Contrasting Patterns of Sequence Evolution at the Functionally Redundant <Emphasis Type="Italic">bric à brac</Emphasis> Paralogs in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Genes with overlapping expression and function may gradually diverge despite retaining some common functions. To test whether such genes show distinct patterns of molecular evolution within species, we examined sequence variation at the bric à brac (bab) locus of Drosophila melanogaster. This locus is composed of two anciently duplicated paralogs, bab1 and bab2, which are involved in patterning the adult abdomen, legs, and ovaries. We have sequenced the 148 kb genomic region spanning the bab1 and bab2 genes from 94 inbred lines of D. melanogaster sampled from a single location. Two non-coding regions, one in each paralog, appear to be under selection. The strongest evidence of directional selection is found in a region of bab2 that has no known functional role. The other region is located in the bab1 paralog and is known to contain a cis-regulatory element that controls sex-specific abdominal pigmentation. The coding region of bab1 appears to be under stronger functional constraint than the bab2 coding sequences. Thus, the two paralogs are evolving under different selective regimes in the same natural population, illuminating the different evolutionary trajectories of partially redundant duplicate genes.     

Prevalence and Acquisition of the Genes for Zoocin A and Zoocin A Resistance in <Emphasis Type="Italic">Streptococcus equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Zoocin A is a streptococcolytic enzyme produced by Streptococcus equi subsp. zooepidemicus strain 4881. The zoocin A gene (zooA) and the gene specifying resistance to zoocin A (zif) are adjacent on the chromosome and are divergently transcribed. Twenty-four S. equi subsp. zooepidemicus strains were analyzed to determine the genetic difference among three previously characterized as zoocin A producers (strains 4881, 9g, and 9h) and the 21 nonproducers. LT-PCR and Southern hybridization studies revealed that none of the nonproducer strains possessed zooA or zif. RAPD and PFGE showed that the 24 strains were a genetically diverse population with eight RAPD profiles. S. equi subsp. zooepidemicus strains 9g and 9h appeared to be genetically identical to each other but quite different from strain 4881. Sequences derived from 4881 and 9g showed that zooA and zif were integrated into the chromosome adjacent to the gene flaR. A comparison of these sequences with the genome sequences of S. equi subsp. zooepidemicus strains H70 and MGCS10565 and S. equi subsp. equi strain 4047 suggests that flaR flanks a region of genome plasticity in this species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation of a <Emphasis Type="Italic">Ve</Emphasis> homolog, <Emphasis Type="Italic">mVe1</Emphasis>, and its relationship to verticillium wilt resistance in <Emphasis Type="Italic">Mentha longifolia</Emphasis> (L.) Huds

As a step toward greater understanding of the genetics of verticillium wilt resistance in plants, we report the sequencing of a candidate wilt resistance gene, mVe1, from the mint diploid model species, Mentha longifolia (Lamiaceae). mVe1 is a putative homolog of tomato (Solanum lycopersicum L.) verticillium wilt (Ve) resistance genes. The mVe1 gene has a coding region of 3,051 bp. The predicted mVe1 protein contains a leucine-rich repeat domain, a common feature of plant disease resistance proteins. We compared 13 mVe1 alleles from three mint species. These alleles shared 96.2–99.6% nucleotide identity. We analyzed four M. longifolia populations segregating with respect to mVe1 alleles and wilt resistance versus susceptibility and found one association between mVe1 genotype and wilt phenotype. We conclude that mVe1 may play a role in mint verticillium wilt resistance, but variation for resistance in our segregating progenies is likely polygenic. Therefore, further investigations of mVe1 and identification of additional candidate genes are both warranted.     

Association Between Levels of Coding Sequence Divergence and Gene Misregulation in <Emphasis Type="Italic">Drosophila</Emphasis> Male Hybrids

Previous studies have shown widespread conservation of gene expression levels between species of the Drosophila melanogaster subgroup as well as a positive correlation between coding sequence divergence and expression level divergence between species. Meanwhile, large-scale misregulation of gene expression level has been described in interspecific sterile hybrids between D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. Using data from gene expression analysis involving D. simulans, D. melanogaster, and their hybrids, we observed a significant positive correlation between protein sequence divergence and gene expression differences between hybrids and their parental species. Furthermore, we demonstrate that underexpressed misregulated genes in hybrids are evolving more rapidly at the protein sequence level than nonmisregulated genes or overexpressed misregulated genes, highlighting the possible effects of sexual and natural selection as male-biased genes and nonessential genes are the principal gene categories affected by interspecific hybrid misregulation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Carlo G. Artieri and Wilfried Haerty contributed equally to this publication.     

Comparative analysis of vertebrate <Emphasis Type="Italic">PEPT1</Emphasis> and <Emphasis Type="Italic">PEPT2</Emphasis> genes

The plasma membrane transport proteins belong to SoLute Carrier 15 (SLC15) family and two members of this family have been characterized extensively in higher vertebrates, namely PEPT1 and PEPT2. Despite many efforts have made to define a pharmacophore model for efficient binding and transporting of substrates, there is not a comprehensive study performed to elucidate the evolutionary mechanisms among the SLC15 family members and to statistically evaluate sequence conservation and functional divergence between members. In this study, we compared and contrasted the rates and patterns of molecular evolution of 2 PEPT genes. Phylogenetic tree assembly with all available vertebrate PEPTs suggests that the PEPTs originated by duplications and diverged from a common protein at the base of the eukaryotic tree. Topological structure demonstrates both members share the similar hydrophobic domains (TMDs), which have been constrained by purifying selection. Although both genes show qualitatively similar patterns, their rates of evolution differ significantly due to an increased rate of synonymous substitutions in the structural domains in one copy, suggesting substantial differences in functional constraint on each gene. Site-specific profiles were established by posterior probability analysis revealing significantly divergent regions mainly locate at the hydrophobic region between predicted transmembrane domains 9 and 10 of the proteins. Thus, these results provide the evidence that several amino acid residues with reduced selective constraints are largely responsible for functional divergence between the paralogous PEPTs. These findings may provide a starting point for further experimental verifications.     

Submolecular unfolding units of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis>-551

Hydrogen exchange rates for backbone amide protons of oxidized Pseudomonas aeruginosa cytochrome c-551 (P. aeruginosa cytochrome c) have been measured in the presence of low concentrations of the denaturant guanidine hydrochloride. Analysis of the data has allowed identification of submolecular unfolding units known as foldons. The highest-energy foldon bears similarity to the proposed folding intermediate for P. aeruginosa cytochrome c. Parallels are seen to the foldons of the structurally homologous horse cytochrome c, although the heme axial methionine-bearing loop has greater local stability in P. aeruginosa cytochrome c, in accord with previous folding studies. Regions of low local stability are observed to correspond with regions that interact with redox partners, providing a link between foldon properties and function. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Overexpression of the Chitosanase Gene in <Emphasis Type="Italic">Fusarium solani</Emphasis> via <Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-Mediated Transformation

To overexpress the chitosanase gene (csn) in F. solani, a vector based on pCAMBIA 1300 was constructed. The csn gene, which is under control of the Aspergillus nidulans gpdA promoter and A. nidulans trpC terminator, was introduced back into the F. solani genome by Agrobacterium tumefaciens-mediated transformation, and the herbicide-resistance gene bar from Streptomyces hygroscopicus was used as the selection marker. Transformants which showed a significant increase in chitosanase production (~2.1-fold than control) were obtained. Southern blot analysis indicated that most transformants had a single-copy T-DNA integration.     

Gene Conversion and Positive Selection Driving the Evolution of the <Emphasis Type="Italic">Caenorhabditis</Emphasis> ssp. ZIM/HIM-8 Protein Family

In C. elegans, four C2H2 zinc-finger proteins (ZIM-1, ZIM-2, ZIM-3, and HIM-8), which are arranged in tandem, mediate chromosome-specific pairing and synapsis during meiosis. The zim/him-8 genes from three Caenorhabditis species were contrasted in an effort to investigate the mechanisms driving their evolution. Here it is shown that the preservation of higher degree of sequence similarity in the N-terminal portion, particularly in several regions within the second exon between paralogous zim genes (especially between zim-1 and zim-3), is due to independent interparalogue gene conversions. However, the evolutionary force is not uniformly strong across species. The present data reveal that more frequent gene conversion events have occurred in C. elegans, whereas only gene conversions between zim-1 and zim-3 are detected in C. remanei. Although gene conversions are predicted to be present among zim-1, zim-2, and zim-3 in C. briggsae, the conversion tracts between zim-1/zim-2 and zim-2/zim-3 are very short. Moreover, positive selection analysis was performed on the basis of the significantly discordant phylogenies reconstructed using the N- and C-terminal sequences, respectively. Several codon sites located in the regions that are supposed not to have experienced gene conversions are predicted to be under the influence of positive selection. In comparison, stronger positive selection has acted on the C-terminal region relative to the N-terminal region. Thus, the zim/him-8 genes that evolve concertedly have also been shown to undergo adaptive diversifying selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Genetic determination of wheat resistance against <Emphasis Type="Italic">Puccinia graminis</Emphasis> (f. sp. <Emphasis Type="Italic">Tritici</Emphasis>) derived from <Emphasis Type="Italic">Aegilops cylindrica,Triticum erebuni</Emphasis>, and amphidiploid 4

Lines of winter soft wheat with introgressed new and effective Sr genes were developed as a result of interspecies hybridization at the Plant Breeding and Genetics Institute. The 85/06 line possesses the SrAc1 gene; the 47/06, 54/06, 82/06, 85/06, 87/06, 238/06, and 367/06 lines carry the SrAc1 and SrAc2 genes originated from Aegilops cylindrica; the 352/06 line has the SrTe1 and SrTe2 genes from Triticum erebuni; and the 12/86-04 line contains the SrAd1 and SrAd2 genes from Amphidipoid 4 (Triticum dicoccoides × Triticum tauschii).     

Multiple alleles at <Emphasis Type="Italic">Early flowering 1</Emphasis> locus making variation in the basic vegetative growth period in rice (<Emphasis Type="Italic">Oryza</Emphasis> <Emphasis Type="Italic">sativa</Emphasis> L.)

A recently established rice breeding program in low latitudes aims to develop varieties with extremely long basic vegetative growth (BVG) periods and weak photoperiod sensitivities. The Taiwanese japonica variety Taichung 65 (T65) harbors a recessive allele ef1 at the Ef1 (Early flowering 1) locus, thereby exhibiting an extremely long BVG period. The previous reported functional allele Ehd1 (Early heading date 1), located on chromosome 10, encodes a B-type response regulator, thereby shortening the BVG period, whereas its nonfunctional allele ehd1 greatly prolongs the BVG period. A conventional analysis using F₂ and F₃ populations and a subsequent CAPS analysis based on the amino acid sequences of Ehd1 and ehd1 showed that Ef1 and Ehd1 were at the same locus. The CAPS analysis also indicated that the Taiwanese japonica varieties with extremely long BVG periods all harbor ef1, but that ef1 does not exist among indica and japonica varieties in the low latitudes. Since ef1 has not been found in any japonica varieties outside Taiwan, this allele might have originated in Taiwan. Sequence analysis revealed that the mutant allele ef1-h, which prolongs the BVG period even more than ef1 does, harbors an mPing insertion in exon 2, which causes the complete loss of gene function. Our results indicate that both ef1 or ef1-h alleles can be used as new gene sources in developing rice varieties with extremely long BVG periods for low latitudes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evolutionary dynamics of the human <Emphasis Type="Italic">ABO</Emphasis> gene

The ABO polymorphism has long been suspected to be under balancing selection. To explore this possibility, we analyzed two datasets: (1) a set of 94 23-Kb sequences in European- and African-Americans produced by the Seattle SNPs project, and (2) a set of 814 2-Kb sequences in O alleles from seven worldwide populations. A phylogenetic analysis of the Seattle sequences showed a complex pattern in which the action of recombination and gene conversion are evident, and in which four main lineages could be individuated. The sequence patterns could be linked to the expected blood group phenotype; in particular, the main mutation giving rise to the null O allele is likely to have appeared at least three times in human evolution, giving rise to allele lineages O02, O01, and O09. However, the genealogy changes along the gene and variations of both numbers of branches and of their time depth were observed, which could result from a combined action of recombination and selection. Several neutrality tests clearly demonstrated deviations compatible with balancing selection, peaking at several locations along the gene. The time depth of the genealogy was also incompatible with neutral evolution, particularly in the region from exons 6 to 7, which codes for most of the catalytic domain. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulatory Evolution of a Duplicated Heterodimer Across Species and Tissues of Allopolyploid Clawed Frogs (<Emphasis Type="Italic">Xenopus</Emphasis>)

Changes in gene expression contribute to reproductive isolation of species, adaptation, and development and may impact the genetic fate of duplicated genes. African clawed frogs (genus Xenopus) offer a useful model for examining regulatory evolution, particularly after gene duplication, because species in this genus are polyploid. Additionally, these species can produce viable hybrids, and expression divergence between coexpressed species-specific alleles in hybrids can be attributed exclusively to cis-acting mechanisms. Here we have explored expression divergence of a duplicated heterodimer composed of the recombination activating genes 1 and 2 (RAG1 and RAG2). Previous work identified a phylogenetically biased pattern of pseudogenization of RAG1 wherein one duplicate—RAG1β—was more likely to become a pseudogene than the other one—RAG1α. In this study we show that ancestral expression divergence between these duplicates could account for this. Using comparative data we demonstrate that regulatory divergence between species and between duplicated genes varies significantly across tissue types. These results have implications for understanding of variables that influence pseudogenization of duplicated genes generated by polyploidization, and for interpretation of the relative contributions of cis versus trans mechanisms to expression divergence at the cellular level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular evolution of <Emphasis Type="Italic">PKD2</Emphasis> gene family in mammals

PKD2 gene encodes a critical cation channel protein that plays important roles in various developmental processes and is usually evolutionarily conserved. In the present study, we analyzed the evolutionary patterns of PKD2 and its homologous genes (PKD2L1, PKD2L2) from nine mammalian species. In this study, we demonstrated the orthologs of PKD2 gene family evolved under a dominant purifying selection force. Our results in combination with the reported evidences from functional researches suggested the entire PKD2 gene family are conserved and perform essential biological roles during mammalian evolution. In rodents, PKD2 gene family members appeared to have evolved more rapidly than other mammalian lineages, probably resulting from relaxation of purifying selection. However, positive selection imposed on synonymous sites also potentially contributed to this case. For the paralogs, our results implied that PKD2L2 genes evolved under a weaker purifying selection constraint than PKD2 and PKD2L1 genes. Interestingly, some loop regions of transmembrane domain of PKD2L2 exhibited higher P _N/P _S ratios than expected, suggesting these regions are more functional divergent in organisms and worthy of special attention. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chun Ye, Huan Sun have contributed equally to this work.     

Regions associated with repression of the barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) <Emphasis Type="Italic">VERNALIZATION1</Emphasis> gene are not required for cold induction

Activity of the VERNALIZATION1 (VRN1) gene is required for flowering in temperate cereals such as wheat and barley. In varieties that require prolonged exposure to cold to flower (vernalization), VRN1 is expressed at low levels and is induced by vernalization to trigger flowering. In other varieties, deletions or insertions in the first intron of the VRN1 gene are associated with increased VRN1 expression in the absence of cold treatment, reducing or eliminating the requirement for vernalization. To characterize natural variation in VRN1, the first intron of the barley (Hordeum vulgare) VRN1 gene (HvVRN1) was assayed for deletions or insertions in a collection of 1,000 barleys from diverse geographical regions. Ten alleles of HvVRN1 containing deletions or insertions in the first intron were identified, including three alleles that have not been described previously. Different HvVRN1 alleles were associated with differing levels of HvVRN1 expression in non-vernalized plants and with different flowering behaviour. Using overlapping deletions, we delineated regions in the HvVRN1 first intron that are associated with low levels of HvVRN1 expression in non-vernalized plants. Deletion of these intronic regions does not prevent induction of HvVRN1 by cold or the maintenance of increased HvVRN1 expression following cold treatment. We suggest that regions within the first intron of HvVRN1 are required to maintain low levels of HvVRN1 expression prior to winter but act independently of the regulatory mechanisms that mediate induction of HvVRN1 by cold during winter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers 1179825, 1179833, 1179836, 1179858.     

Functional analysis and subcellular localization of two geranylgeranyl diphosphate synthases from <Emphasis Type="Italic">Penicillium paxilli</Emphasis>

The filamentous fungus Penicillium paxilli contains two distinct geranylgeranyl diphosphate (GGPP) synthases, GgsA and GgsB (PaxG). PaxG and its homologues in Neotyphodium lolii and Fusarium fujikuroi are associated with diterpene secondary metabolite gene clusters. The genomes of other filamentous fungi including Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae and Fusarium graminearum also contain two or more copies of GGPP synthase genes, although the diterpene metabolite capability of these fungi is not known. The objective of this study was to understand the biological significance of the presence of two copies of GGPP synthases in P. paxilli by investigating their subcellular localization. Using a carotenoid complementation assay and gene deletion analysis, we show that P. paxilli GgsA and PaxG have GGPP synthase activities and that paxG is required for paxilline biosynthesis, respectively. In the ΔpaxG mutant background ggsA was unable to complement paxilline biosynthesis. A GgsA-EGFP fusion protein was localized to punctuate organelles and the EGFP-GRV fusion protein, containing the C-terminus tripeptide GRV of PaxG, was localized to peroxisomes. A truncated PaxG mutant lacking the C-terminus tripeptide GRV was unable to complement a ΔpaxG mutant demonstrating that the tripeptide is functionally important for paxilline biosynthesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.