An antecedent of the MHC-linked genomic region in amphioxus

The MHC genes on human chromosome 6 are located within one of the best-characterised paralogy regions of the human genome. Numerous genes mapping around this location, 6p21, have paralogues at one, two or three other chromosomal locations on HSA 1, 9 and 19. The similarity between these four chromosomal regions suggests the linkages may have adaptive significance, and/or they may be echoes of segmental or genome duplication in human ancestry. Here, we show that six amphioxus cosmids, containing genes orthologous to those from the human MHC-linked paralogy regions, map to a single amphioxus chromosome. The composition of the MHC-linked genomic region, therefore, pre-dates vertebrate origins.     

Origin and evolution of vertebrate ABCA genes: a story from amphioxus

Previous studies showed that the vertebrate ABCA subfamily, one subgroup of the ATP-binding-cassette superfamily, has evolved rapidly in terms of gene duplication and loss. To further uncover the evolutionary history of the ABCA subfamily, we characterized ABCA members of two amphioxus species (Branchiostoma floridae and B. belcheri), the closest living invertebrate relative to vertebrates. Phylogenetic analysis indicated that these two species have the same set of ABCA genes (both containing six members). Five of these genes have clear orthologs in vertebrate, including one cephalochordate-specific duplication and one vertebrate-specific duplication. In addition, it is found that human orthologs of amphioxus ABCA1/4/7 and its neighboring genes mainly localize on chromosome 1, 9, 19 and 5. Considering that most of analyzed amphioxus genes have clear orthologs in zebrafish, we conclude these four human paralogous regions might derive from a common ancestral region by genome duplication occurred prior to teleost/tetrapod split. Therefore, the present results provide new evidence for 2R hypothesis.     

Identification of paralogous HERV-K LTRs on human chromosomes 3, 4, 7 and 11 in regions containing clusters of olfactory receptor genes

A locus harboring a human endogenous retroviral LTR (long terminal repeat) was mapped on the short arm of human chromosome 7 (7p22), and its evolutionary history was investigated. Sequences of two human genome fragments that were homologous to the LTR-flanking sequences were found in human genome databases: (1) an LTR-containing DNA fragment from region 3p13 of the human genome, which includes clusters of olfactory receptor genes and pseudogenes; and (2) a fragment of region 21q22.1 lacking LTR sequences. PCR analysis demonstrated that LTRs with highly homologous flanking sequences could be found in the genomes of human, chimp, gorilla, and orangutan, but were absent from the genomes of gibbon and New World monkeys. A PCR assay with a primer set corresponding to the sequence from human Chr 3 allowed us to detect LTR-containing paralogous sequences on human chromosomes 3, 4, 7, and 11. The divergence times for the LTR-flanking sequences on chromosomes 3 and 7, and the paralogous sequence on chromosome 21, were evaluated and used to reconstruct the order of duplication events and retroviral insertions. (1) An initial duplication event that occurred 14-17 Mya and before LTR insertion - produced two loci, one corresponding to that located on Chr 21, while the second was the ancestor of the loci on chromosomes 3 and 7. (2) Insertion of the LTR (most probably as a provirus) into this ancestral locus took place 13 Mya. (3) Duplication of the LTR-containing ancestral locus occurred 11 Mya, forming the paralogous modern loci on Chr 3 and 7.     

MetaHox gene clusters

Homeobox genes encode important developmental control proteins. The Drosophila fruit fly HOM complex genes are clustered in region 84-89 of chromosome 3. Probably due to large-scale genome duplication events, their human HOX orthologs belong to four paralogous regions. A series of 13 other homeobox genes are also clustered in region 88-94, on the same chromosome of Drosophila. We suggest that they also duplicated during vertebrate evolution and belong to paralogous regions in humans. These regions are on chromosome arms 4p, 5q, 10q, and 2p or 8p. We coined the term "paralogon" to designate paralogous regions in general. We propose to call these genes "meta Hox" genes. Like Hox genes, metaHox genes are present in one cluster in Drosophila and four clusters (metaHox A-D) in humans on the 4p/5q/10q paralogon.     

Complex history of a chromosomal paralogy region: insights from amphioxus aromatic amino acid hydroxylase genes and insulin-related genes

Aromatic amino acid hydroxylase (AAAH) genes and insulin-like genes form part of an extensive paralogy region shared by human chromosomes 11 and 12, thought to have arisen by tetraploidy in early vertebrate evolution. Cloning of a complementary DNA (cDNA) for an amphioxus (Branchiostoma floridae) hydroxylase gene (AmphiPAH) allowed us to investigate the ancestry of the human chromosome 11/12 paralogy region. Molecular phylogenetic evidence reveals that AmphiPAH is orthologous to vertebrate phenylalanine (PAH) genes; the implication is that all three vertebrate AAAH genes arose early in metazoan evolution, predating vertebrates. In contrast, our phylogenetic analysis of amphioxus and vertebrate insulin-related gene sequences is consistent with duplication of these genes during early chordate ancestry. The conclusion is that two tightly linked gene families on human chromosomes 11 and 12 were not duplicated coincidentally. We rationalize this paradox by invoking gene loss in the AAAH gene family and conclude that paralogous genes shared by paralogous chromosomes need not have identical evolutionary histories.     

Conservation of pericentromeric duplications of a 200-kb part of the human 21q22.1 region in primates

We analyzed the conservation of large paralogous regions (more than 200 kb) on human chromosome regions 21q22.1 and 21q11.2 and on pericentromeric regions of chromosomes 2, 13, and 18 in three nonhuman primate species. Orthologous regions were found by FISH analysis of metaphase chromosomes from Gorilla gorilla, Pan troglodytes, and Pongo pygmaeus. Only one orthologous region was detected in chromosomes of P. pygmaeus, showing that the original locus was at 21q22.1 and that the duplication arose after the separation of Asian orangutans from the other hominoids. Surprisingly, the paralogous regions were more highly conserved in gorilla than in chimpanzee. PCR amplification of STSs derived from sequences of the chromosome 21 loci and low-stringency FISH analysis showed that this duplication occurred recently in the evolution of the genome. Different rates of sequence evolution through substitutions or deletions, after the duplication, may have resulted in diversity between closely related primates.     

Phylogenetic tests of the hypothesis of block duplication of homologous genes on human chromosomes 6, 9, and 1

There are 10 gene families that have members on both human chromosome 6 (6p21.3, the location of the human major histocompatibility complex [MHC]) and human chromosome 9 (mostly 9q33-34). Six of these families also have members on mouse chromosome 17 (the mouse MHC chromosome) and mouse chromosome 2. In addition, four of these families have members on human chromosome 1 (1q21-25 and 1p13), and two of these have members on mouse chromosome 1. One hypothesis to explain these patterns is that members of the 10 gene families of human chromosomes 6 and 9 were duplicated simultaneously as a result of polyploidization or duplication of a chromosome segment ("block duplication"). A subsequent block duplication has been proposed to account for the presence of representatives of four of these families on human chromosome 1. Phylogenetic analyses of the 9 gene families for which data were available decisively rejected the hypothesis of block duplication as an overall explanation of these patterns. Three to five of the genes on human chromosomes 6 and 9 probably duplicated simultaneously early in vertebrate history, prior to the divergence of jawed and jawless vertebrates, and shortly after that, all four of the genes on chromosomes 1 and 9 probably duplicated as a block. However, the other genes duplicated at different times scattered over at least 1.6 billion years. Since the occurrence of these clusters of related genes cannot be explained by block duplication, one alternative explanation is that they cluster together because of shared functional characteristics relating to expression patterns.      

The evolutionary origin of human subtelomeric homologies--or where the ends begin

The subtelomeric regions of human chromosomes are comprised of sequence homologies shared between distinct subsets of chromosomes. In the course of developing a set of unique human telomere clones, we identified many clones containing such shared homologies, characterized by the presence of cross-hybridization signals on one or more telomeres in a fluorescence in situ hybridization (FISH) assay. We studied the evolutionary origin of seven subtelomeric clones by performing comparative FISH analysis on a primate panel that included great apes and Old World monkeys. All clones tested showed a single hybridization site in Old World monkeys that corresponded to one of the orthologous human sites, thus indicating the ancestral origin. The timing of the duplication events varied among the subtelomeric regions, from approximately 5 to approximately 25 million years ago. To examine the origin of and mechanism for one of these subtelomeric duplications, we compared the sequence derived from human 2q13--an ancestral fusion site of two great ape telomeric regions--with its paralogous subtelomeric sequences at 9p and 22q. These paralogous regions share large continuous homologies and contain three genes: RABL2B, forkhead box D4, and COBW-like. Our results provide further evidence for subtelomeric-mediated genomic duplication and demonstrate that these segmental duplications are most likely the result of ancestral unbalanced translocations that have been fixed in the genome during recent primate evolution.     

Assembly and analysis of cosmid contigs in the CEA-gene family region of human chromosome 19.

The carcinoembryonic antigen (CEA)-like genes are members of a large gene family which is part of the immunoglobulin superfamily. The CEA family is divided into two major subgroups, the CEA-subgroup and the pregnancy-specific glycoprotein (PSG)-subgroup. In the course of an effort to develop a set of overlapping cosmids spanning human chromosome 19, we identified 245 cosmids in a human chromosome 19 cosmid library (6-7X redundant) by hybridization with an IgC-like domain fragment of the CEA gene. A fluorescence-based restriction enzyme digest fingerprinting strategy was used to assemble 212 probe-positive cosmids, along with 115 additional cosmids from a collection of approximately 8,000 randomly selected cosmids, into five contigs. Two of the contigs contain CEA-subgroup genes while the remaining three contigs contain PSG-subgroup genes. These five contigs range in size from 100 kb to over 300 kb and span an estimated 1 Mb. The CEA-like gene family was determined by fluorescence in situ hybridization to map in the q13.1-q13.2 region of human chromosome 19. Analysis of the two CEA-subgroup contigs provided verification of the contig assembly strategy and insight into the organization of 9 CEA-subgroup genes.     

Two Sequence-Ready Contigs Spanning the Two Copies of a 200-kb Duplication on Human 21q: Partial Sequence and Polymorphisms

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications.     

Genome dynamics of the major histocompatibility complex: insights from genome paralogy

 It has recently become apparent that the human genome contains at least three regions that are paralogous to the major histocompatibility complex (MHC). The number of gene families with copies in the MHC and these paralogous regions is increasing steadily as genome analysis progresses. This review presents the updated listing of the human gene families that constitute the MHC paralogous group. When genes with multiple copies within the MHC, such as class I and class II genes, are counted as single entities, nearly one-third of the genes residing in the HLA complex have paralogous copies in at least one of the three paralogous regions. The review also discusses the long-term genome dynamics of the MHC, taking into account the rapidly accumulating information on the genomic organizations of the MHCs in various model organisms.     

Evolution of the Mhc class I region: the framework hypothesis

 A comparison of the major histocompatibility complex (Mhc) region between human and mouse highlights both stability and differences. The class II and class III regions are orthologous; they probably existed in the ancestor in a similar organization and were not subjected to major rearrangement. The class I genes, by contrast, are definitely paralogous, having been reorganized several times. As long as only class I genes were identified, the class I regions of human and mouse were difficult to compare directly. The identification of non-class I genes has allowed a comparative map to be drawn, which shows that the class I region is orthologous between human and mouse as well. The lack of orthology specifically applies to the class I sequences. However, the comparative map shows that the non-orthologous class I sequences occupy homologous locations with regard to the conserved genes. I propose a model to explain this paradox. The conserved genes may represent samples of a dense "framework" of genes whose alterations are deleterious. The homologous positions occupied by class I genes would thus represent the few permissive places allowing major perturbations. The evolution of the class I sequences, by duplication and deletion, independently in the two species, has taken place within the scope defined by the framework: insertion at the permissive places, and expansion by creation of class I-related DNA by duplication, thus pushing back the boundaries of the framework. Received: 23 March 1998 / Revised: August 14 1998     

The spinal muscular atrophy gene region at 5q13.1 has a paralogous chromosomal region at 6p21.3

Paralogous regions are duplicated segments of chromosomal DNA that have been acquired during the evolution of the genome. Subsequent divergent evolution of the genes within paralogous regions can lead to the formation of gene families. Here, we report the identification of a region on Chromosome (Chr) 6 at 6p21.3 that is paralogous with the Spinal Muscular Atrophy (SMA) gene region on Chr 5 at 5q13.1. Partial characterization of this region identified nine sequences all of which are highly homologous to DNA sequences of the SMA gene region at 5q13.1. These sequences include four β-glucuronidase sequences, two retrotransposon sequences, a novel cDNA, a Sequence Tagged Site (STS), and one that is homologous to exon 9 of the Neuronal Apoptosis Inhibitor Protein (NAIP) gene. The 6p21.3 paralogous SMA region may contain genes that are related to those in the SMA region at 5q13.1; however, a direct association of this region with SMA is unlikely given that no linkage of SMA with Chr 6 has been reported. Received: 12 May 1997 / Accepted: 13 November 1997     

Evolutionary relationships of class II major-histocompatibility-complex genes in mammals

The major histocompatibility complex (MHC) class II molecule consists of noncovalently associated alpha and beta chains. In mammals studied so far, the class II MHC can be divided into a number of regions, each containing one or more alpha-chain genes (A genes) and beta-chain genes (B genes), and it has been known for some time that orthologous relationships exist between genes in corresponding regions from different mammalian species. A phylogenetic analysis of DNA sequences of class II A and B genes confirmed these relationships; but no such orthologous relationship was observed between the B genes of mammals and those of birds. Thus, the class II regions have diverged since the separation of birds and mammals (approximately 300 Mya) but before the radiation of the placental mammalian orders (60-80 Mya). Comparison of the phylogenetic trees for A and B genes revealed an unexpected characteristic of DP-region genes: DPB genes are most closely related to DQB genes, whereas DPA chain genes are most closely related to DRA-chain genes. Thus, the DP region seems to have originated through a recombinational event which brought together a DQB gene and a DRA gene (perhaps approximately 120 Mya). The 5' untranslated region of all class II genes includes sequences which are believed to be important in regulating class II gene expression but which are not conserved in known pseudogenes. These sequences are conserved to an extraordinary degree in the human DQB1 gene and its mouse homologue A beta 1, suggesting that regulation of expression of this locus may play a key role in expression of the entire class II MHC.     

Comparative mapping of the human 9q34 region in Fugu rubripes

Twenty-seven genes have been cloned and mapped in Fugu which have orthologues within the human chromosome 9q34 region. The genes are arranged into five cosmid and BAC contigs which physically map to two different Fugu chromosomes. Considering the gene content of these contigs, it is more probable that a translocation event took place early in the Fugu lineage to split the ancestral 9q34 region onto two chromosomes rather than the alternative hypothesis of a large-scale duplication of the region into two chromosomes with subsequent rapid and dramatic gene loss. There are considerable differences in gene order between the two species, which would appear to be the result of a series of complex chromosome inversions; thus suggesting that there have been no positional constraints on this particular gene set.     

An insight into the phylogenetic history of <Emphasis Type="Italic">HOX</Emphasis> linked gene families in vertebrates

Background  

The human chromosomes 2q, 7, 12q and 17q show extensive intra-genomic homology, containing duplicate, triplicate and quadruplicate paralogous regions centered on the HOX gene clusters. The fact that two or more representatives of different gene families are linked with HOX clusters is taken as evidence that these paralogous gene sets might have arisen from a single chromosomal segment through block or whole chromosome duplication events. This would imply that the constituent genes including the HOX clusters reflect the architecture of a single ancestral block (before vertebrate origin) where all of these genes were linked in a single copy.     

Ancient large-scale genome duplications: phylogenetic and linkage analyses shed light on chordate genome evolution

Paralogous genes from several families were found in four human chromosome regions (4p16, 5q33-35, 8p12-21, and 10q24-26), suggesting that their common ancestral region underwent several rounds of large- scale duplication. Searches in the EMBL databases, followed by phylogenetic analyses, showed that cognates (orthologs) of human duplicated genes can be found in other vertebrates, including bony fishes. In contrast, within each family, only one gene showing the same high degree of similarity with all the duplicated mammalian genes was found in nonvertebrates (echinoderms, insects, nematodes). This indicates that large-scale duplications occurred after the echinoderms/chordates split and before the bony vertebrate radiation. It has been suggested that two rounds of gene duplication occurred in the vertebrate lineage after the separation of Amphioxus and craniate (vertebrates + Myxini) ancestors. Before these duplications, the genes that have led to the families of paralogous genes in vertebrates must have been physically linked in the craniate ancestor. Linkage of some of these genes can be found in the Drosophila melanogaster and Caenorhabditis elegans genomes, suggesting that they were linked in the triploblast Metazoa ancestor.      

Isolation of region-specific cosmids from chromosome 5 by hybridization with microdissection clones.

A method is described for the isolation of chromosome region specific cosmids. The 5q35 region of the long arm of human chromosome 5 was microdissected, digested with MboI, ligated to oligonucleotide adaptors, amplified by the polymerase chain reaction and cloned into a plasmid vector. Inserts which did not contain highly repetitive sequences were used to screen a chromosome 5 cosmid library by direct hybridization. There were 33 positive cosmid clones identified with 4 microclones. Individual cosmid clones were biotinylated and used as probes for fluorescence in situ hybridization to metaphase chromosomes. Of the 33 cosmids that were mapped, 29 localized to q35 and 4 to q34, demonstrating the specificity of the microdissection library and the cosmids.     

Conservation of the MHC-like region throughout evolution

Identification of conserved regions between the genomes of distant species is a crucial step in the reconstruction of the genomic organization of their last common ancestor. Here we confirm for the first time with robust evidence, the existence of a region of conserved synteny between the human genome and the Drosophila genome. This evolutionarily conserved synteny involves the human MHC and paralogous regions, and we identified 19 conserved genes between these two species in a Drosophila genomic region of less than 2 Mb. The statistical analysis of the distribution of these 19 genes between the Drosophila and human genomes shows that it cannot be explained by chance. Our study constitutes a first step towards the reconstruction of the genome of Urbilateria (the ancestor of all bilaterian) and allows for a better understanding of the evolutionary history of our genome as well as other metazoan genomes.