Comparative mapping of human Chromosome 19 with the chicken shows conserved synteny and gives an insight into chromosomal evolution Jacqueline Smith, 1 Ian R. Paton, 1 Frazer Murray, 1 Richard P.M.A. Crooijmans, 2 Martien A.M. Groenen, 2 David W. Burt 1 1 Division of Genomics and Bioinformatics, Roslin Institute, Roslin (Edinburgh), Midlothian EH25 9PS, UK 2 Department of Animal Breeding, Wageningen Institute of Animal Sciences, Wageningen Agricultural University, 6709 PG Wageningen, The Netherlands Received: 24 September 2001 / Accepted: 11 February 2002 Abstract. Human Chromosome 19 (HSA19) is virtually com- pletely sequenced. A complete physical contig map made up of BACs and cosmids is also available for this chromosome. It is, therefore, a rich source of information that we have used as the basis for a comparative mapping study with the chicken. Various orthologs of genes known to map to HSA19 have been mapped in the chicken. Five chicken microchromosomes (two of which were previously undefined) are seen to show conserved synteny with this chromosome, along with individual gene homologs on Chr 1 and another tiny microchromosome. Compared with the mouse, which has 12 chromosomal regions homologous to HSA19, the chicken genotype displays fewer evolutionary rearrangements. The ancestral nature of the chicken karyotype is demonstrated and may prove to be an excellent tool for studying genome evolution. Comparative mapping studies between human and chicken have previously been carried out, and a high level of conserved synteny has been demonstrated (Burt et al. 2000; Suchyta et al. 2001). We have carried out a further, slightly more detailed comparative map- ping project based around one human chromosome and the ho- mologous mouse regions and have looked at the degree of con- servation among all three species. As a result of the Human Ge- nome Project, the complete draft sequence of human Chr 19 is available (http://genome.ucsc.edu), and at the time of writing, 79.6% of that is finished sequence (http://www.ebi.ac.uk/genomes/ mot/). A series of BACs and cosmids have been ordered into a physical contig map of HSA19 (Ashworth et al. 1995), and many genes were thus identified and placed on this map (http://www. bio.llnl.gov/bbrp/genome/html/chrom_map.html; http:// www.ensembl.org/perl/mapview?chr19). The exact order of genes on HSA19 is also, therefore, known. As a result of sequenc- ing the human genome, it would appear that Chr 19 is one of the most gene-dense of the human chromosomes (Wright et al. 2001). We have used the wealth of knowledge available for this chromo- some and have carried out a comparative mapping study between HSA19 and the homologous chicken chromosomes. The chicken karyotype consists of eight large pairs of chro- mosomes (macrochromosomes) and 29 pairs of very small chro- mosomes (microchromosomes) along with the Z and W sex chro- mosomes (Takagi and Sasaki 1974; Tegelstro ¨m and Ryttman 1981; Rodionov 1996). Four genes known to have homologs on HSA19 have previously been mapped in the chicken. AMH, which is found on the human cytogenetic band 19q13.3, was known to map to linkage group E53 (Chr 28). CCNE at human 19q12 was also known to map to linkage group E30 (Chr 11). Similarly, the genes RYR1 (HSA 19q13.1) and TGF1 (HSA 19q13.2) were already identified as being located on chicken linkage group E25 (Smith et al. 2000). In this study we show that five small microchromosomes line up with HSA19. One of the microchromosomes breaks up the conservation between HSA19 and one of the other microchromo- somes, demonstrating six main blocks of conserved synteny (com- pared with 12 between mouse and HSA19) although gene order is not, on the whole, conserved within these homologous regions. Individual gene homologs are also identified on Chr 1 and a tiny microchromosome, previously undefined by any marker (UN). By a combination of screening chicken BAC and cosmid libraries, sequencing, genetic mapping, and fluorescence in situ hybridiza- tion (FISH), HSA19 orthologs have been mapped to the respective chicken chromosomes. Chr 28 (linkage group E53), Chr 11 (E30), linkage group E25, and two newly defined microchromosomes (E64 and E65) all contain genes orthologous to HSA19. Regions where evolutionary breakpoints have occurred have also been identified. Materials and methods PCR screening of BACs. Primers designed around a particular marker were used to screen pools of BAC clones, and individual BACs were identified after two-dimensional PCR screening as described in Crooijmans et al. (2000). Southern blotting. EcoRI-digested cosmid DNA was blotted to a nylon membrane overnight, and the DNA was then fixed by using a UV crosslinker (Stratagene, La Jolla, CA). Hybridization and washing were carried out as described below. Library screening. Probes were labeled with 32 P-dCTP (Prime-a-gene kit, Promega, Southampton, UK) and hybridized to either a chicken grid- ded cosmid library (Buitkamp et al. 1998) or a gridded chicken BAC library (Crooijmans et al. 2000). Hybridization was carried out overnight at 65°C in 10% PEG8000, 7% SDS, 1.5× SSC. Filters were then washed twice in 2× SSC, 0.1% SDS and once in 0.5× SSC, 0.1% SDS and exposed to autoradiographic film for 4 hr at room temperature. Positive clones were ordered from RZPD [Berlin (cosmids)] and HGMP [Hinxton (BACs)]. Fluorescence in situ hybridization. Chicken metaphase chromosome spreads were prepared from chicken embryo fibroblasts after treatment with colcemid solution. Cells were swollen by treatment with hypotonic solution, fixed in methanol:acetic acid fixative (3:1), and dropped onto methanol-cleaned slides and allowed to air dry. Cosmid probes were la- Correspondence to: J. Smith; E-mail: Jacqueline.Smith@bbsrc.ac.uk Mammalian Genome 13, 310–315 (2002). DOI: 10.1007/s00335-001-3071-1 © Springer-Verlag New York Inc. 2002 Incorporating Mouse Genome