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The duplication of entire genomes has long been recognized as having great potential for evolutionary novelties, but the mechanisms underlying their resolution through gene loss are poorly understood. Here we show that in the unicellular eukaryote Paramecium tetraurelia, a ciliate, most of the nearly 40,000 genes arose through at least three successive whole-genome duplications. Phylogenetic analysis indicates that the most recent duplication coincides with an explosion of speciation events that gave rise to the P. aurelia complex of 15 sibling species. We observed that gene loss occurs over a long timescale, not as an initial massive event. Genes from the same metabolic pathway or protein complex have common patterns of gene loss, and highly expressed genes are over-retained after all duplications. The conclusion of this analysis is that many genes are maintained after whole-genome duplication not because of functional innovation but because of gene dosage constraints.

Maize (Zea mays or corn), both a major food source and an important cytogenetic model, evolved from a tetraploid that arose about 4.8 million years ago (Mya). As a result, maize has extensive duplicated regions within its genome. We have sequenced the two copies of one such region, generating 7.8 Mb of sequence spanning 17.4 cM of the short arm of chromosome 1 and 6.6 Mb (25.6 cM) from the long arm of chromosome 9. Rice, which did not undergo a similar whole genome duplication event, has only one orthologous region (4.9 Mb) on the short arm of chromosome 3, and can be used as reference for the maize homoeologous regions. Alignment of the three regions allowed identification of syntenic blocks, and indicated that the maize regions have undergone differential contraction in genic and intergenic regions and expansion by the insertion of retrotransposable elements. Approximately 9% of the predicted genes in each duplicated region are completely missing in the rice genome, and almost 20% have moved to other genomic locations. Predicted genes within these regions tend to be larger in maize than in rice, primarily because of the presence of predicted genes in maize with larger introns. Interestingly, the general gene methylation patterns in the maize homoeologous regions do not appear to have changed with contraction or expansion of their chromosomes. In addition, no differences in methylation of single genes and tandemly repeated gene copies have been detected. These results, therefore, provide new insights into the diploidization of polyploid species.

Thyroid hormone responsive Spot 14 (THRSP) is suggested as a transcription factor involved in the regulation of adipogenic enzymes by 3 thyroid response elements in the promoter region. In the chicken genome, THRSP gene was identified to duplicate into 2 paralogs, THRSPalpha and THRSPbeta. In the current study, cDNA sequences of the duplicated duck THRSP genes were cloned by real-time PCR and rapid amplification of cDNA ends. Duck THRSPalpha and THRSPbeta were predicted to encode peptides with 133 amino acids, which had 74 and 68% sequence identity at cDNA level, 78 and 74% identity at amino acid level to the chicken counterparts, respectively. A high percentage (73.1%) of G and C nucleotides were found in the 3' untranslated region of duck THRSPbeta cDNA. Although a low similarity of peptide composition was shared between ducks and mammals, and a moderate similarity was shared between ducks and chickens, many predicted properties of THRSP, including the pI, subcellular localization and functional domains seemed to be highly conserved. The present study demonstrated that the duck THRSP gene duplicates into the 2 paralogs as in chickens. Phylogenetic analysis indicated that the duplication for THRSP paralogs appeared to have taken place preceding the chicken-duck split, and the diverging rate between THRSP paralogs seemed faster in the chicken genome than that in the duck genome. Expression analysis by real-time quantitative PCR showed that THRSP paralogs in ducks were more actively transcribed in fat tissues (i.e., s.c. fat and abdominal fat) than in liver, and the mRNA concentrations of THRSPbeta were higher than that of THRSPalpha in liver and s.c. fat.

In Arabidopsis, tandemly arrayed genes (TAGs) comprise >10% of the genes in the genome. These duplicated genes represent a rich template for genetic innovation, but little is known of the evolutionary forces governing their generation and maintenance. Here we compare the organization and evolution of TAGs between Arabidopsis and rice, two plant genomes that diverged ~150 million years ago. TAGs from the two genomes are similar in a number of respects, including the proportion of genes that are tandemly arrayed, the number of genes within an array, the number of tandem arrays, and the dearth of TAGs relative to single copy genes in centromeric regions. Analysis of recombination rates along rice chromosomes confirms a positive correlation between the occurrence of TAGs and recombination rate, as found in Arabidopsis. TAGs are also biased functionally relative to duplicated, nontandemly arrayed genes. In both genomes, TAGs are enriched for genes that encode membrane proteins and function in "abiotic and biotic stress" but underrepresented for genes involved in transcription and DNA or RNA binding functions. We speculate that these observations reflect an evolutionary trend in which successful tandem duplication involves genes either at the end of biochemical pathways or in flexible steps in a pathway, for which fluctuation in copy number is unlikely to affect downstream genes. Despite differences in the age distribution of tandem arrays, the striking similarities between rice and Arabidopsis indicate similar mechanisms of TAG generation and maintenance.

Analysis of increasingly saturated sequence databases have shown that gene family sizes are highly skewed with many families being small and few containing many, far-diverged homologs. Additionally, recently published results have identified a structural determinant of mutational plasticity: designability that correlates strongly with gene family size. In this paper, we explore the possible links between the two observations, exploring the possible effect of designability on duplication and divergence. We show that designability has an inverse of expected relationship with strength of selection. More designable domains that should have more mutational plasticity evolve slower. However, we also present evidence that recently duplicated genes have variable probability of locus fixation correlated with strength of selection. As expected, paralogs under stronger evolutionary pressure have a lower failure rate. Finally, we show that probability of pseudogene formation from gene duplication can be directly tied to designability and functional flexibility of the family. We present evidence that gene families with higher designability have diverged farther because of lower probability of pseudogenization. Additionally, mutational plasticity may play an integral role by influencing pseudogenization rate. Either way, we show that considering the failure rate of duplications is integral in understanding the determinants and dynamics of molecular evolution.

The identification and classification of genes and pseudogenes in duplicated regions still constitutes a challenge for standard automated genome annotation procedures. Using an integrated homology and orthology analysis independent of current gene annotation, we have identified 9,484 and 9,017 gene duplicates in human and mouse, respectively. On the basis of the integrity of their coding regions, we have classified them into functional and inactive duplicates, allowing us to define the first consistent and comprehensive collection of 1,811 human and 1,581 mouse unprocessed pseudogenes. Furthermore, of the total of 14,172 human and mouse duplicates predicted to be functional genes, as many as 420 are not included in current reference gene databases and therefore correspond to likely novel mammalian genes. Some of these correspond to partial duplicates with less than half of the length of the original source genes, yet they are conserved and syntenic among different mammalian lineages. The genes and unprocessed pseudogenes obtained here will enable further studies on the mechanisms involved in gene duplication as well as of the fate of duplicated genes.

During phylogenetic analysis of the Nepenthaceae cpDNA trnK intron, it became apparent that a second non-functional copy of the locus was present in most of the investigated taxa. The translocation event was older than the radiation of all recent Nepenthaceae, and the translocated pseudogenized copy was conserved in nearly all members of the plant family. Using single chloroplast PCR and inverse PCR, we could exclude a plastom location for the second copy. Although translocation into the nucleus is possible, mitochondrial localization seems more likely based on these data. In total, the translocated sequence contained at least 3525 base pairs (bp) that were homologous to the Spinacia oleracea chloroplast genome. Comparative phylogenetic analysis of the non-functional copy revealed a high amount of homoplasies compared to topologies from the cpDNA trnK intron phylogenetic reconstruction. Therefore, this copy proved to be insufficient for phylogenetic reconstruction of the family. Since two different paralogs of the non-functional copy were found in one species, it is feasible that different paralogs were conserved in different groups and that paralogous sequences were included in the data matrix. These data demonstrate that phylogenetic analyses of pseudogenized copies of phylogenetically relevant loci should be performed with great caution. In addition, pseudogenized copies can exist in nearly every member of a plant family, and can be PCR-amplified at levels comparable to the specific copy. In this case, the inclusion of such copies can easily remain unnoticed, thus leading to faulty hypotheses.

Human orosomucoid (ORM), or alpha(1)-acid glycoprotein, is known to be controlled by duplicated and triplicated genes on chromosome 9, encoding ORM1 and ORM2 proteins. In this study, the structure and diversity of the ORM gene were investigated in 16 Sub-Saharan Africans, who originated from widely dispersed locations in Africa. The duplicated ORM1-ORM2 gene was observed in all 16 samples. ORM1*S1(2), characterized by an ORM2 gene-specific sequence in intron 5, was common in Africans. Three Africans showed the duplication of the ORM1 gene. The organization of the triplicated ORM1A-ORM1B-ORM2 gene was established in two Africans. The recombination breakpoints resulting in the ORM1 duplication lay within a small genomic interval around exon 1 of the ORM1B gene. The duplication of the ORM2 gene reported previously was not detected in this population sample. Several single-nucleotide polymorphisms were observed in the ORM2 gene. The rearrangement of the ORM gene is likely to occur often in Africans.

Background: The imprint of natural selection on gene sequences is often difficult to detect. A plethora of methods have been devised to detect genetic changes due to selective processes. However, many of those methods depend heavily on underlying assumptions regarding the mode of change of DNA sequences and often require sophisticated mathematical treatments that made them computationally slow. The development of fast and effective methods to detect modifications in the selective constraints of genes is therefore of great interest., Results: We describe UVPAR, a program designed to quickly test for changes in the functional constraints of duplicate genes. Starting with alignments of the proteins encoded by couples of duplicate genes in two different species, UVPAR detects the regions in which modifications of the functional constraints in the paralogs occurred since both species diverged. Sequences can be analyzed with UVPAR in just a few minutes on a standard PC computer. To demonstrate the power of the program, we first show how the results obtained with UVPAR compare to those based on other approaches, using data for vertebrate Hox genes. We then describe a comprehensive study of the RBR family of ubiquitin ligases in which we have performed 529 analyses involving 14 duplicate genes in seven model species. A significant increase in the number of functional shifts was observed for the species Danio rerio and for the gene Ariadne-2., Conclusion: These results show that UVPAR can be used to generate sensitive analyses to detect changes in the selection constraints acting on paralogs. The high speed of the program allows its application to genome-scale analyses.

The question of how duplicate genes are retained in a population remains controversial. The duplication-degeneration-complementation model, which involves no positive selection, stipulates a higher retention rate of duplicate genes in a small population than in a large one. This model has been accepted by many evolutionists. However, we found considerably more retentions and fewer losses of duplicate genes in the mouse genome than in the human genome, although the population size of rodents is in general larger than that of primates. Indeed, in nearly every interval of synonymous divergence between duplicate genes, the number of gene retentions in mouse is larger than that in human. Our findings suggest a more important role of positive selection in duplicate retention than duplication-degeneration-complementation. In addition, certain functional categories show a higher tendency of lineage-specific expansion than expected, suggesting lineage-specific selection or functional bias in retained duplicates.

Duplicons, that is, DNA sequences with minimum length 10 kb and a high sequence similarity, are known to cause unequal homologous recombination, leading to deletions and the reciprocal duplications. In this study, we designed a Multiplex Amplifiable Probe Hybridisation (MAPH) assay containing 63 exon-specific single-copy sequences from within a selection of the 169 regions flanked by duplicons that were identified, at a first pass, in 2001. Subsequently, we determined the frequency of chromosomal rearrangements among patients with developmental delay (DD) and/or congenital malformations (CM). In addition, we tried to identify new regions involved in DD/CM using the same assay. In 105 patients, six imbalances (5.8%) were detected and verified. Three of these were located in microdeletion-related regions, two alterations were polymorphic duplications and the effect of the last alteration is currently unknown. The same study population was tested for rearrangements in regions with no known duplicons nearby, using a set of probes derived from 58 function-selected genes. The latter screening revealed two alterations. As expected, the alteration frequency per unit of DNA is much higher in regions flanked by duplicons (fraction of the genome tested: 5.2%) compared to regions without known duplicons nearby (fraction of the genome tested: 24.5-90.2%). We were able to detect three novel rearrangements, including the previously undescribed reciprocal duplication of the Williams Beuren critical region, a subduplicon alteration within this region and a duplication on chromosome band 16p13.11. Our results support the hypothesis that regions flanked by duplicons are enriched for copy number variations.

Background: While gene duplication is known to be one of the most common mechanisms of genome evolution, the fates of genes after duplication are still being debated. In particular, it is presently unknown whether most duplicate genes preserve (or subdivide) the functions of the parental gene or acquire new functions. One aspect of gene function, that is the expression profile in gene coexpression network, has been largely unexplored for duplicate genes., Results: Here we build a human gene coexpression network using human tissue-specific microarray data and investigate the divergence of duplicate genes in it. The topology of this network is scale-free. Interestingly, our analysis indicates that duplicate genes rapidly lose shared coexpressed partners: after approximately 50 million years since duplication, the two duplicate genes in a pair have only slightly higher number of shared partners as compared with two random singletons. We also show that duplicate gene pairs quickly acquire new coexpressed partners: the average number of partners for a duplicate gene pair is significantly greater than that for a singleton (the latter number can be used as a proxy of the number of partners for a parental singleton gene before duplication). The divergence in gene expression between two duplicates in a pair occurs asymmetrically: one gene usually has more partners than the other one. The network is resilient to both random and degree-based in silico removal of either singletons or duplicate genes. In contrast, the network is especially vulnerable to the removal of highly connected genes when duplicate genes and singletons are considered together., Conclusion: Duplicate genes rapidly diverge in their expression profiles in the network and play similar role in maintaining the network robustness as compared with singletons.

Amphioxus Hairy genes have gone through a number of lineage-specific duplications, resulting in eight members, some of which are differentially expressed in the embryo. In order to gain insights into the evolution and function of this gene family we have compared their genomic structure and searched for conserved non-coding sequence elements. We have found that introns have been lost independently from these genes at least twice and after the duplication events. By carrying out phylogenetic footprinting between paralogues expressed in the embryo, we have found a differential distribution of conserved elements that could explain the limited overlap in expression patterns of Hairy genes in the amphioxus embryo. Furthermore, clustering of RBP-Jk binding sites in these conserved elements suggests that amphioxus Hairy genes are downstream targets of the Notch signaling pathway, as occurs in vertebrates. All of this evidence suggests that amphioxus Hairy genes have gone through a process of subfunctionalization shortly after their duplication, representing an extreme and rapid case of the duplication-degeneration-complementation model.

Holoprosencephaly (HPE) is the most common developmental defect affecting the forebrain and midface in humans. The aetiology of HPE is highly heterogeneous and includes both environmental and genetic factors. Here we report on a boy with mild mental retardation, lobar HPE, epilepsy, mild pyramidal syndrome of the legs, ventricular septal defect, vesicoureteral reflux, preaxial polydactyly, and facial dysmorphisms. Genome-wide tiling path resolution array based comparative genomic hybridisation (array CGH) revealed a de novo copy-number gain at 5q35.1 of 1.24 Mb. Additional multiplex ligation-dependent probe amplification screening of a cohort of 31 patients with HPE for copy-number changes at the 5q35.1 locus did not reveal any additional genomic anomalies. This report defines a novel 1.24 Mb critical interval for HPE and preaxial polydactyly at 5q35.1. The duplicated region encompasses seven genes: RANBP17, TLX3, NPM1, FGF18, FBXW11, STK10, and DC-UbP. Since FBXW11 is relatively highly expressed in fetal brain and is directly involved in proteolytic processing of GLI3, we propose FBXW11 as the most likely candidate gene for the HPE and prexial polydactyly phenotype. Additional research is needed to further establish the role of genes from the 5q35.1 region in brain and limb development and to determine the prevalence of copy number gain in the 5q35.1 region among HPE patients.

Background: Systematic analyses of loss-of-function phenotypes have been carried out for most genes in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster. Although such studies vastly expand our knowledge of single gene function, they do not address redundancy in genetic networks. Developing tools for the systematic mapping of genetic interactions is thus a key step in exploring the relationship between genotype and phenotype., Results: We established conditions for RNA interference (RNAi) in C. elegans to target multiple genes simultaneously in a high-throughput setting. Using this approach, we can detect the great majority of previously known synthetic genetic interactions. We used this assay to examine the redundancy of duplicated genes in the genome of C. elegans that correspond to single orthologs in S. cerevisiae or D. melanogaster and identified 16 pairs of duplicated genes that have redundant functions. Remarkably, 14 of these redundant gene pairs were duplicated before the divergence of C. elegans and C. briggsae 80-110 million years ago, suggesting that there has been selective pressure to maintain the overlap in function between some gene duplicates., Conclusion: We established a high throughput method for examining genetic interactions using combinatorial RNAi in C. elegans. Using this technique, we demonstrated that many duplicated genes can retain redundant functions for more than 80 million years of evolution. This provides strong support for evolutionary models that predict that genetic redundancy between duplicated genes can be actively maintained by natural selection and is not just a transient side effect of recent gene duplication events.

Gene duplication is often cited as a potential mechanism for the evolution of new traits, but this hypothesis has not been thoroughly tested experimentally. A classical model of gene duplication states that after gene duplication one copy of the gene preserves the ancestral function, while the other copy is free to evolve a new function. In an alternative duplication, divergence, and complementation model, duplicated genes are preserved because each copy of the gene loses some, but not all, of its functions through degenerating mutations. This results in the degenerating mutations in one gene being complemented by the other and vice versa. These two models make very different predictions about the function of the preduplication orthologs in closely related species. These predictions have been tested here for several duplicated yeast genes that appeared to be the leading candidates to fit the classical model. Surprisingly, the results show that duplicated genes are maintained because each copy carries out a subset of the conserved functions that were already present in the preduplication gene. Therefore, the results are not consistent with the classical model, but instead fit the duplication, divergence, and complementation model.

Mutations in the leucine-rich, glioma-inactivated 1 gene, LGI1, cause autosomal-dominant lateral temporal lobe epilepsy via unknown mechanisms. LGI1 belongs to a subfamily of leucine-rich repeat genes comprising four members (LGI1-LGI4) in mammals. In this study, both comparative developmental as well as molecular evolutionary methods were applied to investigate the evolution of the LGI gene family and, subsequently, of the functional importance of its different gene members. Our phylogenetic studies suggest that LGI genes evolved early in the vertebrate lineage. Genetic and expression analyses of all five zebrafish lgi genes revealed duplications of lgi1 and lgi2, each resulting in two paralogous gene copies with mostly nonoverlapping expression patterns. Furthermore, all vertebrate LGI1 orthologs experience high levels of purifying selection that argue for an essential role of this gene in neural development or function. The approach of combining expression and selection data used here exemplarily demonstrates that in poorly characterized gene families a framework of evolutionary and expression analyses can identify those genes that are functionally most important and are therefore prime candidates for human disorders.

Partial and complete genome duplications occurred during evolution and resulted in the creation of new genes and gene families. We identified a novel and intricate human gene family located primarily in regions of segmental duplications on human chromosome 1. We named it NBPF, for neuroblastoma breakpoint family, because one of its members is disrupted by a chromosomal translocation in a neuroblastoma patient. The NBPF genes have a repetitive structure with high intragenic and intergenic sequence similarity in both coding and noncoding regions. These similarities might expose these genomic regions to illegitimate recombination, resulting in structural variation in the NBPF genes. The encoded proteins contain a highly conserved domain of unknown function, which we have named the NBPF repeat. In silico analysis combined with the isolation of multiple full-length cDNA clones showed that several members of this gene family are abundantly expressed in a large variety of tissues and cell lines. Strikingly, no discernable orthologues could be identified in the completed genomes of fruit fly, nematode, mouse, or rat, but sequences with low homology could be isolated from the draft canine and bovine genomes. Interestingly, this gene family shows primate-specific duplications that result in species-specific arrays of NBPF homologous sequences. Overall, this novel NBPF family reflects the continuous evolution of primate genomes that resulted in large physiological differences, and its potential role in this process is discussed.

Much recent work has explored molecular and population-genetic constraints on the rate of protein sequence evolution. The best predictor of evolutionary rate is expression level, for reasons that have remained unexplained. Here, we hypothesize that selection to reduce the burden of protein misfolding will favor protein sequences with increased robustness to translational missense errors. Pressure for translational robustness increases with expression level and constrains sequence evolution. Using several sequenced yeast genomes, global expression and protein abundance data, and sets of paralogs traceable to an ancient whole-genome duplication in yeast, we rule out several confounding effects and show that expression level explains roughly half the variation in Saccharomyces cerevisiae protein evolutionary rates. We examine causes for expression's dominant role and find that genome-wide tests favor the translational robustness explanation over existing hypotheses that invoke constraints on function or translational efficiency. Our results suggest that proteins evolve at rates largely unrelated to their functions and can explain why highly expressed proteins evolve slowly across the tree of life.

Gene duplication is the fundamental source of new genes. Biases in duplication have profound implications for the dynamics of gene content during evolution. In this article, we compare genes arising from whole gene duplication (WGD), smaller scale duplication (SSD) and singletons in Saccharomyces cerevisiae. Our results demonstrate that genes duplicated by WGD and SSD are similarly biased with respect to codon bias and evolutionary rate, although differing significantly in their functional constituency.

We identified 15 regions of >1 Mb in the human genome composed of large ancient local duplications corresponding to gene deserts. We detected these intrachromosomal duplications in mouse and dog but not in chicken; they present as patches of similarity as low as 60%. These findings suggest that some human gene deserts originated from duplications of segments lacking genes in a mammalian common ancestor.

In bacteria, magnesium uptake is mainly mediated by the well-characterized CorA type of membrane proteins. In recent years, functional homologues have been characterized in the inner mitochondrial membrane of yeast and mammals (the MRS2/LPE10 type), in the plasma membrane of yeast (the ALR/MNR type) and, as an extended family of proteins, in the model plant Arabidopsis thaliana. Despite generally low sequence similarity, individual proteins can functionally complement each other over large phylogenetic distances. All these proteins are characterized by a universally conserved Gly-Met-Asn (GMN) motif at the end of the first of two conserved transmembrane domains near the C-terminus. Mutations of the GMN motif are known to abolish Mg(2+) transport, but the naturally occurring variants GVN and GIN may be associated with the transport of other divalent cations, such as zinc and cadmium, respectively. We refer to this whole class of proteins as the 2-TM-GxN type. The functional membrane channel is thought to be formed by oligomers containing four or five subunits. The wealth of sequence data now available allows us to explore the evolutionary diversification of the basic 2-TM-GxN model within the so-called metal ion transporter (MIT) superfamily. Here we report phylogenetic analyses on more than 360 homologous protein sequences derived from genomic sequences from representatives of all three domains of life. Independent gene duplications have occurred in fungi, plants and proteobacteria at different phylogenetic depths. Moreover, there is ample evidence for several instances of horizontal gene transfer of members of the 2-TM-GxN superfamily in Eubacteria and Archaea. Only single genes of the MRS2 type have been identified in vertebrate genomes. In contrast, 15 members are found in the model plant Arabidopsis thaliana, which appear to have arisen by at least four independent founder events before the diversification of flowering plants. Phylogenetic clade assignment seems to correlate with alterations in the highly conserved sequence around the GMN motif. This presumably forms an integral part of the pore surface, and changes in its structure may result in altered transport capacities for different divalent cations.

Four classes of apple allergens (Mal d 1, -2, -3 and -4) have been reported. By using PCR cloning and sequencing approaches, we obtained genomic sequences of Mal d 2 (thaumatin-like protein) and Mal d 4 (profilin) from the cvs Prima and Fiesta, the two parents of a European reference mapping population. Two copies of the Mal d 2 gene (Mal d 2.01 A and Mal d 2.01 B) were identified, which primarily differed in the length of a single intron (378 or 380 nt) and in one amino acid in the signal peptide. Both Mal d 2.01 A and Mal d 2.01 B were mapped at identical position on linkage group 9. Genomic characterization of four Mal d 4 genes (Mal d 4.01 A and B, Mal d 4.02 A and Mal d 4.03 A) revealed their complete gDNA sequences which varied among genes in length from 862 to 2,017 nt. They all contained three exons of conserved length: 123, 138, and 135 nt. Mal d 4.01 appeared to be duplicated in two copies and located on linkage group 9. Mal d 4.02 A and Mal d 4.03 A were single copy genes located on linkage group 2 and 8, respectively.

The Arabidopsis RPM1 protein confers resistance to disease caused by Pseudomonas syringae strains delivering either the AvrRpm1 or AvrB type III effector proteins into host cells. We characterized two closely related RPM1-interacting proteins, RIN2 and RIN3. RIN2 and RIN3 encode RING-finger type ubiquitin ligases with six apparent transmembrane domains and an ubiquitin-binding CUE domain. RIN2 and RIN3 are orthologs of the mammalian autocrine motility factor receptor, a cytokine receptor localized in both plasma membrane caveolae and the endoplasmic reticulum. RIN2 is predominantly localized to the plasma membrane, as are RPM1 and RPS2. The C-terminal regions of RIN2 and RIN3, including the CUE domain, interact strongly with an RPM1 N-terminal fragment and weakly with a similar domain from the Arabidopsis RPS2 protein. RIN2 and RIN3 can dimerize through their C-terminal regions. The RING-finger domains of RIN2 and RIN3 encode ubiquitin ligases. Inoculation with P. syringae DC3000(avrRpm1) or P. syringae DC3000(avrRpt2) induces differential decreases of RIN2 mobility in SDS-PAGE and disappearance of the majority of RIN2. A rin2 rin3 double mutant expresses diminished RPM1- and RPS2-dependent hypersensitive response (HR), but no alteration of pathogen growth. Thus, the RIN2/RIN3 RING E3 ligases apparently act on a substrate that regulates RPM1- and RPS2-dependent HR.

Genome doubling, or polyploidy, is a major factor accounting for duplicate genes found in most eukaryotic genomes. Polyploidy has considerable effects on duplicate gene expression, including silencing and up- or downregulation of one of the duplicated genes. These changes can arise with the onset of polyploidization or within several generations after polyploid formation and they can have epigenetic causal factors. Many expression alterations are organ-specific. Specific genes can be independently and repeatedly silenced during polyploidization, whereas patterns for other genes appear to be more stochastic. Three recent reports have provided intriguing new insights into the patterns, timing and mechanisms of gene expression changes that accompany polyploidy in plants.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The recently identified AHR repressor (AHRR) forms a negative feedback loop with the AHR. We investigated AHRR structure, function, evolution, and regulation in zebrafish, a powerful model in developmental biology and toxicology. We identified and cloned two distinct AHRR cDNAs that encode predicted proteins of 550 (AHRR1) and 573 (AHRR2) amino acids. The ahrr1 and ahrr2 genes map to zebrafish chromosomes 24 and 2, respectively, both of which share conserved synteny with human chromosome 5, the location of human AHRR. Mapping and phylogenetic analysis show that AHRR1 and AHRR2 are co-orthologs of the mammalian AHRR. In transient transfection assays, AHRR1 and AHRR2 repressed constitutive and TCDD-inducible transactivation by AHR2. Expression of both AHRR mRNAs was induced in ZF-L cells by AHR agonists but not by non-agonists. TCDD induced AHRR1 and AHRR2 expression in a dose-dependent manner in ZF-L cells, with EC50 values similar to those for induction of CYP1A. Both AHRRs were expressed and induced by TCDD in zebrafish embryos. Thus, zebrafish possess duplicate AHR-regulated AHRR paralogs that act in a negative feedback loop to repress the AHR signaling pathway.

Transcription factors (TFs), which are central to the regulation of gene expression, are usually members of multigene families. In plants, they are involved in diverse processes such as developmental control and elicitation of defense and stress responses. To investigate if differences exist in the expansion patterns of TF gene families between plants and other eukaryotes, we first used Arabidopsis (Arabidopsis thaliana) TFs to identify TF DNA-binding domains. These DNA-binding domains were then used to identify related sequences in 25 other eukaryotic genomes. Interestingly, among 19 families that are shared between animals and plants, more than 14 are larger in plants than in animals. After examining the lineage-specific expansion of TF families in two plants, eight animals, and two fungi, we found that TF families shared among these organisms have undergone much more dramatic expansion in plants than in other eukaryotes. Moreover, this elevated expansion rate of plant TF is not simply due to higher duplication rates of plant genomes but also to a higher degree of expansion compared to other plant genes. Further, in many Arabidopsis-rice (Oryza sativa) TF orthologous groups, the degree of lineage-specific expansion in Arabidopsis is correlated with that in rice. This pattern of parallel expansion is much more pronounced than the whole-genome trend in rice and Arabidopsis. The high rate of expansion among plant TF genes and their propensity for parallel expansion suggest frequent adaptive responses to selection pressure common among higher plants.

Gene duplication is the main source of evolutionary novelties. However, the problem with duplicates is that the purifying selection overlooks deleterious mutations in the redundant sequence, which therefore, instead of gaining a new function, often degrades into a functionless pseudogene. This risk of functional loss instead of gain is much higher for small populations of higher organisms with a slow and complex development. We propose that it is the epigenetic tissue/stage-complementary silencing of duplicates that makes them exposable to the purifying selection, thus saving them from pseudogenization and opening the way towards new function(s). Our genome-wide analyses of gene duplicates in several eukaryotic species combined with the phylogenetic comparison of vertebrate alpha- and beta-globin gene clusters strongly support this epigenetic complementation (EC) model. The distinctive condition for a new duplicate to survive by the EC mechanism seems to be its repositioning to an ectopic site, which is accompanied by changes in the rate and direction of mutagenesis. The most distinguished in this respect is the human genome. In this review, we extend and discuss the data on the EC- and repositioning-dependent fate of gene duplicates with the special emphasis on the problem of detecting brief postduplication period of adaptive evolution driven by positive selection. Accordingly, we propose a new CpG-focused measure of selection that is insensitive to translocation-caused biases in mutagenesis.

Gene duplication plays a fundamental role in evolution by providing the genetic material from which novel functions can arise. Newly duplicated genes can be maintained by subfunctionalization (the duplicated genes perform different aspects of the original gene's function) and/or neofunctionalization (one of the genes acquires a novel function). PLENA in Antirrhinum and AGAMOUS in Arabidopsis are the canonical C-function genes that are essential for the specification of reproductive organs. These functionally equivalent genes encode closely related homeotic MADS-box transcription factors. Using genome synteny, we confirm phylogenetic analyses showing that PLENA and AGAMOUS are nonorthologous genes derived from a duplication in a common ancestor. Their respective orthologs, SHATTERPROOF in Arabidopsis and FARINELLI in Antirrhinum, have undergone independent subfunctionalization via changes in regulation and protein function. Surprisingly, the functional divergence between PLENA and FARINELLI, is morphologically manifest in both transgenic Antirrhinum and Arabidopsis. This provides a clear illustration of a random evolutionary trajectory for gene functions after a duplication event. Different members of a duplicated gene pair have retained the primary homeotic functions in different lineages, illustrating the role of chance in evolution. The differential ability of the Antirrhinum genes to promote male or female development provides a striking example of subfunctionalization at the protein level.

Recent studies on leaf development demonstrate that the mechanism on the adaxial-abaxial polarity pattern formation could be well conserved among the far-related species, in which PHANTASTICA (PAHN)-like genes play important roles. In this study, we explored the conservation and diversity on functions of PHAN-like genes during the compound leaf development in Lotus japonicus, a papilionoid legume. Two PHAN-like genes in L. japonicus, LjPHANa and LjPHANb, were found to originate from a gene duplication event and displayed different expression patterns during compound leaf development. Two mutants, reduced leaflets1 (rel1) and reduced leaflets3 (rel3), which exhibited decreased adaxial identity of leaflets and reduced leaflet initiation, were identified and investigated. The expression patterns of both LjPHANs in rel mutants were altered and correlated with abnormalities of compound leaves. Our data suggest that LjPHANa and LjPHANb play important but divergent roles in regulating adaxial-abaxial polarity of compound leaves in L. japonicus.

The cellular retinoic acid-binding protein type I (CRABPI) is encoded by a single gene in mammals. We have characterized two crabp1 genes in zebrafish, designated crabp1a and crabp1b. These two crabp1 genes share the same gene structure as the mammalian CRABP1 genes and encode proteins that show the highest amino acid sequence identity to mammalian CRABPIs. The zebrafish crabp1a and crabp1b were assigned to linkage groups 25 and 7, respectively. Both linkage groups show conserved syntenies to a segment of the human chromosome 15 harboring the CRABP1 locus. Phylogenetic analysis suggests that the zebrafish crabp1a and crabp1b are orthologs of the mammalian CRABP1 genes that likely arose from a teleost fish lineage-specific genome duplication. Embryonic whole mount in situ hybridization detected zebrafish crabp1b transcripts in the posterior hindbrain and spinal cord from early stages of embryogenesis. crabp1a mRNA was detected in the forebrain and midbrain at later developmental stages. In adult zebrafish, crabp1a mRNA was localized to the optic tectum, whereas crabp1b mRNA was detected in several tissues by RT-PCR but not by tissue section in situ hybridization. The differential and complementary expression patterns of the zebrafish crabp1a and crabp1b genes imply that subfunctionalization may be the mechanism for the retention of both crabp1 duplicated genes in the zebrafish genome.

Motivation: One major area of interest in analyzing oligonucleotide gene array data is identifying differentially expressed genes. A challenge to biostatisticians is to develop an approach to summarizing probe-level information that adequately reflects the true expression level while accounting for probe variation, chip variation and interaction effects. Various statistical tools, such as MAS and RMA, have been developed to address this issue. In these approaches, the probe level expression data are summarized into gene level data, which are then used for downstream statistical analysis. Since probe variation is often larger than chip variation and there is also a potential interaction effect between probe affinity and treatment effect, strategies such as a gene level analysis, may not be optimal. In this study, we propose a procedure to analyze probe level data for selecting differentially expressed genes under two treatment conditions (groups) with a small number of replicates. The probe level discrepancy between two groups can be measured by a difference of the percentiles of probe perfect-match (PM) ranks or of probe PM weighted ranks. The difference is then compared with a pre-specified threshold to determine differentially expressed genes. The probe level approach takes into account non-homogenous treatment effects and reduces possible cross-hybridization effects across a set of probes., Results: The proposed approach is compared with MAS and RMA using two benchmark gene array datasets. Positive predictivity and sensitivity are used for evaluation. Results show the proposed approach has higher positive predictivity and higher sensitivity., Availability: Available on request from the authors., Contact: dtchen@uab.edu.

Fifteen Rps genes confer resistance against the oomycete pathogen Phytophthora sojae, which causes root and stem rot disease in soybean. We have isolated a disease resistance gene-like sequence from the genomic region containing Rps1-k. Four classes of cDNA of the sequence were isolated from etiolated hypocotyl tissues that express the Rps1-k-encoded Phytophthora resistance. Sequence analyses of a cDNA clone showed that the sequence is a member of the coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type of disease resistance genes. It showed 36% identity to the recently cloned soybean resistance gene Rpg1-b, which confers resistance against Pseudomonas syringae pv. glycinea, and 56% and 38% sequence identity to putative resistance gene sequences from lotus and Medicago truncatula, respectively. The soybean genome contains about 38 copies of the sequence. Most of these copies are clustered in approximately 600 kb of contiguous DNA of the Rps1-k region. We have identified a recombinant that carries both rps1-k- and Rps1-k-haplotype-specific allelomorphs of two Rps1-k-linked molecular markers. An unequal crossover event presumably led to duplication of alleles for these two physically linked molecular markers. We hypothesize that the unequal crossing over was one of the mechanisms involved in tandem duplication of CC-NBS-LRR sequences in the Rps1-k region.

Homologous proteins occurring through gene duplication may give rise to novel functions through mutations affecting protein sequence or expression. Comparison of such homologues allows insight into how morphological traits evolve. However, it is often unclear which changes are key to determining new functions. To address these ideas, we have studied a system where two homologues have evolved clear and opposite functions in controlling a major developmental switch. In plants, flowering is a major developmental transition that is critical to reproductive success. Arabidopsis phosphatidylethanolamine-binding protein homologues TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) are key controllers of flowering, determining when and where flowers are made, but as opposing functions: TFL1 is a repressor, FT is an activator. We have uncovered a striking molecular basis for how these homologous proteins have diverged. Although <60% identical, we have shown that swapping a single amino acid is sufficient to convert TFL1 to FT function and vice versa. Therefore, these key residues may have strongly contributed to the selection of these important functions over plant evolution. Further, our results suggest that TFL1 and FT are highly conserved in biochemical function and that they act as repressors or activators of flowering through discrimination of structurally related interactors by a single residue.

In the 14 years since the first higher-level bird phylogenies based on DNA sequence data, avian phylogenetics has witnessed the advent and maturation of the genomics era, the completion of the chicken genome and a suite of technologies that promise to add considerably to the agenda of avian phylogenetics. In this review, we summarize current approaches and data characteristics of recent higher-level bird studies and suggest a number of as yet untested molecular and analytical approaches for the unfolding tree of life for birds. A variety of comparative genomics strategies, including adoption of objective quality scores for sequence data, analysis of contiguous DNA sequences provided by large-insert genomic libraries, and the systematic use of retroposon insertions and other rare genomic changes all promise an integrated phylogenetics that is solidly grounded in genome evolution. The avian genome is an excellent testing ground for such approaches because of the more balanced representation of single-copy and repetitive DNA regions than in mammals. Although comparative genomics has a number of obvious uses in avian phylogenetics, its application to large numbers of taxa poses a number of methodological and infrastructural challenges, and can be greatly facilitated by a 'community genomics' approach in which the modest sequencing throughputs of single PI laboratories are pooled to produce larger, complementary datasets. Although the polymerase chain reaction era of avian phylogenetics is far from complete, the comparative genomics era-with its ability to vastly increase the number and type of molecular characters and to provide a genomic context for these characters-will usher in a host of new perspectives and opportunities for integrating genome evolution and avian phylogenetics.

In this study the molecular evolution of duplicated HoxA genes in zebrafish and fugu has been investigated. All 18 duplicated HoxA genes studied have a higher non-synonymous substitution rate than the corresponding genes in either bichir or paddlefish, where these genes are not duplicated. The higher rate of evolution is not due solely to a higher non-synonymous-to-synonymous rate ratio but to an increase in both the non-synonymous as well as the synonymous substitution rate. The synonymous rate increase can be explained by a change in base composition, codon usage, or mutation rate. We found no changes in nucleotide composition or codon bias. Thus, we suggest that the HoxA genes may experience an increased mutation rate following cluster duplication. In the non-Hox nuclear gene RAG1 only an increase in non-synonymous substitutions could be detected, suggesting that the increased mutation rate is specific to duplicated Hox clusters and might be related to the structural instability of Hox clusters following duplication. The divergence among paralog genes tends to be asymmetric, with one paralog diverging faster than the other. In fugu, all b-paralogs diverge faster than the a-paralogs, while in zebrafish Hoxa-13a diverges faster. This asymmetry corresponds to the asymmetry in the divergence rate of conserved non-coding sequences, i.e., putative cis-regulatory elements. These results suggest that the 5' HoxA genes in the same cluster belong to a co-evolutionary unit in which genes have a tendency to diverge together.

Recent analysis of complete eukaryotic genome sequences has revealed that gene duplication has been rampant. Moreover, next to a continuous mode of gene duplication, in many eukaryotic organisms the complete genome has been duplicated in their evolutionary past. Such large-scale gene duplication events have been associated with important evolutionary transitions or major leaps in development and adaptive radiations of species. Here, we present an evolutionary model that simulates the duplication dynamics of genes, considering genome-wide duplication events and a continuous mode of gene duplication. Modeling the evolution of the different functional categories of genes assesses the importance of different duplication events for gene families involved in specific functions or processes. By applying our model to the Arabidopsis genome, for which there is compelling evidence for three whole-genome duplications, we show that gene loss is strikingly different for large-scale and small-scale duplication events and highly biased toward certain functional classes. We provide evidence that some categories of genes were almost exclusively expanded through large-scale gene duplication events. In particular, we show that the three whole-genome duplications in Arabidopsis have been directly responsible for >90% of the increase in transcription factors, signal transducers, and developmental genes in the last 350 million years. Our evolutionary model is widely applicable and can be used to evaluate different assumptions regarding small- or large-scale gene duplication events in eukaryotic genomes.

Zebrafish have two red, LWS-1 and LWS-2, and four green, RH2-1, RH2-2, RH2-3 and RH2-4, opsin genes encoding photopigments with distinct absorption spectra. Occurrence of opsin subtypes by gene duplication is characteristic of fish but little is known whether the subtypes are expressed differently in the retina, either spatially or temporally. Here we show by in situ hybridization the dynamic expression patterns of the opsin subtypes in the zebrafish retina. Expression of red type opsins is initiated with the shorter-wavelength subtype LWS-2, followed by the longer-wavelength subtype LWS-1. In the adult retina, LWS-2 was expressed in the central to dorsal area and LWS-1 in the ventral and peripheral areas. Expression patterns of green type opsins were similar to those of the red type opsins. The expression started with the shortest wavelength subtype RH2-1 followed by the longer wavelength ones, and in the adult retina, the shorter wavelength subtypes (RH2-1 and RH2-2) were expressed in the central to dorsal area and longer wavelength subtypes (RH2-3 and RH2-4) in the ventral and peripheral areas. These results provide the framework for subsequent studies of opsin gene regulation and for probing functional rationale of the developmental changes by using the power of zebrafish genetics.

We have used sequence information and gene location to identify NPY family genes in the pufferfish, Takifugu rubripes (fugu), and zebrafish. Fugu has two copies of NPY, presumably resulting from the ray-fin fish tetraploidization. Zebrafish has probably lost one of the copies. Both species have two copies of PYY, the second of which was previously named PY. The two fugu NPY genes are predominantly expressed in brain. The two PYY genes are expressed in a broad range of tissues including brain and gonads. Thus, the NPY system appears to be more complex in teleosts than in tetrapods.

Teleost fishes have three distinct oestrogen receptor (ER) subtypes: ER-alpha, ER-beta a (or ER-gamma) and ER-beta b. ER-beta a and ER-beta b arose from a duplication of an ancestral ER-beta gene early in the teleost lineage. Here, we describe the distribution of the three ER mRNAs in the hypothalamus and cerebellum of the Atlantic croaker to address two issues: the specific functions of multiple ERs in the neuroendocrine system and the evolution and fate of duplicated genes. ER-alpha was detected in nuclei of the preoptic area (POA) and hypothalamus previously shown to possess ER-alphas in teleosts. AcER-beta b, but not ER-beta a, labelling was detected in the magnocellular neurons of the POA, nucleus posterior tuberis, the nucleus recessus posterior and cerebellum. By contrast, acER-beta a, but not ER-beta b, was detected in the dorsal anterior parvocellular POA and suprachiasmatic nucleus. Both ER-betas were found in posterior parvocellular and ventral anterior POA nuclei, the ventral hypothalamus, and periventricular dorsal hypothalamus. The differences we observed in ER subtype mRNA distribution within well-characterized brain nuclei suggest that ER-beta a and ER-beta b have distinct functions in the neuroendocrine control of reproduction and behaviour, and provide evidence that the teleost ER-beta paralogues have partitioned functions of the ancestral ER-beta gene they shared with tetrapods.

In the sensory receptors of both the eye and the ear, specialized apical structures have evolved to detect environmental stimuli such as light and sound. Despite the morphological divergence of these specialized structures and differing transduction mechanisms, the receptors appear to rely in part on a shared group of genes for function. For example, mutations in Usher (USH) genes cause a syndrome of visual and acoustic-vestibular deficits in humans. Several of the affected genes have been identified, including the USH1F gene, which encodes protocadherin 15 (PCDH15). Pcdh15 mutant mice also have both auditory and vestibular defects, although visual defects are not evident. Here we show that zebrafish have two closely related pcdh15 genes that are required for receptor-cell function and morphology in the eye or ear. Mutations in pcdh15a cause deafness and vestibular dysfunction, presumably because hair bundles of inner-ear receptors are splayed. Vision, however, is not affected in pcdh15a mutants. By contrast, reduction of pcdh15b activity using antisense morpholino oligonucleotides causes a visual defect. Optokinetic and electroretinogram responses are reduced in pcdh15b morpholino-injected larvae. In electron micrographs, morphant photoreceptor outer segments are improperly arranged, positioned perpendicular to the retinal pigment epithelium and are clumped together. Our results suggest that both cadherins act within their respective transduction organelles: Pcdh15a is necessary for integrity of the stereociliary bundle, whereas Pcdh15b is required for alignment and interdigitation of photoreceptor outer segments with the pigment epithelium. We conclude that after a duplication of pcdh15, one gene retained an essential function in the ear and the other in the eye.

The Prader-Willi/Angelman Critical Region (PWACR; Chromosome 15q11-13) is of interest as a potential locus for genes conferring susceptibility to autism spectrum disorders (ASD). This report describes a female proband referred for evaluation of a possible ASD. Genetic analyses indicated that the proband, her father and one of her sisters, carried a paternally derived interstitial duplication involving 15q11-13. The proband showed evidence of ASD (PDD-NOS), borderline mental retardation, mild hypotonia and joint laxity. Her father and her sister were of normal intelligence and neither was thought to have an ASD, although speech/language difficulties and some autistic type behaviours were reported to have been present early in the development of the sister. This is one of the first reports of a child with a paternal duplication and an autism spectrum disorder. More research is required to determine whether paternally derived duplications that involve 15q11-13 are associated with developmental impairments.

Among the major proteins in the rat seminal vesicle secretion, transglutaminase catalyzed the cross-links among RSVS I-III. Six peptide sequences determined from the trypsin digests of RSVS III were confirmed in the protein sequence derived from two paralogs, RSVS III(alpha) and RSVS III(beta) gene, in rat 3q42. Their transcription units are organized with the first exon encoding a signal peptide, and the second a secreted protein, whereas the third encompasses a 3'-non-translated nucleotide that shares common features of rapidly evolving substrates of transglutaminase (REST) gene family. These two genes have 99% identity in their coding region and both express in adult rats with the same transglutaminase cross-linking site, manifesting functional preservation. All of REST genes reported thus far for human and Muridae were mapped in a chromosomal locus between KCNS1 and SLPI suggesting the locus as an active evolving region. The molecular evolution of this gene family is discussed.

Human pericentromeric sequences are enriched for recent sequence duplications. The continual creation and shuffling of these duplications can create novel intron-exon structures and it has been suggested that these regions have a function as gene nurseries. However, these sequences are also rich in satellite repeats which can repress transcription, and analyses of chromosomes 10 and 21 have suggested that they are transcript poor. Here, we investigate the relationship between pericentromeric duplication and transcription by analyzing the in silico transcriptional profiles within the proximal 1.5 Mb of genomic sequence on all human chromosome arms in relation to duplication status. We identify an approximately 5x excess of transcripts specific to cancer and/or testis in pericentromeric duplications compared to surrounding single copy sequence, with the expression of >50% of all transcripts in duplications being restricted to these tissues. We also identify an approximately 5x excess of transcripts in duplications which contain large quantities of interspersed repeats. These results indicate that the transcriptional profiles of duplicated and single copy sequences within pericentromeric DNA are distinct, suggesting that pericentromeric instability is unlikely to represent a common route for gene creation but may have a disproportionate effect upon genes whose function is restricted to the germ line., (Copyright (c) 2005 S. Karger AG, Basel.)

Human beta-defensin (HBD)-2 is a small cationic peptide with a broad range of antimicrobial activity. In this study, multiple copies of the hBD2 gene were linked in tandem, and a number of different Escherichia coli expression vectors were evaluated, including pQE-30, pBV220, pET-28a(+), and pGEX-4T-2. No expression of multiple joined genes was detectable in the pQE-30 expression system, whereas in pBV220 with one or two joined hBD2 genes and in pET-28a(+) with one, two, or four copies, target proteins were expressed at a low level. Only when pGEX-4T-2 was applied as expression plasmid with one or two joined hBD2 genes were target proteins expressed in high level, and the expressed fusion proteins account for 26 and 16% of the total insoluble proteins, respectively. In the pGEX-4T-2 and pET-28a(+) expression systems, the effects of multiple joined genes on the growth of host strains and plasmid stability were examined. Host cells containing plasmid carrying fewer copies of hBD2 genes were faster in cell growth. Plasmid stability decreased with an increase in multiple joined genes, which was especially noticeable in the pET-28a(+) system. Furthermore, the presence of glucose in culture medium brought about a positive effect on plasmid stability when using pET28-nhBD2 as expression vectors.

Background: Quantification of intracellular metabolite fluxes by 13C-tracer experiments is maturing into a routine higher-throughput analysis. The question now arises as to which mutants should be analyzed. Here we identify key experiments in a systems biology approach with a genome-scale model of Saccharomyces cerevisiae metabolism, thereby reducing the workload for experimental network analyses and functional genomics., Results: Genome-scale 13C flux analysis revealed that about half of the 745 biochemical reactions were active during growth on glucose, but that alternative pathways exist for only 51 gene-encoded reactions with significant flux. These flexible reactions identified in silico are key targets for experimental flux analysis, and we present the first large-scale metabolic flux data for yeast, covering half of these mutants during growth on glucose. The metabolic lesions were often counteracted by flux rerouting, but knockout of cofactor-dependent reactions, as in the adh1, ald6, cox5A, fum1, mdh1, pda1, and zwf1 mutations, caused flux responses in more distant parts of the network. By integrating computational analyses, flux data, and physiological phenotypes of all mutants in active reactions, we quantified the relative importance of 'genetic buffering' through alternative pathways and network redundancy through duplicate genes for genetic robustness of the network., Conclusions: The apparent dispensability of knockout mutants with metabolic function is explained by gene inactivity under a particular condition in about half of the cases. For the remaining 207 viable mutants of active reactions, network redundancy through duplicate genes was the major (75%) and alternative pathways the minor (25%) molecular mechanism of genetic network robustness in S. cerevisiae.

We studied the age distribution of duplicate genes in each of four eukaryotic genomes: human, Arabidopsis thaliana, Caenorhabditis elegans, and Drosophila melanogaster. The four distributions differ greatly from each other, contrary to the previous proposal of a universal L-shaped distribution in all eukaryotic genomes studied. Indeed, only the distribution in humans is L-shaped. The distribution in Arabidopsis is consistent with the hypothesis of an ancient genome duplication with no recent burst of duplication events, while the distribution in C. elegans is nearly uniform. We also applied a nonparametric method to the human distribution to show that the rate of loss of duplicate genes decreases over time, contrary to the proposal of an exponential decay. One possible explanation of the decreasing rate of loss of duplicate genes over time could be rapid functional divergence between duplicate genes, providing an advantage for the retention of both duplicates.

Histones are the major protein component of nucleosomes, and de novo histone synthesis is essential for packaging newly replicated DNA into chromatin. As a result, histone gene expression is exquisitely and functionally coupled with DNA replication. Vastly divergent organisms such as yeast, fly and human all demonstrate the phylogenetically conserved propensity to maintain clustering of histone genes at one or more genomic loci. Although specific mechanisms are unclear, clustering is presumed to be important for common stringent transcriptional control of these genes at the G1/S phase transition. In this study, we describe a genomic duplication of the human histone gene cluster located at chromosome 1q21, which effectively doubles the previously known size and gene number of that cluster. The duplication persists in all examined tissues and cell lines, and the duplicated genes are transcriptionally active. Levels of messenger RNAs for duplicated histone H4 genes are high relative to those for non-duplicated H4 genes. Our data suggest that transcriptionally robust histone H4 genes may have been preferentially duplicated during evolution.

Vernalization, the requirement of a long exposure to low temperatures to induce flowering, is an essential adaptation of plants to cold winters. We have shown recently that the vernalization gene VRN-1 from diploid wheat Triticum monococcum is the meristem identity gene APETALA1, and that deletions in its promoter were associated with spring growth habit. In this study, we characterized the allelic variation at the VRN-1 promoter region in polyploid wheat. The Vrn-A1a allele has a duplication including the promoter region. Each copy has similar foldback elements inserted at the same location and is flanked by identical host direct duplications (HDD). This allele was found in more than half of the hexaploid varieties but not among the tetraploid lines analyzed here. The Vrn-A1b allele has two mutations in the HDD region and a 20-bp deletion in the 5' UTR compared with the winter allele. The Vrn-A1b allele was found in both tetraploid and hexaploid accessions but at a relatively low frequency. Among the tetraploid wheat accessions, we found two additional alleles with 32 bp and 54 bp deletions that included the HDD region. We found no size polymorphisms in the promoter region among the winter wheat varieties. The dominant Vrn-A1 allele from two spring varieties from Afghanistan and Egypt ( Vrn-A1c allele) and all the dominant Vrn-B1 and Vrn-D1 alleles included in this study showed no differences from their respective recessive alleles in promoter sequences. Based on these results, we concluded that the VRN-1 genes should have additional regulatory sites outside the promoter region studied here.