Initial sequencing and analysis of the human genome - Vitagenes

articlescopies of the rDNA tandem repeats in the draft genome sequence,owing to the deliberate bias in the initial phase of the sequencingeffort against sequencing BAC clones whose restriction fragment®ngerprints showed them to contain primarily tandemly repeatedsequence. Sequence similarity analysis with the BLASTN computerprogram does, however, detect hundreds of rDNA-derived sequencefragments dispersed throughout the complete genome, includingone `full-length' copy of an individual 5.8S rRNA gene not associatedwith a true tandem repeat unit (Table 20).The 5S rDNA genes also occur in tandem arrays, the largest ofwhich is on chromosome 1 between 1q41.11 and 1q42.13, close tothe telomere 265,266 . There are 200±300 true 5S genes in thesearrays 265,267 . The number of 5S-related sequences in the genome,including numerous dispersed pseudogenes, is classically cited as2,000 (refs 252, 254). The long tandem array on chromosome 1 isnot yet present in the draft genome sequence because there are noEcoRI or HindIII sites present, and thus it was not cloned in themost heavily utilized BAC libraries (Table 1). We expect to recover itduring the ®nishing stage. We do detect four individual copies of 5SrDNA by our search criteria ($ 95% identity and $ 95% fulllength). We also ®nd many more distantly related dispersedsequences (520 at P # 0.001), which we interpret as probablepseudogenes (Table 20).Small nucleolar RNA genes. Eukaryotic rRNA is extensively processedand modi®ed in the nucleolus. Much of this activity isdirected by numerous snoRNAs. These come in two families: C/Dbox snoRNAs (mostly involved in guiding site-speci®c 29-O-ribosemethylations of other RNAs) and H/ACA snoRNAs (mostlyinvolved in guiding site-speci®c pseudouridylations) 247,248 . Wecompiled a set of 97 known human snoRNA gene sequences; 84of these (87%) have at least one copy in the draft genome sequence(Table 20), almost all as single-copy genes.It is thought that all 29-O-ribose methylations and pseudouridylationsin eukaryotic rRNA are guided by snoRNAs. There are105±107 methylations and around 95 pseudouridylations in humanrRNA 268 . Only about half of these have been tentatively assigned toknown guide snoRNAs. There are also snoRNA-directedmodi®cations on other stable RNAs, such as U6 (ref. 269), andthe extent of this is just beginning to be explored. Sequencesimilarity has so far proven insuf®cient to recognize all snoRNAgenes. We therefore expect that there are many unrecognizedsnoRNA genes that are not detected by BLAST queries.Spliceosomal RNAs and other ncRNA genes. We also looked forcopies of other known ncRNA genes. We found at least one copy of21 (95%) of 22 known ncRNAs, including the spliceosomalsnRNAs. There were multiple copies for several ncRNAs, asexpected; for example, we ®nd 44 dispersed genes for U6 snRNA,and 16 for U1 snRNA (Table 20).For some of these RNA genes, homogeneous multigene familiesthat occur in tandem arrays are again under-represented owing tothe restriction enzymes used in constructing the BAC libraries and,in some instances, the decision to delay the sequencing of BACclones with low complexity ®ngerprints indicative of tandemlyrepeated DNA. The U2 RNA genes are located at the RNU2 locus,a tandem array of 10±20 copies of nearly identical 6.1-kb units at17q21±q22 (refs 270±272). Similarly, the U3 snoRNA genes(included in the aggregate count of C/D snoRNAs in Table 20) areclustered at the RNU3 locus at 17p11.2, not in a tandem array, but ina complex inverted repeat structure of about 5±10 copies perhaploid genome 273 . The U1 RNA genes are clustered with about30 copies at the RNU1 locus at 1p36.1, but this cluster is thought tobe loose and irregularly organized; no two U1 genes have beencloned on the same cosmid 271 . In the draft genome sequence, we seesix copies of U2 RNA that meet our criteria for true genes, three ofwhich appear to be in the expected position on chromosome 17. ForU3, so far we see one true copy at the correct place on chromosome17p11.2. For U1, we see 16 true genes, 6 of which are looselyclustered within 0.6 Mb at 1p36.1 and another 6 are elsewhere onchromosome 1. Again, these and other clusters will be a matter forthe ®nishing process.Table 20 Known non-coding RNA genes in the draft genome sequenceRNA gene* Number expected² Number found³ Number ofrelated genes§FunctiontRNA 1,310 497 324 Protein synthesisSSU (18S) rRNA 150±200 0 40 Protein synthesis5.8S rRNA 150±200 1 11 Protein synthesisLSU (28S) rRNA 150±200 0 181 Protein synthesis5S rRNA 200±300 4 520 Protein synthesisU1 ,30 16 134 Spliceosome componentU2 10±20 6 94 Spliceosome componentU4 ?? 4 87 Spliceosome componentU4atac ?? 1 20 Component of minor (U11/U12) spliceosomeU5 ?? 1 31 Spliceosome componentU6 ?? 44 1,135 Spliceosome componentU6atac ?? 4 32 Component of minor (U11/U12) spliceosomeU7 1 1 3 Histone mRNA 39 processingU11 1 0 6 Component of minor (U11/U12) spliceosomeU12 1 1 0 Component of minor (U11/U12) spliceosomeSRP (7SL) RNA 4 3 773 Component of signal recognition particle (protein secretion)RNAse P 1 1 2 tRNA 59 end processingRNAse MRP 1 1 6 rRNA processingTelomerase RNA 1 1 4 Template for addition of telomereshY1 1 1 353 Component of Ro RNP, function unknownhY3 1 25 414 Component of Ro RNP, function unknownhY4 1 3 115 Component of Ro RNP, function unknownhY5 (4.5S RNA) 1 1 9 Component of Ro RNP, function unknownVault RNAs 3 3 1 Component of 13-MDa vault RNP, function unknown7SK 1 1 330 UnknownH19 1 1 2 UnknownXist 1 1 0 Initiation of X chromosome inactivation (dosage compensation)Known C/D snoRNAs 81 69 558 Pre-rRNA processing or site-speci®c ribose methylation of rRNAKnown H/ACA snoRNAs 16 15 87 Pre-rRNA processing or site-speci®c pseudouridylation of rRNA...................................................................................................................................................................................................................................................................................................................................................................* Known ncRNA genes (or gene families, such as the C/D and H/ACA snoRNA families); reference sequences were extracted from GenBank and used to probe the draft genome sequence.² Number of genes that were expected in the human genome, based on previous literature (note that earlier experimental techniques probably tend to overestimate copy number, by counting closely relatedpseudogenes).³ The copy number of `true' full-length genes identi®ed in the draft genome sequence.§ The copy number of other signi®cantly related copies (pseudogenes, fragments, paralogues) found. Except for the 497 true tRNA genes, all sequence similarities were identi®ed by WashU BLASTN 2.0MP(W. Gish, unpublished; http://blast.wustl.edu), with parameters `-kap wordmask = seg B = 50000 W = 8' and the default +5/-4 DNA scoring matrix. True genes were operationally de®ned as BLAST hitswith $ 95% identity over $ 95% of the length of the query. Related sequences were operationally de®ned as all other BLAST hits with P-values # 0.001.NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd895