Gene transfer and genome plasticity in Thermotoga maritima, a model hyperthermophilic species

The genome sequence of the hyperthermophilic bacterium Thermotoga maritima MSB8 presents evidence for lateral gene transfer events between bacterial and archaeal species. To estimate the extent of genomic diversity across the order Thermotogales, a comparative genomic hybridization study was initiated to compare nine Thermotoga strains to the sequenced T. maritima MSB8. Many differences could be associated with substrate utilization patterns, which are most likely a reflection of the environmental niche that these individual species occupy. A detailed analysis of some of the predicted variable regions demonstrates many examples of the deletion/insertion of complete cassettes of genes and of gene rearrangements and insertions of DNA within genes, with the C or N terminus being retained. Although the mechanism for gene transfer in this lineage remains to be elucidated, this analysis suggests possible associations with repetitive elements and highlights the possible benefits of rampant genetic exchange to these species.     

Promoter trapping identifies real genes in C. elegans

Promoter trapping involved screening uncharacterized fragments of C. elegans genomic DNA for C. elegans promoter activity. By sequencing the ends of these DNA fragments and locating their genomic origin using the available genome sequence data, promoter trapping has now been shown to identify real promoters of real genes, exactly as anticipated. Developmental expression patterns have thereby been linked to gene sequence, allowing further inferences on gene function to be drawn. Some expression patterns generated by promoter trapping include subcellular details. Localization to the surface of particular cells or even particular aspects of the cell surface was found to be consistent with the genes, now associated with these patterns, encoding membrane-spanning proteins. Data on gene expression patterns are easier to generate and characterize than mutant phenotypes and may provide the best means of interpreting the large quantity of sequence data currently being generated in genome projects.     

Promoter trapping identifies real genes in C. elegans

Promoter trapping involved screening uncharacterized fragments of C. elegans genomic DNA for C. elegans promoter activity. By sequencing the ends of these DNA fragments and locating their genomic origin using the available genome sequence data, promoter trapping has now been shown to identify real promoters of real genes, exactly as anticipated. Developmental expression patterns have thereby been linked to gene sequence, allowing further inferences on gene function to be drawn. Some expression patterns generated by promoter trapping include subcellular details. Localization to the surface of particular cells or even particular aspects of the cell surface was found to be consistent with the genes, now associated with these patterns, encoding membrane-spanning proteins. Data on gene expression patterns are easier to generate and characterize than mutant phenotypes and may provide the best means of interpreting the large quantity of sequence data currently being generated in genome projects. Received: 12 June 1998 / Accepted: 21 August 1998     

The malaria genome sequencing project: complete sequence of Plasmodium falciparum chromosome 2

An international consortium has been formed to sequence the entire genome of the human malaria parasite Plasmodium falciparum. We sequenced chromosome 2 of clone 3D7 using a shotgun sequencing strategy. Chromosome 2 is 947 kb in length, has a base composition of 80.2% A + T, and contains 210 predicted genes. In comparison to the Saccharomyces cerevisiae genome, chromosome 2 has a lower gene density, a greater proportion of genes containing introns, and nearly twice as many proteins containing predicted non-globular domains. A group of putative surface proteins was identified, rifins, which are encoded by a gene family comprising up to 7% of the protein-encoding gene in the genome. The rifins exhibit considerable sequence diversity and may play an important role in antigenic variation. Sixteen genes encoded on chromosome 2 showed signs of a plastid or mitochondrial origin, including several genes involved in fatty acid biosynthesis. Completion of the chromosome 2 sequence demonstrated that the A + T-rich genome of P. falciparum can be sequenced by the shotgun approach. Within 2-3 years, the sequence of almost all P. falciparum genes will have been determined, paving the way for genetic, biochemical, and immunological research aimed at developing new drugs and vaccines against malaria.     

Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation

Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus.     

Changes in twelve homoeologous genomic regions in soybean following three rounds of polyploidy

With the advent of high-throughput sequencing, the availability of genomic sequence for comparative genomics is increasing exponentially. Numerous completed plant genome sequences enable characterization of patterns of the retention and evolution of genes within gene families due to multiple polyploidy events, gene loss and fractionation, and differential evolutionary pressures over time and across different gene families. In this report, we trace the changes that have occurred in 12 surviving homoeologous genomic regions from three rounds of polyploidy that contributed to the current Glycine max genome: a genome triplication before the origin of the rosids (~130 to 240 million years ago), a genome duplication early in the legumes (~58 million years ago), and a duplication in the Glycine lineage (~13 million years ago). Patterns of gene retention following the genome triplication event generally support predictions of the Gene Balance Hypothesis. Finally, we find that genes in networks with a high level of connectivity are more strongly conserved than those with low connectivity and that the enrichment of these highly connected genes in the 12 highly conserved homoeologous segments may in part explain their retention over more than 100 million years and repeated polyploidy events.     

Characterization of genes with tissue-specific differential expression patterns in Populus

Like many plants, Populus has an evolutionary history in which several, both recent and more ancient, genome duplication events have occurred and, therefore, constitutes an excellent model system for studying the functional evolution of genes. In the present study, we have focused on the properties of genes with tissue-specific differential expression patterns in poplar. We identified the genes by analyzing digital expression profiles derived by mapping 90,000+ expressed sequence tags (ESTs) from 18 sources to the predicted genes of Populus. Our sequence analysis suggests that tissue-specific differentially expressed genes have less diverged paralogs than average, indicating that gene duplication events is an important event in the pathway leading to this type of expression pattern. The functional analysis showed that genes coding for proteins involved in processes of functional importance for the specific tissue(s) in which they are expressed and genes coding for regulatory or responsive proteins are most common among the differentially expressed genes, demonstrating that the expression differentiation process is under strong selective pressure. Thus, our data supports a model where gene duplication followed by gene specialization or expansion of the regulatory and responsive networks leads to tissue-specific differential expression patterns. We have also searched for clustering of genes with similar expression pattern into gene-expression neighborhoods within the Populus genome. However, we could not detect any major clustering among the analyzed genes with highly specific expression patterns. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila heterochromatin protein 1 (HP1) gene family

Heterochromatin is the gene-poor, satellite-rich eukaryotic genome compartment that supports many essential cellular processes. The functional diversity of proteins that bind and often epigenetically define heterochromatic DNA sequence reflects the diverse functions supported by this enigmatic genome compartment. Moreover, heterogeneous signatures of selection at chromosomal proteins often mirror the heterogeneity of evolutionary forces that act on heterochromatic DNA. To identify new such surrogates for dissecting heterochromatin function and evolution, we conducted a comprehensive phylogenomic analysis of the Heterochromatin Protein 1 gene family across 40 million years of Drosophila evolution. Our study expands this gene family from 5 genes to at least 26 genes, including several uncharacterized genes in Drosophila melanogaster. The 21 newly defined HP1s introduce unprecedented structural diversity, lineage-restriction, and germline-biased expression patterns into the HP1 family. We find little evidence of positive selection at these HP1 genes in both population genetic and molecular evolution analyses. Instead, we find that dynamic evolution occurs via prolific gene gains and losses. Despite this dynamic gene turnover, the number of HP1 genes is relatively constant across species. We propose that karyotype evolution drives at least some HP1 gene turnover. For example, the loss of the male germline-restricted HP1E in the obscura group coincides with one episode of dramatic karyotypic evolution, including the gain of a neo-Y in this lineage. This expanded compendium of ovary- and testis-restricted HP1 genes revealed by our study, together with correlated gain/loss dynamics and chromosome fission/fusion events, will guide functional analyses of novel roles supported by germline chromatin.     

From 2R to 3R: evidence for a fish-specific genome duplication (FSGD)

An important mechanism for the evolution of phenotypic complexity, diversity and innovation, and the origin of novel gene functions is the duplication of genes and entire genomes. Recent phylogenomic studies suggest that, during the evolution of vertebrates, the entire genome was duplicated in two rounds (2R) of duplication. Later, approximately 350 mya, in the stem lineage of ray-finned (actinopterygian) fishes, but not in that of the land vertebrates, a third genome duplication occurred-the fish-specific genome duplication (FSGD or 3R), leading, at least initially, to up to eight copies of the ancestral deuterostome genome. Therefore, the sarcopterygian (lobe-finned fishes and tetrapods) genome possessed originally only half as many genes compared to the derived fishes, just like the most-basal and species-poor lineages of extant fishes that diverged from the fish stem lineage before the 3R duplication. Most duplicated genes were secondarily lost, yet some evolved new functions. The genomic complexity of the teleosts might be the reason for their evolutionary success and astounding biological diversity.     

Phylogeny and molecular evolution of the DMC1 gene in the polyploid genus Leymus (Triticeae: Poaceae) and its diploid relatives

The level and pattern of nucleotide variation in duplicate genes provide important information on the evolutionary history of polyploids and divergent processes between homoeologous loci within lineages. Leymus, a group of allopolyploid species with the NsXm genomes, is a perennial genus with a diverse array of morphology, ecology, and distribution in Triticeae. To estimate the phylogeny and molecular evolution of a single-copy DMC1 gene in Leymus and its diploid relatives,DMC1 homoeologous sequences were isolated from the sampled Leymus species and were analyzed with those from 30 diploid taxa representing 18 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that: (i) different Leymus species might derive their Ns genome from different Psathyrostachys species; (ii) Pseudoroegneria has contributed to the nuclear genome of some Leymus species, which might result from recurrent hybridization or incomplete lineage sorting; (iii) the Xm genome origin of Leymus could differ among species; (iv) rapid radiation and multiple origin might account for the rich diversity, numbers of species, and wide ecological adaptation of Leymus species; and (v) the DMC1 sequence diversity of the Ns genome in Leymus species was lower than that in the Psathyrostachys diploids, while the level of DMC1 sequence diversity in Leymus was higher than that in diploid Pseudoroegneria. Our results provide new insight on the evolutionary dynamics of duplicate DMC1 genes, polyploid speciation, and the phylogeny of Leymus species.     

The plasticity of global proteome and genome expression analyzed in closely related W3110 and MG1655 strains of a well-studied model organism, Escherichia coli-K12

The use of Escherichia coli as a model organism has provided a great deal of basic information in biomolecular sciences. Examining trait differences among closely related strains of the same species addresses a fundamental biological question: how much diversity is there at the single species level? The main aim of our research was to identify significant differences in the activities of groups of genes between two laboratory strains of an organism closely related in genome structure. We demonstrate that despite strict and controlled growth conditions, there is high plasticity in the global proteome and genome expression in two closely related E. coli K12 sub-strains (W3110 and MG1655), which differ insignificantly in genome structure. The growth patterns of these two sub-strains were very similar in a well-equipped bioreactor, and their genome structures were shown to be almost identical by DNA microarray. However, detailed profiling of protein and gene expression by 2-dimensional gel electrophoresis and microarray analysis showed many differentially expressed genes and proteins, combinations of which were highly correlated. The differentially regulated genes and proteins belonged to the following functional categories: genes regulated by sigma subunit of RNA polymerase (RpoS), enterobactin-related genes, and genes involved in central metabolism. Genes involved in central cell metabolism - the glycolysis pathway, the tricarboxylic acid cycle and the glyoxylate bypass - were differentially regulated at both the mRNA and proteome levels. The strains differ significantly in central metabolism and thus in the generation of precursor metabolites and energy. This high plasticity probably represents a universal feature of metabolic activities in closely related species, and has the potential to reveal differences in regulatory networks. We suggest that unless care is taken in the choice of strains for any validating experiment, the results might be misleading.     

Nature and Extent of Genetic Diversity of Dengue Viruses Determined by 454 Pyrosequencing

Dengue virus (DENV) populations are characteristically highly diverse. Regular lineage extinction and replacement is an important dynamic DENV feature, and most DENV lineage turnover events are associated with increased incidence of disease. The role of genetic diversity in DENV lineage extinctions is not understood. We investigated the nature and extent of genetic diversity in the envelope (E) gene of DENV serotype 1 representing different lineages histories. A region of the DENV genome spanning the E gene was amplified and sequenced by Roche/454 pyrosequencing. The pyrosequencing results identified distinct sub-populations (haplotypes) for each DENV-1 E gene. A phylogenetic tree was constructed with the consensus DENV-1 E gene nucleotide sequences, and the sequences of each constructed haplotype showed that the haplotypes segregated with the Sanger consensus sequence of the population from which they were drawn. Haplotypes determined through pyrosequencing identified a recombinant DENV genome that could not be identified through Sanger sequencing. Nucleotide level sequence diversities of DENV-1 populations determined from SNP analysis were very low, estimated from 0.009–0.01. There were also no stop codon, frameshift or non-frameshift mutations observed in the E genes of any lineage. No significant correlations between the accumulation of deleterious mutations or increasing genetic diversity and lineage extinction were observed (p>0.5). Although our hypothesis that accumulation of deleterious mutations over time led to the extinction and replacement of DENV lineages was ultimately not supported by the data, our data does highlight the significant technical issues that must be resolved in the way in which population diversity is measured for DENV and other viruses. The results provide an insight into the within-population genetic structure and diversity of DENV-1 populations.     

Host specificity of septicemic Escherichia coli: human and avian pathogens

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are the cause of a diverse spectrum of invasive human and animal infections, often leading to septicemia. ExPEC strains contain virulence factors that enable them to survive in the host blood and tissues. Most of these virulence factors are distributed in ExPEC strains in a host-independent fashion. Genomic analyses of these strains provide evidence for numerous recombinational events and horizontal gene transfer, as well as for a high diversity of virulence factors. In studies of human and avian septicemic strains of serotypes O2 and O78 it appears that there is a positive correlation between virulence, invasiveness and clonal origin. Yet, it is clear that clonal division in these strains, as well as distribution of virulence factors, is independent of the host and closely related clones reside in different hosts. Although the possibility exists that ExPEC strains do have a certain degree of host specificity, which is not obvious from genomic studies, it is clear that the similarity of virulence factors presents a significant zoonotic risk.     

Gain and loss of multiple genes during the evolution of Helicobacter pylori

Sequence diversity and gene content distinguish most isolates of Helicobacter pylori. Even greater sequence differences differentiate distinct populations of H. pylori from different continents, but it was not clear whether these populations also differ in gene content. To address this question, we tested 56 globally representative strains of H. pylori and four strains of Helicobacter acinonychis with whole genome microarrays. Of the weighted average of 1,531 genes present in the two sequenced genomes, 25% are absent in at least one strain of H. pylori and 21% were absent or variable in H. acinonychis. We extrapolate that the core genome present in all isolates of H. pylori contains 1,111 genes. Variable genes tend to be small and possess unusual GC content; many of them have probably been imported by horizontal gene transfer. Phylogenetic trees based on the microarray data differ from those based on sequences of seven genes from the core genome. These discrepancies are due to homoplasies resulting from independent gene loss by deletion or recombination in multiple strains, which distort phylogenetic patterns. The patterns of these discrepancies versus population structure allow a reconstruction of the timing of the acquisition of variable genes within this species. Variable genes that are located within the cag pathogenicity island were apparently first acquired en bloc after speciation. In contrast, most other variable genes are of unknown function or encode restriction/modification enzymes, transposases, or outer membrane proteins. These seem to have been acquired prior to speciation of H. pylori and were subsequently lost by convergent evolution within individual strains. Thus, the use of microarrays can reveal patterns of gene gain or loss when examined within a phylogenetic context that is based on sequences of core genes.     

Genomics of speciation and introgression in Princess cichlid fishes from Lake Tanganyika

How variation in the genome translates into biological diversity and new species originate has endured as the mystery of mysteries in evolutionary biology. African cichlid fishes are prime model systems to address speciation‐related questions for their remarkable taxonomic and phenotypic diversity, and the possible role of gene flow in this process. Here, we capitalize on genome sequencing and phylogenomic analyses to address the relative impacts of incomplete lineage sorting, introgression and hybrid speciation in the Neolamprologus savoryi‐complex (the ‘Princess cichlids’) from Lake Tanganyika. We present a time‐calibrated species tree based on whole‐genome sequences and provide strong evidence for incomplete lineage sorting in the early phases of diversification and multiple introgression events affecting different stages. Importantly, we find that the Neolamprologus chromosomes show centre‐to‐periphery biases in nucleotide diversity, sequence divergence, GC content, incomplete lineage sorting and rates of introgression, which are likely modulated by recombination density and linked selection. The detection of heterogeneous genomic landscapes has strong implications on the genomic mechanisms involved in speciation. Collinear chromosomal regions can be protected from gene flow and harbour incompatibility genes if they reside in lowly recombining regions, and coupling can evolve between nonphysically linked genomic regions (chromosome centres in particular). Simultaneously, higher recombination towards chromosome peripheries makes these more dynamic, evolvable regions where adaptation polymorphisms have a fertile ground. Hence, differences in genome architecture could explain the levels of taxonomic and phenotypic diversity seen in taxa with collinear genomes and might have contributed to the spectacular cichlid diversity observed today.     

Genome diversification in phylogenetic lineages I and II of Listeria monocytogenes: identification of segments unique to lineage II populations

Thirteen different serotypes of Listeria monocytogenes can be distinguished on the basis of variation in somatic and flagellar antigens. Although the known virulence genes are present in all serotypes, greater than 90% of human cases of listeriosis are caused by serotypes 1/2a, 1/2b, and 4b and nearly all outbreaks of food-borne listeriosis have been caused by serotype 4b strains. Phylogenetic analysis of these three common clinical serotypes places them into two different lineages, with serotypes 1/2b and 4b belonging to lineage I and 1/2a belonging to lineage II. To begin examining evolution of the genome in these serotypes, DNA microarray analysis was used to identify lineage-specific and serotype-specific differences in genome content. A set of 44 strains representing serotypes 1/2a, 1/2b, and 4b was probed with a shotgun DNA microarray constructed from the serotype 1/2a strain 10403s. Clones spanning 47 different genes in 16 different contiguous segments relative to the lineage II 1/2a genome were found to be absent in all lineage I strains tested (serotype 4b and 1/2b) and an additional nine were altered exclusively in 4b strains. Southern hybridization confirmed that conserved alterations were, in all but two loci, due to absence of the segments from the genome. Genes within these contiguous segments comprise five functional categories, including genes involved in synthesis of cell surface molecules and regulation of virulence gene expression. Phylogenetic reconstruction and examination of compositional bias in the regions of difference are consistent with a model in which the ancestor of the two lineages had the 1/2 somatic serotype and the regions absent in the lineage I genome arose by loss of ancestral sequences.     

Full-length sequence analysis of the HLA-DRB1 locus suggests a recent origin of alleles

The HLA region harbors some of the most polymorphic loci in the human genome. Among them is the class II locus HLA-DRB1, with more than 400 known alleles. The age of the polymorphism and the rate at which new alleles are generated at HLA loci has caused much controversy over the years. Previous studies have mostly been restricted to the 270 base pairs that constitute the second exon and represent the most variable part of the gene. Here, we investigate the evolutionary history of the HLA-DRB1 locus on the basis of an analysis of 15 genomic full-length alleles (10-15 kb). In addition, the variation in 49 complete coding sequences and 322 exon 2 sequences were analyzed. When excluding exon 2 from the analysis, the diversity at the synonymous sites was found to be similar to the intron diversity. The overall diversity in noncoding region was also similar to the genome average. The DRB1*03 lineage has been found in human, chimpanzee, bonobo, gorilla, and orangutan. An ancestral "proto HLA-DRB1*03 lineage" appeared to have diverged in the last 5 million years into the human-specific lineages *08, *11, *13, and *14. With exception to exon 2, both the coding- and the noncoding diversity suggests a recent origin (<1 million years ago) for most of the alleles at the HLA-DRB1 locus. Sites encoding for amino acids involved in antigen binding [antigen recognizing sites (ARS)] appear to have a more ancient origin. Taken together, the recent origin of most alleles, the high diversity between allelic lineages, and the ancient origin of sequence motifs in exon 2, is consistent with a relatively rapid generation of novel alleles by gene conversion like events.