SCP1, a 356,023 bp linear plasmid adapted to the ecology and developmental biology of its host, Streptomyces coelicolor A3(2).

The sequencing of the entire genetic complement of Streptomyces coelicolor A3(2) has been completed with the determination of the 365,023 bp sequence of the linear plasmid SCP1. Remarkably, the functional distribution of SCP1 genes somewhat resembles that of the chromosome: predicted gene products/functions include ECF sigma factors, antibiotic biosynthesis, a gamma-butyrolactone signalling system, members of the actinomycete-specific Wbl class of regulatory proteins and 14 secreted proteins. Some of these genes are among the 18 that contain a TTA codon, making them targets for the developmentally important tRNA encoded by the bldA gene. RNA analysis and gene fusions showed that one of the TTA-containing genes is part of a large bldA-dependent operon, the gene products of which include three proteins isolated from the spore surface by detergent washing (SapC, D and E), and several probable metabolic enzymes. SCP1 shows much evidence of recombinational interactions with other replicons and transposable elements during its history. For example, it has two sets of partitioning genes (which may explain why an integrated copy of SCP1 partially suppressed the defective partitioning of a parAB-deleted chromosome during sporulation). SCP1 carries a cluster of probable transfer determinants and genes encoding likely DNA polymerase III subunits, but it lacks an obvious candidate gene for the terminal protein associated with its ends. This may be related to atypical features of its end sequences.

The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129.

Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium's nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.

Conservation of long-range synteny and microsynteny between the genomes of two distantly related nematodes.

BACKGROUND: Comparisons between the genomes of the closely related nematodes Caenorhabditis elegans and Caenorhabditis briggsae reveal high rates of rearrangement, with a bias towards within-chromosome events. To assess whether this pattern is true of nematodes in general, we have used genome sequence to compare two nematode species that last shared a common ancestor approximately 300 million years ago: the model C. elegans and the filarial parasite Brugia malayi. RESULTS: An 83 kb region flanking the gene for Bm-mif-1 (macrophage migration inhibitory factor, a B. malayi homolog of a human cytokine) was sequenced. When compared to the complete genome of C. elegans, evidence for conservation of long-range synteny and microsynteny was found. Potential C. elegans orthologs for II of the 12 protein-coding genes predicted in the B. malayi sequence were identified. Ten of these orthologs were located on chromosome I, with eight clustered in a 2.3 Mb region. While several, relatively local, intrachromosomal rearrangements have occurred, the order, composition, and configuration of two gene clusters, each containing three genes, was conserved. Comparison of B. malayi BAC-end genome survey sequence to C. elegans also revealed a bias towards intrachromosome rearrangements. CONCLUSIONS: We suggest that intrachromosomal rearrangement is a major force driving chromosomal organization in nematodes, but is constrained by the interdigitation of functional elements of neighboring genes.

Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2).

Streptomyces coelicolor is a representative of the group of soil-dwelling, filamentous bacteria responsible for producing most natural antibiotics used in human and veterinary medicine. Here we report the 8,667,507 base pair linear chromosome of this organism, containing the largest number of genes so far discovered in a bacterium. The 7,825 predicted genes include more than 20 clusters coding for known or predicted secondary metabolites. The genome contains an unprecedented proportion of regulatory genes, predominantly those likely to be involved in responses to external stimuli and stresses, and many duplicated gene sets that may represent 'tissue-specific' isoforms operating in different phases of colonial development, a unique situation for a bacterium. An ancient synteny was revealed between the central 'core' of the chromosome and the whole chromosome of pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. The genome sequence will greatly increase our understanding of microbial life in the soil as well as aiding the generation of new drug candidates by genetic engineering.

Secondary DNA structure analysis of the coding strand switch regions of five Leishmania major Friedlin chromosomes.

As part of the EULEISH international genome project, a region of 74,674 nucleotides from chromosome 21 of Leishmania major Friedlin was subcloned and sequenced; and 31 new coding sequences were predicted. Of particular interest was a unique coding strand switching region covering 1.6 kb of DNA; and this was subjected to further investigation. Bioinformatic analysis of this region revealed an unusually high AT composition, a lack of putative hairpins and a strong curvature of the DNA in agreement with the structural characteristics of similar regions of other Leishmania chromosomes. These observations and a comparison with the secondary DNA structure of four other Leishmania chromosomes and chromosomes of different organisms could suggest a functional role of this region in transcription and mitotic division.

Whole genome comparison of Campylobacter jejuni human isolates using a low-cost microarray reveals extensive genetic diversity.

Campylobacter jejuni is the leading cause of bacterial food-borne diarrhoeal disease throughout the world, and yet is still a poorly understood pathogen. Whole genome microarray comparisons of 11 C. jejuni strains of diverse origin identified genes in up to 30 NCTC 11168 loci ranging from 0.7 to 18.7 kb that are either absent or highly divergent in these isolates. Many of these regions are associated with the biosynthesis of surface structures including flagella, lipo-oligosaccharide, and the newly identified capsule. Other strain-variable genes of known function include those responsible for iron acquisition, DNA restriction/modification, and sialylation. In fact, at least 21% of genes in the sequenced strain appear dispensable as they are absent or highly divergent in one or more of the isolates tested, thus defining 1300 C. jejuni core genes. Such core genes contribute mainly to metabolic, biosynthetic, cellular, and regulatory processes, but many virulence determinants are also conserved. Comparison of the capsule biosynthesis locus revealed conservation of all the genes in this region in strains with the same Penner serotype as strain NCTC 11168. By contrast, between 5 and 17 NCTC 11168 genes in this region are either absent or highly divergent in strains of a different serotype from the sequenced strain, providing further evidence that the capsule accounts for Penner serotype specificity. These studies reveal extensive genetic diversity among C. jejuni strains and pave the way toward identifying correlates of pathogenicity and developing improved epidemiological tools for this problematic pathogen.

Genome sequence of Yersinia pestis, the causative agent of plague.

The Gram-negative bacterium Yersinia pestis is the causative agent of the systemic invasive infectious disease classically referred to as plague, and has been responsible for three human pandemics: the Justinian plague (sixth to eighth centuries), the Black Death (fourteenth to nineteenth centuries) and modern plague (nineteenth century to the present day). The recent identification of strains resistant to multiple drugs and the potential use of Y. pestis as an agent of biological warfare mean that plague still poses a threat to human health. Here we report the complete genome sequence of Y. pestis strain CO92, consisting of a 4.65-megabase (Mb) chromosome and three plasmids of 96.2 kilobases (kb), 70.3 kb and 9.6 kb. The genome is unusually rich in insertion sequences and displays anomalies in GC base-composition bias, indicating frequent intragenomic recombination. Many genes seem to have been acquired from other bacteria and viruses (including adhesins, secretion systems and insecticidal toxins). The genome contains around 150 pseudogenes, many of which are remnants of a redundant enteropathogenic lifestyle. The evidence of ongoing genome fluidity, expansion and decay suggests Y. pestis is a pathogen that has undergone large-scale genetic flux and provides a unique insight into the ways in which new and highly virulent pathogens evolve.

Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18.

Salmonella enterica serovar Typhi (S. typhi) is the aetiological agent of typhoid fever, a serious invasive bacterial disease of humans with an annual global burden of approximately 16 million cases, leading to 600,000 fatalities. Many S. enterica serovars actively invade the mucosal surface of the intestine but are normally contained in healthy individuals by the local immune defence mechanisms. However, S. typhi has evolved the ability to spread to the deeper tissues of humans, including liver, spleen and bone marrow. Here we have sequenced the 4,809,037-base pair (bp) genome of a S. typhi (CT18) that is resistant to multiple drugs, revealing the presence of hundreds of insertions and deletions compared with the Escherichia coli genome, ranging in size from single genes to large islands. Notably, the genome sequence identifies over two hundred pseudogenes, several corresponding to genes that are known to contribute to virulence in Salmonella typhimurium. This genetic degradation may contribute to the human-restricted host range for S. typhi. CT18 harbours a 218,150-bp multiple-drug-resistance incH1 plasmid (pHCM1), and a 106,516-bp cryptic plasmid (pHCM2), which shows recent common ancestry with a virulence plasmid of Yersinia pestis.

Analysis of 41 kb of the DNA sequence from the right arm of chromosome II of Schizosaccharomyces pombe.

We report the complete sequence of cosmid c18A7 (41 046 bp insert), located on the right arm of chromosome II of the Schizosaccharomyces pombe genome. The sequence, which partially overlaps with cosmids SPBC4F6 and SPBC336, contains 16 open reading frames (ORFs) capable of coding for proteins of at least 100 amino acid residues in length (one partial) and one small nucleolar RNA (snoRNA). Four known genes were found: swi10 (encoding a mating-type switching protein also involved in nucleotide excision repair); dim1 (encoding a dimethyladenosine transferase); arf1 (encoding ADP-ribosylation factor 1); and pol3 (cdc6) the partial fragment, encoding the 125 kDa catalytic subunit of the DNA polymerase type B. Six ORFs similar to known proteins were found. They include a transporter of the major facilitator superfamily class, a vacuolar sorting protein, an asparagine synthase, a nuclear protein, a reticulum oxidoreductin and a heat shock protein. Each protein product of the other six ORFs has conserved domains and can be assigned a molecular, but not a biological, function. The sequence has been submitted to the EMBL database under Accession No. AL080287.

Genomics of Mycobacterium bovis.

The imminent completion of the genome sequence of Mycobacterium bovis will reveal the genetic blueprint for this most successful pathogen. Comparative analysis with the genome sequences of M. tuberculosis and M. bovis BCG promises to expose the genetic basis for the phenotypic differences between the tubercle bacilli, offering unparalleled insight into the virulence factors of the M. tuberculosis complex. Initial analysis of the sequence data has already revealed a novel deletion from M. bovis, as well as identifying variation in members of the PPE family of proteins. As the study of bacterial pathogenicity enters the postgenomic phase, the genome sequence of M. bovis promises to serve as a cornerstone of mycobacterial genetics.

From first base: the sequence of the tip of the X chromosome of Drosophila melanogaster, a comparison of two sequencing strategies.

We present the sequence of a contiguous 2.63 Mb of DNA extending from the tip of the X chromosome of Drosophila melanogaster. Within this sequence, we predict 277 protein coding genes, of which 94 had been sequenced already in the course of studying the biology of their gene products, and examples of 12 different transposable elements. We show that an interval between bands 3A2 and 3C2, believed in the 1970s to show a correlation between the number of bands on the polytene chromosomes and the 20 genes identified by conventional genetics, is predicted to contain 45 genes from its DNA sequence. We have determined the insertion sites of P-elements from 111 mutant lines, about half of which are in a position likely to affect the expression of novel predicted genes, thus representing a resource for subsequent functional genomic analysis. We compare the European Drosophila Genome Project sequence with the corresponding part of the independently assembled and annotated Joint Sequence determined through "shotgun" sequencing. Discounting differences in the distribution of known transposable elements between the strains sequenced in the two projects, we detected three major sequence differences, two of which are probably explained by errors in assembly; the origin of the third major difference is unclear. In addition there are eight sequence gaps within the Joint Sequence. At least six of these eight gaps are likely to be sites of transposable elements; the other two are complex. Of the 275 genes in common to both projects, 60% are identical within 1% of their predicted amino-acid sequence and 31% show minor differences such as in choice of translation initiation or termination codons; the remaining 9% show major differences in interpretation.

A superfamily of variant genes encoded in the subtelomeric region of Plasmodium vivax.

The malarial parasite Plasmodium vivax causes disease in humans, including chronic infections and recurrent relapses, but the course of infection is rarely fatal, unlike that caused by Plasmodium falciparum. To investigate differences in pathogenicity between P. vivax and P. falciparum, we have compared the subtelomeric domains in the DNA of these parasites. In P. falciparum, subtelomeric domains are conserved and contain ordered arrays of members of multigene families, such as var, rif and stevor, encoding virulence determinants of cytoadhesion and antigenic variation. Here we identify, through the analysis of a continuous 155,711-base-pair sequence of a P. vivax chromosome end, a multigene family called vir, which is specific to P. vivax. The vir genes are present at about 600-1,000 copies per haploid genome and encode proteins that are immunovariant in natural infections, indicating that they may have a functional role in establishing chronic infection through antigenic variation.

Massive gene decay in the leprosy bacillus.

Leprosy, a chronic human neurological disease, results from infection with the obligate intracellular pathogen Mycobacterium leprae, a close relative of the tubercle bacillus. Mycobacterium leprae has the longest doubling time of all known bacteria and has thwarted every effort at culture in the laboratory. Comparing the 3.27-megabase (Mb) genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus with that of Mycobacterium tuberculosis (4.41 Mb) provides clear explanations for these properties and reveals an extreme case of reductive evolution. Less than half of the genome contains functional genes but pseudogenes, with intact counterparts in M. tuberculosis, abound. Genome downsizing and the current mosaic arrangement appear to have resulted from extensive recombination events between dispersed repetitive sequences. Gene deletion and decay have eliminated many important metabolic activities including siderophore production, part of the oxidative and most of the microaerophilic and anaerobic respiratory chains, and numerous catabolic systems and their regulatory circuits.

Subtelomeric sequence from the right arm of Schizosaccharomyces pombe chromosome I contains seven permease genes.

The sequence has been determined of 80 888 bp of contiguous subtelomeric DNA, including the isp5 gene, from the right arm of chromosome I of Schizosaccharomyces pombe; 27 open reading frames (ORFs) longer than 100 codons are present, giving a density of one gene per 3.0 kb. Seven of the predicted proteins are members of the major facilitator superfamily (MFS) of transport proteins, including four amino acid permease homologues, bringing this family of amino acid permease sequences to 17 in Sz. pombe, and a phylogenetic analysis is presented. Also encoded is an allantoate permease homologue, a sulphate permease homologue and a probable urea active transporter. Predicted non-membrane proteins include a 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), a class III aminotransferase, serine acetyltransferase, protein-L-isoaspartate O-methyltransferase, alpha-glucosidase, alpha-galactosidase, esterase/lipase, oxidoreductase of the short-chain dehydrogenase/reductase (SDR) family, aldehyde dehydrogenase, formamidase, amidase, flavohaemoprotein, a putative translation initiation inhibitor and a protein with similarity to a filamentous fungal conidiation-specific protein. The remaining six ORFs are likely to encode proteins, either because they have sequence similarity with hypothetical proteins or because they are known to be transcribed. Introns are scarce in the sequenced region: only three ORFs contain introns, with only one having multiple introns. The sequenced region also contains a single Tf1 transposon long terminal repeat (LTR). The sequence is derived from cosmid clones c869, c922 and c1039 and has been submitted to the EMBL database under entries SPAC869 (Accession No. AL132779), SPAC922 (AL133522) and SPAC1039 (AL133521).

A re-annotation of the Saccharomyces cerevisiae genome.

Discrepancies in gene and orphan number indicated by previous analyses suggest that S. cerevisiae would benefit from a consistent re-annotation. In this analysis three new genes are identified and 46 alterations to gene coordinates are described. 370 ORFs are defined as totally spurious ORFs which should be disregarded. At least a further 193 genes could be described as very hypothetical, based on a number of criteria. It was found that disparate genes with sequence overlaps over ten amino acids (especially at the N-terminus) are rare in both S. cerevisiae and Sz. pombe. A new S. cerevisiae gene number estimate with an upper limit of 5804 is proposed, but after the removal of very hypothetical genes and pseudogenes this is reduced to 5570. Although this is likely to be closer to the true upper limit, it is still predicted to be an overestimate of gene number. A complete list of revised gene coordinates is available from the Sanger Centre (S. cerevisiae reannotation: ftp://ftp/pub/yeast/SCreannotation).

The decaying genome of Mycobacterium leprae.

Everything that we need to know about Mycobacterium leprae, a close relative of the tubercle bacillus, is encrypted in its genome. Inspection of the 3.27 Mb genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus identified 1,605 genes encoding proteins and 50 genes for stable RNA species. Comparison with the genome sequence of Mycobacterium tuberculosis revealed an extreme case of reductive evolution, since less than half of the genome contains functional genes while inactivated or pseudogenes are highly abundant. The level of gene duplication was approximately 34% and, on classification of the proteins into families, the largest functional groups were found to be involved in the metabolism and modification of fatty acids and polyketides, transport of metabolites, cell envelope synthesis and gene regulation. Reductive evolution, gene decay and genome downsizing have eliminated entire metabolic pathways, together with their regulatory circuits and accessory functions, particularly those involved in catabolism. This may explain the unusually long generation time and account for our inability to culture the leprosy bacillus.

Artemis: sequence visualization and annotation.

SUMMARY: Artemis is a DNA sequence visualization and annotation tool that allows the results of any analysis or sets of analyses to be viewed in the context of the sequence and its six-frame translation. Artemis is especially useful in analysing the compact genomes of bacteria, archaea and lower eukaryotes, and will cope with sequences of any size from small genes to whole genomes. It is implemented in Java, and can be run on any suitable platform. Sequences and annotation can be read and written directly in EMBL, GenBank and GFF format. AVAILABITLTY: Artemis is available under the GNU General Public License from http://www.sanger.ac.uk/Software/Artemis

Prevalence of small inversions in yeast gene order evolution.

Gene order evolution in two eukaryotes was studied by comparing the Saccharomyces cerevisiae genome sequence to extensive new data from whole-genome shotgun and cosmid sequencing of Candida albicans. Gene order is substantially different between these two yeasts, with only 9% of gene pairs that are adjacent in one species being conserved as adjacent in the other. Inversion of small segments of DNA, less than 10 genes long, has been a major cause of rearrangement, which means that even where a pair of genes has been conserved as adjacent, the transcriptional orientations of the two genes relative to one another are often different. We estimate that about 1,100 single-gene inversions have occurred since the divergence between these species. Other genes that are adjacent in one species are in the same neighborhood in the other, but their precise arrangement has been disrupted, probably by multiple successive multigene inversions. We estimate that gene adjacencies have been broken as frequently by local rearrangements as by chromosomal translocations or long-distance transpositions. A bias toward small inversions has been suggested by other studies on animals and plants and may be general among eukaryotes.