Widespread conservation of genetic redundancy during a billion years of eukaryotic evolution

Genetic redundancy means that two genes can perform the same function. Using a comprehensive phylogenetic analysis, we show here in both Saccharomyces cerevisiae and Caenorhabditis elegans that genetic redundancy is not just a transient consequence of gene duplication, but is often an evolutionary stable state. In multiple examples, genes have retained redundant functions since the divergence of the animal, plant and fungi kingdoms over a billion years ago. The stable conservation of genetic redundancy contrasts with the more rapid evolution of genetic interactions between unrelated genes and can be explained by theoretical models including a 'piggyback' mechanism in which overlapping redundant functions are co-selected with nonredundant ones.     

Pathogenic Parkinson’s disease mutations across the functional domains of LRRK2 alter the autophagic/lysosomal response to starvation

LRRK2 is one of the most important genetic contributors to Parkinson’s disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations.     

Emerging novel functions of RNAs, and binary phenotype?

Loss-of-function technology has been one of the most popular knockout tools for the study of gene function in cell and developmental biology. This technology employs two basic approaches for elimination of the protein of interest. The morpholino antisense oligonucleotides approach relies on inhibiting translation of the given protein without degrading the cognate mRNA. The antisense deoxynucleotides and siRNA approach acts via removal of the mRNA template, which then prevents protein translation. In the latest approach, as well as in these genetic knockout approaches that eliminate or alter the level of mRNA transcribed from the gene of interest, the assumption is and always has been that the only relevant function of mRNA is to make a protein, and, thus, the effect of removing mRNA equals the effect of removing its protein function. However, the most recent studies of different biological systems point to completely novel and unexpected functions of the subpopulation of localized RNAs and suggest that, at least in some cases, the normal cell or embryo phenotype is in fact binary i.e. depends not only on the function of the protein but also on the autonomous function of its mRNA.     

Interpretation of phenotype in genetically engineered mice.

BACKGROUND AND PURPOSE: In mice, genetic engineering involves two general approaches-addition of an exogenous gene, resulting in transgenic mice, and use of knockout mice, which have a targeted mutation of an endogenous gene. The advantages of these approaches is that questions can be asked about the function of a particular gene in a living mammalian organism, taking into account interactions among cells, tissues, and organs under normal, disease, injury, and stress situations. METHODS: Review of the literature concentrating principally on knockout mice and questions of unexpected phenotypes, lack of phenotype, redundancy, and effect of genetic background on phenotype will be discussed. CONCLUSION: There is little gene redundancy in mammals; knockout phenotypes exist even if none are immediately apparent; and investigating phenotypes in colonies of mixed genetic background may reveal not only more phenotypes, but also may lead to better understanding of the molecular or cellular mechanism underlying the phenotype and to discovery of modifier gene(s).     

Ligand receptor interactions in the Wnt signaling pathway in Drosophila

Secreted Wnt proteins have numerous signaling functions during development, mediated by Frizzled molecules that act as Wnt receptors on the cell surface. In the genome of Drosophila, seven Wnt genes (including wingless; wg), and five frizzled genes have been identified. Relatively little is known about signaling and binding specificities of different Wnt and Frizzled proteins. We have developed an assay to determine the strength of binding between membrane-tethered Wnts and ligand binding domains of the Frizzled receptors. We found a wide spectrum of binding affinities, reflecting known genetic interactions. Most Wnt proteins can bind to multiple Frizzleds and vice versa, suggesting redundancy in vivo. In an extension of these experiments, we tested whether two different subdomains of the Wg protein would by themselves bind to Frizzled and generate a biological response. Whereas these two separate domains are secreted from cells, suggesting that they form independently folded parts of the protein, they were only able to evoke a response when co-transfected, indicating that both are required for function. In addition to the Frizzleds, members of the LRP family (represented by the arrow gene in Drosophila) are also necessary for Wnt signal transduction and have been postulated to act as co-receptors. We have therefore examined whether a soluble form of the Arrow molecule can bind to Wingless and Frizzled, but no interactions were detected.     

The role of domain redundancy in genetic robustness against null mutations

A key question in molecular genetics is why severe gene mutations often do not result in a detectable abnormal phenotype. Alternative networks are known to be a gene compensation mechanism. Gene redundancy, i.e. the presence of a duplicate gene (or paralog) elsewhere in the genome, also underpins many cases of gene dispensability. Here, we investigated the role of partial duplicate genes on dispensability, where a partial duplicate is defined as a gene that has no paralog but which codes for a protein made of domains, each of which belongs to at least another protein. The rationale behind this investigation is that, as a partial duplicate codes for a domain redundant protein, we hypothesised that its deletion might have a less severe phenotypic effect than the deletion of other genes. This prompted us to (re)address the topic of gene dispensability by focusing on domain redundancy rather than on gene redundancy. Using fitness data of single-gene deletion mutants of Saccharomyces cerevisiae, we will show that domain redundancy is a compensation mechanism, the strength of which is lower than that of gene redundancy. Finally, we shall discuss the molecular basis of this new compensation mechanism.     

Distributed robustness versus redundancy as causes of mutational robustness

A biological system is robust to mutations if it continues to function after genetic changes in its parts. Such robustness is pervasive on different levels of biological organization, from macromolecules to genetic networks and whole organisms. I here ask which of two possible causes of such robustness are more important on a genome-wide scale, for systems whose parts are genes, such as metabolic and genetic networks. The first of the two causes is redundancy of a system's parts: A gene may be dispensable if the genome contains redundant, back-up copies of the gene. The second cause, distributed robustness, is more poorly understood. It emerges from the distributed nature of many biological systems, where many (and different) parts contribute to system functions. I will here discuss evidence suggesting that distributed robustness is equally or more important for mutational robustness than gene redundancy. This evidence comes from the functional divergence of redundant genes, as well as from large-scale gene deletion studies. I also ask whether one can quantify the extent to which redundancy or distributed robustness contribute to mutational robustness.     

Gene duplication, the evolution of novel gene functions, and detecting functional divergence of duplicates in silico

Duplication of genes increases the amount of genetic material on which evolution can work and has been considered of major importance for the development of biological novelties or to explain important transitions that have occurred during biological evolution. Recently, much research has been devoted to the study of the evolutionary and functional divergence of duplicated genes. Since the majority of genes are part of gene families, there is considerable interest in predicting differences in function between duplicates and assessing the functional redundancy of genes within gene families. In this review, we discuss the strengths and limitations of both older and novel approaches to investigate the evolution of duplicated genes in silico.     

Evolution of genetic redundancy for advanced players

An ever expanding database on the sequence organization and repetition of genic and non-genic components of nuclear and organelle genomes reveals that the vast majority of sequences are subject to one or other mechanism of DNA turnover (gene conversion, unequal crossing over, slippage, retrotransposition, transposition and others). Detailed studies, using novel methods of experimental detection and analytical procedures, show that such mechanisms can operate one on top of another and that wide variations in their unit lengths, biases, polarities and rates create bizarre and complex patterns of genetic redundancy. The ability of these mechanisms to operate both within and between chromosomes implies that realistic models of the evolutionary dynamics of redundancy, and of the potential interaction with natural selection in a sexual species, need to consider the diffusion of variant repeats across multiple chromosome lineages, in a population context. Recently, important advances in both experimental and analytical approaches have been made along these lines. There is increasing awareness that genetic redundancy and turnover induces a molecular co-evolution between functionally interacting genetic systems in order to maintain essential functions.     

Characterization of dFMR1, a Drosophila melanogaster homolog of the fragile X mental retardation protein

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by loss of FMR1 gene activity due to either lack of expression or expression of a mutant form of the protein. In mammals, FMR1 is a member of a small protein family that consists of FMR1, FXR1, and FXR2. All three members bind RNA and contain sequence motifs that are commonly found in RNA-binding proteins, including two KH domains and an RGG box. The FMR1/FXR proteins also contain a 60S ribosomal subunit interaction domain and a protein-protein interaction domain which mediates homomer and heteromer formation with each family member. Nevertheless, the specific molecular functions of FMR1/FXR proteins are unknown. Here we report the cloning and characterization of a Drosophila melanogaster homolog of the mammalian FMR1/FXR gene family. This first invertebrate homolog, termed dfmr1, has a high degree of amino acid sequence identity/similarity with the defined functional domains of the FMR1/FXR proteins. The dfmr1 product binds RNA and is similar in subcellular localization and embryonic expression pattern to the mammalian FMR1/FXR proteins. Overexpression of dfmr1 driven by the UAS-GAL4 system leads to apoptotic cell loss in all adult Drosophila tissues examined. This phenotype is dependent on the activity of the KH domains. The ability to induce a dominant phenotype by overexpressing dfmr1 opens the possibility of using genetic approaches in Drosophila to identify the pathways in which the FMR1/FXR proteins function.     

Functional overlap and regulatory links shape genetic interactions between signaling pathways

To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy, and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multiprocess control.     

Switching the flip: protein phosphatase roles in signaling pathways

Protein phosphatases are the obligate partners of protein kinases in cellular control circuitry. Several specific roles of phosphatases have been revealed in plant systems through recent work capitalizing on functional genomics, interaction screens and activation-tagging approaches. Historically, the redundancy of genes that encode phosphatase functions has impeded genetic analysis in plant systems, and relatively few elucidated signaling pathways include protein phosphatases that have clearly defined substrates. Functional genomics, interaction and suppression screens, activation tagging and phosphoproteomics now hold great promise for identifying phosphatases that are active in signaling and regulation, and for revealing their in vivo substrates. This level of analysis will be essential for a full understanding of the effects of reversible protein phosphorylation in modulating and integrating cellular activities.     

The plant-specific G protein γ subunit AGG3 influences organ size and shape in Arabidopsis thaliana

? Control of organ size and shape by cell proliferation and cell expansion is a fundamental developmental process, but the mechanisms that set the size and shape of determinate organs are largely unknown in plants. ? Molecular, genetic, cytological and biochemical approaches were used to characterize the roles of the Arabidopsis thaliana G protein γ subunit (AGG3) gene in organ growth. ? Here, we describe A. thaliana AGG3, which promotes petal growth by increasing the period of cell proliferation. Both the N-terminal region and the C-terminal domains of AGG3 are necessary for the function of AGG3. By contrast, analysis of a series of AGG3 derivatives with deletions in specific domains showed that the deletion of any of these domains cannot completely abolish the function of AGG3. The GFP-AGG3 fusion protein is localized to the plasma membrane. The predicted transmembrane domain plays an important role in the plasma membrane localization of AGG3. Genetic analyses revealed that AGG3 action requires a functional G protein α subunit (GPA1) and G protein β subunit (AGB1). ? Our findings demonstrate that AGG3, GPA1 and AGB1 act in the same genetic pathway to influence organ size and shape in A. thaliana.     

Kallmann's syndrome: molecular pathogenesis

Kallmann's syndrome (KS) is a genetic condition characterised by hypogonadotrophic hypogonadism (HH) and anosmia; although these are the defining features of the condition, additional neurological and non-neurological sequel may also occur depending on the specific mode of inheritance. KS affects about 1 in 8000 males and 1 in 40,000 females, with most presentations being of the 'sporadic' type. Of the inherited forms, hitherto, only the gene responsible for the X-linked form (X-KS), namely KAL-1, has been identified and the encoded protein, anosmin-1, consists primarily of a whey acidic protein (WAP) and fibronectin-like type III (FnIII) domains which appear to mediate distinctly different protein functions. The WAP/FnIII combination is conserved in anosmins across species and recent studies in rodents and in Caenorhabditis elegans demonstrate that anosmin functions in both axonal targeting and branching. Screening for loci that modify these phenotypes in C. elegans has identified heparan-6-O-sulphotransferase as a key interactor mediating anosmin-1 function. Furthermore, over-expression and loss of function of the C. elegans Kal-1 gene disrupt epidermal morphogenesis, resulting in ventral enclosure and male tail formation defects. These findings provide novel insights into the molecular pathogenesis of X-KS.     

Bindin,a multifunctional sperm ligand and the evolution of new species

Sea urchin sperm species-specifically adhere to the egg surface during fertilization. The protein which mediates this adhesion is known as bindin and cDNAs have recently been cloned and sequenced from several different species. Bindin proteins contain a highly conserved central domain flanked by much more highly divergent amino- and carboxyl-terminal domains. Investigations of the structure and function relationships indicate that the conserved domains may participate in membrane fusion and sulfated fucan binding activities, which may be conserved functions of bindin. The species-specific adhesion activity appears to be duplicated in both the amino- and carboxyl-terminal domain and may correspond to repeated sequence motifs found in these domains. The duplication of these sequence motifs and the redundancy of the adhesive domains may be important for the molecular mechanism of bindin evolution during speciation.     

Characterization of wheat DArT markers: genetic and functional features

Diversity array technology (DArT) markers are largely used for mapping, genetic diversity, and association mapping studies. For years, they have been used as anonymous genomic markers, as their sequences were not known. As the sequences of 2,000 wheat DArT clones are now available, this study was designed to analyze these sequences with bioinformatic approaches, and to study the genetic features of a subset of 291 markers positioned on the A and B genomes in three durum wheat genetic maps. A set of 1,757 non-redundant sequences was identified, and used as queries for similarity searches. Analysis of the genetic positions of markers corresponding to nearly identical sequences indicates that redundancy of sequences is one of the factors that explains the clustering of these markers in specific genomic regions. Of a total of 1,124 DArT clones (64?%) that represent putatively expressed sequences, putative functions are proposed for more than 700 of them. Of note, many clones correspond to genes that are related to disease resistance, as characterized by leucine-rich repeat domains, and 40 of these clones are positioned in the three genetic maps presented in this study. Finally, DArT markers have been used to find syntenic regions in the Brachypodium and rice genomes. In conclusion, the analyses herein presented contribute to explain the main features of DArT markers observed in genetic maps, as clustering in short chromosome regions. Moreover, the attribution of putative gene functions for more than 700 sequences makes these markers an optimal tool for collinearity studies or for the identification of candidate genes.     

Perspectives on Systematic Analyses of Gene Function in Arabidopsis thaliana: New Tools, Topics and Trends

Since the sequencing of the nuclear genome of Arabidopsis thaliana ten years ago, various large-scale analyses of gene function have been performed in this model species. In particular, the availability of collections of lines harbouring random T-DNA or transposon insertions, which include mutants for almost all of the ~27,000 A. thaliana genes, has been crucial for the success of forward and reverse genetic approaches. In the foreseeable future, genome-wide phenotypic data from mutant analyses will become available for Arabidopsis, and will stimulate a flood of novel in-depth gene-function analyses. In this review, we consider the present status of resources and concepts for systematic studies of gene function in A. thaliana. Current perspectives on the utility of loss-of-function and gain-of-function mutants will be discussed in light of the genetic and functional redundancy of many A. thaliana genes.