enes on the basis of a lack of introns and, most importantly, on the presence of multiple in-frame stop codons. Given the inability of transcripts from these genes to produce a complete protein, it is unlikely that any pseudogenes were incorrectly classified. Secondly, for EST matches, only those that showed evidence of splicing were used - that is, those that were non-contiguous with genomic sequence, showed consensus splice sites, and represented essentially perfect matches (>95% identity) to the genomic sequence. This eliminates many of the artifacts common to cDNA libraries. A survey of the EST database  for fifty of the known chromosome 21 genes found that forty-three were present as spliced ESTs, six were present only as unspliced ESTs (five of these were intronless genes), and one was not present in dbEST.
Finally, the criterion of consistent exon prediction required that two of the three coding-exon prediction programs (Grail, Genscan and MZEF) agreed on the location of an exon, and that a minimum of three consistent exons were found within < 60 kb, with introns <30 kb. It is noteworthy that the coding regions of intronless genes were well predicted but only as single exons. Such exons tend to be very large - greater than a kilobase (kb) in length - in contrast to typical coding exons that average 100-150 base pairs (bp). After making exceptions for, and including, large single-coding-exon genes, by these criteria, all but one of the 107 known genes could be identified by exon prediction. This included very large genes, such as DSCAM, which spans >800 kb, and GRIK1, which spans >400 kb, both of which were well predicted through at least some of their coding regions.
The important conclusion here is that each of the 107 genes previously known to map to chromosome 21 would have been identified, in the absence of protein similarities, by the criteria of EST matches plus exon prediction. These criteria do not, in most cases, define a complete gene structure, but they do successfully indicate the presence of a gene. Thus, unless novel genes have very different characteristics, it is reasonable to expect a similarly high level of success in their identification. Using these criteria a further 118 genes were identified.
What is likely to have been missed? First, there are gaps in the sequence of 21q. They are few (three) and small (<50 kb each), however, and therefore cannot harbor large numbers of genes. Second, genes that would not be identified would have to possess the following features: no similarity to any known protein; consistently very large introns (>30-60 kb), so that patterns of predicted exons would not be scored; and long intronless 3' untranslated regions (UTRs) or restricted and/or low expression levels, so that no spliced EST is present in dbEST. It is certainly possible that some number of genes with such characteristics exist; that they represent a significant proportion of chromosome 21 genes is unlikely, however. The distal one third of 21q is the most gene-rich (and GC-rich); but intergenic distances here are not large enough to accommodate additional genes with uniformly large introns. So, unless coding exons in these genes are for some reason not recognized, such genes would be scored on the basis of patterns of predicted exons. The proximal two thirds, in contrast, is uniformly AT-rich and does have large segments lacking gene features; indeed, there is one segment of approximately 7 Mb that harbors only seven genes. Here there is room for numerous genes that have large introns and restricted expression. One argument against this is a biological one: an individual who is monosomic for this region has only mild phenotypic abnormalities . A second argument is a general scarcity of any consistent exon prediction in the region, regardless of 'intron' size. If there are many coding exons within this region, they must also be largely unrecognized by prediction programs. Together, these data suggest that the total of 225 genes is likely to be reliable: false negatives should be few.
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