Multi-species comparative mapping in silico using the COMPASS strategy

MOTIVATION: The completion of human and mouse genome sequences provides a valuable resource for decoding other mammalian genomes. The comparative mapping by annotation and sequence similarity (COMPASS) strategy takes advantage of the resource and has been used in several genome-mapping projects. It uses existing comparative genome maps based on conserved regions to predict map locations of a sequence. An automated multiple-species COMPASS tool can facilitate in the genome sequencing effort and comparative genomics study of other mammalian species. RESULTS: The prerequisite of COMPASS is a comparative map table between the reference genome and the predicting genome. We have built and collected comparative maps among five species including human, cattle, pig, mouse and rat. Cattle-human and pig-human comparative maps were built based on the positions of orthologous markers and the conserved synteny groups between human and cattle and human and pig genomes, respectively. Mouse-human and rat-human comparative maps were based on the conserved sequence segments between the two genomes. With a match to human genome sequences, the approximate location of a query sequence can be predicted in cattle, pig, mouse and rat genomes based on the position of the match relatively to the orthologous markers or the conserved segments. AVAILABILITY: The COMPASS-tool and databases are available at http://titan.biotec.uiuc.edu/COMPASS/     

Comparative human-mouse-rat sequence analysis of the ICAM gene cluster on HSA 19p13.2 and a 185-kb porcine region from SSC 2q

The human intercellular adhesion molecule gene (ICAM) cluster is located in a GC-rich and gene-rich region on HSA 19p13.2. We determined the complete DNA sequence of a 185-kb porcine bacterial artificial chromosome (BAC) clone containing parts of the ICAM gene cluster. We used the porcine sequence for a detailed comparative analysis between human, pig, mouse and rat. The 185 kb of porcine sequence covered 220 kb of homologous sequence in the human genome, which adds to the growing evidence that the porcine genome is somewhat smaller than the human genome. The genomic sequences of the four species showed a high level of conserved synteny and no rearrangements in gene order were observed. During evolution, the ICAM3 gene was inactivated by mutation in the mouse and rat genome, whereas it is still present in the human and pig genome. The loss of Icam3 in rodent genomes might be relevant for rodent-specific properties of the T-cell-mediated immune response. All the other investigated genes are conserved across all four investigated sequences.     

Evolution of human immunoglobulin kappa J region genes

Immunoglobulin kappa chain variable region genes are assembled from two discontinuous DNA segments, a V and a J gene. The J region genes, in addition to encoding amino acid positions 96-108 of the kappa polypeptide chain, also provide sequences required for both DNA and RNA splicing reactions. For purposes of evolutionary comparison and to establish the complexity of the kappa J region locus in man, we have determined an approximately 3000 basepair nucleotide sequence in a cloned human DNA fragment that encodes the germline distinct J region segments. Significant blocks of homology have been tightly maintained between this region and an analogous segment of the mouse genome. In particular, the short sequences, GGTTTTTGT and CACTGTG, thought to be involved in V-J recombination, are the most highly conserved regions (97% homology). In addition, from heteroduplex data and computer analysis of the nucleotide sequences, it is clear that the mouse J3 sequence, a pseudogene, is not present in the human cluster. This can be explained by a duplication event in the mouse J region gene cluster that may have been the result of unequal crossing over between homologous chromosomes.     

Direct selection of DNA sequences conserved between species.

An essential requirement in the analysis of genomes is the identification of functionally important sequence elements, which are often evolutionarily conserved. We describe here the development of a novel procedure for the selective isolation of conserved sequences which is based on hybridization of PCR-amplifiable DNA fragments from the whole genome of one species to biotinylated DNA from a genomic region of another species. The interspecies DNA hybrids are immobilized and the PCR-amplifiable DNA fragments are eluted, amplified and after further hybridization-amplification rounds cloned. This method was used for the generation of sublibraries of conserved sequences from mouse and pig DNA from regions corresponding to cosmids from the human Xq28 region. Mouse and pig homologs of sequences containing exons of known human genes, as well as exons from novel genes have been identified.     

AuberGene--a sensitive genome alignment tool

MOTIVATION: The accumulation of genome sequences will only accelerate in the coming years. We aim to use this abundance of data to improve the quality of genomic alignments and devise a method which is capable of detecting regions evolving under weak or no evolutionary constraints. RESULTS: We describe a genome alignment program AuberGene, which explores the idea of transitivity of local alignments. Assessment of the program was done based on a 2 Mbp genomic region containing the CFTR gene of 13 species. In this region, we can identify 53% of human sequence sharing common ancestry with mouse, as compared with 44% found using the usual pairwise alignment. Between human and tetraodon 93 orthologous exons are found, as compared with 77 detected by the pairwise human-tetraodon comparison. AuberGene allows the user to (1) identify distant, previously undetected, conserved orthogonal regions such as ORFs or regulatory regions; (2) identify neutrally evolving regions in related species which are often overlooked by other alignment programs; (3) recognize false orthologous genomic regions. The increased sensitivity of the method is not obtained at the cost of reduced specificity. Our results suggest that, over the CFTR region, human shares 10% more sequence with mouse than previously thought ( approximately 50%, instead of 40% found with the pairwise alignment).     

Isolation, chromosomal localization, and nucleotide sequence of the human HOX 1.4 homeobox

We have isolated a 14-kb DNA sequence containing a single homeobox from a low-stringency screen of a human genomic phage library by using heterologous homeobox sequences as probes. Chromosomal mapping of this clone using in situ hybridization to metaphase chromosomes and a panel of mouse x human somatic cell hybrids localized it to human chromosome 7p13-p15 in the region of the HOX 1 locus. We have sequenced the homeobox and show it has 100% identity to the deduced amino acid sequence of the mouse Hox-1.4 homeobox. We detect no restriction fragment length polymorphisms with the 14-kb clone, which is devoid of any moderately repetitive DNA sequences. This implies an inability of this region to tolerate change in sequence, consistent with a function highly conserved throughout evolution. The regions in the human genome where homeobox-containing loci reside share patterns of organization and sequence and have other gene loci in common, implying evolutionary constraints over these regions and providing clues on how they may have evolved.     

Human and mouse LSP1 genes code for highly conserved phosphoproteins

With use of the mouse LSP1 cDNA we isolated a human homologue of the mouse LSP1 gene from a human CTL cDNA library. The predicted protein sequence of human LSP1 is compared with the predicted mouse LSP1 protein sequence and regions of homology are identified in order to predict structural features of the LSP1 protein that might be important for its function. Both the human and mouse LSP1 proteins consist of two domains, an N-terminal acidic domain and a C-terminal basic domain. The C-terminal domains of the mouse and human LSP1 proteins are highly conserved and include several conserved, putative serine/threonine phosphorylation sites. Immunoprecipitation of LSP1 protein from 32P-orthophosphate-loaded cells show that both the mouse and human LSP1 proteins are phosphoproteins. The sequences of the putative Ca2(+)-binding sites present in the N-terminal domain of the mouse LSP1 protein are not conserved in the human LSP1 protein; however, a different Ca2(+)-binding site may exist in the human protein, indicating a functional conservation rather than a strict sequence conservation of the two proteins. The expression of the human LSP1 gene follows the same pattern as the expression of the mouse LSP1 gene. Southern analysis of human genomic DNA shows multiple LSP1-related fragments of varying intensity in contrast to the simple pattern found after similar analysis of mouse genomic DNA. By using different parts of the human LSP1 cDNA as a probe, we show that most of these multiple bands contain sequences homologous to the conserved C-terminal region of the LSP1 cDNA. This suggests that there are several LSP1-related genes present in the human genome.     

Identification and functional validation of a 5' upstream regulatory sequence in the human tyrosinase gene homologous to the locus control region of the mouse tyrosinase gene

Comparison analysis of the sequences of the mouse and human genomes has proven a powerful approach in identifying functional regulatory elements within the non-coding regions that are conserved through evolution between homologous mammalian loci. Here, we applied computational analysis to identify regions of homology in the 5' upstream sequences of the human tyrosinase gene, similar to the locus control region (LCR) of the mouse tyrosinase gene, located at -15 kb. We detected several stretches of homology within the first 30 kb 5' tyrosinase gene upstream sequences of both species that include the proximal promoter sequences, the genomic region surrounding the mouse LCR, and further upstream segments. We cloned and sequenced a 5' upstream regulatory sequence found between -8 and -10 kb of the human tyrosinase locus (termed h5'URS) homologous to the mouse LCR sequences, and confirmed the presence of putative binding sites at -9 kb, homologous to those described in the mouse tyrosinase LCR core. Finally, we functionally validated the presence of a tissue-specific enhancer in the h5'URS by transient transfection analysis in human and mouse cells, as compared with homologous DNA sequences from the mouse tyrosinase locus. Future experiments in cells and transgenic animals will help us to understand the in vivo relevance of this newly described h5'URS sequence as a potentially important regulatory element for the correct expression of the human tyrosinase gene.     

Homology between a 173-kb Region from Mouse Chromosome 10, Telomeric to the Ifng Locus, and Human Chromosome 12q15

We sequenced a 173-kb region of mouse chromosome 10, telomeric to the Ifng locus, and compared it with the human homologous sequence located on chromosome 12q15 using various sequence analysis programs. This region has a low density of genes: one gene was detected in the mouse and the human sequences and a second gene was detected only in the human sequence. The mouse gene and its human orthologue, which are expressed in the immune system at a low level, produce a noncoding mRNA. Nonexpressed sequences show a higher degree of conservation than exons in this genomic region. At least three of these conserved sequences are also conserved in a third mammalian species (sheep or cow).     

Conserved fragments of transposable elements in intergenic regions: evidence for widespread recruitment of MIR- and L2-derived sequences within the mouse and human genomes

We analysed the distribution of transposable elements (TEs) in 100 aligned pairs of orthologous intergenic regions from the mouse and human genomes. Within these regions, conserved segments of high similarity between the two species alternate with segments of low similarity. Identifiable TEs comprise 40-60% of segments of low similarity. Within such segments, a particular copy of a TE found in one species has no orthologue in the other. Overall, TEs comprise only approximately 20 % of conserved segments. However, TEs from two families, MIR and L2, are rather common within conserved segments. Statistical analysis of the distributions of TEs suggests that a majority of the MIR and L2 elements present in murine intergenic regions have human orthologues. These elements must have been present in the common ancestor of human and mouse and have remained under substantial negative selection that prevented their divergence beyond recognition. If so, recruitment of MIR- and L2-derived sequences to perform a function that increases host fitness is rather common, with at least two such events per host gene. The central part of the MIR consensus sequence is over-represented in conserved segments given its background frequency in the genome, suggesting that it is under the strongest selective constraint.     

Comparative sequence analysis of a single-gene conserved segment in mouse and human

Comparative mapping and sequencing of the mouse and human genomes have defined large, conserved chromosomal segments in which gene content and order are highly conserved. These regions span megabase-sized intervals and together comprise the vast majority of both genomes. However, the evolutionary relationships among the small remaining portions of these genomes are not as well characterized. Here we describe the sequencing and annotation of a 341-kb region of mouse Chr 2 containing nine genes, including biliverdin reductase A (Blvra), and its comparison with the orthologous regions of the human and rat genomes. These analyses reveal that the known conserved synteny between mouse Chromosome (Chr) 2 and human Chr 7 reflects an interval containing one gene (Blvra/BLVRA) that is, at most, just 34 kb in the mouse genome. In the mouse, this segment is flanked proximally by genes orthologous to human chromosome 15q21 and distally by genes orthologous to human Chr 2q11. The observed differences between the human and mouse genomes likely resulted from one or more rearrangements in the rodent lineage. In addition to the resulting changes in gene order and location, these rearrangements also appear to have included genomic deletions that led to the loss of at least one gene in the rodent lineage. Finally, we also have identified a recent mouse-specific segmental duplication. These finding illustrate that small genomic regions outside the large mouse–human conserved segments can contain a single gene as well as sequences that are apparently unique to one genome. The nucleotide sequence data reported in this paper have been submitted to GenBank and assigned the accession numbers AC074224 and AC074041.     

Identification and Functional Validation of a 5′ Upstream Regulatory Sequence in the Human Tyrosinase Gene Homologous to the Locus Control Region of the Mouse Tyrosinase Gene

Comparison analysis of the sequences of the mouse and human genomes has proven a powerful approach in identifying functional regulatory elements within the non‐coding regions that are conserved through evolution between homologous mammalian loci. Here, we applied computational analysis to identify regions of homology in the 5′ upstream sequences of the human tyrosinase gene, similar to the locus control region (LCR) of the mouse tyrosinase gene, located at ?15 kb. We detected several stretches of homology within the first 30 kb 5′ tyrosinase gene upstream sequences of both species that include the proximal promoter sequences, the genomic region surrounding the mouse LCR, and further upstream segments. We cloned and sequenced a 5′ upstream regulatory sequence found between ?8 and ?10 kb of the human tyrosinase locus (termed h5′URS) homologous to the mouse LCR sequences, and confirmed the presence of putative binding sites at ?9 kb, homologous to those described in the mouse tyrosinase LCR core. Finally, we functionally validated the presence of a tissue‐specific enhancer in the h5′URS by transient transfection analysis in human and mouse cells, as compared with homologous DNA sequences from the mouse tyrosinase locus. Future experiments in cells and transgenic animals will help us to understand the in vivo relevance of this newly described h5′URS sequence as a potentially important regulatory element for the correct expression of the human tyrosinase gene.     

Annotating regulatory DNA based on man-mouse genomic comparison

Non-coding DNA segments that are conserved between the human and mouse genomic sequence are good indicators of possible regulatory sequences. Here we report on a systematic approach to delineate such conserved elements from upstream regions of orthologous gene pairs from man and mouse. We focus on orthologous genes in order to maximize our chances to find functionally similar regulatory elements. The identification of conserved elements is effected using the Waterman-Eggert local suboptimal alignment algorithm. We have modified an implementation of this algorithm such that it integrates the determination of statistical significance for the local suboptimal alignments. This has the effect of outputting a dynamically determined number of suboptimal alignments that are deemed statistically significant. Comparison with experimentally determined annotation shows a striking enrichement of regulatory sites among the conserved regions. Furthermore, the conserved regions tend to cover the promotor region described in the EPD database.