frequently several million base pairs and as, at a rough estimate, the average density of genes transcribed in a given tissue is only four per one million base pairs, the isolation of a disease gene would be facilitated by focussing efforts on expressed sequences. Specifically, identifying sequences from the appropriate chromosomal region expressed in the affected tissue could be the initial step in a strategy to isolate a disease gene.
The problem now is to find the needle in the haystack, to identify within a genomic region those sequences encoding transcripts. Three approaches are commonly used to screen for coding sequences in cloned genomic material. First, genomic clones can be used directly as hybridization probes on blots of mRNA or of cDNA libraries. Genomic fragments cloned into lambda vectors, cosmid vectors, and even YAC vectors(@ have been used successfully as hybridization probes. Second, as protein coding sequences are generally more conserved during evolution than noncoding sequences, genomic clones can be used as hybridization probes on blots of genomic DNAs of other mammalian species (Zoo blots)('). Conserved sequences, which are probably protein coding sequences, hybridize at a higher stringency with genomic DNAs of other mammalian species than do less conserved, putative non-coding sequences. Third, genomic clones can be assayed for the presence of HTF islands, rare regions having high content of unmethylated CpG residues, which have a high frequency of associated genes@). Genomic clones are routinely tested for HTF islands by digestion with restriction enzymes whose recognition sequences include unmethylated CpG residues. The association is stronger for genes expressed ubiquitously than for genes whose expression is restricted to specific tissues. Each of these strategies is feasible for screening small numbers of genomic clones, but in order to screen larger chromosomal regions, whole chromosomes, or ultimately the entire genome for expressed sequences, additional approaches need to be developed. Recently, several approaches aimed at systematically identifying and recovering transcribed sequences from specific chromosomal regions have been reported; these will be reviewed here.