The Genom of Homo sapiens.pdf

266 ROE ET AL.noncoding regions. On the basis of these observations, wehave begun examining the gene expression profiles ofthese conserved coding regions by whole-mount in situhybridization in the zebrafish model system. Thus,through these two approaches, we will have taken the firststeps to determine the genomic features of the full repertoireof higher eukaryote genes, their expression profiles,and eventually the function of their gene products.COMPARATIVE GENOMIC SEQUENCINGMuch of our comparative genomic sequencing hasbeen concentrated on regions of the chimpanzee, baboon,cow, and mouse genomes orthologous to the upper half ofhuman Chromosome 22. The regions of sequence orthologousvertebrate clones and the chimp Chromosome 23whole-genome sequence (WGS) reads that have homologywith human Chromosome 22 and are aligned with theorthologous regions of human Chromosome 22 areshown below in Figure 1. At present, our combined assemblyof 208,000 WGS reads and our BAC-based sequencesresults in a 33,077,854-bp assembly of chimpanzeeChromosome 23 with ~1,305 gaps and over 98%identity to human Chromosome 22, while similar regionsof the baboon are ~92% identical.This observation is confirmed by the percentage identityplot (PIP) (Schwartz et al. 2000, 2003) shown in Figure2, where it is also shown that human Chromosome 22contains retroviral and alu sequences that are not presentin the orthologous regions of the baboon and chimpanzee.The completed sequences of these two primate chromosomeswill allow us to discover additional differencesthat will further illuminate human-specific and chimpspecificgenetic features that likely will include additionalinsertions, deletions, and even possible gene duplications.These results are in agreement with those reported inthe excellent comparative sequencing study from EricGreen’s laboratory (Thomas et al. 2003) and a recent publicationfrom the laboratory of Sakaki and Okada(Ohshima et al. 2003). However, at present, only relativelysmall regions from selected species have beencompared. Thus, additional comparative sequencingfrom numerous species is needed to obtain the full pictureof these observations on the broader genomic scale.WHOLE-MOUNT IN SITU HYBRIDIZATIONSTUDIES IN THE ZEBRAFISH SYSTEMBecause several hundred genes predicted on humanChromosome 22 have an unknown function but are expressedin one or more tissues and developmental stages,or have neither a known function nor known expressionprofile, it was critical to develop rapid, high-throughputmethods to investigate the expression profiles of the zebrafishorthologs of these classes of human genes. To facilitatethe automation of these analyses, we recentlyhave developed optimized conditions for 96-well microtiter-basedwhole-mount in situ hybridization in zebrafishusing the digoxigenin-labeled (DIG-labeled),small (~100 base), ssDNA unidirectional PCR probes(Kitazawa et al. 1999; Knuchel et al. 2000) producedfrom exon-specific PCR products similar to those usedfor high-throughput in situ hybridization in Drosophila(Tautz and Pfeifle 1989; Patel and Goodman 1992) andCaenorhabditis elegans (Seydoux and Fire 1995). In theexperiment shown in Figure 3, a cDNA clone correspondingto dJ508I15.C22.5, a human Chromosome-22-5000 10000 15000 20000 25000 30000 35000 40000 45000 50000Chimp whole genome shotgun coverageChimp BAC clones matchesBaboon BAC clones matchesCow BAC clonesMouse BAC clonesFigure 1. Schematic representation of the chimpanzee WGS and our sequenced chimpanzee, baboon, cow, and mouse BACs alignedwith the orthologous regions in human Chromosome 22.