We have conducted a comprehensive search for conserved elements in vertebrate genomes, using genome-wide multiple alignments of five vertebrate species (human, mouse, rat, chicken, and Fugu rubripes). Parallel searches have been performed with multiple alignments of four insect species (three species of Drosophila and Anopheles gambiae), two species of Caenorhabditis, and seven species of Saccharomyces. Conserved elements were identified with a computer program called phastCons, which is based on a two-state phylogenetic hidden Markov model (phylo-HMM). PhastCons works by fitting a phylo-HMM to the data by maximum likelihood, subject to constraints designed to calibrate the model across species groups, and then predicting conserved elements based on this model. The predicted elements cover roughly 3%-8% of the human genome (depending on the details of the calibration procedure) and substantially higher fractions of the more compact Drosophila melanogaster (37%-53%), Caenorhabditis elegans (18%-37%), and Saccharaomyces cerevisiae (47%-68%) genomes. From yeasts to vertebrates, in order of increasing genome size and general biological complexity, increasing fractions of conserved bases are found to lie outside of the exons of known protein-coding genes. In all groups, the most highly conserved elements (HCEs), by log-odds score, are hundreds or thousands of bases long. These elements share certain properties with ultraconserved elements, but they tend to be longer and less perfectly conserved, and they overlap genes of somewhat different functional categories. In vertebrates, HCEs are associated with the 3Ј UTRs of regulatory genes, stable gene deserts, and megabase-sized regions rich in moderately conserved noncoding sequences. Noncoding HCEs also show strong statistical evidence of an enrichment for RNA secondary structure.

CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing. Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis. In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array. Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase. Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation. These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.

this diversity is located in discrete gene clusters that are spread throughout the different genomes. In contrast to this diversity, these enteric microorganisms exhibit marked synteny in their largescale genomic organization, bearing in mind that E. coli and S. enterica diverged about 100 Myr ago 28. The conserved genes may be a re¯ection of the basic lifestyle of the bacteria, requiring intestine colonization, environmental survival and transmission. The unique gene clusters probably contribute to adaptation to environmental niches and to pathogenicity. The pseudogene complement of S. typhi has implications for our understanding of the tight host restriction of this organism, and raises the question of whether it may be possible to eradicate S. typhi and typhoid fever altogether. M Methods Salmonella typhi CT18 was isolated in December 1993, at the Mekong Delta region of Vietnam, from a 9-year-old girl who was suffering from typhoid. The strain was isolated from blood using routine culture methods 23 , and after serological and metabolic con-®rmation of the strain as S. typhi it was immediately frozen in glycerol at-70 8C. The genome sequence was obtained from 97,000 end sequences (giving 7.9´coverage) derived from several pUC18 genomic shotgun libraries (with insert sizes ranging from 1.4 to 4.0 kb) using dye terminator chemistry on ABI377 automated sequencers. This was supplemented with 0.7´sequence coverage from M13mp18 libraries with similar insert sizes. End sequences from a larger insert plasmid (pSP64; 1.9´clone coverage, 10±14-kb insert size) and lambda (lambda-FIX-II; 0.4´clone coverage, 20±22-kb insert size) libraries were used as a scaffold, and the ®nal assembly was veri®ed by comparison with restriction-enzyme digest patterns using pulsed-®eld gel electrophoresis (data not shown). Total sequence coverage was 9.1´. The sequence was assembled, ®nished and annotated as described 29 , using Artemis 30 to collate data and facilitate annotation. In addition we used a gene®nder that was trained speci®cally for S. typhi, which uses a hidden Markov model with modules for the coding region, start and stop codons, and the ribosome-binding site (T.S.L. and A.K., unpublished data). The genome and proteome sequences of S. typhi and S. typhimurium or E. coli were compared in parallel to identify deletions and insertions using the Artemis Comparison Tool (ACT) (K. Rutherford, unpublished data; see also http://www.sanger.ac.uk/Software/ ACT/). Pseudogenes had one or more mutations that would ablate expression, and were identi®ed by direct comparison with S. typhimurium; each of the inactivating mutations was subsequently checked against the original sequencing data.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.