Introduction

The introduction of fluorescence in situ hybridization (FISH) to comparative karyotype analysis (1) marked a paradigm shift from the analysis of chromosome morphology toward chromosomal DNA content. The use of human chromosome-specific probes in cross-species chromosome painting experiments for the first time allowed the secure identification of chromosomal homologies between species at a resolution of 3-5 Mb within primates and 5-10 Mb when comparing human with nonprimate mammals. A second landmark was the establishment of a reproducible Zoo-FISH protocol (2) for the analysis of any mammalian species with human probes. In addition, chromosome-specific "painting" probes were established from several nonhuman primates as well as from other mammalian and vertebrate species by fluorescence-activated chromosome sorting and subsequent degenerate oligonucleotide-primed polymerase chain reaction amplification (3), for example, from lemurs (4), mouse (5), and chicken (6). The availability of chromosome paint probes from nonhuman species for Zoo-FISH experiments allowed the assembly of comparative chromosome maps in a reciprocal way (7) (Fig. 1). This approach is particularly helpful when analyzing distantly related species in which the hybridization efficiency is low. Moreover, when performing reverse painting to human chromosomes, evolutionary breakpoints can be localized on the human sequence map for a subsequent detailed characterization of evolutionary breakpoints. Further development of this strategy led to the concept of multidirectional chromosome painting (8), where members of a species group of interest are systematically analyzed with human paint probes and in addition with paint probes of a species from the targeted species group.

Cross-species chromosome painting certainly has a number of limitations: apart from the limited resolution, intrachromosomal rearrangements usually escape detection. For the construction of more detailed comparative genome maps, several complementary methods have been established, among others Zoo-FISH employing chromosome bar codes and subregional DNA probes, comparative radiation hybrid mapping and, ultimately, whole genome sequencing.

In order to obtain high-resolution comparative chromosome maps, Zoo-FISH with vector-cloned DNA probes can be performed. Bacterial artificial chromosome (BAC) libraries from numerous species are publicly available (http://bacpac.chori.org), among them human, chicken, mouse, rat, cat, pig, and cow, and several nonhuman primates (chimpanzee, orangutan, gibbon, macaque, squirrel monkey, lemur, and others). Because human, mouse, and rat genome projects are essentially complete, "tile path" BAC probes from these species provide an excellent source for comparative FISH mapping studies with a direct link to the genome sequence (www.ensembl.org). For FISH analysis of distantly related species, human BAC probes can be selected from genomic regions with high evolutionary sequence conservation between human and mouse (9).

Alternatively, somatic cell hybrid panels may serve a template for physical mapping studies in order to assemble interspecies homology maps. High-resolution radiation hybrid panels have been established, for example, for the rhesus macaque (10). For chimpanzee, gorilla, and orangutan, lower resolution panels are available (11).

The rapid progress of the human, mouse, and rat genome projects already provides a detailed and comprehensive insight into the ancestral mammalian genome organization. Although the major mammalian evolutionary breakpoints are hidden among the noise that stems from the extreme genome reshuffling in rodents, it is increasingly possible to read the human/mouse sequence alignment like cross-species chromosome painting data. With other more recently

Fig. 1. (A) The principle of reciprocal chromosome painting. (B,C) Reciprocal chromosome painting between human and orangutan: (B) in situ hybridization of human chromosome 2 probe to an orangutan metaphase delineates two homologs. (C) Reverse painting of the orangutan 2q homolog to human chromosomes hybridizes to the 2q13-qter segment.

Fig. 1. (A) The principle of reciprocal chromosome painting. (B,C) Reciprocal chromosome painting between human and orangutan: (B) in situ hybridization of human chromosome 2 probe to an orangutan metaphase delineates two homologs. (C) Reverse painting of the orangutan 2q homolog to human chromosomes hybridizes to the 2q13-qter segment.

launched genome projects (i.e., chicken, dog, and chimpanzee [12]) advancing even faster, the view becomes increasingly clear. For example, the fusion point of human chromosome 2 in 2q13 can readily be identified on the human/chimpanzee comparative sequence map (www.ensembl.org) at the junction of chimpanzee chromosome 12 and 13 homologous sequences.

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