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PubMed2002

Molecular basis of adaptive convergence in experimental populations of RNA viruses.

Characterizing the molecular basis of adaptation is one of the most important goals in modern evolutionary genetics. Here, we report a full-genome sequence analysis of 21 independent populations of vesicular stomatitis ribovirus evolved on the same cell type but under different demographic regimes. Each demographic regime differed in the effective viral population size. Evolutionary convergences are widespread both at synonymous and nonsynonymous replacements as well as in an intergenic region. We also found evidence for epistasis among sites of the same and different loci. We explain convergences as the consequence of four factors: (1) environmental homogeneity that supposes an identical challenge for each population, (2) structural constraints within the genome, (3) epistatic interactions among sites that create the observed pattern of covariation, and (4) the phenomenon of clonal interference among competing genotypes carrying different beneficial mutations. Using these convergences, we have been able to estimate the fitness contribution of the identified mutations and epistatic groups. Keeping in mind statistical uncertainties, these estimates suggest that along with several beneficial mutations of major effect, many other mutations got fixed as part of a group of epistatic mutations.

Epistasis, Genetic↗

PubMed2004

Isolation and location of three homoeologous dihydroflavonol-4-reductase (DFR) genes of wheat and their tissue-dependent expression.

DFR is involved in an important step in the flavonoid biosynthesis pathway upstream of anthocyanin, proanthocyanidin, and phlobaphene production, which contributes to the pigmentation of various plant tissues. Full genomic sequences of three DFRs were isolated in hexaploid wheat. Loci of TaDFRs were found in a more proximal region of the long arm of chromosomes of homoeologous group 3 than the R gene for red grain colour of wheat. These DFRs were designated TaDFR-A, TaDFR-B, and TaDFR-D on chromosome 3A, 3B, and 3D, respectively. In the 5' upstream region of DFR genes, two or three combinations of a G box core element and a putative binding site for a Myb-type transcription factor, P, of maize were found. Expression of DFR reached a maximal level in red grain of wheat cv. Chinese Spring (CS) at 5 d post-anthesis (DPA) and decreased gradually in the grain coat tissue from 10 to 20 DPA, in contrast to a very low expression level of DFR in white wheat grain during the same period. These DFRs differed in their expression. TaDFR-B and -D were expressed predominantly in grains. In developing leaves, DFR expression was light-responsive, and TaDFR-B was more up-regulated in leaves and roots than the other two.

Alcohol Oxidoreductases↗

PubMed2003

A selective sweep driven by pyrimethamine treatment in southeast asian malaria parasites.

Malaria parasites (Plasmodium falciparum) provide an excellent system in which to study the genomic effects of strong selection in a recombining eukaryote because the rapid spread of resistance to multiple drugs during the last the past 50 years has been well documented, the full genome sequence and a microsatellite map are now available, and haplotype data can be easily generated. We examined microsatellite variation around the dihydrofolate reductase (dhfr) gene on chromosome 4 of P. falciparum. Point mutations in dhfr are known to be responsible for resistance to the antimalarial drug pyrimethamine, and resistance to this drug has spread rapidly in Southeast (SE) Asia after its introduction in 1970s. We genotyped 33 microsatellite markers distributed across chromosome 4 in 61 parasites from a location on the Thailand/Myanmar border. We observed minimal microsatellite length variation in a 12-kb (0.7-cM) region flanking the dhfr gene and diminished variation for approximately 100 kb (6 cM), indicative of a single origin of resistant alleles. Furthermore, we found the same or similar microsatellite haplotypes flanked resistant dhfr alleles sampled from 11 parasite populations in five SE Asian countries indicating recent invasion of a single lineage of resistant dhfr alleles in locations 2000 km apart. Three features of these data are of especially interest. (1). Pyrimethamine resistance is generally assumed to have evolved multiple times because the genetic basis is simple and resistance can be selected easily in the laboratory. Yet our data clearly indicate a single origin of resistant dhfr alleles sampled over a large region of SE Asia. (2). The wide valley ( approximately 6 cM) of reduced variation around dhfr provides "proof-of-principle" that genome-wide association may be an effective way to locate genes under strong recent selection. (3). The width of the selective valley is consistent with predictions based on independent measures of recombination, mutation, and selection intensity, suggesting that we have reasonable estimates of these parameters. We conclude that scanning the malaria parasite genome for evidence of recent selection may prove an extremely effective way to locate genes underlying recently evolved traits such as drug resistance, as well as providing an opportunity to study the dynamics of selective events that have occurred recently or are currently in progress.

Alleles↗

PubMed2004

An evolutionary model for protein-coding regions with conserved RNA structure.

Here we present a model of nucleotide substitution in protein-coding regions that also encode the formation of conserved RNA structures. In such regions, apparent evolutionary context dependencies exist, both between nucleotides occupying the same codon and between nucleotides forming a base pair in the RNA structure. The overlap of these fundamental dependencies is sufficient to cause "contagious" context dependencies which cascade across many nucleotide sites. Such large-scale dependencies challenge the use of traditional phylogenetic models in evolutionary inference because they explicitly assume evolutionary independence between short nucleotide tuples. In our model we address this by replacing context dependencies within codons by annotation-specific heterogeneity in the substitution process. Through a general procedure, we fragment the alignment into sets of short nucleotide tuples based on both the protein coding and the structural annotation. These individual tuples are assumed to evolve independently, and the different tuple sets are assigned different annotation-specific substitution models shared between their members. This allows us to build a composite model of the substitution process from components of traditional phylogenetic models. We applied this to a data set of full-genome sequences from the hepatitis C virus where five RNA structures are mapped within the coding region. This allowed us to partition the effects of selection on different structural elements and to test various hypotheses concerning the relation of these effects. Of particular interest, we found evidence of a functional role of loop and bulge regions, as these were shown to evolve according to a different and more constrained selective regime than the nonpairing regions outside the RNA structures. Other potential applications of the model include comparative RNA structure prediction in coding regions and RNA virus phylogenetics.

Base Sequence↗

PubMed2001

Homologous recombination in GB virus C/hepatitis G virus.

Analysis of 33 GB virus C/hepatitis G virus (GBV-C/HGV) full or nearly full genome sequences revealed several putative inter- and intrasubtype recombinants. The breakpoints of the recombinant regions were mapped using a maximum-likelihood method, and the statistical significance for each region was tested using Monte Carlo simulation. The results were highly significant and provided evidence for the existence of complex mosaic genomes showing as many as nine recombination events, with breakpoints in the 5' UTR and in all of the coding regions except the short NS4b gene. Recombination was confirmed by separate phylogenetic analysis of the various recombinant regions and by Sawyer's runs test. Taken together, these findings demonstrate for the first time that recombination is common in natural populations of GBV-C and that it takes place both within and between subtypes. The wide-ranging implications of such nonclonal history for reconstructing the spread and timescale of GBV-C evolution are discussed.

5' Untranslated Regions↗

PubMed2002

A strong effect of AT mutational bias on amino acid usage in Buchnera is mitigated at high-expression genes.

The advent of full genome sequences provides exceptionally rich data sets to explore molecular and evolutionary mechanisms that shape divergence among and within genomes. In this study, we use multivariate analysis to determine the processes driving genome-wide patterns of amino usage in the obligate endosymbiont Buchnera and its close free-living relative Escherichia coli. In the AT-rich Buchnera genome, the primary source of variation in amino acid usage differentiates high- and low-expression genes. Amino acids of high-expression Buchnera genes are generally less aromatic and use relatively GC-rich codons, suggesting that selection against aromatic amino acids and against amino acids with AT-rich codons is stronger in high-expression genes. Selection to maintain hydrophobic amino acids in integral membrane proteins is a primary factor driving protein evolution in E. coli but is a secondary factor in Buchnera. In E. coli, gene expression is a secondary force driving amino acid usage, and a correlation with tRNA abundance suggests that translational selection contributes to this effect. Although this and previous studies demonstrate that AT mutational bias and genetic drift influence amino acid usage in Buchnera, this genome-wide analysis argues that selection is sufficient to affect the amino acid content of proteins with different expression and hydropathy levels.

Adenine↗

PubMed2006

A silent mutation (2939G>A, exon 6; CYP2D6*59) leading to impaired expression and function of CYP2D6.

We analyzed CYP2D6 in two individuals characterized by impaired sparteine oxidation (intermediate metabolizer phenotype) and genotype 2D62/4 (1661G>C; 2850C>T; 4180G>C) usually associated with normal function. Full genomic sequencing and haplotype analysis confirmed the previously identified silent mutation 2939G>A in exon 6 (former allele variant 2D62J, now termed 2D659), as well as an additional novel 2291G>A change in intron 4. Transient expression in Huh7 hepatoma cells of the entire CYP2D6 gene of constructs carrying either both or only the 2939G>A change resulted in about three-fold reduced levels of mRNA, immunoreactive 2D6 protein and propafenone hydroxylase activity. These data demonstrate profound effects of a silent mutation on expression and function of CYP2D6, resulting in impaired drug oxidation phenotype. The 2939G>A single nucleotide polymorphism in exon 6 was present heterozygously in two individuals out of 308 (0.65%), corresponding to an allele frequency of 0.3%. Genotyping for this mutation thus improves phenotype-genotype correlation for CYP2D6 and may help to predict adverse drug treatment events.

Base Sequence↗

PubMed2006

Orf virus infection and host immunity.

PURPOSE OF REVIEW: A summary of recent advances in our knowledge of the biology of orf virus is presented to illustrate the interaction of a zoonotic pathogen with host skin. This is providing novel and interesting data on the viral mechanism of skin infection and the host response. RECENT FINDINGS: The full genome sequences of two parapoxviruses (orf virus and bovine papular stomatitis virus) have recently been published, defining the parapoxvirus genus at the molecular genetic level. This, along with more detailed characterization of viral immuno-modulatory proteins, is providing an insight into the acquisition of host genes and the mechanism of pathogenesis. A new chemokine-binding protein has been discovered with unique features. Structure-function analysis of the viral granulocyte/macrophage colony-stimulating factor inhibitory factor has revealed a similarity to type 1 cytokine receptors. The viral vascular endothelial growth factor-E stimulates angiogenesis in the skin without the side effects seen with cellular vascular endothelial growth factor-A, and may have therapeutic potential. Finally, orf virus is proving useful both as an immuno-modulator and as a vector for the expression of foreign antigens in non-permissive species. SUMMARY: Orf virus infection provokes a vigorous skin immune response. However, the virus has acquired a range of immuno-modulatory/pathogenesis-related genes that function to limit (at least transiently) the effectiveness of host immunity. With the advent of the orf virus genome sequence, the study of this dynamic process will provide important insights into virus pathogenesis and the host skin immune response to infection.

Animals↗

PubMed2006

Evidence of ancient papillomavirus recombination.

An open question amongst papillomavirus taxonomists is whether recombination has featured in the evolutionary history of these viruses. Since the onset of the global AIDS epidemic, the question is somewhat less academic, because immune-compromised human immunodeficiency virus patients are often co-infected with extraordinarily diverse mixtures of human papillomavirus (HPV) types. It is expected that these conditions may facilitate the emergence of HPV recombinants, some of which might have novel pathogenic properties. Here, a range of rigorous analyses is applied to full-genome sequences of papillomaviruses to provide convincing statistical and phylogenetic evidence that evolutionarily relevant papillomavirus recombination can occur.

Evolution, Molecular↗

PubMed2001

cis element/transcription factor analysis (cis/TF): a method for discovering transcription factor/cis element relationships.

We report a simple new algorithm, cis/TF, that uses genomewide expression data and the full genomic sequence to match transcription factors to their binding sites. Most previous computational methods discovered binding sites by clustering genes having similar expression patterns and then identifying over-represented subsequences in the promoter regions of those genes. By contrast, cis/TF asserts that B is a likely binding site of a transcription factor T if the expression pattern of T is correlated to the composite expression patterns of all genes containing B, even when those genes are not mutually correlated. Thus, our method focuses on binding sites rather than genes. The algorithm has successfully identified experimentally-supported transcription factor binding relationships in tests on several data sets from Saccharomyces cerevisiae.

Algorithms↗

PubMed2005

Discovering functional transcription-factor combinations in the human cell cycle.

With the completion of full genome sequences and advancement in high-throughput technologies, in silico methods have been successfully used to integrate diverse data sources toward unraveling the combinatorial nature of transcriptional regulation. So far, almost all of these studies are restricted to lower eukaryotes such as budding yeast. We describe here a computational search for functional transcription-factor (TF) combinations using phylogenetically conserved sequences and microarray-based expression data. Taking into account both orientational and positional constraints, we investigated the overrepresentation of binding sites in the vicinity of one another and whether these combinations result in more coherent expression profiles. Without any prior biological knowledge, the search led to the discovery of several experimentally established TF associations, as well as some novel ones. In particular, we identified a regulatory module controlling cell cycle-dependent transcription of G2-M genes and expanded its functional generality. We also detected many homotypic combinations, supporting the importance of binding-site density in transcriptional regulation of higher eukaryotes.

G2 Phase↗

PubMed2004

Solution of the quasispecies model for an arbitrary gene network.

In this paper, we study the equilibrium behavior of Eigen's quasispecies equations for an arbitrary gene network. We consider a genome consisting of N genes, so that the full genome sequence sigma may be written as sigma= sigma1sigma2...sigmaN, where sigma(i) are sequences of individual genes. We assume a single fitness peak model for each gene, so that gene i has some "master" sequence sigma(i,0) for which it is functioning. The fitness landscape is then determined by which genes in the genome are functioning and which are not. The equilibrium behavior of this model may be solved in the limit of infinite sequence length. The central result is that, instead of a single error catastrophe, the model exhibits a series of localization to delocalization transitions, which we term an "error cascade." As the mutation rate is increased, the selective advantage for maintaining functional copies of certain genes in the network disappears, and the population distribution delocalizes over the corresponding sequence spaces. The network goes through a series of such transitions, as more and more genes become inactivated, until eventually delocalization occurs over the entire genome space, resulting in a final error catastrophe. This model provides a criterion for determining the conditions under which certain genes in a genome will lose functionality due to genetic drift. It also provides insight into the response of gene networks to mutagens. In particular, it suggests an approach for determining the relative importance of various genes to the fitness of an organism, in a more accurate manner than the standard "deletion set" method. The results in this paper also have implications for mutational robustness and what C.O. Wilke termed "survival of the flattest."

Animals↗

PubMed2005

Nitrogen deprivation stimulates symbiotic gland development in Gunnera manicata.

Gunnera is the only genus of angiosperms known to host cyanobacteria and the only group of land plants that hosts cyanobacteria intracellularly. Motile filaments of cyanobacteria, known as hormogonia, colonize Gunnera plants through cells in the plant's specialized stem glands. It is commonly held that Gunnera plants always possess functional glands for symbiosis. We found, however, that stem gland development did not occur when Gunnera manicata plants were grown on nitrogen (N)-replete medium but, rather, was initiated at predetermined positions when plants were deprived of combined N. While N status was the main determinant for gland development, an exogenous carbon source (sucrose) accelerated the process. Furthermore, a high level of sucrose stimulated the formation of callus-like tissue in place of the gland under N-replete conditions. Treatment of plants with the auxin transport inhibitor 1-naphthylphthalamic acid prevented gland development on N-limited medium, most likely by preventing resource reallocation from leaves to the stem. Optimized conditions were found for in vitro establishment of the Nostoc-Gunnera symbiosis by inoculating mature glands with hormogonia from Nostoc punctiforme, a cyanobacterium strain for which the full genome sequence is available. In contrast to uninoculated plants, G. manicata plants colonized by N. punctiforme were able to continue their growth on N-limited medium. Understanding the nature of the Gunnera plant's unusual adaptation to an N-limited environment may shed light on the evolution of plant-cyanobacterium symbioses and may suggest a route to establish productive associations between N-fixing cyanobacteria and crop plants.

Biological Transport, Active↗

PubMed2006

Plant metabolomics: from holistic hope, to hype, to hot topic.

In a short time, plant metabolomics has gone from being just an ambitious concept to being a rapidly growing, valuable technology applied in the stride to gain a more global picture of the molecular organization of multicellular organisms. The combination of improved analytical capabilities with newly designed, dedicated statistical, bioinformatics and data mining strategies, is beginning to broaden the horizons of our understanding of how plants are organized and how metabolism is both controlled but highly flexible. Metabolomics is predicted to play a significant, if not indispensable role in bridging the phenotype-genotype gap and thus in assisting us in our desire for full genome sequence annotation as part of the quest to link gene to function. Plants are a fabulously rich source of diverse functional biochemicals and metabolomics is also already proving valuable in an applied context. By creating unique opportunities for us to interrogate plant systems and characterize their biochemical composition, metabolomics will greatly assist in identifying and defining much of the still unexploited biodiversity available today.

Biotechnology↗

PubMed2004

Multiple polymorphic loci for molecular typing of strains of Mycobacterium leprae.

The need for molecular tools for the differentiation of isolates of Mycobacterium leprae, the organism that causes leprosy, is urgent in view of the continuing high levels of new case detection, despite years of aggressive chemotherapy and the consequent reduction in the prevalence of leprosy. The slow onset of leprosy and the reliance on physical examination for detection of disease have restricted the epidemiological tracking necessary to understand and control transmission. Two genetic loci in several isolates of M. leprae have previously been demonstrated to contain variable-number tandem repeats (VNTRs). On the basis of these reports and the availability of the full genome sequence, multiple-locus VNTR analysis for strain typing has been undertaken. A panel of 11 short tandem repeat (STR) loci with repeat units of 1, 2, 3, 6, 12, 18, 21, and 27 bp from four clinical isolates of M. leprae propagated in armadillo hosts were screened by PCR. Fragment length polymorphisms were detected at 9 of the 11 loci by agarose gel electrophoresis. Sequencing of representative DNA products confirmed the presence of VNTRs between isolates. The application of nine new polymorphic STRs in conjunction with automated methods for electrophoresis and size determination allows greater discrimination between isolates of M. leprae and enhances the potential of this technique to track the transmission of leprosy.

Animals↗

PubMed2006

Resistance to alpha/beta interferon is a determinant of West Nile virus replication fitness and virulence.

The emergence of West Nile virus (WNV) in the Western Hemisphere is marked by the spread of pathogenic lineage I strains, which differ from typically avirulent lineage II strains. To begin to understand the virus-host interactions that may influence the phenotypic properties of divergent lineage I and II viruses, we compared the genetic, pathogenic, and alpha/beta interferon (IFN-alpha/beta)-regulatory properties of a lineage II isolate from Madagascar (MAD78) with those of a new lineage I isolate from Texas (TX02). Full genome sequence analysis revealed that MAD78 clustered, albeit distantly, with other lineage II strains, while TX02 clustered with emergent North American isolates, more specifically with other Texas strains. Compared to TX02, MAD78 replicated at low levels in cultured human cells, was highly sensitive to the antiviral actions of IFN in vitro, and demonstrated a completely avirulent phenotype in wild-type mice. In contrast to TX02 and other pathogenic forms of WNV, MAD78 was defective in its ability to disrupt IFN-induced JAK-STAT signaling, including the activation of Tyk2 and downstream phosphorylation and nuclear translocation of STAT1 and STAT2. However, replication of MAD78 was rescued in cells with a nonfunctional IFN-alpha/beta receptor (IFNAR). Consistent with this finding, the virulence of MAD78 was unmasked upon infection of mice lacking IFNAR. Thus, control of the innate host response and IFN actions is a key feature of WNV pathogenesis and replication fitness.

Animals↗

PubMed1999

Dengue virus structural differences that correlate with pathogenesis.

The understanding of dengue virus pathogenesis has been hampered by the lack of in vitro and in vivo models of disease. The study of viral factors involved in the production of severe dengue, dengue hemorrhagic fever (DHF), versus the more common dengue fever (DF), have been limited to indirect clinical and epidemiologic associations. In an effort to identify viral determinants of DHF, we have developed a method for comparing dengue type 2 genomes (reverse transcriptase PCR in six fragments) directly from patient plasma. Samples for comparison were selected from two previously described dengue type 2 genotypes which had been shown to be the cause of DF or DHF. When full genome sequences of 11 dengue viruses were analyzed, several structural differences were seen consistently between those associated with DF only and those with the potential to cause DHF: a total of six encoded amino acid charge differences were seen in the prM, E, NS4b, and NS5 genes, while sequence differences observed within the 5' nontranslated region (NTR) and 3' NTR were predicted to change RNA secondary structures. We hypothesize that the primary determinants of DHF reside in (i) amino acid 390 of the E protein, which purportedly alters virion binding to host cells; (ii) in the downstream loop (nucleotides 68 to 80) of the 5' NTR, which may be involved in translation initiation; and (iii) in the upstream 300 nucleotides of the 3' NTR, which may regulate viral replication via the formation of replicative intermediates. The significance of four amino acid differences in the nonstructural proteins NS4b and NS5, a presumed transport protein and the viral RNA polymerase, respectively, remains unknown. This new approach to the study of dengue virus genome differences should better reflect the true composition of viral RNA populations in the natural host and permit their association with pathogenesis.

3' Untranslated Regions↗

PubMed2003

Mosaic genomes of the six major primate lentivirus lineages revealed by phylogenetic analyses.

To clarify the origin and evolution of the primate lentiviruses (PLVs), which include human immunodeficiency virus types 1 and 2 as well as their simian relatives, simian immunodeficiency viruses (SIVs), isolated from several host species, we investigated the phylogenetic relationships among the six supposedly nonrecombinant PLV lineages for which the full genome sequences are available. Employing bootscanning as an exploratory tool, we located several regions in the PLV genome that seem to have uncertain or conflicting phylogenetic histories. Phylogeny reconstruction based on distance and maximum-likelihood algorithms followed by a number of statistical tests confirms the existence of at least five putative recombinant fragments in the PLV genome with different clustering patterns. Split decomposition analysis also shows that phylogenetic relationships among PLVs may be better represented by network-based graphs, such as the ones produced by SplitsTree. Our findings not only imply that the six so-called pure PLV lineages have in fact mosaic genomes but also make more unlikely the hypothesis of cospeciation of SIVs and their simian hosts.

Algorithms↗