Comparative sequence analyses reveal rapid and divergent evolutionary changes of the WFDC locus in the primate lineage

The initial comparison of the human and chimpanzee genome sequences revealed 16 genomic regions with an unusually high density of rapidly evolving genes. One such region is the whey acidic protein (WAP) four-disulfide core domain locus (or WFDC locus), which contains 14 WFDC genes organized in two subloci on human chromosome 20q13. WAP protease inhibitors have roles in innate immunity and/or the regulation of a group of endogenous proteolytic enzymes called kallikreins. In human, the centromeric WFDC sublocus also contains the rapidly evolving seminal genes, semenogelin 1 and 2 (SEMG1 and SEMG2). The rate of SEMG2 evolution in primates has been proposed to correlate with female promiscuity and semen coagulation, perhaps related to post-copulatory sperm competition. We mapped and sequenced the centromeric WFDC sublocus in 12 primate species that collectively represent four different mating systems. Our analyses reveal a 130-kb region with a notably complex evolutionary history that has included nested duplications, deletions, and significant interspecies divergence of both coding and noncoding sequences; together, this has led to striking differences of this region among primates and between primates and rodents. Further, this region contains six closely linked genes (WFDC12, PI3, SEMG1, SEMG2, SLPI, and MATN4) that show strong patterns of adaptive selection, although an unambiguous correlation between gene mutation rates and mating systems could not be established.     

Identification of a hypervariable region containing new Legionella pneumophila Icm/Dot translocated substrates by using the conserved icmQ regulatory signature

Legionella pneumophila is an intracellular pathogen that has been shown to utilize the Icm/Dot type IV secretion system for pathogenesis. This system was shown to be composed of Icm/Dot complex components, accessory proteins, and a large number of translocated substrates. In this study, comparison of the icmQ regulatory regions from many Legionella species revealed a conserved regulatory sequence that includes the icmQ −10 promoter element. Mutagenesis of this conserved regulatory element indicated that each of the nucleotides in it affects the level of expression of the icmQ gene but not in a uniform fashion. A genomic analysis discovered that four additional genes in L. pneumophila contain this conserved regulatory sequence, which was found to function similarly in these genes as well. Examination of these four genes indicated that they are dispensable for intracellular growth, but two of them were found to encode new Icm/Dot translocated substrates (IDTS). Comparison of the genomic regions encoding these two IDTS among the four available L. pneumophila genomic sequences indicated that one of these genes is located in a hypervariable genomic region, which was shown before to contain an IDTS-encoding gene. Translocation analysis that was performed for nine proteins encoded from this hypervariable genomic region indicated that six of them are new IDTS which are translocated into host cells in an Icm/Dot-dependent manner. Furthermore, a bioinformatic analysis indicated that additional L. pneumophila genomic regions that contain several neighboring IDTS-encoding genes are hypervariable in gene content.     

Chromosomal localization and complete genomic sequence of the murine autoimmune regulator gene (Aire)

We have recently cloned the murine autoimmune regulator (Aire) gene, the homologue of human AIRE responsible for the autoimmune polyglandular syndrome type 1 (APS1) or autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). Here, we report the genomic sequence (18,413 bp) for the entire Aire gene and its 5' flanking region, which contains putative regulatory sequences. Comparison of the genomic and cDNA sequences indicates that the Aire gene is composed of 14 exons and the coding sequence shares high similarities between mouse and human. The sizes of the homologous introns in the two species are conserved; however, the introns do not share significant sequence homologies except the sequences near the splice donor and acceptor sites. Sequence analyses of the 5' regulatory region and the complete coding region in three mouse strains (B6, NOD and SJL) did not reveal any sequence variation, suggesting sequence conservation between different inbred mouse strains. Using one of the six microsatellite markers identified by genomic sequencing and a B6 x Cast backcross mapping panel, we mapped the mouse Aire gene to chromosome 10, a syntenic region containing the Cdl18 and Pfkl genes on human chromosome 21q22.     

Comparative genome sequence analysis of the Bpa/Str region in mouse and Man

The progress of human and mouse genome sequencing programs presages the possibility of systematic cross-species comparison of the two genomes as a powerful tool for gene and regulatory element identification. As the opportunities to perform comparative sequence analysis emerge, it is important to develop parameters for such analyses and to examine the outcomes of cross-species comparison. Our analysis used gene prediction and a database search of 430 kb of genomic sequence covering the Bpa/Str region of the mouse X chromosome, and 745 kb of genomic sequence from the homologous human X chromosome region. We identified 11 genes in mouse and 13 genes and two pseudogenes in human. In addition, we compared the mouse and human sequences using pairwise alignment and searches for evolutionary conserved regions (ECRs) exceeding a defined threshold of sequence identity. This approach aided the identification of at least four further putative conserved genes in the region. Comparative sequencing revealed that this region is a mosaic in evolutionary terms, with considerably more rearrangement between the two species than realized previously from comparative mapping studies. Surprisingly, this region showed an extremely high LINE and low SINE content, low G+C content, and yet a relatively high gene density, in contrast to the low gene density usually associated with such regions.     

Reduced purifying selection prevails over positive selection in human copy number variant evolution

Copy number variation is a dominant contributor to genomic variation and may frequently underlie an individual’s variable susceptibilities to disease. Here we question our previous proposition that copy number variants (CNVs) are often retained in the human population because of their adaptive benefit. We show that genic biases of CNVs are best explained, not by positive selection, but by reduced efficiency of selection in eliminating deleterious changes from the human population. Of four CNV data sets examined, three exhibit significant increases in protein evolutionary rates. These increases appear to be attributable to the frequent coincidence of CNVs with segmental duplications (SDs) that recombine infrequently. Furthermore, human orthologs of mouse genes, which, when disrupted, result in pre- or postnatal lethality, are unusually depleted in CNVs. Together, these findings support a model of reduced purifying selection (Hill–Robertson interference) within copy number variable regions that are enriched in nonessential genes, allowing both the fixation of slightly deleterious substitutions and increased drift of CNV alleles. Additionally, all four CNV sets exhibited increased rates of interspecies chromosomal rearrangement and nucleotide substitution and an increased gene density. We observe that sequences with high G+C contents are most prone to copy number variation. In particular, frequently duplicated human SD sequence, or CNVs that are large and/or observed frequently, tend to be elevated in G+C content. In contrast, SD sequences that appear fixed in the human population lie more frequently within low G+C sequence. These findings provide an overarching view of how CNVs arise and segregate in the human population.     

Selection against Robertsonian fusions involving housekeeping genes in the house mouse: integrating data from gene expression arrays and chromosome evolution

Monobrachial homology resulting from Robertsonian (Rb) fusions is thought to contribute to chromosomal speciation through underdominance. Given the karyotypic diversity characterizing wild house mouse populations [Mus musculus domesticus, (MMU)], variation that results almost exclusively from Rb fusions (diploid numbers range from 22 to 40) and possibly whole arm reciprocal translocations (WARTs), this organism represents an excellent model for testing hypotheses of chromosomal evolution. Previous studies of chromosome size and recombination rates have failed to explain the bias for certain chromosomes to be involved more frequently than others in these rearrangements. Here, we show that the pericentromeric region of one such chromosome, MMU19, which is infrequently encountered as a fusion partner in wild populations, is significantly enriched for housekeeping genes when compared to other chromosomes in the genome. These data suggest that there is selection against breakpoints in the pericentromeric region and provide new insights into factors that constrain chromosomal reorganizations in house mice. Given the anticipated increase in vertebrate whole genome sequences, the examination of gene content and expression profiles of the pericentromeric regions of other mammalian lineages characterized by Rb fusions (i.e., other rodents, bats, and bovids, among others) is both achievable and crucial to developing broadly applicable models of chromosome evolution.     

Comparative analyses among the <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>, <Emphasis Type="Italic">Trichomonas tenax</Emphasis>, and <Emphasis Type="Italic">Tritrichomonas foetus</Emphasis> 5S ribosomal RNA genes

The 5S ribosomal RNA (5S rRNA) is an essential component of ribosomes. Throughout evolution, variation is found among 5S rRNA genes regarding their chromosomal localization, copy number, and intergenic regions. In this report, we describe and compare the gene sequences, motifs, genomic copy number, and chromosomal localization of the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S rRNA genes. T. vaginalis and T. foetus have a single type of 5S rRNA-coding region, whereas two types were found in T. tenax. The sequence identities among the three organisms are between 94 and 97%. The intergenic regions are more divergent in sequence and size with characteristic species-specific motifs. The T. foetus 5S rRNA gene has larger and more complex intergenic regions, which contain either an ubiquitin gene or repeated sequences. The 5S rRNA genes were located in Trichomonads chromosomes by fluorescent in situ hybridization. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers FJ492747, FJ492748, FJ492749, FJ492750, and FJ492751.     

Activation-induced cytidine deaminase induces DNA break repair events more frequently in the Ig switch region than other sites in the mammalian genome

Activation-induced cytidine deaminase (AID) produces DNA breaks in immunoglobulin genes during antibody diversification. Double-stranded breaks (DSB) in the switch region mediate class switch recombination, and contribute to gene conversion and somatic hypermutation in the variable regions. However, the relative extent to which AID induces DSB in these regions or between these and other actively expressed sequences is unknown. Here, we exploited an enhancer-trap plasmid that identifies DSB in actively expressed loci to investigate the frequency and position of AID-induced vector integration events in mouse hybridoma cells. Compared to control cells, wild-type AID stimulates plasmid integration into the genome by as much as 29-fold. Southern and digestion-circularization PCR analysis revealed non-uniformity in the integration sites, with biases of 30- and 116-fold for the immunoglobulin kappa light chain and mu heavy chain genes, respectively. Further, within the immunoglobulin mu gene, 73% of vector integrations map to the mu switch region, an enhancement of five- and 12-fold compared to the adjacent heavy chain variable and mu gene constant regions, respectively. Thus, among potential highly transcribed genes in mouse hybridoma cells, the immunoglobulin heavy and light chain genes are important AID targets, with the immunoglobulin mu switch region being preferred compared to other genomic sites.     

Comparative analysis of human and mouse 3' Igh regulatory regions identifies distinctive structural features

Immunoglobulin heavy chain (Igh) locus rearrangements are controlled in part by an approximately 30 b complex 3' regulatory region located 3' of C alpha: this region contains several enhancers. We report here the comparison of the genomic sequences of the 3' regulatory region and further downstream sequences from mouse, rat, human and chimpanzee. Only short segments of homology were detected in the 3' regulatory region, and these were located in the vicinity of the known 3' enhancers. The nearest highly conserved segment is the nearest non-Igh gene, hole, which is located approximately 62 kb downstream of mouse C alpha. Analysis of murine 3' Igh sequences by single nucleotide polymorphism (SNP) and restriction fragment length polymorphism (RFLP) detected a transition region (high to low SNP or RFLP density) approximately 120 kb downstream of mouse C alpha. Although there is only limited sequence identity between rodent and primate 3' Igh regulatory regions, all of these regulatory regions contain a palindrome and locally repetitive elements. Locally repetitive elements in primates comprise blocks of "switch-like" sequences that differ from the families of inverted and tandem repeats that are present in rodents. We propose that together with enhancers, these "conserved" structural features are essential for the activity of the 3' Igh regulatory region in vivo.     

Comparative genomic analysis of the MHC: the evolution of class I duplication blocks, diversity and complexity from shark to man

Summary: The major histocompatibility complex (MHC) genomic region is composed of a group of linked genes involved functionally with the adaptive and innate immune systems. The class I and class II genes are intrinsic features of the MHC and have been found in all the jawed vertebrates studied so far. The MHC genomic regions of the human and the chicken (B locus) have been fully sequenced and mapped, and the mouse MHC sequence is almost finished. Information on the MHC genomic structures (size, complexity, genic and intergenic composition and organization, gene order and number) of other vertebrates is largely limited or nonexistent. Therefore, we are mapping, sequencing and analyzing the MHC genomic regions of different human haplotypes and at least eight nonhuman species. Here, we review our progress with these sequences and compare the human MHC structure with that of the nonhuman primates (chimpanzee and rhesus macaque), other mammals (pigs, mice and rats) and nonmammalian vertebrates such as birds (chicken and quail), bony fish (medaka, pufferfish and zebrafish) and cartilaginous fish (nurse shark). This comparison reveals a complex MHC structure for mammals and a relatively simpler design for nonmammalian animals with a hypothetical prototypic structure for the shark. In the mammalian MHC, there are two to five different class I duplication blocks embedded within a framework of conserved nonclass I and/or nonclass II genes. With a few exceptions, the class I framework genes are absent from the MHC of birds, bony fish and sharks. Comparative genomics of the MHC reveal a highly plastic region with major structural differences between the mammalian and nonmammalian vertebrates. Additional genomic data are needed on animals of the reptilia, crocodilia and marsupial classes to find the origins of the class I framework genes and examples of structures that may be intermediate between the simple and complex MHC organizations of birds and mammals, respectively.     

Identification of an enhancer region in the mouse ephA8 locus directing expression to the anterior region of the dorsal mesencephalon.

Eph receptors and ephrins are dynamically expressed in a wide range of regions of the vertebrate during embryogenesis. The dorsal mesencephalon appears to be segmented into two broad regions demarcated by the mutually exclusive expression of EphA receptors and ephrinA ligands. It is of considerable interest to elucidate how these expression domains are established in the development of the mesencephalon. In this study, we used a transgenic approach to define the cis-acting DNA regulatory elements involved in the anterior mesencephalon-specific expression of the mouse ephA8 gene. Our analyses of the temporal and spatial expression patterns of various ephA8/lacZ gene fusions in transgenic mice revealed that the 10-kb genomic DNA 5' immediately upstream of the ephA8 coding sequence is capable of directing lacZ expression in an ephA8-specific manner. Further deletion analyses of the ephA8 genomic region led to the identification of a 1-kb enhancer region, which directs expression in the embryo to the anterior region of the developing midbrain. This ephA8-specific regulatory DNA sequences can now be used in biochemical analyses to identify proteins modulating the anterior differentiation of the optic tectum, and in functional analyses to direct the expression of other developmentally important genes to this region.     

Haplotype variation in structure and expression of a gene cluster associated with a quantitative trait locus for improved yield in rice

By constructing nearly isogenic lines (NILs) that differ only at a single quantitative trait locus (QTL), we fine-mapped the yield-improving QTL qGY2-1 to a 102.9-kb region on rice chromosome 2. Comparison analysis of the genomic sequences in the mapped QTL region between the donor (Dongxiang wild rice, Oryza rufipogon Griff.) and recurrent (Guichao2, Oryza sativa ssp. indica) parents used for the development of NILs identified the haplotypes of a leucine-rich repeat receptor kinase gene cluster, which showed extensive allelic variation. The sequences between genes in the cluster had a very high rate of divergence. More importantly, the genes themselves also differed between two haplotypes: Only 92% identity was observed for one allele, and another allele was found to have completely lost its allelic counterpart in Guichao2. The other six shared genes all showed >98% identity, and four of these exhibited obvious regulatory variation. The same haplotype segments also differed in length (43.9-kb in Guichao2 vs. 52.6-kb in Dongxiang wild rice). Such extensive sequence variation was also observed between orthologous regions of indica (cv. 93-11) and japonica (cv. Nipponbare) subspecies of Oryza sativa. Different rates of sequence divergence within the cluster have resulted in haplotype variability in 13 rice accessions. We also detected allelic expression variation in this gene cluster, in which some genes gave unequal expression of alleles in hybrids. These allelic variations in structure and expression suggest that the leucine-rich repeat receptor kinase gene cluster identified in our study should be a particularly good candidate for the source of the yield QTL.