Orthologs of seven genes (AKT1, CDC42BPB, DIO3, EIF5, JAG2, KLC, NDUFB1) from human chromosome 14q32 map to distal chicken chromosome 5.

Although birds and mammals diverged approximately 300 million years ago, evolutionarily conserved chromosomal segments of similar gene contents can be discerned in the human and chicken genomes (Burt et al. 1995, 1999; Girard-Santosuosso 1997). The resolution of the human–chicken comparative map is limited by the number of genes that have been mapped in the chicken genome and is defined by approximately 350 loci (Groenen et al. 2000; Schmid et al. 2000). In comparison, there are nearly 6000 loci that define the comparative map of human and mouse (Mouse Genome Informatics, The Jackson Laboratory). It is, therefore, possible that chromosomal regions where gene content and order appear to be conserved between chicken and mammals may contain hitherto undetected rearrangements and adventitious segments from elsewhere in the genome. A more detailed human–chicken comparative map will enable accurate selection of candidates for mapped chicken phenotypes of clinical or veterinary interest. It is also of interest in the context of the chicken as an intermediate model organism since it has genome size approximately one-third that of mammals (Tiersch and Wachtel 1991). We demonstrate here how two recent developments can be exploited to increase the resolution of the comparative map in targeted regions of the chicken genome. These are the public availability of human genome sequence (International Human Genome Sequencing Consortium 2001) and the increasing amount of chicken EST (expressed sequence tag) sequence data being deposited in public databases (Tirunagaru et al. 2000; Abdrakhmanov et al. 2000). As a pilot study, we have analyzed human genome sequence and chicken EST databases to find additional chicken genes likely to map to an approximately 4-Mb region on distal chicken Chr 5. This region is of interest because it has a high probability of containing a gene (SAL1) implicated in resistance to visceral infection by Salmonella typhimurium in young chicks (Mariani et al. 2001). The region is defined as the genetic interval of 8.4 cM that lies between the chicken microsatellite loci MCW29 and ADL166 and was known to contain three genes, DNCH1, CKB, and HSPCAL4, that have been mapped to 14q32 (Mariani et al. 2001). Together with TGFRB (transforming growth factor, beta 3) and CALM1 (calmodulin 1), these three genes define a conserved segment that appears to extend from TGFRB to CKB on chicken Chr 5, from q24–q32 on human Chr 14, and to cover the distal 20 cM of mouse Chr 12. Initially, human cDNA sequence data for DNCHC1 (Accession no. AB002323) and for CKB (Accession no. X15334) were chosen as entry points to identify BAC clones and contigs in the finished and unfinished human sequence data deposited in GENBANK (accessed through the NCBI website, http:// www.nlm.nih.gov). Publicly available sequence data from these and neighboring but non-contiguous BAC contigs (representing a total of 4.3 Mb of human genomic sequence) were analyzed and annotated by using the NIX software available through the MRC HGMP Resource Centre website (http://www.hgmp.mrc.ac.uk). Most of the genes identified were confirmed as lying in this region from human radiation hybrid maps (Deloukas et al. 1998), the human genome sequence annotated by ENSEMBL (http:// www.ensembl.org) and the mouse genetic map for Chr 12 (D’Eustachio and Riblet 1998). In total, 22 known genes, in addition to DNCHC1 and CKB, were identified from six nonoverlapping sequenced BAC contigs or, in the case of AKT1, from mouse genomic information. In addition, over 50 ESTs and several unidentified cDNAs were noted, but these were not pursued further. The complete cDNA sequence data from each of the known human genes was used to search for cloned chicken homologs by a stepwise approach. Initially, all sequences were screened by using the BLASTn and BLASTx algorithms to search for matches in the Gallus gallus sequences deposited in the EMBL database. If no positives were found, searches were conducted against appropriate subsections of the EMBL database with the FASTA algorithm (accessed through HGMP Resource Centre). Finally, chicken EST databases held at Hamburg (http://genetics.uni. hamburg.de/dt40Est), the University of Delaware (http://www. udelest.edu), and the Roslin Institute (http://www.roslin. bbsrc.ac.uk) were searched for matches to the human gene sequences. A decision to follow up putative matches was based on the extent, degree, and specificity of homology between the chicken and human genes. A reciprocal screen was performed with the DNA sequence from the potential chicken homolog to confirm that it most closely resembled the gene encoded by the human locus in 14q32 and not another, similar gene. Ten genes were found to have potential chicken homologs, but only seven were mapped to distal Chr 5 (see Table 1 and below). The simplest explanations for the fact that three chicken genes (TNFAIP2, MARK3, PPP2RC5) could not be mapped to Chr 5 is that the putative chicken homolog was not in fact a true ortholog and encoded either a different member of the same gene family or a closely related gene. To determine the map position of each chicken gene, we analyzed a limited panel of chicken × Chinese hamster radiation hybrids that had been constructed previously in the laboratory (Haynes and Bumstead, unpublished data). Briefly, this panel consists of 39 hybrid cell clones derived from fusions between chicken embryo fibroblasts after irradiation at 3000 rads (RH 1-11), 5000 rads (RH 12-24) or 10,000 rads (RH 25-39), to the HPRT-deficient Chinese hamster cell line Wg3h by using standard protocols (Kwok et al. 1998). The entire panel has been analyzed for the presence/absence of approximately 100 chicken loci representing at least 24 chicken linkage groups, with all hybrids positive for at least one chicken locus. The hybrid cells have a low (∼16.7%) Correspondence to: Y. Boyd; email: yvonne.boyd@bbsrc.ac.uk Mammalian Genome 13, 120–122 (2002). DOI: 10.1007/s00335-001-3066-y