Orthologous Pairs Research Articles

Of Mice and Men: Divergence of Gene Expression Patterns in Kidney

Since the development of methods for homologous gene recombination, mouse models have played a central role in research in renal pathophysiology. However, many published and unpublished results show that mice with genetic changes mimicking human pathogenic mutations do not display the human phenotype. These functional differences may stem from differences in gene expression between mouse and human kidneys. However, large scale comparison of gene expression networks revealed conservation of gene expression among a large panel of human and mouse tissues including kidneys. Because renal functions result from the spatial integration of elementary processes originating in the glomerulus and the successive segments constituting the nephron, we hypothesized that differences in gene expression profiles along the human and mouse nephron might account for different behaviors. Analysis of SAGE libraries generated from the glomerulus and seven anatomically defined nephron segments from human and mouse kidneys allowed us to identify 4644 pairs of gene orthologs expressed in either one or both species. Quantitative analysis shows that many transcripts are present at different levels in the two species. It also shows poor conservation of gene expression profiles, with less than 10% of the 4644 gene orthologs displaying a higher conservation of expression profiles than the neutral expectation (p<0.05). Accordingly, hierarchical clustering reveals a higher degree of conservation of gene expression patterns between functionally unrelated kidney structures within a given species than between cognate structures from the two species. Similar findings were obtained for sub-groups of genes with either kidney-specific or housekeeping functions. Conservation of gene expression at the scale of the whole organ and divergence at the level of its constituting sub-structures likely account for the fact that although kidneys assume the same global function in the two species, many mouse “models” of human pathologies do not display the expected phenotype.

In Silico Classification of Proteins from Acidic and Neutral Cytoplasms

Protein acidostability is a common problem in biopharmaceutical and other industries. However, it remains a great challenge to engineer proteins for enhanced acidostability because our knowledge of protein acidostabilization is still very limited. In this paper, we present a comparative study of proteins from bacteria with acidic (AP) and neutral cytoplasms (NP) using an integrated statistical and machine learning approach. We construct a set of 393 non-redundant AP-NP ortholog pairs and calculate a total of 889 sequence based features for these proteins. The pairwise alignments of these ortholog pairs are used to build a residue substitution propensity matrix between APs and NPs. We use Gini importance provided by the Random Forest algorithm to rank the relative importance of these features. A scoring function using the 10 most significant features is developed and optimized using a hill climbing algorithm. The accuracy of the score function is 86.01% in predicting AP-NP ortholog pairs and is 76.65% in predicting non-ortholog AP-NP pairs, suggesting that there are significant differences between APs and NPs which can be used to predict relative acidostability of proteins. The overall trends uncovered in the study can be used as general guidelines for designing acidostable proteins. To best of our knowledge, this work represents the first systematic comparative study of the acidostable proteins and their non-acidostable orthologs.

The Diversity of Class II Transposable Elements in Mammalian Genomes Has Arisen from Ancestral Phylogenetic Splits during Ancient Waves of Proliferation through the Genome

DNA transposons make up 3% of the human genome, approximately the same percentage as genes. However, because of their inactivity, they are often ignored in favor of the more abundant, active, retroelements. Despite this relative ignominy, there are a number of interesting questions to be asked of these transposon families. One particular question relates to the timing of proliferation and inactivation of elements in a family. Does an ongoing process of turnover occur, or is the process more akin to a life cycle for the family, with elements proliferating rapidly before deactivation at a later date? We answer this question by tracing back to the most recent common ancestor (MRCA) of each modern transposon family, using two different methods. The first method identifies the MRCA of the species in which a family of transposon fossils can still be found, which we assume will have existed soon after the true origin date of the transposon family. The second method uses molecular dating techniques to predict the age of the MRCA element from which all elements found in a modern genome are descended. Independent data from five pairs of species are used in the molecular dating analysis: human–chimpanzee, human–orangutan, dog–panda, dog–cat, and cow–pig. Orthologous pairs of elements from host species pairs are included, and the divergence dates of these species are used to constrain the analysis. We discover that, in general, the times to element common ancestry for a given family are the same for the different species pairs, suggesting that there has been no order-specific process of turnover. Furthermore, for most families, the ages of the common ancestor of the host species and of that of the elements are similar, suggesting a life cycle model for the proliferation of transposons. Where these two ages differ, in families found only in Primates and Rodentia, for example, we find that the host species date is later than that of the common ancestor of the elements, implying that there may be large deletions of elements from host species, examples of which were found in their ancestors.

Arabidopsis Molybdopterin Biosynthesis Protein Cnx5 Collaborates with the Ubiquitin-like Protein Urm11 in the Thio-modification of tRNA

The thio-modification of tRNA that occurs in virtually all organisms affects the accuracy and efficiency of protein translation and is therefore biologically important. However, the molecular mechanism responsible for this tRNA modification in plants is largely unclear. We demonstrate here that Arabidopsis sulfurtransferase Cnx5, a ubiquitin-activating enzyme-like (UBA) protein involved in molybdopterin (MPT) biosynthesis, is strictly required for the thio-modification of cytosolic tRNAs in vivo. A previously uncharacterized ubiquitin-like (Ubl) protein Urm11 is also essential for tRNA thio-modification in Arabidopsis. When expressed in Saccharomyces cerevisiae, Cnx5 and Urm11 can substitute for the corresponding yeast orthologs ScUba4 and ScUrm1, respectively, in the thio-modification of yeast cytosolic tRNAs. However, another Ubl protein, Cnx7 of Arabidopsis, which is involved in MPT biosynthesis in conjunction with Cnx5, cannot replace yeast ScUrm1. Interestingly, the expression of a mutant form of Cnx7 in which the carboxyl-terminal six amino acids are substituted by those of Urm11 can significantly restore the thio-modification of tRNAs in the yeast urm1Δ mutant. These findings suggest that in Arabidopsis the common UBA protein Cnx5 collaborates with two functionally differentiated Ubl proteins, Urm11 and Cnx7, in the thio-modification of tRNA and MPT biosynthesis, respectively. Phylogenetic analysis revealed that although most eukaryotes contained a Cnx5-Urm11 ortholog pair and the tRNA thio-modification some fungi, including S. cerevisiae, had lost the Cnx7 ortholog and the ability to synthesize the molybdenum cofactor.

Functional evolution of mammalian odorant receptors.

The mammalian odorant receptor (OR) repertoire is an attractive model to study evolution, because ORs have been subjected to rapid evolution between species, presumably caused by changes of the olfactory system to adapt to the environment. However, functional assessment of ORs in related species remains largely untested. Here we investigated the functional properties of primate and rodent ORs to determine how well evolutionary distance predicts functional characteristics. Using human and mouse ORs with previously identified ligands, we cloned 18 OR orthologs from chimpanzee and rhesus macaque and 17 mouse-rat orthologous pairs that are broadly representative of the OR repertoire. We functionally characterized the in vitro responses of ORs to a wide panel of odors and found similar ligand selectivity but dramatic differences in response magnitude. 87% of human-primate orthologs and 94% of mouse-rat orthologs showed differences in receptor potency (EC50) and/or efficacy (dynamic range) to an individual ligand. Notably dN/dS ratio, an indication of selective pressure during evolution, does not predict functional similarities between orthologs. Additionally, we found that orthologs responded to a common ligand 82% of the time, while human OR paralogs of the same subfamily responded to the common ligand only 33% of the time. Our results suggest that, while OR orthologs tend to show conserved ligand selectivity, their potency and/or efficacy dynamically change during evolution, even in closely related species. These functional changes in orthologs provide a platform for examining how the evolution of ORs can meet species-specific demands.

Structural homology in the Solanaceae: analysis of genomic regions in support of synteny studies in tomato, potato and pepper

We have analysed the structural homology in euchromatin regions of tomato, potato and pepper with special attention for the long arm of chromosome 2 (2L). Molecular organization and colinear junctions were delineated using multi-color BAC FISH analysis and comparative sequence alignment. We found large-scale rearrangements including inversions and segmental translocations that were not reported in previous comparative studies. Some of the structural rearrangements are specific for the tomato clade, and differentiate tomato from potato, pepper and other Solanaceous species. Although local gene vicinity is largely preserved, there are many small-scale synteny perturbations. Gene adjacency in the aligned segments was frequently disrupted for 47% of the ortholog pairs as a result of gene and LTR retrotransposon insertions, and occasionally by single gene inversions and translocations. Our data also suggests that long distance intra-chromosomal rearrangements and local gene rearrangements have evolved frequently during speciation in the Solanum genus, and that small changes are more prevalent than large-scale differences. The occurrence of sonata and harbinger transposable elements and other repeats near or at junction breaks is considered in the light of repeat-mediated rearrangements and a reconstruction scenario for an ancestral 2L topology is discussed.

Transcriptome sequencing of wild chickpea as a rich resource for marker development

The transcriptome of cultivated chickpea (Cicer arietinum L.), an important crop legume, has recently been sequenced. Here, we report sequencing of the transcriptome of wild chickpea, C.reticulatum (PI489777), the progenitor of cultivated chickpea, by GS-FLX 454 technology. The optimized assembly of C.reticulatum transcriptome generated 37 265 transcripts in total with an average length of 946 bp. A total of 4072 simple sequence repeats (SSRs) could be identified in these transcript sequences, of which at least 561 SSRs were polymorphic between C.arietinum and C.reticulatum. In addition, a total of 36 446 single-nucleotide polymorphisms (SNPs) were identified after optimization of probability score, quality score, read depth and consensus base ratio. Several of these SSRs and SNPs could be associated with tissue-specific and transcription factor encoding transcripts. A high proportion (92-94%) of polymorphic SSRs and SNPs identified between the two chickpea species were validated successfully. Further, the estimation of synonymous substitution rates of orthologous transcript pairs suggested that the speciation event for divergence of C.arietinum and C.reticulatum may have happened approximately 0.53 million years ago. The results of our study provide a rich resource for exploiting genetic variations in chickpea for breeding programmes.

Functionally Orthologous Viral and Cellular MicroRNAs Studied by a Novel Dual-Fluorescent Reporter System

Recent research raised the possibility that some viral microRNAs (miRNAs) may function as orthologs of cellular miRNAs. In the present work, to study the functional orthologous relationships of viral and cellular miRNAs, we first constructed a dual-fluorescent protein reporter vector system for the easy determination of miRNA function. By expressing the miRNAs and the indicator and internal control fluorescent proteins individually from a single vector, this simple reporter system can be used for miRNA functional assays that include visualizing miRNA activity in live cells. Sequence alignments indicated that the simian virus 40 (SV40) encoded miRNA sv40-mir-S1-5p contains a seed region identical to that of the human miRNA hsa-miR423-5p. Using the new reporter system, it was found that sv40-mir-S1-5p and hsa-miR423-5p downregulate the expression of common artificial target mRNAs and some predicted biological targets of hsa-miR423-5p, demonstrating that they are functional orthologs. The human immunodeficiency virus 1 (HIV-1) encoded hiv1-miR-N367 also contains a seed sequence identical to that of the human miRNA hsa-miR192. Functional assays showed that hiv1-miR-N367 and hsa-miR192 could downregulate common artificial and predicted biological targets, suggesting that these miRNAs may also act as functional orthologs. Thus, this study presents a simple and universal system for testing miRNA function and identifies two new pairs of functional orthologs, sv40-mir-S1-5p and hsa-miR423-5p as well as hiv-1-miR-N367 and hsa-miR192. These findings also expand upon our current knowledge of functional homology and imply that a more general phenomenon of orthologous relationships exists between viral and cellular miRNAs.

Alternative Splicing of a Multi-Drug Transporter from Pseudoperonospora cubensis Generates an RXLR Effector Protein That Elicits a Rapid Cell Death

Pseudoperonospora cubensis, an obligate oomycete pathogen, is the causal agent of cucurbit downy mildew, a foliar disease of global economic importance. Similar to other oomycete plant pathogens, Ps. cubensis has a suite of RXLR and RXLR-like effector proteins, which likely function as virulence or avirulence determinants during the course of host infection. Using in silico analyses, we identified 271 candidate effector proteins within the Ps. cubensis genome with variable RXLR motifs. In extending this analysis, we present the functional characterization of one Ps. cubensis effector protein, RXLR protein 1 (PscRXLR1), and its closest Phytophthora infestans ortholog, PITG_17484, a member of the Drug/Metabolite Transporter (DMT) superfamily. To assess if such effector-non-effector pairs are common among oomycete plant pathogens, we examined the relationship(s) among putative ortholog pairs in Ps. cubensis and P. infestans. Of 271 predicted Ps. cubensis effector proteins, only 109 (41%) had a putative ortholog in P. infestans and evolutionary rate analysis of these orthologs shows that they are evolving significantly faster than most other genes. We found that PscRXLR1 was up-regulated during the early stages of infection of plants, and, moreover, that heterologous expression of PscRXLR1 in Nicotiana benthamiana elicits a rapid necrosis. More interestingly, we also demonstrate that PscRXLR1 arises as a product of alternative splicing, making this the first example of an alternative splicing event in plant pathogenic oomycetes transforming a non-effector gene to a functional effector protein. Taken together, these data suggest a role for PscRXLR1 in pathogenicity, and, in total, our data provide a basis for comparative analysis of candidate effector proteins and their non-effector orthologs as a means of understanding function and evolutionary history of pathogen effectors.

Transcriptome analysis reveals the time of the fourth round of genome duplication in common carp (Cyprinus carpio)

BackgroundCommon carp (Cyprinus carpio) is thought to have undergone one extra round of genome duplication compared to zebrafish. Transcriptome analysis has been used to study the existence and timing of genome duplication in species for which genome sequences are incomplete. Large-scale transcriptome data for the common carp genome should help reveal the timing of the additional duplication event.ResultsWe have sequenced the transcriptome of common carp using 454 pyrosequencing. After assembling the 454 contigs and the published common carp sequences together, we obtained 49,669 contigs and identified genes using homology searches and an ab initio method. We identified 4,651 orthologous pairs between common carp and zebrafish and found 129,984 paralogous pairs within the common carp. An estimation of the synonymous substitution rate in the orthologous pairs indicated that common carp and zebrafish diverged 120 million years ago (MYA). We identified one round of genome duplication in common carp and estimated that it had occurred 5.6 to 11.3 MYA. In zebrafish, no genome duplication event after speciation was observed, suggesting that, compared to zebrafish, common carp had undergone an additional genome duplication event. We annotated the common carp contigs with Gene Ontology terms and KEGG pathways. Compared with zebrafish gene annotations, we found that a set of biological processes and pathways were enriched in common carp.ConclusionsThe assembled contigs helped us to estimate the time of the fourth-round of genome duplication in common carp. The resource that we have built as part of this study will help advance functional genomics and genome annotation studies in the future.

Investigating the molecular basis of local adaptation to thermal stress: population differences in gene expression across the transcriptome of the copepod Tigriopus californicus

BackgroundGeographic variation in the thermal environment impacts a broad range of biochemical and physiological processes and can be a major selective force leading to local population adaptation. In the intertidal copepod Tigriopus californicus, populations along the coast of California show differences in thermal tolerance that are consistent with adaptation, i.e., southern populations withstand thermal stresses that are lethal to northern populations. To understand the genetic basis of these physiological differences, we use an RNA-seq approach to compare genome-wide patterns of gene expression in two populations known to differ in thermal tolerance.ResultsObserved differences in gene expression between the southern (San Diego) and the northern (Santa Cruz) populations included both the number of affected loci as well as the identity of these loci. However, the most pronounced differences concerned the amplitude of up-regulation of genes producing heat shock proteins (Hsps) and genes involved in ubiquitination and proteolysis. Among the hsp genes, orthologous pairs show markedly different thermal responses as the amplitude of hsp response was greatly elevated in the San Diego population, most notably in members of the hsp70 gene family. There was no evidence of accelerated evolution at the sequence level for hsp genes. Among other sets of genes, cuticle genes were up-regulated in SD but down-regulated in SC, and mitochondrial genes were down-regulated in both populations.ConclusionsMarked changes in gene expression were observed in response to acute sub-lethal thermal stress in the copepod T. californicus. Although some qualitative differences were observed between populations, the most pronounced differences involved the magnitude of induction of numerous hsp and ubiquitin genes. These differences in gene expression suggest that evolutionary divergence in the regulatory pathway(s) involved in acute temperature stress may offer at least a partial explanation of population differences in thermal tolerance observed in Tigriopus.

Calculating orthologs in bacteria and Archaea: a divide and conquer approach.

Among proteins, orthologs are defined as those that are derived by vertical descent from a single progenitor in the last common ancestor of their host organisms. Our goal is to compute a complete set of protein orthologs derived from all currently available complete bacterial and archaeal genomes. Traditional approaches typically rely on all-against-all BLAST searching which is prohibitively expensive in terms of hardware requirements or computational time (requiring an estimated 18 months or more on a typical server). Here, we present xBASE-Orth, a system for ongoing ortholog annotation, which applies a “divide and conquer” approach and adopts a pragmatic scheme that trades accuracy for speed. Starting at species level, xBASE-Orth carefully constructs and uses pan-genomes as proxies for the full collections of coding sequences at each level as it progressively climbs the taxonomic tree using the previously computed data. This leads to a significant decrease in the number of alignments that need to be performed, which translates into faster computation, making ortholog computation possible on a global scale. Using xBASE-Orth, we analyzed an NCBI collection of 1,288 bacterial and 94 archaeal complete genomes with more than 4 million coding sequences in 5 weeks and predicted more than 700 million ortholog pairs, clustered in 175,531 orthologous groups. We have also identified sets of highly conserved bacterial and archaeal orthologs and in so doing have highlighted anomalies in genome annotation and in the proposed composition of the minimal bacterial genome. In summary, our approach allows for scalable and efficient computation of the bacterial and archaeal ortholog annotations. In addition, due to its hierarchical nature, it is suitable for incorporating novel complete genomes and alternative genome annotations. The computed ortholog data and a continuously evolving set of applications based on it are integrated in the xBASE database, available at http://www.xbase.ac.uk/.

A pair of orthologs of a leucine-rich repeat receptor kinase-like disease resistance gene family regulates rice response to raised temperature

BackgroundRice Xa3/Xa26 disease-resistance gene encodes a leucine-rich repeat (LRR) receptor kinase-type protein against Xanthomonas oryzae pv. oryzae (Xoo) and belongs to a multigene family. However, the functions of most genes in this family are unknown.ResultsHere we report that two orthologs of this family, the NRKe from rice variety Nipponbare and 9RKe from variety 93-11 at the RKe locus, have similar functions although they encode different proteins. This pair of orthologs could not mediate resistance to Xoo, but they were transcriptionally induced by raised temperature. Transcriptional activation of NRKe or 9RKe resulted in the formation of temperature-sensitive lesion mimics, which were spots of dead cells associated with accumulation of superoxides, in different organs of the transgenic plants. These plants were more sensitive to high temperature shock than wild-type controls. Transgenic plants carrying a chimeric protein consisting of the LRR domain of NRKe and the kinase domain of Xa3/Xa26 developed the same lesion mimics as the NRKe-transgenic plants, whereas transgenic plants carrying another chimeric protein consisting of the LRR domain of Xa3/Xa26 and the kinase domain of NRKe were free of lesion mimic. All the transgenic plants carrying a chimeric protein were susceptible to Xoo.ConclusionThese results suggest that the RKe locus is involved in rice response to raised temperature. The LRR domain of RKe protein appears to be important to sense increased temperature. The RKe-involved temperature-related pathway and Xa3/Xa26-mediated disease-resistance pathway may partially overlap.

Comparative Studies on Human and Rat Basic Helix-loop-helix Proteins

The basic Helix-Loop-Helix (bHLH) proteins are a family of transcription factors that are found in almost all eukaryotic genomes. This family of proteins regulates a variety of biological processes including embryonic development. Here we describe the catalog of human, mouse and rat bHLH proteins. Extensive in silico studies identified 121 human, 121 mouse and 120 rat bHLH proteins. Human has orthologs for 119 rat bHLH proteins. Two bHLH proteins are found only in human, while human lacks one rodent specific bHLH protein, MYCS. Fifty bHLH protein families are present in all three lineages. Forty nine bHLH proteins contain 16 additional domains. Orthologous bHLH pairs vary in sequence conservation along their length, creating a map of functionally important regions for every bHLH protein pair. Many species-specific sequence inserts exist. This study provides a comprehensive overview of bHLH proteins.

Selection-Driven Divergence After Gene Duplication in Arabidopsis thaliana

Gene duplications are one of the most important mechanisms for the origin of evolutionary novelties. Even though various models of the fate of duplicated genes have been established, current knowledge about the role of divergent selection after gene duplication is rather limited. In this study, we analyzed sequence divergence in response to neo- and subfunctionalization of segmentally duplicated genes in the genome of Arabidopsis thaliana. We compared the genomes of A. thaliana and the poplar Populus trichocarpa to identify orthologous pairs of genes and their corresponding inparalogs. Maximum-likelihood analyses of the nonsynonymous and synonymous substitution rate ratio [Formula: see text] of pairs of A. thaliana inparalogs were used to detect differences in the evolutionary rates of protein coding sequences. We analyzed 1,924 A. thaliana paralogous pairs and our results indicate that around 6.9% show divergent ω values between the lineages for a fraction of sites. We observe an enrichment of regulatory sequences, a reduced level of co-expression and an increased number of substitutions that can be attributed to positive selection based on an McDonald-Kreitman type of analysis. Taken together, these results show that selection after duplication contributes substantially to gene novelties and hence functional divergence in plants.

An integrative approach to ortholog prediction for disease-focused and other functional studies

BackgroundMapping of orthologous genes among species serves an important role in functional genomics by allowing researchers to develop hypotheses about gene function in one species based on what is known about the functions of orthologs in other species. Several tools for predicting orthologous gene relationships are available. However, these tools can give different results and identification of predicted orthologs is not always straightforward.ResultsWe report a simple but effective tool, the Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT; http://www.flyrnai.org/diopt), for rapid identification of orthologs. DIOPT integrates existing approaches, facilitating rapid identification of orthologs among human, mouse, zebrafish, C. elegans, Drosophila, and S. cerevisiae. As compared to individual tools, DIOPT shows increased sensitivity with only a modest decrease in specificity. Moreover, the flexibility built into the DIOPT graphical user interface allows researchers with different goals to appropriately 'cast a wide net' or limit results to highest confidence predictions. DIOPT also displays protein and domain alignments, including percent amino acid identity, for predicted ortholog pairs. This helps users identify the most appropriate matches among multiple possible orthologs. To facilitate using model organisms for functional analysis of human disease-associated genes, we used DIOPT to predict high-confidence orthologs of disease genes in Online Mendelian Inheritance in Man (OMIM) and genes in genome-wide association study (GWAS) data sets. The results are accessible through the DIOPT diseases and traits query tool (DIOPT-DIST; http://www.flyrnai.org/diopt-dist).ConclusionsDIOPT and DIOPT-DIST are useful resources for researchers working with model organisms, especially those who are interested in exploiting model organisms such as Drosophila to study the functions of human disease genes.

The non-random clustering of non-synonymous substitutions and its relationship to evolutionary rate

BackgroundProtein sequences are subject to a mosaic of constraint. Changes to functional domains and buried residues, for example, are more apt to disrupt protein structure and function than are changes to residues participating in loops or exposed to solvent. Regions of constraint on the tertiary structure of a protein often result in loose segmentation of its primary structure into stretches of slowly- and rapidly-evolving amino acids. This clustering can be exploited, and existing methods have done so by relying on local sequence conservation as a signature of selection to help identify functionally important regions within proteins. We invert this paradigm by leveraging the regional nature of protein structure and function to both illuminate and make use of genome-wide patterns of local sequence conservation.ResultsOur hypothesis is that the regional nature of structural and functional constraints will assert a positive autocorrelation on the evolutionary rates of neighboring sites, which, in a pairwise comparison of orthologous proteins, will manifest itself as the clustering of non-synonymous changes across the amino acid sequence. We introduce a dispersion ratio statistic to test this and related hypotheses. Using genome-wide interspecific comparisons of orthologous protein pairs, we reveal a strong log-linear relationship between the degree of clustering and the intensity of constraint. We further demonstrate how this relationship varies with the evolutionary distance between the species being compared. We provide some evidence that proteins with a history of positive selection deviate from genome-wide trends.ConclusionsWe find a significant association between the evolutionary rate of a protein and the degree to which non-synonymous changes cluster along its primary sequence. We show that clustering is a non-redundant predictor of evolutionary rate, and we speculate that conflicting signals of clustering and constraint may be indicative of a historical period of relaxed selection.

Domain architecture conservation in orthologs

BackgroundAs orthologous proteins are expected to retain function more often than other homologs, they are often used for functional annotation transfer between species. However, ortholog identification methods do not take into account changes in domain architecture, which are likely to modify a protein's function. By domain architecture we refer to the sequential arrangement of domains along a protein sequence.To assess the level of domain architecture conservation among orthologs, we carried out a large-scale study of such events between human and 40 other species spanning the entire evolutionary range. We designed a score to measure domain architecture similarity and used it to analyze differences in domain architecture conservation between orthologs and paralogs relative to the conservation of primary sequence. We also statistically characterized the extents of different types of domain swapping events across pairs of orthologs and paralogs.ResultsThe analysis shows that orthologs exhibit greater domain architecture conservation than paralogous homologs, even when differences in average sequence divergence are compensated for, for homologs that have diverged beyond a certain threshold. We interpret this as an indication of a stronger selective pressure on orthologs than paralogs to retain the domain architecture required for the proteins to perform a specific function. In general, orthologs as well as the closest paralogous homologs have very similar domain architectures, even at large evolutionary separation.The most common domain architecture changes observed in both ortholog and paralog pairs involved insertion/deletion of new domains, while domain shuffling and segment duplication/deletion were very infrequent.ConclusionsOn the whole, our results support the hypothesis that function conservation between orthologs demands higher domain architecture conservation than other types of homologs, relative to primary sequence conservation. This supports the notion that orthologs are functionally more similar than other types of homologs at the same evolutionary distance.

Genome-Wide Gene Expression Profiling of Fertilization Competent Mycelium in Opposite Mating Types in the Heterothallic Fungus Podospora anserina

BackgroundMating-type loci in yeasts and ascomycotan filamentous fungi (Pezizomycotina) encode master transcriptional factors that play a critical role in sexual development. Genome-wide analyses of mating-type-specification circuits and mating-type target genes are available in Saccharomyces cerevisiae and Schizosaccharomyces pombe; however, no such analyses have been performed in heterothallic (self-incompatible) Pezizomycotina. The heterothallic fungus Podospora anserina serves as a model for understanding the basic features of mating-type control. Its mat+ and mat− mating types are determined by dissimilar allelic sequences. The mat− sequence contains three genes, designated FMR1, SMR1 and SMR2, while the mat+ sequence contains one gene, FPR1. FMR1 and FPR1 are the major regulators of fertilization, and this study presents a genome-wide view of their target genes and analyzes their target gene regulation.Methodology/Principal FindingsThe transcriptomic profiles of the mat+ and mat− strains revealed 157 differentially transcribed genes, and transcriptomic analysis of fmr1− and fpr1− mutant strains was used to determine the regulatory actions exerted by FMR1 and FPR1 on these differentially transcribed genes. All possible combinations of transcription repression and/or activation by FMR1 and/or FPR1 were observed. Furthermore, 10 additional mating-type target genes were identified that were up- or down-regulated to the same level in mat+ and mat− strains. Of the 167 genes identified, 32 genes were selected for deletion, which resulted in the identification of two genes essential for the sexual cycle. Interspecies comparisons of mating-type target genes revealed significant numbers of orthologous pairs, although transcriptional profiles were not conserved between species.Conclusions/SignificanceThis study represents the first comprehensive genome-wide analysis of mating-type direct and indirect target genes in a heterothallic filamentous fungus. Mating-type transcription factors have many more target genes than are found in yeasts and exert a much greater diversity of regulatory actions on target genes, most of which are not directly related to mating.

Regulation of DNA Replication through Sld3-Dpb11 Interaction Is Conserved from Yeast to Humans

Cyclin-dependent kinases (CDKs) play crucial roles in promoting DNA replication and preventing rereplication in eukaryotic cells [1-4]. In budding yeast, CDKs promote DNA replication by phosphorylating two proteins, Sld2 and Sld3, which generates binding sites for pairs of BRCT repeats (breast cancer gene 1 [BRCA1] C terminal repeats) in the Dpb11 protein [5, 6]. The Sld3-Dpb11-Sld2 complex generated by CDK phosphorylation is required for the assembly and activation of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase. In response to DNA replication stress, the interaction between Sld3 and Dpb11 is blocked by the checkpoint kinase Rad53 [7], which prevents late origin firing [7, 8]. Here we show that the two key CDK sites in Sld3 are conserved in the human Sld3-related protein Treslin/ticrr and are essential for DNA replication. Moreover, phosphorylation of these two sites mediates interaction with the orthologous pair of BRCT repeats in the human Dpb11 ortholog, TopBP1. Finally, we show that DNA replication stress prevents the interaction between Treslin/ticrr and TopBP1 via the Chk1 checkpoint kinase. Our results indicate that Treslin/ticrr is a genuine ortholog of Sld3 and that the Sld3-Dpb11 interaction has remained a critical nexus of S phase regulation through eukaryotic evolution.