strains.

Collapse of duplicated regions

The human genome contains many large duplicated regions, estimated to comprise roughly 5% of the genome⁵⁹, with nearly identical sequence. If such regions are also common in the mouse genome, they might collapse into a single copy in the WGS assembly. Such artefactual collapse could be detected as regions with unusually high read coverage, compared with the average depth of 7.4-fold in long assembled contigs. We searched for contigs that were >20 kb in size and contained >10 kb of sequence in which the read coverage was at least twofold higher than the average. Such regions comprised only a tiny fraction (<0.0001) of the total assembly, of which only half had been anchored to a chromosome. None of these windows had coverage exceeding the average by more than threefold. This may indicate that the mouse genome contains fewer large regions of near-exact duplication than the human. Alternatively, regions of near-exact duplication may have been systematically excluded by the WGS assembly programme. This issue is better addressed through hierarchical shotgun than WGS sequencing and will be examined more carefully in the course of producing a finished mouse genome sequence.

Unplaced reads and large tandem repeats

We expected that highly repetitive regions of the genome would not be assembled or would not be anchored on the chromosomes. Indeed, 5.9 million of the 33.6 million passing reads were not part of anchored sequence, with 88% of these not assembled into sequence contigs and 12% assembled into small contigs but not chromosomally localized.

A striking example of unassembled sequence is a large region on mouse chromosome 1 that contains a tandem expansion of sequence containing the Sp100-rs gene fusion. This region is highly variable among mouse species and even laboratory strains, with estimated lengths ranging from 6 to 200 Mb^{60, 61}. The bulk of this region was not reliably assembled in the draft genome sequence. The individual sequence reads together were found to contain 493-fold coverage of the Sp100-rs gene, suggesting that there are roughly 60 copies in the B6 genome (corresponding to a region of about 6 Mb). This is consistent with an estimate of 50 copies in B6 obtained by Southern blotting⁶².

We also examined centromeric sequences, including the euchromatin-proximal major satellite repeat (234 bases) and the telomere-proximal minor repeat (120 bases) found on some chromosomes^{63, 64}. (Note that mouse chromosomes are all acrocentric, meaning that the centromere is adjacent to one telomere.) The minor satellite was poorly represented among the sequence reads (present in about 24,000 reads or <0.1% of the total) suggesting that this satellite sequence is difficult to isolate in the cloning systems used. The major satellite was found in about 3.6% of the reads; this is also lower than previous estimates based on density gradient experiments, which found that major satellites comprise about 5.5% of the mouse genome, or approximately 8 Mb per chromosome⁶⁵.

Evaluation of WGS assembly strategy

The WGS assembly described here involved only random reads, without any additional map-based information. By many criteria, the assembly is of very high quality. The N50 supercontig size of 16.9 Mb far exceeds that achieved by any previous WGS assembly, and the agreement with genome-wide maps is excellent. The assembly quality may be due to several factors, including the use of high-quality libraries, the variety of insert lengths in multiple libraries, the improved assembly algorithms, and the inbred nature of the mouse strain (in contrast to the polymorphisms in the human genome sequences). Another contributing factor may be that the mouse differs from the human in having less recent segmental duplication to confound assembly.

Notwithstanding the high quality of the draft genome sequence, we are mindful that it contains many gaps, small misassemblies and nucleotide errors. It is likely that these could not all be resolved by further WGS sequencing, therefore directed sequencing will be needed to produce a finished sequence. The results also suggest that WGS sequencing may suffice for large genomes for which only draft sequence is required, provided that they contain minimal amounts of sequence associated with recent segmental duplications or large, recent interspersed repeat elements.

Adding finished sequence

As a final step, we enhanced the WGS sequence assembly by substituting available finished BAC-derived sequence from the B6 strain. In total, we replaced 3,528 draft sequence contigs with 48.2 Mb of finished sequence from 210 finished BACs available at the time of the assembly. The resulting draft genome sequence, MGSCv3, was submitted to the public databases and is freely available in electronic form through various sources (see below).

The sequence data and assemblies have been freely available throughout the course of the project. The next step of the project, which is already underway, is to convert the draft sequence into a finished sequence. As the MGSC produces additional BAC assemblies and finished sequence, we plan to continue to revise and release enhanced versions of the genome sequence en route to a completely finished sequence⁶⁶, thereby providing a permanent foundation for biomedical research in the twenty-first century.

Genome expansion and contraction

The projected total length of the euchromatic portion of the mouse genome (2.5 Gb) is about 14% smaller than that of the human genome (2.9 Gb). To investigate the source of this difference, we examined the relative size of intervals between consecutive orthologous landmarks in the human and mouse genomes. The mouse/human ratio has a mean at 0.91 for autosomes, but varies widely, with the mouse interval being larger than the human in 38% of cases (Fig. 6). Chromosome X, by contrast, shows no net relative expansion or contraction, with a mouse/human ratio of 1.03 (Fig. 6 and Table 4). What accounts for the smaller size of the mouse genome? We address this question below in the sections on repeat sequences and on genome evolution.

Figure 6: Size ratio of mouse to human for orthologous 100-kb windows.

For each 100-kb region of the mouse genome, the size ratio to the related segment of the human genome was determined. The frequency of the various ratios is plotted on a logarithmic scale for both the autosomes (blue line) and the X chromosome (red line). The ratio for autosomes shows a mean of 0.91 but the ratio varies widely, with the mouse genome larger for 38% of the intervals. The X chromosome by contrast has a mean ratio of just over 1.0. Indeed, chromosome X is slightly smaller in human.

High resolution image and legend (36K)

(G+C) content

The overall distribution of local (G+C) content is significantly different between the mouse and human genomes (Fig. 7). Such differences have been noted in biochemical studies^{78, 79, 80, 81} and in comparative analyses of fourfold degenerate sites in codons of mouse and human genes^{82, 83, 84, 85}, but the availability of nearly complete genome sequences provides the first detailed picture of the phenomenon.

Figure 7: Distribution of (G+C) content in the mouse (blue) and human (red) genomes.

Mouse has a higher mean (G+C) content than human (42% compared with 41%), but human has a larger fraction of windows with either high or low (G+C) content. The distribution was determined using the unmasked genomes in 20-kb non-overlapping windows, with the fraction of windows (y axis) in each percentage bin (x axis) plotted for both human and mouse.

High resolution image and legend (31K)

The mouse has a slightly higher overall (G+C) content than the human (42% compared with 41%), but the distribution is tighter. When local (G+C) content is measured in 20-kb windows across the genome, the human genome has about 1.4% of the windows with (G+C) content >56% and 1.3% with (G+C) content <33%. Such extreme deviations are virtually absent in the mouse genome. The contrast is even seen at the level of entire chromosomes. The human has extreme outliers with respect to (G+C) content (the most extreme being chromosome 19), whereas the mouse chromosomes tend to be far more uniform (Fig. 8).

Figure 8: (G+C) content and density of CpG islands shows more variability in human (red) than mouse (blue) chromosomes.

a, The (G+C) content for each of the mouse chromosomes is relatively similar, whereas human chromosomes show more variation; chromosomes 16, 17, 19 and 22 have higher (G+C) content, and chromosome 13 lower (G+C) content. b, Similarly, the density of CpG islands is relatively homogenous for all mouse chromosomes and more variable in human, with the same exceptions. Note that the mouse and human chromosomes are matched by chromosome number, not by regions of conserved synteny.

High resolution image and legend (37K)

There is a strong positive correlation in local (G+C) content between orthologous regions in the mouse and human genomes (Fig. 9), but with the mouse regions showing a clear tendency to be less extreme in (G+C) content than the human regions. This tendency is not uniform, with the most extreme differences seen at the tails of the distribution.

Figure 9: Comparison of (G+C) and gene content in mouse and human.

a, Scatter plot of mouse (y axis) compared with human (x axis) (G+C) content for all non-overlapping orthologous 100-kb windows. In general, (G+C) content is correlated between the two species, but very few mouse windows have a (G+C) content over 55%, even where the related human window has over 60% (G+C) content. b, Average mouse (G+C) content of 100-kb syntenic windows binned by human (G+C) content (1% intervals). The red line indicates median values with standard deviation and 5% (green) and 95% (blue) confidence intervals. The black line indicates identical (G+C) content in orthologous segments. c–e, Gene content increases with (G+C) content when comparing (G+C) and gene content in 320-kb non-overlapping, unmasked windows for mouse (blue lines) and human (red lines). c, Cumulative proportions of genes (solid lines) and genome (dashed lines) having (G+C) content below a given level. The tighter distribution of (G+C) content in mouse results in the curve for mouse crossing that for human at 45–46% for both genes and total sequence. The tendency for both genomes to be gene-poor at low (G+C) content and gene-rich at high (G+C) content is shown directly in d, which shows the fraction of genes residing within the portion of the genome having (G+C) content below a given level (for example, the half of the genome with the lowest (G+C) content contains 25% of the genes). e, The average number of genes per window is plotted against the (G+C) content of the window for both genomes, showing that the gene density in mouse reaches the same level as in human but at a lower level of (G+C) content.

High resolution image and legend (63K)

In mammalian genomes, there is a positive correlation between gene density and (G+C) content^{81, 86, 87, 88, 89}. Given the differences in (G+C) content between human and mouse, we compared the distribution of genes—using the sets of orthologous mouse and human genes described below—with respect to (G+C) content for both genomes (Fig. 9). The density of genes differed markedly when expressed in terms of absolute (G+C) content, but was nearly identical when expressed in terms of percentiles of (G+C) content (Fig. 9). For example, both species have 75–80% of genes residing in the (G+C)-richest half of their genome. Mouse and human thus show similar degrees of homogeneity in the distribution of genes, despite the overall differences in (G+C) content. Notably, the mouse shows similar extremes of gene density despite being less extreme in (G+C) content.

What accounts for the differences in (G+C) content between mouse and human? Does it reflect altered selection for (G+C) content^{90, 91}, altered mutational or repair processes^{92, 93, 94}, or possibly both? Data from additional species will probably be needed to address these issues. Any explanation will need to account for various mysterious phenomena. For example, although overall (G+C) content in mouse is slightly higher than in human (42% compared with 41%), the (G+C) content of chromosome X is slightly lower (39.0% compared with 39.4%). The effect is even more pronounced if one excludes lineage-specific repeats (see below), thereby focusing primarily on shared DNA. In that case, mouse autosomes have an overall (G+C) content that is 1.5% higher than human autosomes (41.2% compared with 39.7%) whereas mouse chromosome X has a (G+C) content that is 1% lower than human chromosome X (37.8% compared with 36.8%).

CpG islands

In mammalian genomes, the palindromic dinucleotide CpG is usually methylated on the cytosine residue. Methyl-CpG is mutated by deamination to TpG, leading to approximately fivefold under-representation of CpG across the human^{1, 95} and mouse genomes. In some regions of the genome that have been implicated in gene regulation, CpG dinucleotides are not methylated and thus are not subject to deamination and mutation. Such regions, termed CpG islands, are usually a few hundred nucleotides in length, have high (G+C) content and above average representation of CpG dinucleotides.

We applied a computer program that attempts to recognize CpG islands on the basis of (G+C) and CpG content of arbitrary lengths of sequence^{96, 97} to the non-repetitive portions of human and mouse genome sequences (see Supplementary Information). The mouse genome contains fewer CpG islands than the human genome (about 15,500 compared with 27,000), which is qualitatively consistent with previous reports⁹⁸. The absolute number of islands identified depends on the precise definition of a CpG island used, but the ratio between the two species remains fairly constant.

The reason for the smaller number of predicted CpG islands in mouse may relate simply to the smaller fraction of the genome with extremely high (G+C) content⁹⁹ and its effect on the computer algorithm. Approximately 10,000 of the predicted CpG islands in each species show significant sequence conservation with CpG islands in the orthologous intervals in the other species, falling within the orthologous landmarks described above. Perhaps these represent functional CpG islands, a proposition that can now be tested experimentally⁸⁴.

Mouse has accumulated more new repetitive sequence than human

Approximately 46% of the human genome can be recognized currently as interspersed repeats resulting from insertions of transposable elements that were active in the last 150–200 million years. The total fraction of the human genome derived from transposons may be considerably larger, but it is not possible to recognize fossils older than a certain age because of the high degree of sequence divergence. Because only 37.5% of the mouse genome is recognized as transposon-derived (Table 5), it is tempting to conclude that the smaller size of the mouse genome is due to lower transposon activity since the divergence of the human and mouse lineages. Closer analysis, however, shows that this is not the case. As we discuss below, transposition has been more active in the mouse lineage. The apparent deficit of transposon-derived sequence in the mouse genome is mostly due to a higher nucleotide substitution rate, which makes it difficult to recognize ancient repeat sequences.

Table 5: Composition of interspersed repeats in the mouse genome

Full table

Lineage-specific versus ancestral repeats

Interspersed repeats can be divided into lineage-specific repeats (defined as those introduced by transposition after the divergence of mouse and human) and ancestral repeats (defined as those already present in a common ancestor). Such a division highlights the fact that transposable elements have been more active in the mouse lineage than in the human lineage. Approximately 32.4% of the mouse genome (about 818 Mb) but only 24.4% of the human genome (about 695 Mb) consists of lineage-specific repeats (Table 5). Contrary to initial appearances, transposon insertions have added at least 120 Mb more transposon-derived sequence to the mouse genome than to the human genome since their divergence. This observation is consistent with the previous report that the rate of transposition in the human genome has fallen markedly over the past 40 million years^{1, 100}.

The overall lower interspersed repeat density in mouse is the result of an apparent lack of ancestral repeats: they comprise only 5% of the mouse genome compared with 22% of the human genome. The ancestral repeats recognizable in mouse tend to be those of more recent origin, that is, those that originated closest to the mouse–human divergence. This difference may be due partly to a higher deletion rate of non-functional DNA in the mouse lineage, so that more of the older interspersed repeats have been lost. However, the deficit largely reflects a much higher neutral substitution rate in the mouse lineage than in the human lineage, rendering many older ancestral repeats undetectable with available computer programs.

Higher substitution rate in mouse lineage

The hypothesis that the neutral substitution rate is higher in mouse than in human was suggested as early as 1969 (refs 101–103). The idea has continued to be challenged on the basis that the apparent differences may be due to inaccuracies in mammalian phylogenies^{104, 105}. The explanation, however, remains unclear, with some attributing it to generation time^{101, 106} and others pointing to a closer correlation with body size^{107, 108}.

Ancestral repeats provide a powerful measure of neutral substitution rates, on the basis of comparing thousands of current copies to the inferred consensus sequence of the ancestral element. The large copy number and ubiquitous distribution of ancestral repeats overcome issues of local variation in substitution rates (see below). Most notably, differences in divergence levels are not affected by phylogenetic assumptions, as the time spent by an ancestral repeat family in either lineage is necessarily identical.

The median divergence levels of 18 subfamilies of interspersed repeats that were active shortly before the human–rodent speciation (Table 6) indicates an approximately twofold higher average substitution rate in the mouse lineage than in the human lineage, corresponding closely to an early estimate by Wu and Li¹⁰⁹. In human, the least-diverged ancestral repeats have about 16% mismatch to their consensus sequences, which corresponds to approximately 0.17 substitutions per site. In contrast, mouse repeats have diverged by at least 26–27% or about 0.34 substitutions per site, which is about twofold higher than in the human lineage. The total number of substitutions in the two lineages can be estimated at 0.51. Below, we obtain an estimate of a combined rate of 0.46–0.47 substitutions per site, on the basis of an analysis that counts only substitutions since the divergence of the species (see Supplementary Information concerning the methods used).

Table 6: Divergence levels of interspersed repeats predating the human–mouse speciation

Full table

Assuming a speciation time of 75 Myr, the average substitution rates would have been 2.2 10^-9 and 4.5 10^-9 in the human and mouse lineages, respectively. This is in accord with previous estimates of neutral substitution rates in these organisms. (Reports of highly similar substitution rates in human and mouse lineages relied on a much earlier divergence time of rodents from other mammals¹⁰⁴.)

Comparison of ancestral repeats to their consensus sequence also allows an estimate of the rate of occurrence of small (<50 bp) insertions and deletions (indels). Both species show a net loss of nucleotides (with deleted bases outnumbering inserted bases by at least 2–3-fold), but the overall loss owing to small indels in ancestral repeats is at least twofold higher in mouse than in human. This may contribute a small amount (1–2%) to the difference in genome size noted above.

It should be noted that the roughly twofold higher substitution rate in mouse represents an average rate since the time of divergence, including an initial period when the two lineages had comparable rates. Comparison with more recent relatives (mouse–rat and human–gibbon, each about 20–25 Myr) indicate that the current substitution rate per year in mouse is probably much higher, perhaps about fivefold higher (see Supplementary Information). Also, note that these estimates refer to substitution rate per year, rather than per generation. Because the human generation time is much longer than that of the mouse (by at least 20-fold), the substitution rate is greater in human than mouse when measured per generation.

Higher substitution rate obscures old repeats

We measured the impact of the higher substitution rate in mouse on the ability to detect ancestral repeats in the mouse genome. By computer simulation, the ability of the RepeatMasker¹⁰⁰ program to detect repeats was found to fall off rapidly for divergence levels above about 37%. If we simulate the events in the mouse lineage by adjusting the ancestral repeats in the human genome for the higher substitution levels that would have occurred in the mouse genome, the proportion of the genome that would still be recognizable as ancestral repeats falls to only 6%. This is in close agreement with the proportion actually observed for the mouse. Thus, the current analysis of repeated sequences allows us to see further back into human history (roughly 150–200 Myr) than into mouse history (roughly 100–120 Myr).

A higher rate of interspersed repeat insertion does not explain the larger size of the human genome. Below, we suggest that the explanation lies in a higher rate of large deletions in the mouse lineage.

Comparison of mouse and human repeats

All mammals have essentially the same four classes of transposable elements: (1) the autonomous long interspersed nucleotide element (LINE)-like elements; (2) the LINE-dependent, short RNA-derived short interspersed nucleotide elements (SINEs); (3) retrovirus-like elements with long terminal repeats (LTRs); and (4) DNA transposons. The first three classes procreate by reverse transcription of an RNA intermediate (retroposition), whereas DNA transposons move by a cut-and-paste mechanism of DNA sequence (see refs 1, 100 for further information about these classes).

A comparison of these repeat classes in the mouse and human genomes can be enlightening. On the one hand, differences between the two species reveal the dynamic nature of transposable elements; on the other hand, similarities in the location of lineage-specific elements point to common biological factors that govern insertion and retention of interspersed repeats.

Differences between mouse and human

The most notable difference is in the changing rate of transposition over time: the rate has remained fairly constant in mouse, but markedly increased to a peak at about 40 Myr in human, and then plummeted. This phenomenon was noted in our initial analysis of the human genome; the availability of the mouse genome sequence now confirms and sharpens the observation (Fig. 10). Beyond this overall tendency, there are specific differences in each of the four repeat classes.

Figure 10: Age distribution of interspersed repeats in the mouse and human genomes.

This is an update of Fig. 18 in the IHGC human genome paper¹. a, b, Distribution for mouse and human of copies of each repeat class in bins corresponding to 1% increments in substitution level calculated using Jukes–Cantor formula (K = -3/4 ln(1 - D_rest^*4/3)) (see Supplementary Information for definition). The first bin for mouse is artificially low because the WGS assembly used for mouse excludes a larger percentage of very recent repeats. c, d, Interspersed repeats grouped into bins of approximately equal time periods after adjusting for the different rates of substitution in the two genomes. On average, the substitution level has been twofold higher in the mouse than in the human lineage (Table 6), but the difference was initially less and has increased over time. The present rates may differ over fourfold. The activity of transposable elements in the mouse lineage has been quite uniform compared with the human lineage, where an overall decline was interrupted temporarily by a burst of Alu activity. The apparent absence of <2% diverged interspersed repeats in mouse is primarily due to the shotgun sequencing strategy; long, closely similar interspersed repeats very often were not assembled. This is supported by an up to tenfold higher concentration of young L1 and ERV elements at the edges of gaps. The gradually decreasing density of repeats beyond a 30% substitution level reflects in part the limits of the detection method.

High resolution image and legend (80K)

The first class that we discuss is LINEs. Copies of LINE1 (L1) form the single largest fraction of interspersed repeat sequence in both human and mouse. No other LINE seems to have been active in either lineage. The extant L1 elements in both species derive from a common ancestor (L1MA6 in Table 6) by means of a series of subfamilies defined primarily by the rapidly evolving 3' non-coding sequences¹¹⁰. The L1 5'-untranslated regions (UTRs) in both lineages have been even more variable, occasionally through acquisition of entirely new sequences¹¹¹. Indeed, the three active subfamilies in mouse, which are otherwise >97% identical, have unrelated or highly diverged 5' ends^{112, 113, 114}. L1 seems to have remained highly active in mouse, whereas it has declined in the human lineage. Goodier and co-workers¹¹³ estimated that the mouse genome contains at least 3,000 potentially active elements (full-length with two intact open reading frames (ORFs)). The current draft sequence of the mouse genome contains only 400 young, full-length elements; of these only 12 have two intact ORFs. This is probably a reflection of the WGS shotgun approach used to assemble the genome. Indeed, most of the young elements in the draft genome sequence are incomplete owing to internal sequence gaps, reflecting the difficulty that WGS assembly has with highly similar repeat sequences. This is a notable limitation of the draft sequence.

The second repeat class is SINEs. Whereas only a single SINE (Alu) was active in the human lineage, the mouse lineage has been exposed to four distinct SINEs (B1, B2, ID, B4). Each is thought to rely on L1 for retroposition, although none share sequence similarity, as is the rule for other LINE–SINE pairs^{115, 116}. The mouse B1 and human Alu SINEs are unique among known SINEs in being derived from 7SL RNA; they probably have a common origin¹¹⁷. The mouse B2 is typical among SINEs in having a transfer RNA-derived promoter region. Recent ID elements seem to be derived from a neuronally expressed RNA gene called BC1, which may itself have been recruited from an earlier SINE. This subfamily is minor in mouse, with 2–4,000 copies, but has expanded rapidly in rat where it has produced more than 130,000 copies since the mouse–rat speciation¹¹⁸. Both B2 and ID closely resemble Ala-tRNA, but seem to have independent origins. The B4 family resembles a fusion between B1 and ID^{119, 120}. We found that 25% of the 75,000 identified ID elements were located within 50 bp of a B1 element of similar orientation, suggesting that perhaps most older ID elements are mislabelled or truncated B4 SINEs.

More rodent-specific SINEs are present in the mouse genome than Alu SINEs in human (1.4 and 1.1 million, respectively), but they occupy a smaller portion of the genome (7.6% and 10.7%, respectively) because of their smaller sizes. The existence of four families in mouse provides independent opportunities to investigate the properties of SINEs (see below).

The third repeat class is LTR elements. All interspersed LTR-containing elements in mammals are derivatives of the vertebrate-specific retrovirus clade of retrotransposons. The earliest infectious retroviruses probably originated from endogenous retroviral-like (ERV) elements that acquired mechanisms for horizontal transmission¹²¹, whereas many current endogenous retroviral elements have probably arisen from infection by retroviruses.

Endogenous retroviruses fall into three classes (I–III), which show a markedly dissimilar evolutionary history in human and mouse (see Fig. 10). Notably, ERVs are nearly extinct in human whereas all three classes have active members in mouse.

Class III accounts for 80% of recognized LTR element copies predating the human–mouse speciation. This class includes the non-autonomous MaLRs: with 388,000 recognizable copies in mouse, it is the single most successful LTR element. It is still active in mouse (represented by MERVL and the MT and ORR1 MaLRs), but died out some 50 Myr in human¹²².

Copies of class II elements are tenfold denser in mouse than in human. Among the active class II elements in mouse are two abundant and active groups, the intracisternal-A particles (IAP) and the early-transposons (ETn). About 15% of all spontaneous mouse mutants have an allele associated with IAP or ETn insertion, demonstrating the functional consequences of class I element activity in mice. A third active class, the mouse mammary tumour virus, is present in only a few copies¹²³ (see Supplementary Information). In human, there is evidence for at most a few active elements (HERVK10 and HERBK113 (ref. 124)). No class II ERVs are known to predate the human–mouse speciation.

In contrast, class I element copies are fourfold more common in the human than the mouse genome (although it is possible that some have not yet been recognized in mouse). In mouse, this class includes active ERVs, such as the murine leukaemia virus, MuRRS, MuRVY and VL30 (several of which have caused insertional mutations in mouse)—no similar activity is known to exist in human. It is unclear why the class I ERVs have been more successful in the human lineage whereas the class II ERVs have flourished in the mouse lineage.

The fourth repeat class is the DNA transposons. Although most transposable elements have been more active in mouse than human, DNA transposons show the reverse pattern. Only four lineage-specific DNA transposon families could be identified in mouse (the mariner element MMAR1, and the hAT elements URR1, RMER30 and RChar1), compared with 14 in the primate lineage.

For evolutionary survival, DNA transposons are thought to depend on frequent horizontal transfer to new host genomes by means of vectors such as viruses and other intracellular parasites^{116, 125}. The mammalian immune system probably forms a large obstacle to the successful invasion of DNA transposons. Perhaps the rodent germ line has been harder to infiltrate by horizontal transfer than the primate genome. Alternatively, it is possible that highly diverged families active in early rodent evolution have not been detected yet. Notably, most copies in the human genome were deposited early in primate evolution.

An interesting case is the mariner element, which seems to have infiltrated independently both the rodent and human genomes. The mariner element is represented by elements (MMAR1 in mouse and HSMAR1 in human) that are 97% identical. The average substitution level outside CpG sites of HSMAR1 is 8% and of MMAR1 is 22%, both well below the divergence of elements predating the human–mouse speciation (Table 6).

Some of the above differences in the nature of interspersed repeats in human and mouse could reflect systematic factors in mouse and human biology, whereas others may represent random fluctuations. Deeper understanding of the biology of transposable elements and detailed knowledge of interspersed repeat populations in other mammals should clarify these issues.

Similar repeats accumulate in orthologous locations

One of the most notable features about repeat elements is the contrast in the genomic distribution of LINEs and SINEs. Whereas LINEs are strongly biased towards (A+T)-rich regions, SINEs are strongly biased towards (G+C)-rich regions. The contrast is all the more notable because both elements are inserted into the genome through the action of the same endonuclease^{126, 127}.

Such preferences were studied in detail in the initial analysis of the human genome¹, and essentially equivalent preferences are seen in the mouse genome (Fig. 11). With the availability of two mammalian genomes, however, it is possible to extend this analysis to explore whether (A+T) and (G+C) content are truly causative factors or merely reflections of an underlying biological process.

Figure 11: Density of interspersed repeat classes at different (G+C) content in the mouse (a) and human (b) genomes.

In both species, there is a strong increase in SINE density and a decrease in L1 density with increasing (G+C) content, with the latter particularly marked in the mouse. Another notable contrast is that in mouse, overall interspersed repeat density gradually decreases 2.5-fold with increasing (G+C) content, whereas in human the overall repeat density remains quite uniform. This reflects both the abundance of L1 elements in the mouse (G+C)-poor regions and the unusually high density of Alu in human (G+C)-rich regions.

High resolution image and legend (70K)

Towards that end, we studied the insertion of lineage-specific repeat elements in orthologous segments in the human and mouse genomes (Fig. 12). Each insertion represents a new, independent event occurring in one lineage, and thus any correlation between the two species reflects underlying proclivity to insert or retain repeats in particular regions. Visual inspection reveals a strong correlation in the sites of lineage-specific repeats of the various classes (Fig. 12). Lineage-specific repeats also correlate with other genomic features, as discussed in the section on genome evolution.

Figure 12: Conservation of (G+C) content and convergence of interspersed repeat distribution between the human and mouse genomes.

For each mouse chromosome, its (G+C) content is depicted as a greyscale (centre, right), with darker shades indicating (G+C)-richer regions. Rodent-specific repeats are shown as cumulative histograms (far right), with red, green and blue indicating SINEs, LINEs and other repeats, respectively. The (G+C) content of the orthologous human sequence is similarly shown (centre, left) as well as the primate-specific repeats (far left). Gaps in the human sequence appear opposite those regions of the mouse genome lacking assigned conserved syntenic segments. Note the correlation in (G+C) and repeat content between orthologous regions of the two genomes. Many abrupt shifts in (G+C) content and repeat density are clearly associated with syntenic breaks, which are therefore more likely to be breaks associated with the rodent lineage⁴⁵.

High resolution image and legend (148K)

The correlation of local lineage-specific SINE density is extremely strong (Fig. 13a). Moreover, local SINE density in one species is better predicted by SINE density in the other species than it is by local (G+C) content (Table 7). The local density of each distinct rodent-specific type of SINE is a strong predictor of Alu density at the orthologous locus in human, although the Alu equivalent B1 SINEs show the strongest correlation (r² = 0.784) (Table 7).

Figure 13: Correlation of order-specific SINEs and LINEs in human and mouse orthologous regions.

SINE and LINE densities were calculated for 4,126 orthologous pairs with a constant size of 500 kb in mouse. a, b, Strong linear correlation of Alu density in human, and both the Alu-like B1 SINEs (a) and the unrelated B2 SINEs (b) densities in mouse. These correlations are stronger than the correlation of SINE density with (G+C) level (c). d, The relationship of LINE1 density in human and mouse orthologous regions is not linear, reflecting the more extreme bias of LINE1 for (A+T)-rich DNA in mouse.

High resolution image and legend (64K)

Table 7: Predicted repeat density in human based on mouse

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We interpret these results to mean that SINE density is influenced by genomic features that are correlated with (G+C) content but that are distinct from (G+C) content per se. The fact that (G+C) content alone does not determine SINE density is consistent with the observation that some (G+C)-rich regions of the human genome are not Alu rich^{128, 129}.

Lineage-specific LINE density is also clearly correlated between mouse and human (Fig. 13b), although the relationship does not seem to be linear and it is not as strong (Spearman rank analysis, r² = 0.45). (G+C) content seems to contribute as an independent variable (increasing r² to 0.52), suggesting that (G+C) content itself directly affects LINE integration.

Genomic outliers

In addition to examining the general correlation in repeat density between mouse and human, we also considered some of the extreme examples. In the human genome, the four homeobox clusters (HOXA, HOXB, HOXC and HOXD) are by far the most repeat-poor regions of the human genome, with repeat content in the range of 1%. These same four regions are exceptions in the mouse genome as well. The strong selective constraints against insertion in these regions probably reflect dense, long-range regulatory information across this developmentally important gene cluster. Other repeat-poor loci in the human genome¹ (about 100-kb regions on human chromosomes 1p36, 8q21 and 18q22) have independently remained repeat-poor in mouse (3.6, 6.5 and 7%, respectively) over roughly 75 million years of evolution; we speculate that this similarly reflects dense regulatory information in the region.

Conversely, we searched the mouse genome for repeat-poor regions of at least 100 kb. Again, the outliers show a clear tendency to be repeat-poor in human (see Supplementary Information). A notable feature is that in half of the selected loci the repeat-poor region is confined almost exactly to the extent of a single gene. Figure 14 shows this for the Zfhx1b locus, and also shows coincidence of exclusion of interspersed repeats with high conservation between human and mouse.

Figure 14: The zinc-finger homeobox 1b (Zfhx1b) loci in human and mouse are both repeat poor.

The repeat content for mouse (blue) and human (red) in 50-kb windows is shown for a 1-Mb region surrounding the Zfhx1b gene (green). Dotted lines indicate genome average for repeat content in mouse (blue) and human (red). The repeat-poor regions (<10% repeat content in mouse and human) coincide with the location of the 150-kb-long gene and regions of high conservation between human and mouse.

High resolution image and legend (56K)

LINE elements prefer sex chromosomes

A conspicuous feature of the repeat distribution is that LINE elements in both human and mouse show a preference for accumulating on sex chromosomes (Figs 12 and 15). Mouse chromosome X contains almost twice the density of lineage-specific L1 copies as the mouse autosomes (28.5% compared with 14.6%). Human sex chromosomes show an even stronger bias (17.5% on X and 18.0% on Y compared with 7.5% for the autosomes). The enrichment is still highly significant even after accounting for the generally higher (A+T) content of the sex chromosomes (Fig. 15).

Figure 15: Comparison of L1 characteristics of autosomes and sex chromosomes as a function of (G+C) content in mouse (blue) and human (red).

Error bars depict standard deviation over all autosomes (circles). Diamonds, X chromosomes; squares, human Y chromosome. The mouse Y chromosome is not represented in the whole-genome assembly, and too little clone-based information is available to be included. a, The number of lineage-specific L1 copies per megabase declines 13- to 20-fold from lowest to highest (G+C) content. This relationship is stronger in mouse and on the sex chromosomes. Note that, for the same (G+C) content, L1 density is 1.5- to twofold higher on the sex chromosomes. b, The average length of lineage-specific L1 copies peaks at around the 39% (G+C) level, where it is three- (human) to fourfold (mouse) higher than in the (G+C)-richest regions. The average length in mouse is underestimated owing to the bias against full-length young elements in the shotgun assembly. On average, L1 copies are longer on human Y than on either X chromosome or the autosomes.

High resolution image and legend (71K)

The higher density of L1 on sex chromosomes had been noted in early hybridization experiments^{130, 131} and has led to the suggestion that L1 copies may help facilitate X inactivation^{132, 133}. For chromosome Y, the accumulation probably reflects a greater tolerance for insertion (owing to the paucity of genes) and the inability to purge deleterious mutations by recombination. Consistent with the latter explanation, chromosome Y also shows a threefold higher density of full-length L1 copies (which are rapidly eliminated elsewhere in the genome¹³⁴) and an overall excess of LTR element insertions. Chromosome X shows an excess of L1 copies, but not a marked excess of either full-length L1 or LTR copies. The explanation for this preferential accumulation of L1 elements on chromosome X in both the mouse and human lineages remains unclear.

Simple sequence repeats

Mammalian genomes are scattered with simple sequence repeats (SSRs), consisting of short perfect or near-perfect tandem repeats that presumably arise through slippage during DNA replication. SSRs have had a particularly important role as genetic markers in linkage studies in both mouse and human, because their lengths tend to be polymorphic in populations and can be readily assayed by PCR. It is possible that such SSRs, arising as they do through replication errors, would be largely equivalent between mouse and human; however, there are impressive differences between the two species¹³⁵.

Overall, mouse has 2.25–3.25-fold more short SSRs (1–5 bp unit) than human (Table 8); the precise ratio depends on the percentage identity required in defining a tandem repeat. The mouse seems to represent an exception among mammals on the basis of comparison with the small amount of genomic sequence available from dog (4 Mb) and pig (5 Mb), both of which show proportions closer to human¹³⁶ (E. Green, unpublished data; Table 8).

Table 8: Density of short SSRs in mouse compared with other mammals

Full table

The analysis can be refined, however, by excluding transposable elements that contain SSRs at their 3' ends. For example, 90% of A-rich SSRs in human are provided by or spawned from poly(A) tails of Alu and L1 elements, and 15% of (CA)_n-like SSRs in mouse are contained in B2 element tails. When these sources are eliminated, the contrast between mouse and human grows to roughly fourfold.

The reason for the greater density of SSRs in mouse is unknown. Table 9 shows that SSRs of >20 bp are not only more frequent, but are generally also longer in the mouse than in the human genome, suggesting that this difference is due to extension rather than to initiation. The equilibrium distribution of SSR length has been proposed¹³⁷ to be determined by slippage between exact copies of the repeat during meiotic recombination¹³⁸. The shorter lengths of SSRs in human may result from the higher rate of point substitutions per generation (see above), which disrupts the exactness of the repeats.

Table 9: Frequency of different SSRs in mouse and human

Full table

Apart from the absolute number of SSRs, there are also some marked differences in the frequency of certain SSR classes (Table 9)¹³⁶. The most extreme is the tetramer (ACAG)n, which is 20-fold more common in mouse than human (even after eliminating copies associated with B2 and B4 SINEs); the sequence does not occur in large clusters, but rather is distributed throughout the genome. In general, SSRs in which one strand is a polypurine tract and the other a polypyrimidine tract are much more common and extended in mouse than human. For the six such di-, tri- and tetramer SSRs (AG, AAG, AGG, AAAG, AAGG, AGGG), copies with at least 20 bp and 95% identity are 1.6-fold longer and tenfold more common in mouse than human.

Analysis of the distribution of SSRs across chromosomes also reveals an interesting feature common to both organisms (see Supplementary Information). In both human and mouse, there is a nearly twofold increase in density of SSRs near the distal ends of chromosome arms. Because mouse chromosomes are acrocentric, they show the effect only at one end. The increased density of SSRs in telomeric regions may reflect the tendency towards higher recombination rates in subtelomeric regions¹.