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Genome sequence comparison and scenarios for gene rearrangements: a test case.

As large portions of related genomes are being sequenced, methods for comparing complete or nearly complete genomes, as opposed to comparing individual genes, are becoming progressively more important. A major, widespread phenomenon in genome evolution is the rearrangement of genes and gene blocks. There is, however, no consistent method for genome sequence comparison combined with the reconstruction of the evolutionary history of highly rearranged genomes. We developed a schema for genome sequence comparison that includes three successive steps: (i) comparison of all proteins encoded in different genomes and generation of genomic similarity plots; (ii) construction of an alphabet of conserved genes and gene blocks; and (iii) generation of most parsimonious genome rearrangement scenarios. The approach is illustrated by a comparison of the herpesvirus genomes that constitute the largest set of relatively long, complete genome sequences available to date. Herpesviruses have from 70 to about 200 genes; comparison of the amino acid sequences encoded in these genes results in an alphabet of about 30 conserved genes comprising 7 conserved blocks that are rearranged in the genomes of different herpesviruses. Algorithms to analyze rearrangements of multiple genomes were developed and applied to the derivation of most parsimonious scenarios of herpesvirus evolution under different evolutionary models. The developed approaches to genome comparison will be applicable to the comparative analysis of bacterial and eukaryotic genomes as soon as their sequences become available.

DNA, Viral↗

Sequencing strategies for parasite genomes.

Recent advances in the field of sequencing have enabled the determination of the complete nucleotide sequence of a large number of complex genomes. The complete genome sequence of the parasite Plasmodium falciparum has been published recently, and many other parasite genome initiatives are underway. Parasite genomes vary in size, nucleotide composition, polymorphism level, content, and distribution of repetitive elements. These genomic features affect the performance of sequencing strategies. As a consequence, each of the ongoing parasite genome projects has adopted distinct sequencing approaches. The degree of completeness and accuracy desired as well as available funds should be considered carefully when choosing the most appropriate sequencing strategy.

Animals↗

The complete nucleotide sequence of Passiflora latent virus and its phylogenetic relationship to other carlaviruses.

A virus identified as Passiflora latent virus (PLV) was isolated from passion fruit plants. Particle morphology, host range and serological properties suggested that this virus belongs to the genus Carlavirus. The complete genomic sequence of PLV was determined by sequencing overlapping cDNA fragments. The genome consisted of 8386 nt, excluding the poly (A) tail and contained six open reading frames, typical of carlaviruses. The overall similarities of the predicted amino acid sequence of PLV to those of other carlaviruses ranged from 25 to 73%. Phylogenetic analysis indicated that PLV was closely related to lily symptomless virus and blueberry scorch virus. This is the first report of the complete nucleotide sequence and genome structure of PLV.

Base Sequence↗

Comprehensive comparative analysis of kinesins in photosynthetic eukaryotes.

BACKGROUND: Kinesins, a superfamily of molecular motors, use microtubules as tracks and transport diverse cellular cargoes. All kinesins contain a highly conserved approximately 350 amino acid motor domain. Previous analysis of the completed genome sequence of one flowering plant (Arabidopsis) has resulted in identification of 61 kinesins. The recent completion of genome sequencing of several photosynthetic and non-photosynthetic eukaryotes that belong to divergent lineages offers a unique opportunity to conduct a comprehensive comparative analysis of kinesins in plant and non-plant systems and infer their evolutionary relationships. RESULTS: We used the kinesin motor domain to identify kinesins in the completed genome sequences of 19 species, including 13 newly sequenced genomes. Among the newly analyzed genomes, six represent photosynthetic eukaryotes. A total of 529 kinesins was used to perform comprehensive analysis of kinesins and to construct gene trees using the Bayesian and parsimony approaches. The previously recognized 14 families of kinesins are resolved as distinct lineages in our inferred gene tree. At least three of the 14 kinesin families are not represented in flowering plants. Chlamydomonas, a green alga that is part of the lineage that includes land plants, has at least nine of the 14 known kinesin families. Seven of ten families present in flowering plants are represented in Chlamydomonas, indicating that these families were retained in both the flowering-plant and green algae lineages. CONCLUSION: The increase in the number of kinesins in flowering plants is due to vast expansion of the Kinesin-14 and Kinesin-7 families. The Kinesin-14 family, which typically contains a C-terminal motor, has many plant kinesins that have the motor domain at the N terminus, in the middle, or the C terminus. Several domains in kinesins are present exclusively either in plant or animal lineages. Addition of novel domains to kinesins in lineage-specific groups contributed to the functional diversification of kinesins. Results from our gene-tree analyses indicate that there was tremendous lineage-specific duplication and diversification of kinesins in eukaryotes. Since the functions of only a few plant kinesins are reported in the literature, this comprehensive comparative analysis will be useful in designing functional studies with photosynthetic eukaryotes.

Algal Proteins↗

Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana.

The genome of the model plant Arabidopsis thaliana has been sequenced by an international collaboration, The Arabidopsis Genome Initiative. Here we report the complete sequence of chromosome 5. This chromosome is 26 megabases long; it is the second largest Arabidopsis chromosome and represents 21% of the sequenced regions of the genome. The sequence of chromosomes 2 and 4 have been reported previously and that of chromosomes 1 and 3, together with an analysis of the complete genome sequence, are reported in this issue. Analysis of the sequence of chromosome 5 yields further insights into centromere structure and the sequence determinants of heterochromatin condensation. The 5,874 genes encoded on chromosome 5 reveal several new functions in plants, and the patterns of gene organization provide insights into the mechanisms and extent of genome evolution in plants.

Animals↗

Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses.

Many members of the genus Flavivirus are the agents of important diseases of humans, livestock, and wildlife. Currently, no complete genome sequence is available for the three African viruses, Bagaza, Zika, and Kedougou viruses, each representing a distinct virus subgroup according to the latest virus classification. In this study, we obtained a complete genome sequence of each of those three viruses and characterized the open reading frames (ORFs) with respect to gene sizes, cleavage sites, potential glycosylation sites, distribution of cysteine residues, and unique motifs. The sequences of the three viruses were then scanned across the entire length of the ORF against available sequences of other African flaviviruses and selected reference viruses for genetic relatedness. The data collectively indicated that Kedougou virus was close to dengue viruses but nonetheless distinct, while Bagaza virus shared genetic relatedness with West Nile virus in several genomic regions. In the non-coding regions, it was found that a particular organizational pattern of conserved sequences in the 3' terminal region generally correlated with the current virus grouping.

3' Untranslated Regions↗

Identification of 5' capped structure and 3' terminal sequence of hepatitis E virus isolated from Morocco.

AIM: To examine 5' and 3' terminal sequences of hepatitis E virus (HEV) isolated from Morocco, to confirm 5' methylated cap structure of the genome, and to investigate whether the 3' UTR can be used to distinguish HEV genotypes instead of HEV complete genome sequence. METHODS: RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) was employed to obtain the 5' and 3' terminal sequences of HEV Morocco strain. The 3' UTR sequence of the Morocco strain was compared with that of the other 29 HEV strains using the DNAStar software. RESULTS: The 5' PCR product was obtained only from the RLM-RACE based on the capped RNA template. The 5' UTR of the Morocco strain had 26 nucleotides, and the 3' UTR had 65 nucleotides upstream to the polyA. The 5' UTR between HEV strains had only point mutations of nucleotides. The phylogenetic tree based on the sequences of 3' UTR was not the same as that based on the complete sequences. CONCLUSION: The genome of HEV Morocco strain was methylated cap structure. The 3' terminal sequence can not be used for distinguishing HEV genotype for all HEV strains in place of the whole HEV genome sequence.

3' Untranslated Regions↗

The complete mitochondrial genome sequence and characterization of single-nucleotide polymorphisms in the control region of the Asian seabass (Lates calcarifer).

We determined the complete mtDNA nucleotide sequence of Lates calcarifer using the shotgun sequencing method. The mitochondrial DNA (mtDNA) was 16,535 base pairs (bp) in length, and contained 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and one major noncoding control region (CR). The CR was unusually short at only 768 bp. A striking feature of the mitochondrial genome was the high G+C content (46.1%), which is among the highest in fish. The gene order was identical to that of a typical vertebrate. Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes of 30 fish species representing 14 suborders clearly showed Lates calcarifer was located in the cluster of fish species from the order Perciformes, supporting the traditional systematic classification. We characterized single-nucleotide polymorphisms (SNPs) in the CR by sequencing the complete CR of 25 individuals obtained from Australia and Singapore. A total of 68 SNPs were detected. Eighteen SNPs were fixed with alternative nucleotides in Australian and Singapore seabass, and these SNPs could be used for differentiating fish from the two countries.

Animals↗

Malaria research in the post-genomic era.

Within the next few years, the complete genomic sequences of Plasmodium falciparum, and potentially several other Plasmodium spp, will be available to researchers worldwide. These complete genomic sequence data are certain to provide the foundation for nearly all malaria research in the next decades, as discussed here by Dan Carucci.

Animals↗

Genome sequence of Yersinia pestis KIM.

We present the complete genome sequence of Yersinia pestis KIM, the etiologic agent of bubonic and pneumonic plague. The strain KIM, biovar Mediaevalis, is associated with the second pandemic, including the Black Death. The 4.6-Mb genome encodes 4,198 open reading frames (ORFs). The origin, terminus, and most genes encoding DNA replication proteins are similar to those of Escherichia coli K-12. The KIM genome sequence was compared with that of Y. pestis CO92, biovar Orientalis, revealing homologous sequences but a remarkable amount of genome rearrangement for strains so closely related. The differences appear to result from multiple inversions of genome segments at insertion sequences, in a manner consistent with present knowledge of replication and recombination. There are few differences attributable to horizontal transfer. The KIM and E. coli K-12 genome proteins were also compared, exposing surprising amounts of locally colinear "backbone," or synteny, that is not discernible at the nucleotide level. Nearly 54% of KIM ORFs are significantly similar to K-12 proteins, with conserved housekeeping functions. However, a number of E. coli pathways and transport systems and at least one global regulator were not found, reflecting differences in lifestyle between them. In KIM-specific islands, new genes encode candidate pathogenicity proteins, including iron transport systems, putative adhesins, toxins, and fimbriae.

Bacteriophages↗

Kelp fly virus: a novel group of insect picorna-like viruses as defined by genome sequence analysis and a distinctive virion structure.

The complete genomic sequence of kelp fly virus (KFV), originally isolated from the kelp fly, Chaetocoelopa sydneyensis, has been determined. Analyses of its genomic and structural organization and phylogeny show that it belongs to a hitherto undescribed group within the picorna-like virus superfamily. The single-stranded genomic RNA of KFV is 11,035 nucleotides in length and contains a single large open reading frame encoding a polypeptide of 3,436 amino acids with 5' and 3' untranslated regions of 384 and 343 nucleotides, respectively. The predicted amino acid sequence of the polypeptide shows that it has three regions. The N-terminal region contains sequences homologous to the baculoviral inhibitor of apoptosis repeat domain, an inhibitor of apoptosis commonly found in animals and in viruses with double-stranded DNA genomes. The second region contains at least two capsid proteins. The third region has three sequence motifs characteristic of replicase proteins of many plant and animal viruses, including a helicase, a 3C chymotrypsin-like protease, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the replicase motifs shows that KFV forms a distinct and distant taxon within the picorna-like virus superfamily. Cryoelectron microscopy and image reconstruction of KFV to a resolution of 15 A reveals an icosahedral structure, with each of its 12 fivefold vertices forming a turret from the otherwise smooth surface of the 20-A-thick capsid. The architecture of the KFV capsid is unique among the members of the picornavirus superfamily for which structures have previously been determined.

Amino Acid Sequence↗

A phylogenetic survey of recombination frequency in plant RNA viruses.

The severe economic consequences of emerging plant viruses highlights the importance of studies of plant virus evolution. One question of particular relevance is the extent to which the genomes of plant viruses are shaped by recombination. To this end we conducted a phylogenetic survey of recombination frequency in a wide range of positive-sense RNA plant viruses, utilizing 975 capsid gene sequences and 157 complete genome sequences. In total, 12 of the 36 RNA virus species analyzed showed evidence for recombination, comprising 17% of the capsid gene sequence alignments and 44% of the genome sequence alignments. Given the conservative nature of our analysis, we propose that recombination is a relatively common process in some plant RNA viruses, most notably the potyviruses.

Computational Biology↗

The relevance of genetic analysis to dairy bacteria: building upon our heritage.

Lactic acid bacteria (LAB) are essential for the manufacture of fermented dairy products. Studies on the physiology, biochemistry and genetics of these microorganisms over the last century have contributed considerably to the improvement of fermentation processes and have resulted in better and safer products. Nevertheless, the potential of LAB is far from being maximized. The sophistication of biotechnologies and the availability of complete genome sequences have opened the door to the metabolic engineering of LAB. In this regard, the recent publication of the complete genome sequences of two Streptococcus thermophilus strains will provide a key tool to facilitate the genetic manipulation of this important dairy species.

Editorial↗

Evidence for symmetric chromosomal inversions around the replication origin in bacteria.

BACKGROUND: Whole-genome comparisons can provide great insight into many aspects of biology. Until recently, however, comparisons were mainly possible only between distantly related species. Complete genome sequences are now becoming available from multiple sets of closely related strains or species. RESULTS: By comparing the recently completed genome sequences of Vibrio cholerae, Streptococcus pneumoniae and Mycobacterium tuberculosis to those of closely related species - Escherichia coli, Streptococcus pyogenes and Mycobacterium leprae, respectively - we have identified an unusual and previously unobserved feature of bacterial genome structure. Scatterplots of the conserved sequences (both DNA and protein) between each pair of species produce a distinct X-shaped pattern, which we call an X-alignment. The key feature of these alignments is that they have symmetry around the replication origin and terminus; that is, the distance of a particular conserved feature (DNA or protein) from the replication origin (or terminus) is conserved between closely related pairs of species. Statistically significant X-alignments are also found within some genomes, indicating that there is symmetry about the replication origin for paralogous features as well. CONCLUSIONS: The most likely mechanism of generation of X-alignments involves large chromosomal inversions that reverse the genomic sequence symmetrically around the origin of replication. The finding of these X-alignments between many pairs of species suggests that chromosomal inversions around the origin are a common feature of bacterial genome evolution.

Bacteria↗

Comparison of archaeal and bacterial genomes: computer analysis of protein sequences predicts novel functions and suggests a chimeric origin for the archaea.

Protein sequences encoded in three complete bacterial genomes, those of Haemophilus influenzae, Mycoplasma genitalium and Synechocystis sp., and the first available archaeal genome sequence, that of Methanococcus jannaschii, were analysed using the BLAST2 algorithm and methods for amino acid motif detection. Between 75% and 90% of the predicted proteins encoded in each of the bacterial genomes and 73% of the M. jannaschii proteins showed significant sequence similarity to proteins from other species. The fraction of bacterial and archaeal proteins containing regions conserved over long phylogenetic distances is nearly the same and close to 70%. Functions of 70-85% of the bacterial proteins and about 70% of the archaeal proteins were predicted with varying precision. This contrasts with the previous report that more than half of the archaeal proteins have no homologues and shows that, with more sensitive methods and detailed analysis of conserved motifs, archaeal genomes become as amenable to meaningful interpretation by computer as bacterial genomes. The analysis of conserved motifs resulted in the prediction of a number of previously undetected functions of bacterial and archaeal proteins and in the identification of novel protein families. In spite of the generally high conservation of protein sequences, orthologues of 25% or less of the M. jannaschii genes were detected in each individual completely sequenced genome, supporting the uniqueness of archaea as a distinct domain of life. About 53% of the M. jannaschii proteins belong to families of paralogues, a fraction similar to that in bacteria with larger genomes, such as Synechocystis sp. and Escherichia coli, but higher than that in H. influenzae, which has approximately the same number of genes as M. jannaschii. Certain groups of proteins, e.g. molecular chaperones and DNA repair enzymes, thought to be ubiquitous and represented in the minimal gene set derived by bacterial genome comparison, are missing in M. jannaschii, indicating massive non-orthologous displacement of genes responsible for essential functions. An unexpectedly large fraction of the M. jannaschii gene products, 44%, shows significantly higher similarity to bacterial than to eukaryotic proteins, compared with 13% that have eukaryotic proteins as their closest homologues (the rest of the proteins show approximately the same level of similarity to bacterial and eukaryotic homologues or have no homologues). Proteins involved in translation, transcription, replication and protein secretion are most closely related to eukaryotic proteins, whereas metabolic enzymes, metabolite uptake systems, enzymes for cell wall biosynthesis and many uncharacterized proteins appear to be 'bacterial'. A similar prevalence of proteins of apparent bacterial origin was observed among the currently available sequences from the distantly related archaeal genus, Sulfolobus. It is likely that the evolution of archaea included at least one major merger between ancestral cells from the bacterial lineage and the lineage leading to the eukaryotic nucleocytoplasm.

Algorithms↗

The complete sequence of the bovine torovirus genome.

Viruses in the family Coronaviridae have elicited new interest, with the outbreaks caused by SARS-HCoV in 2003 and the recent discovery of a new human coronavirus, HCoV-NL63. The genus Torovirus, within the family Coronaviridae, is less well characterized, in part because toroviruses cannot yet be grown in cell culture (except for the Berne virus). In this study, we determined the sequence of the complete genome of Breda-1 (BoTV-1), a bovine torovirus. This is the first complete torovirus genome sequence to be reported. BoTV-1 RNA was amplified using long RT-PCR and the amplicons sequenced. The genome has a length of 28.475 kb and consisted mainly of the replicase gene ( approximately 20.2 kb) which contains two large overlapping ORFs, ORF1a and ORF1b, encoding polyproteins pp1a and pp1b, respectively. Sequence analysis identified conserved domains within the predicted sequences of pp1a and pp1b. Sequence alignments and protein secondary structure prediction data suggest the presence of a 3C-like serine protease domain with similarity to the arterivirus 3C-like serine protease and a single papain-like cysteine protease domain with similarity to the picornavirus leader protease. The ADRP (APPR-1'') domain - unique to the Coronaviridae - was also located in BoTV pp1a. In addition, several hydrophobic domains were identified that are typical of a nidovirus replicase. Within the pp1b sequence the polymerase and helicase domains were identified, as well as sequences predicted to be involved in ribosomal frameshifting, including the conserved slippery sequence UUUAAAC and two potential pseudoknot structures.

Amino Acid Sequence↗

Hepatitis C virus variants from Thailand classifiable into five novel genotypes in the sixth (6b), seventh (7c, 7d) and ninth (9b, 9c) major genetic groups.

Nine (10%) out of 90 hepatitis C virus (HCV) isolates from hepatitis patients and commercial blood donors in Thailand were not classifiable into any of genotypes I/1a, II/1b, III/2a, IV/2b, V/3a or VI/3b by RT-PCR with type-specific primers deduced from the HCV core gene. These isolates were sequenced over a 1.6 kb stretch of the 5'-terminal sequence and 1.1 kb of the 3'-terminal sequence covering 30% of the entire genome. Based on two-by-two comparison and phylogenetic analyses of the nine Thailand isolates among themselves and with known full or partial sequences of previously reported HCV isolates, the Thailand isolates were classified into five genotypes not reported previously, viz. 6b, 7c, 7d, 9b and 9c. Along with HCV isolates reported already, they make at least nine major genetic groups of HCV which further break down into at least 28 genotypes with sequence similarity in the E1 gene (576 bp) of < or = 80%. As many more HCV isolates of distinct genotypes are expected to be found throughout the world, it will become increasingly difficult to classify them by comparison of any partial sequences of the genome. Complete sequence data will be required for the full characterization and classification of HCV genotypes.

Amino Acid Sequence↗

An expanded role for microbial physiology in metabolic engineering and functional genomics: moving towards systems biology.

Microbial physiology has traditionally played a very important role in both fundamental research and in industrial applications of microorganisms. The classical approach in microbial physiology has been to analyze the role of individual components (genes or proteins) in the overall cell function. With the progress in molecular biology it has become possible to optimize industrial fermentations through introduction of directed genetic modification - an approach referred to as metabolic engineering. Furthermore, as a consequence of large sequencing programs the complete genomic sequence has become available for an increasing number of microorganisms. This has resulted in substantial research efforts in assigning function to all identified open reading frames - referred to as functional genomics. In both metabolic engineering and functional genomics there is a trend towards application of a macroscopic view on cell function, and this leads to an expanded role of the classical approach applied in microbial physiology. With the increased understanding of the molecular mechanisms it is envisaged that in the future it will be possible to describe the interaction between all the components in the system (the cell), also at the quantitative level, and this is the goal of systems biology. Clearly this will have a significant impact on microbial physiology as well as on metabolic engineering.

Aspergillus↗