The Effect of Recent Admixture on Inference of Ancient Human Population History

Despite the widespread study of genetic variation in admixed human populations, such as African-Americans, there has not been an evaluation of the effects of recent admixture on patterns of polymorphism or inferences about population demography. These issues are particularly relevant because estimates of the timing and magnitude of population growth in Africa have differed among previous studies, some of which examined African-American individuals. Here we use simulations and single-nucleotide polymorphism (SNP) data collected through direct resequencing and genotyping to investigate these issues. We find that when estimating the current population size and magnitude of recent growth in an ancestral population using the site frequency spectrum (SFS), it is possible to obtain reasonably accurate estimates of the parameters when using samples drawn from the admixed population under certain conditions. We also show that methods for demographic inference that use haplotype patterns are more sensitive to recent admixture than are methods based on the SFS. The analysis of human genetic variation data from the Yoruba people of Ibadan, Nigeria and African-Americans supports the predictions from the simulations. Our results have important implications for the evaluation of previous population genetic studies that have considered African-American individuals as a proxy for individuals from West Africa as well as for future population genetic studies of additional admixed populations.STUDIES of archeological and genetic data show that anatomically modern humans originated in Africa and more recently left Africa to populate the rest of the world (Tishkoff and Williams 2002; Barbujani and Goldstein 2004; Garrigan and Hammer 2006; Reed and Tishkoff 2006; Campbell and Tishkoff 2008; Jakobsson et al. 2008; Li et al. 2008). Given the central role Africa has played in the origin of diverse human populations, understanding patterns of genetic variation and the demographic history of populations within Africa is important for understanding the demographic history of global human populations. The availability of large-scale single-nucleotide polymorphism (SNP) data sets coupled with recent advances in statistical methodology for inferring parameters in population genetic models provides a powerful means of accomplishing these goals (Keinan et al. 2007; Boyko et al. 2008; Lohmueller et al. 2009; Nielsen et al. 2009).It is important to realize that studies of African demographic history using genetic data have come to qualitatively different conclusions regarding important parameters. Some recent studies have found evidence for ancient (>100,000 years ago) two- to fourfold growth in African populations (Adams and Hudson 2004; Marth et al. 2004; Keinan et al. 2007; Boyko et al. 2008). Other studies have found evidence of very recent growth (Pluzhnikov et al. 2002; Akey et al. 2004; Voight et al. 2005; Cox et al. 2009; Wall et al. 2009) or could not reject a model with a constant population size (Pluzhnikov et al. 2002; Voight et al. 2005). It is unclear why studies found such different parameter estimates. However, these studies all differ from each other in the amount of data considered, the types of data used (e.g., SNP genotypes vs. full resequencing), the genomic regions studied (e.g., noncoding vs. coding SNPs), and the types of demographic models considered (e.g., including migration vs. not including migration postseparation of African and non-African populations).Another important way in which studies of African demographic history differ from each other is in the populations sampled. Some studies have focused on genetic data from individuals sampled from within Africa (Pluzhnikov et al. 2002; Adams and Hudson 2004; Voight et al. 2005; Keinan et al. 2007; Cox et al. 2009; Wall et al. 2009), while other studies included American individuals with African ancestry (Adams and Hudson 2004; Akey et al. 2004; Marth et al. 2004; Boyko et al. 2008). While there is no clear correspondence between those studies which sampled native African individuals (as opposed to African-Americans) and particular growth scenarios, it is clear from previous studies that African-American populations do differ from African populations in their recent demographic history. In particular, genetic studies suggest that there is wide variation in the degree of European admixture in most African-American individuals in the United States and that they have, on average, ∼80% African ancestry and 20% European ancestry (Parra et al. 1998; Pfaff et al. 2001; Falush et al. 2003; Patterson et al. 2004; Tian et al. 2006; Lind et al. 2007; Reiner et al. 2007; Price et al. 2009; Bryc et al. 2010). Furthermore, both historical records and genetic evidence suggest that the admixture process began quite recently, within the last 20 generations (Pfaff et al. 2001; Patterson et al. 2004; Seldin et al. 2004; Tian et al. 2006). Recent population admixture can alter patterns of genetic variation in a discernible and predictable way. For example, recently admixed populations will exhibit correlation in allele frequencies (i.e., linkage disequilibrium) among markers that differ in frequency between the parental populations. This so-called admixture linkage disequilibrium (LD) (Chakraborty and Weiss 1988) can extend over long physical distances (Lautenberger et al. 2000) and decays exponentially with time the since the admixture process began (i.e., recently admixed populations typically exhibit LD over a longer physical distance than anciently admixed populations).While it is clear that African-American populations have a different recent demographic history than do African populations from within Africa and that admixture tracts can be identified in admixed individuals (Falush et al. 2003; Patterson et al. 2004; Tang et al. 2006; Sankararaman et al. 2008a,b; Price et al. 2009; Bryc et al. 2010), the effect that admixture has on other patterns of genetic variation remains unclear. For example, Xu et al. (2007) found similar LD decay patterns when comparing African-American and African populations. It is also unclear whether the recent admixture affects our ability to reconstruct ancient demographic events (such as expansions that predate the spread of humans out of Africa) from whole-genome SNP data. Most studies of demographic history have summarized the genome-wide SNP data by allele frequency or haplotype summary statistics. If these summary statistics are not sensitive to the recent European admixture, then the African-American samples may yield estimates of demographic parameters that are close to the true demographic parameters for the ancestral, unsampled, African populations. This would suggest that the differences in growth parameter estimates obtained from African populations cannot be explained by certain studies sampling African-American individuals and others sampling African individuals from within Africa. However, if these statistics are sensitive to recent admixture, then they may give biased estimates of growth parameters.Here, we examine the effect of recent admixture on the estimation of population demography. In particular, we estimate growth parameters from simulated data sets using SNP frequencies as well as a recently developed haplotype summary statistic (Lohmueller et al. 2009). We compare the demographic parameter estimates made from the admixed and nonadmixed populations and find that some parameter estimates are qualitatively similar between the two populations when inferred using allele frequencies. Inferences of growth using haplotype-based approaches appear to be more sensitive to recent admixture than inferences based on SNP frequencies. We discuss implications that our results have for interpreting studies of demography in admixed populations.     

The Drosophila Claudin Kune-kune Is Required for Septate Junction Organization and Tracheal Tube Size Control

The vertebrate tight junction is a critical claudin-based cell–cell junction that functions to prevent free paracellular diffusion between epithelial cells. In Drosophila, this barrier is provided by the septate junction, which, despite being ultrastructurally distinct from the vertebrate tight junction, also contains the claudin-family proteins Megatrachea and Sinuous. Here we identify a third Drosophila claudin, Kune-kune, that localizes to septate junctions and is required for junction organization and paracellular barrier function, but not for apical-basal polarity. In the tracheal system, septate junctions have a barrier-independent function that promotes lumenal secretion of Vermiform and Serpentine, extracellular matrix modifier proteins that are required to restrict tube length. As with Sinuous and Megatrachea, loss of Kune-kune prevents this secretion and results in overly elongated tubes. Embryos lacking all three characterized claudins have tracheal phenotypes similar to any single mutant, indicating that these claudins act in the same pathway controlling tracheal tube length. However, we find that there are distinct requirements for these claudins in epithelial septate junction formation. Megatrachea is predominantly required for correct localization of septate junction components, while Sinuous is predominantly required for maintaining normal levels of septate junction proteins. Kune-kune is required for both localization and levels. Double- and triple-mutant combinations of Sinuous and Megatrachea with Kune-kune resemble the Kune-kune single mutant, suggesting that Kune-kune has a more central role in septate junction formation than either Sinuous or Megatrachea.EPITHELIA are essential for separating physiologically distinct body compartments and regulating trafficking between them. For proper function, it is imperative that epithelia maintain effective barriers against free paracellular diffusion. To this end, epithelial cells contain occluding junctions, which regulate paracellular permeability. In vertebrates, this is accomplished by tight junctions (TJ), structures that are characterized by regions of close membrane apposition between adjacent cells known as “kissing points” (Tsukita and Furuse 2002). While the TJ is made up of at least 40 different components (Schneeberger and Lynch 2004), the core proteins responsible for the paracellular barrier are the claudins (Angelow et al. 2008).Claudins are four-transmembrane domain proteins that form homo- and heterophilic interactions within the same cell (Furuse et al. 1999; Blasig et al. 2006) and with claudins in adjacent cells (Furuse et al. 1999), thereby establishing the paracellular seal. There are 24 members of the claudin family in mammals, many of which display distinct, tissue-specific expression patterns (Kiuchi-Saishin et al. 2002; Angelow et al. 2008). Mutations in several claudins can cause significant paracellular permeability defects in mice. For example, mutations in claudin-14 increase TJ permeability in the organ of Corti and cause deafness (Ben-Yosef et al. 2003), while loss of claudin-1 compromises epidermal barrier function (Furuse et al. 2002).In Drosophila, primary (ectodermally derived) epithelia lack discernable TJs and instead use pleated septate junctions (SJ) for the paracellular barrier (Baumgartner et al. 1996; Lamb et al. 1998; Genova and Fehon 2003; Paul et al. 2003). However, despite sharing a common barrier function, vertebrate TJs and invertebrate SJs differ in several ways. While vertebrate TJs are positioned apical to adherens junctions (AJ) and contain conserved apical polarity proteins, SJs are basal to AJs and contain conserved basolateral polarity proteins (reviewed in Tepass 2003; Wu and Beitel 2004). In addition, SJs do not contain kissing points, but rather ladder-like septa that span the intermembrane space (Lane and Swales 1982; Tepass and Hartenstein 1994).Beyond their general epithelial barrier function, SJs are also required for several tissue-specific processes. Glial cells, for example, ensheath nerve fibers and use SJs to maintain the blood–brain barrier (Auld et al. 1995; Baumgartner et al. 1996; Schwabe et al. 2005). In the embryonic tracheal system, SJs are required for the apical secretion of the lumenal matrix modifying proteins, Vermiform (Verm) and Serpentine (Serp), which act through undefined pathways to restrict tube length (Wang et al. 2006). This secretory pathway appears to be specific for Verm and Serp, since other apical proteins are secreted normally in SJ mutants. SJ proteins have also been shown to play a role in morphogenesis of the heart tube, even though this tissue lacks typical SJ septa (Yi et al. 2008).Although SJs have clear differences from vertebrate TJs, SJs contain at least two claudins, Megatrachea (Mega) and Sinuous (Sinu), both of which are required for the paracellular barrier (Behr et al. 2003; Wu et al. 2004; Stork et al. 2008). In this article, we identify a third claudin, Kune-kune (Kune), that is an integral SJ protein. Like the other claudins, Kune is required for maintaining epithelial paracellular barrier and tracheal tube size control and is not required for apical-basal polarity. We also find that, of all three characterized claudins, Kune has a more severe SJ phenotype, suggesting that it is a more central player in SJ organization and function than previously characterized Drosophila claudins.     

In Planta Mutagenesis Determines the Functional Regions of the Wheat Puroindoline Proteins

In planta analysis of protein function in a crop plant could lead to improvements in understanding protein structure/function relationships as well as selective agronomic or end product quality improvements. The requirements for successful in planta analysis are a high mutation rate, an efficient screening method, and a trait with high heritability. Two ideal targets for functional analysis are the Puroindoline a and Puroindoline b (Pina and Pinb, respectively) genes, which together compose the wheat (Triticum aestivum L.) Ha locus that controls grain texture and many wheat end-use properties. Puroindolines (PINs) together impart soft texture, and mutations in either PIN result in hard seed texture. Studies of the PINs'' mode of action are limited by low allelic variation. To create new Pin alleles and identify critical function-determining regions, Pin point mutations were created in planta via EMS treatment of a soft wheat. Grain hardness of 46 unique PIN missense alleles was then measured using segregating F₂:F₃ populations. The impact of individual missense alleles upon PIN function, as measured by grain hardness, ranged from neutral (74%) to intermediate to function abolishing. The percentage of function-abolishing mutations among mutations occurring in both PINA and PINB was higher for PINB, indicating that PINB is more critical to overall Ha function. This is contrary to expectations in that PINB is not as well conserved as PINA. All function-abolishing mutations resulted from structure-disrupting mutations or from missense mutations occurring near the Tryptophan-rich region. This study demonstrates the feasibility of in planta functional analysis of wheat proteins and that the Tryptophan-rich region is the most important region of both PINA and PINB.NATURAL selection has captured a relatively small subset of potentially useful protein sequences. Unraveling the critical features of proteins via understanding the process of their evolution is a powerful approach for proteins present in many diverse species (Bashford et al. 1987; Hampsey et al. 1988). However, this approach is not feasible for the wheat puroindolines (PINs) that are present only in hexaploid wheat and related species (Massa and Morris 2006). The PINs are unique in structure in having a tryptophan-rich domain and are members of the protease inhibitor/seed storage/lipid transfer protein family (PF00234) (Finn et al. 2008).The tryptophan-rich domain has been hypothesized to control PIN function (Giroux and Morris 1997), but there is no unbiased direct evidence for this since previous studies have focused on the tryptophan box alone (Evrard et al. 2008). A nonbiased approach would consist of random mutagenesis followed by functional analysis (Bowie et al. 1990). This approach has been used extensively for proteins that can be expressed in vitro using either random (Tarun et al. 1998; Guo et al. 2004; Smith and Raines 2006; Georgelis et al. 2007) or site-directed mutations (Miyahara et al. 2008; Osmani et al. 2008). However, functional analysis of many plant proteins in vitro may not be comparable to in planta analysis. In the case of puroindolines, there is no in vitro assay that properly mimics the synergistic binding of PINA and PINB to starch granules or is as easy to measure as grain hardness. Therefore, creation and analysis of a large number of new alleles in wheat in planta is an ideal approach to dissect PIN function.The absence of high-throughput transformation and/or functional screening methods in most crop plants is the largest obstacle in the way of in planta protein functional analysis. However, high-throughput in vitro random or targeted mutagenesis followed by functional analysis has been demonstrated in Arabidopsis thaliana (Dunning et al. 2007) and Nicotiana benthamiana (Boter et al. 2007). Traditional in planta mutagenesis followed by analysis of loss-of-function mutations has been used to clone unknown genes (Xiong et al. 2001) or to define function for candidate genes (Haralampidis et al. 2001; Qi et al. 2006). A high-throughput in planta functional approach for PINA and PINB seems attractive for three reasons. First, the EMS mutation rate in wheat is higher than in any other plant (Slade et al. 2005; Feiz et al. 2009a). Second, PINs control the vast majority of variation in grain hardness (Campbell et al. 1999). Finally, a small-scale preliminary study indicated the feasibility of this approach (Feiz et al. 2009a).PINA and PINB are cysteine-rich proteins unique in having a tryptophan-rich domain (Blochet et al. 1993) and together compose the wheat Hardness (Ha) locus (Giroux and Morris 1998; Wanjugi et al. 2007a). Ha is located on chromosome 5DS and is the major determinant of wheat endosperm texture (Mattern et al. 1973; Law et al. 1978; Campbell et al. 1999). Soft texture (Ha) results when both Pin genes are wild type (Pina-D1a, Pinb-D1a) while hard texture (ha) results from mutations in either Pin (Giroux and Morris 1997, 1998). Transgenic studies in rice (Krishnamurthy and Giroux 2001), wheat (Beecher et al. 2002; Martin et al. 2006), and corn (Zhang et al. 2009) have demonstrated that Pin mutations are causative to hard grain texture. PINA and PINB are not functionally interchangeable and control grain hardness via cooperative binding to starch granules (Hogg et al. 2004; Swan et al. 2006; Wanjugi et al. 2007a; Feiz et al. 2009b). PIN binding to starch granules is mediated by polar lipids (Greenblatt et al. 1995) and PIN abundance is correlated with seed polar lipid content (Feiz et al. 2009b). Variation in PIN function affects grain hardness along with nearly all end product quality traits (Hogg et al. 2005; Martin et al. 2007, 2008; Wanjugi et al. 2007b; Feiz et al. 2008). Determining PINs'' function-determining regions could lead to greater knowledge of their mode of action and to wheat quality improvements. Current PIN functional analyses have been limited to in vitro tests of binding to each other (Ziemann et al. 2008) or to yeast membranes (Evrard et al. 2008).Here, we report the creation and functional analysis in planta of new alleles of PINA and PINB. This is the first successful in planta functional analysis of a crop plant protein.     

Deletion of a Novel F-Box Protein,MUS-10, in Neurospora crassa Leads to Altered Mitochondrial Morphology,Instability of mtDNA and Senescence

While mitochondria are renowned for their role in energy production, they also perform several other integral functions within the cell. Thus, it is not surprising that mitochondrial dysfunction can negatively impact cell viability. Although mitochondria have received an increasing amount of attention in recent years, there is still relatively little information about how proper maintenance of mitochondria and its genomes is achieved. The Neurospora crassa mus-10 mutant was first identified through its increased sensitivity to methyl methanesulfonate (MMS) and was thus believed to be defective in some aspect of DNA repair. Here, we report that mus-10 harbors fragmented mitochondria and that it accumulates deletions in its mitochondrial DNA (mtDNA), suggesting that the mus-10 gene product is involved in mitochondrial maintenance. Interestingly, mus-10 begins to senesce shortly after deletions are visualized in its mtDNA. To uncover the function of MUS-10, we used a gene rescue approach to clone the mus-10 gene and discovered that it encodes a novel F-box protein. We show that MUS-10 interacts with a core component of the Skp, Cullin, F-box containing (SCF) complex, SCON-3, and that its F-box domain is essential for its function in vivo. Thus, we provide evidence that MUS-10 is part of an E3 ubiquitin ligase complex involved in maintaining the integrity of mitochondria and may function to prevent cellular senescence.THE mus-10 mutant was isolated from a screen aimed at identifying Neurospora crassa strains that were sensitive to MMS and therefore likely to lack proper DNA repair mechanisms (Kafer and Perlmutter 1980). Epistasis analyses involving mus-10 suggested that it belonged to the uvs-6 epistasis group, which functions in recombination repair (Kafer and Perlmutter 1980; Kafer 1983). However, mus-10 did not display several phenotypes common to other members of the uvs-6 epistasis group: chromosomal instability, a high sensitivity to histidine, and the inability to produce viable ascospores in homozygous crosses (Newmeyer et al. 1978; Newmeyer and Galeazzi 1978; Kafer and Perlmutter 1980; Kafer 1981; Schroeder 1986; Watanabe et al. 1997; Handa et al. 2000; Sakuraba et al. 2000). Furthermore, the frequencies of spontaneous and radiation-induced mutation observed in mus-10 were similar to those of a wild-type strain (Kafer 1981). Past efforts to uncover the nature of these discrepancies or the function of the mus-10 gene product have been uninformative.The majority of cellular ATP is produced in mitochondria through aerobic respiration, which couples electron flow through respiratory complexes within the mitochondrial inner membrane with oxidative phosphorylation. Besides their role in ATP synthesis, mitochondria are also involved in many other cellular processes including beta-oxidation (Bartlett and Eaton 2004), calcium homeostasis (Gunter et al. 2004; Rimessi et al. 2008), production of iron-sulfur clusters (Zheng et al. 1998; Gerber and Lill 2002; Lill and Muhlenhoff 2005; Rouault and Tong 2005), and apoptosis (Green 2005; Antignani and Youle 2006; Xu and Shi 2007). Although virtually all mitochondrial proteins are encoded within the nucleus, a small number of proteins are encoded by mitochondrial DNA (mtDNA). The integrity of the mitochondrial genome may affect cell survival as mutations in mtDNA accumulate in patients suffering from severe neurological diseases including Alzheimer''s, Huntington''s and Parkinson''s, as well as several types of cancer (Chatterjee et al. 2006; Higuchi 2007; Krishnan et al. 2007; Reeve et al. 2008). The number of mtDNA mutations also increases with age, suggesting a link between mitochondrial dysfunction and ageing (Cortopassi and Arnheim 1990; Corral-Debrinski et al. 1992; Cortopassi et al. 1992; Simonetti et al. 1992; Reeve et al. 2008). Contrary to the single genome in the nucleus, there are several copies of mtDNA in each mitochondrion. Thus, defects in a few mitochondrial genomes do not necessarily lead to mitochondrial dysfunction. Many patients suffering from mitochondrial diseases exhibit heteroplasmy, a phenomenon in which a mixture of wild-type and mutant mtDNAs exist in a single cell. The ratio of wild-type to mutant mtDNAs is critical in determining the penetrance of the genetic defect, where mutant loads >60% are required to cause respiratory chain dysfunction within an individual cell (Boulet et al. 1992; Chomyn et al. 1992; Sciacco et al. 1994).Even though N. crassa strains are generally deemed immortal if they can be subcultured ∼50 times, a wild-type strain was recently reported to senesce after 12,000 hr of growth, implying that this fungus undergoes natural or programmed ageing (Maheshwari and Navaraj 2008; Kothe et al. 2010). However, replicative life span is also influenced by genetic background as certain mutations can cause progressive deterioration of growth, ultimately leading to death. One such example is the nuclear-encoded natural death (nd), which when mutant causes a senescence phenotype correlating with the accumulation of multiple mtDNA deletions (Sheng 1951; Seidel-Rogol et al. 1989). The deletions of mtDNA in nd occurred between two 70- to 701-bp direct repeats, suggesting that the nd gene product regulates recombination, repair, or replication of mtDNA (Bertrand et al. 1993). Another nuclear mutation, senescence (sen), was isolated from N. intermedia and introgressed into N. crassa (Navaraj et al. 2000). Deletions were also observed in the mtDNA of sen mutants, but unlike those occurring in nd were flanked by 6- to 10-bp repeats typically associated with GC-rich palindromic sequences (D''Souza et al. 2005). The nature of the sequences that flanked the mtDNA deletions in these two mutants supported the existence of two distinct systems of mtDNA recombination in N. crassa: a general system of homologous recombination (system I) and a site-specific mechanism (system II), mediated in part by nd and sen, respectively (Bertrand et al. 1993; D''Souza et al. 2005). The nd and sen mutations have been mapped to linkage groups I and V, respectively, but neither gene has been cloned and the precise function of their gene products remains unclear. Two ultraviolet (UV)-sensitive mutants, uvs-4 and uvs-5, are thought to undergo senescence, but unfortunately, these strains have not been studied in great detail (Schroeder 1970; Perkins et al. 1993; Hausner et al. 2006). Premature senescence has also been observed in cytoplasmic mutants of N. crassa including the E35 and ER-3 stopper mutants that harbor large mtDNA deletions, as well as strains that accumulate mitochondrial plasmids capable of inserting into mtDNA through homologous recombination (de Vries et al. 1986; Akins et al. 1989; Myers et al. 1989; Niagro and Mishra 1989; Court et al. 1991; Alves and Videira 1998).While trying to establish the role of MUS-10 in DNA repair, we discovered that the mus-10 mutant exhibited a shortened life span, an abnormal mitochondrial morphology and mtDNA instability. We cloned the mus-10 gene through its ability to complement the MMS sensitivity of the mus-10 mutant and revealed that it encoded a novel F-box protein. This suggested that MUS-10 is part of an Skp, Cullin, F-box containing (SCF) E3 ubiquitin ligase complex that targets proteins for degradation by the 26S proteasome. The data we present in this article offer proof that an SCF complex can regulate both mitochondrial maintenance and cellular senescence.     

Genetic Control of Photoperiod Sensitivity in Maize Revealed by Joint Multiple Population Analysis

Variation in maize for response to photoperiod is related to geographical adaptation in the species. Maize possesses homologs of many genes identified as regulators of flowering time in other species, but their relation to the natural variation for photoperiod response in maize is unknown. Candidate gene sequences were mapped in four populations created by crossing two temperate inbred lines to two photoperiod-sensitive tropical inbreds. Whole-genome scans were conducted by high-density genotyping of the populations, which were phenotyped over 3 years in both short- and long-day environments. Joint multiple population analysis identified genomic regions controlling photoperiod responses in flowering time, plant height, and total leaf number. Four key genome regions controlling photoperiod response across populations were identified, referred to as ZmPR1–4. Functional allelic differences within these regions among phenotypically similar founders suggest distinct evolutionary trajectories for photoperiod adaptation in maize. These regions encompass candidate genes CCA/LHY, CONZ1, CRY2, ELF4, GHD7, VGT1, HY1/SE5, TOC1/PRR7/PPD-1, PIF3, ZCN8, and ZCN19.MAIZE (Zea mays L. subsp. mays) was domesticated in southern Mexico and its center of diversity is in tropical Latin America (Goodman 1999; Matsuoka et al. 2002), where precipitation rates and day lengths cycle annually. The presumed ancestor of maize, teosinte (Zea mays L. subsp. parviglumis), likely evolved photoperiod sensitivity to synchronize its reproductive phases to the wetter, short-day growing season (Ribaut et al. 1996; Campos et al. 2006). A critical event in the postdomestication evolution of maize was its spread from tropical to temperate regions of the Americas (Goodman 1988), requiring adaptation to longer day lengths. The result of this adaptation process is manifested today as a major genetic differentiation between temperate and tropical maize (Liu et al. 2003) and substantially reduced photoperiod sensitivity of temperate maize (Gouesnard et al. 2002). Tropical maize exhibits delayed flowering time, increased plant height, and a greater total leaf number when grown in temperate latitudes with daily dark periods <11 hr (Allison and Daynard 1979; Warrington and Kanemasu 1983a,b). Identifying the genes underlying maize photoperiod sensitivity will provide insight into the postdomestication evolution of maize and may reduce barriers to the use of diverse tropical germplasm resources for improving temperate maize production (Holland and Goodman 1995; Liu et al. 2003; Ducrocq et al. 2009).Natural variation at key genes in flowering time pathways is related to adaptation and evolution of diverse plant species (Caicedo et al. 2004; Shindo et al. 2005; Turner et al. 2005; Cockram et al. 2007; Izawa 2007; Slotte et al. 2007). Identification of some of the genes controlling adaptation in numerous plant species relied on regulatory pathways elucidated in Arabidopsis (Simpson and Dean 2002). Many key genes in the Arabidopsis flowering time regulatory pathways are conserved across diverse plant species (Kojima et al. 2002; Hecht et al. 2007; Kwak et al. 2008), but their functions have diverged, resulting in unique regulatory pathways in some phylogenetic groups (Colasanti and Coneva 2009). For example, FRI and FLC control most natural variation for vernalization response in Arabidopsis (Caicedo et al. 2004; Shindo et al. 2005), but wheat and barley appear to lack homologs of these genes and regulate vernalization response with different genes (Yan et al. 2004).Maize exhibits tremendous natural variation for flowering time (Gouesnard et al. 2002; Camus-Kulandaivelu et al. 2006), for which numerous QTL have been identified (Chardon et al. 2004). In contrast, only a few flowering time mutants are known and only a handful of flowering time genes, including DWARF8 (D8), DELAYED FLOWERING1 (DLF1), VEGETATIVE TO GENERATIVE TRANSITION1 (VGT1), and INDETERMINATE GROWTH1 (ID1), have been cloned in maize (Thornsberry et al. 2001; Colasanti et al. 2006; Muszynski et al. 2006; Salvi et al. 2007; Colasanti and Coneva 2009). Variation at or near D8 and VGT1 is related to latitudinal adaptation, but these genes do not appear to regulate photoperiod responses and account for only a limited proportion of the standing flowering time variation in maize (Camus-Kulandaivelu et al. 2006, 2008; Ducrocq et al. 2008; Buckler et al. 2009).Quantitative trait loci (QTL) mapping was a key first step to identifying the genes underlying natural variation for flowering time in Arabidopsis (Koornneef et al. 2004). Photoperiodic QTL have been mapped previously in individual biparental maize mapping populations (Koester et al. 1993; Moutiq et al. 2002; Wang et al. 2008; Ducrocq et al. 2009). Such studies are informative with respect to the parents from which the populations were derived, but often do not reflect the genetic heterogeneity of broader genetic reference populations (Holland 2007).Association mapping (Thornsberry et al. 2001; Ersoz et al. 2007) and combined analysis of multiple biparental crosses (Rebaï et al. 1997; Rebaï and Goffinet 2000; Blanc et al. 2006; Verhoeven et al. 2006; Yu et al. 2008) represent alternative approaches to understanding the variation in genetic control for complex traits among diverse germplasm. Association mapping has limited power to identify genes that affect traits closely associated with population structure, such as flowering time in maize (Camus-Kulandaivelu et al. 2006; Ersoz et al. 2007). In contrast, joint QTL analysis of multiple populations is not hindered by the associations between causal genes and population structure. Combined QTL analysis of multiple mapping populations provides improved power to detect QTL, more precise estimation of their effects and positions, and better understanding of their functional allelic variation and distribution across more diverse germplasm compared to single-population mapping (Rebaï et al. 1997; Wu and Jannink 2004; Jourjon et al. 2005; Blanc et al. 2006; Verhoeven et al. 2006; Yu et al. 2008; Buckler et al. 2009). Joint analysis also provides a direct test of the importance of higher-order epistatic interactions between founder alleles at individual loci with genetic backgrounds (Jannink and Jansen 2001; Blanc et al. 2006). In this study, joint analysis of multiple populations was used to test directly the hypothesis that diverse tropical maize lines carry functionally similar alleles at key photoperiod loci, which would imply genetic homogeneity for a common set of mutations and a shared evolutionary pathway for photoperiod insensitivity.The objective of this study was to integrate candidate gene analyses with photoperiod QTL mapping across multiple maize populations. We tested candidate floral regulators known from other species for associations with natural variation for photoperiod response in maize. We analyzed flowering time in four interrelated recombinant inbred line (RIL) populations, each derived from crosses between temperate and tropical maize parents (Figure 1), in both long- and short-day environments to characterize their responses to distinct photoperiods. Joint population analysis provided high resolution of many QTL positions, permitting robust testing of underlying candidate genes. We directly and indirectly mapped homologs of flowering time candidates genes from Arabidopsis, rice, and barley on a dense consensus genetic map of these four populations, permitting identification of homologs that colocalize with genome regions associated with variation for photoperiod response. These mapping families are being integrated into the maize nested association mapping (NAM) population (Buckler et al. 2009; McMullen et al. 2009) because they were genotyped with the maize NAM map SNP markers, they involve the common parent B73, and their seed and genotypic information (File S1 cont.) are publicly available. Their availability further expands the genetic diversity represented by the maize NAM population and enhances this valuable public community resource.Open in a separate windowFigure 1.—Factorial mating of two temperate (B73 and B97) and two tropical (CML254 and Ki14) inbred maize lines to create four related recombinant inbred line mapping populations.     

Estimating the Parameters of Selection on Nonsynonymous Mutations in Drosophila pseudoobscura and D. miranda

We present the results of surveys of diversity in sets of >40 X-linked and autosomal loci in samples from natural populations of Drosophila miranda and D. pseudoobscura, together with their sequence divergence from D. affinis. Mean silent site diversity in D. miranda is approximately one-quarter of that in D. pseudoobscura; mean X-linked silent diversity is about three-quarters of that for the autosomes in both species. Estimates of the distribution of selection coefficients against heterozygous, deleterious nonsynonymous mutations from two different methods suggest a wide distribution, with coefficients of variation greater than one, and with the average segregating amino acid mutation being subject to only very weak selection. Only a small fraction of new amino acid mutations behave as effectively neutral, however. A large fraction of amino acid differences between D. pseudoobscura and D. affinis appear to have been fixed by positive natural selection, using three different methods of estimation; estimates between D. miranda and D. affinis are more equivocal. Sources of bias in the estimates, especially those arising from selection on synonymous mutations and from the choice of genes, are discussed and corrections for these applied. Overall, the results show that both purifying selection and positive selection on nonsynonymous mutations are pervasive.SURVEYS of DNA sequence diversity and divergence are shedding light on a number of questions in evolutionary genetics (for recent reviews, see Akey 2009; Sella et al. 2009). Two of the most important questions of this kind concern the distribution of selection coefficients against deleterious mutations affecting protein sequences and the proportion of amino acid sequence differences between related species that have been fixed by positive selection. Several different methods have been proposed for studying each of these questions, using different features of data on polymorphism and divergence at nonsynonymous and silent sites.For example, the parameters of the distribution of selection coefficients against deleterious amino acid mutations have been estimated by contrasting the numbers of nonsynonymous and silent within-species polymorphisms and fixed differences between species (Sawyer and Hartl 1992; Bustamante et al. 2002; Piganeau and Eyre-Walker 2003; Sawyer et al. 2007); by fitting the frequency spectra of nonsynonymous and silent variants to models of selection, mutation, and drift (Akashi 1999; Eyre-Walker et al. 2006; Keightley and Eyre-Walker 2007; Kryukov et al. 2007; Boyko et al. 2008; Eyre-Walker and Keightley 2009); or by comparing levels of nonsynonymous and silent diversities between species with different population sizes (Loewe and Charlesworth 2006; Loewe et al. 2006). The results of these different approaches generally agree in suggesting that there is a wide distribution of selection coefficients against nonsynonymous mutations and that the mean selection coefficient against heterozygous carriers of such mutations is very small. The results imply that a typical individual from a human population carries several hundred weakly deleterious mutations (Eyre-Walker et al. 2006; Kryukov et al. 2007; Boyko et al. 2008); for a typical Drosophila population, with its much higher level of variability, the number is probably an order of magnitude greater (Loewe et al. 2006; Keightley and Eyre-Walker 2007).The presence of this large load of slightly deleterious mutations in human and natural populations, most of which are held at low frequencies by natural selection, has many implications. From the point of view of understanding human genetic disease, it means that we have to face the likelihood that susceptibility to a disease can be influenced by variants at many loci, each with small effects (Kryukov et al. 2007). The pervasive presence of deleterious mutations throughout the genome contributes to inbreeding depression (Charlesworth and Willis 2009) and may mean that the effective population size is reduced by background selection effects, even in regions of the genome with normal levels of genetic recombination (Loewe and Charlesworth 2007). Their presence may contribute so strongly to Hill–Robertson effects (Hill and Robertson 1966; Felsenstein 1974) that they cause severely reduced levels of diversity and adaptation in low-recombination regions of the genome (Charlesworth et al. 2010) and create a selective advantage to maintaining nonzero levels of recombination (Keightley and Otto 2006; Charlesworth et al. 2010). In addition, having an estimate of the distribution of selection coefficients against deleterious nonsynonymous mutations allows their contribution to between-species divergence to be predicted, providing a way of estimating the fraction of fixed nonsynonymous differences caused by positive selection (Loewe et al. 2006; Boyko et al. 2008; Eyre-Walker and Keightley 2009).It is thus important to collect data that shed light on the properties of selection against nonsynonymous mutations in a wide range of systems and also to compare the results from different methods of estimation, since they are subject to different sources of difficulty and biases. In a previous study, we proposed the use of a comparison between two related species with different effective population sizes for this purpose (Loewe and Charlesworth 2006; Loewe et al. 2006), using Drosophila miranda and D. pseudoobscura as material. These are well suited for this type of study, as they are closely related, live together in similar habitats, and yet have very different levels of silent nucleotide diversity, indicating different effective population sizes (N_e). This study was hampered by our inability to compare the same set of loci across the two species and by the small number of loci that could be used. We here present the results of a much larger study of DNA variation at X-linked and autosomal loci for these two species, using D. affinis as a basis for estimating divergence. We compare the results, applying the method of Loewe et al. (2006) with that of Eyre-Walker and Keightley (2009) for estimating the distribution of deleterious selection coefficients and with McDonald–Kreitman test-based methods for estimating the proportion of nonsynonymous differences fixed by positive selection. While broadly confirming the conclusions from earlier studies, we note some possible sources of bias and describe methods for minimizing their effects.     

Using RNA Interference to Identify Specific Modifiers of a Temperature-Sensitive,Embryonic-Lethal Mutation in the Caenorhabditis elegans Ubiquitin-Like Nedd8 Protein Modification Pathway E1-Activating Gene rfl-1

The essential Caenorhabditis elegans gene rfl-1 encodes one subunit of a heterodimeric E1-activating enzyme in the Nedd8 ubiquitin-like protein conjugation pathway. This pathway modifies the Cullin scaffolds of E3 ubiquitin ligases with a single Nedd8 moiety to promote ligase function. To identify genes that influence neddylation, we used a synthetic screen to identify genes that, when depleted with RNAi, enhance or suppress the embryonic lethality caused by or198ts, a temperature-sensitive (ts) mutation in rfl-1. We identified reproducible suppressor and enhancer genes and employed a systematic specificity analysis for each modifier using four unrelated ts embryonic lethal mutants. Results of this analysis highlight the importance of specificity controls in identifying genetic interactions relevant to a particular biological process because 8/14 enhancers and 7/21 suppressors modified lethality in other mutants. Depletion of the strongest specific suppressors rescued the early embryonic cell division defects in rfl-1(or198ts) mutants. RNAi knockdown of some specific suppressors partially restored Cullin neddylation in rfl-1(or198ts) mutants, consistent with their gene products normally opposing neddylation, and GFP fusions to several suppressors were detected in the cytoplasm or the nucleus, similar in pattern to Nedd8 conjugation pathway components in early embryonic cells. In contrast, depletion of the two strongest specific enhancers did not affect the early embryonic cell division defects observed in rfl-1(or198ts) mutants, suggesting that they may act at later times in other essential processes. Many of the specific modifiers are conserved in other organisms, and most are nonessential. Thus, when controlled properly for specificity, modifier screens using conditionally lethal C. elegans mutants can identify roles for nonessential but conserved genes in essential processes.UBIQUITIN-mediated proteolysis regulates many biological processes (Nandi et al. 2006). In the early Caenorhabditis elegans embryo, these include oocyte maturation, cell cycle progression, cell polarization, and cell fate patterning, all of which require the timely destruction of maternally expressed proteins (Bowerman and Kurz 2006; Greenstein and Lee 2006). One C. elegans protein targeted for proteolysis early in embryogenesis is MEI-1, the AAA-ATPase subunit of the microtubule-severing complex called katanin (Mains et al. 1990; Dow and Mains 1998; Srayko et al. 2000; Kurz et al. 2002; Pintard et al. 2003a; Xu et al. 2003). Katanin is a heterodimer of two subunits called p60 and p80 in vertebrates and MEI-1 and MEI-2 in C. elegans. Katanin in C. elegans is required for proper assembly and function of the small, barrel-shaped meiotic spindles (Albertson and Thomson 1993; McNally et al. 2006) and must be degraded after meiotic divisions to permit assembly of the much larger first mitotic spindle in the one-cell zygote. In mutants that fail to degrade katanin after the completion of meiosis, the first mitotic spindle is fragmented and mis-oriented, cytokinesis is defective, and the embryos die without hatching (Dow and Mains 1998; Srayko et al. 2000; Kurz et al. 2002).The katanin subunit MEI-1 is targeted for poly-ubiquitylation and proteolytic destruction by a Cullin-based E3 ligase (Kurz et al. 2002). This complex includes the Cullin scaffolding protein CUL-3 and a substrate-specific adaptor called MEL-26 that binds to CUL-3 through a BTB domain and to MEI-1 through a MATH domain (Pintard et al. 2003b). Cullin 3-based E3 ligases in mammals also utilize substrate-specific adaptor proteins that, like MEL-26, have both a Cullin-binding BTB/POZ domain and another protein–protein interaction domain that binds to the substrate (Geyer et al. 2003; Cullinan et al. 2004; Angers et al. 2006). While MEI-1/Katanin downregulation by the CUL-3/MEL-26 E3 ligase is essential at most growth temperatures, a mel-26 null mutation is viable at the low growth temperature of 15° (Lu and Mains 2007). This bypass of mel-26 at 15° depends at least in part on the anaphase-promoting complex and its targeting of MEI-1 for proteolytic degradation (Lu and Mains 2007). Phosphorylation by the kinase MBK-2 primes MEI-1 for proteolysis (Quintin et al. 2003; Stitzel et al. 2007) and also promotes the downregulation of MEI-1 by the anaphase-promoting complex (Lu and Mains 2007).CUL-3 is the only C. elegans Cullin thus far identified that requires modification by the ubiquitin-like protein Nedd8 (Bowerman and Kurz 2006). In contrast, C. elegans CUL-2 is required for progression through meiosis and for the localized degradation of cell fate determinants in one-cell-stage embryos (Liu et al. 2004; Sonneville and Gonczy 2004), but neddylation-defective mutants do not exhibit these early defects (Bowerman and Kurz 2006). Cullin neddylation is mediated by the Nedd8 protein conjugation pathway, which begins with a heterodimeric E1-activating enzyme consisting of ULA-1 and RFL-1 (Uba3p in budding yeast) and also includes the E2-conjugating enzyme UBC-12 (Jones and Candido 2000; Srayko et al. 2000; Kurz et al. 2002) and the E3 ligase DCN-1 (Kurz et al. 2005).The downregulation of MEI-1/katanin by the CUL-3/MEL-26 E3 ligase requires a balance of both CUL-3 neddylation, which is mediated by the Nedd8 conjugation pathway, and deneddylation, which is mediated by the conserved COP-9 Signalosome (Pintard et al. 2003a). Other Cullin-based E3 ubiquitin ligases also require a balance of neddylation and deneddylation (Lyapina et al. 2001; Schwechheimer et al. 2001; Bornstein et al. 2006; Hetfeld et al. 2008). Deneddylation may modulate activation of the E3 ligase and thereby prevent the premature degradation of substrate adaptor proteins that also can become poly-ubiquitylated and degraded as a result of E3 ligase function.To identify additional factors that influence neddylation, and the downregulation of MEI-1/katanin after the completion of meiosis in C. elegans, we report here our use of RNA interference (RNAi) to reduce gene functions in a temperature-sensitive (ts) neddylation-defective mutant, rfl-1(or198ts). The discovery of RNAi and its systemic properties in C. elegans have made it possible to systematically target C. elegans genes for depletion by feeding worms bacterial strains that express double-strand RNAs corresponding to C. elegans gene sequences (Fire et al. 1998; Timmons et al. 2001; Feinberg and Hunter 2003; Baugh et al. 2005; Lehner et al. 2006; van Haaften et al. 2006). Furthermore, chemical mutagenesis screens have identified temperature-sensitive mutations in many essential C. elegans genes, which can be used for synthetic screens by choosing intermediate-growth temperatures that sensitize the genetic background and also optimize visual scoring of embryonic viability. Recently, genomewide RNAi screens have been used to identify C. elegans genes that, when reduced in function, restore viability to temperature-sensitive, embryonic-lethal mutants (Labbe et al. 2006; O''Rourke et al. 2007). Because a loss of suppressor function restores mutant viability, the suppressors may negatively regulate either the wild-type gene product or the process that requires the wild-type gene product.Here we report our identification of C. elegans genes that, when reduced in function by feeding RNAi, reproducibly suppressed or enhanced rfl-1(or198ts) embryonic lethality. Most suppressors were specific for rfl-1(or198ts), while specific enhancement was less common. Many of the rfl-1-specific suppressors and enhancers are conserved but appear nonessential. GFP fusions to several specific suppressors exhibit localization patterns that resemble those known for neddylation pathway components, and depletion of some of these partially restored CUL-3 neddylation in rfl-1(or198ts) mutants. In addition to identifying possible roles for conserved genes in cullin neddylation, we report the first quantitative analysis of specificity for both the enhancement and the suppression of a conditionally lethal mutant in C. elegans. Our results highlight the importance of testing genetic modifiers of conditionally lethal mutants for locus specificity.     

Distinct Regulation of Mlh1p Heterodimers in Meiosis and Mitosis in Saccharomyces cerevisiae

Mlh1p forms three heterodimers that are important for mismatch repair (Mlh1p/Pms1p), crossing over during meiosis (Mlh1p/Mlh3p), and channeling crossover events into a specific pathway (Mlh1p/Mlh2p). All four proteins contain highly conserved ATPase domains and Pms1p has endonuclease activity. Studies of the functional requirements for Mlh1p/Pms1p in Saccharomyces cerevisae revealed an asymmetric contribution of the ATPase domains to repairing mismatches. Here we investigate the functional requirements of the Mlh1p and Mlh3p ATPase domains in meiosis by constructing separation of function mutations in Mlh3p. These mutations are analogous to mutations of Mlh1p that have been shown to lead to loss of ATP binding and/or ATP hydrolysis. Our data suggest that ATP binding by Mlh3p is required for meiotic crossing over while ATP hydrolysis is dispensable. This has been seen previously for Mlh1p. However, when mutations that affect ATP hydrolysis by both Mlh3p and Mlh1p are combined within a single cell, meiotic crossover frequencies are reduced. These observations suggest that the function of the Mlh1p/Mlh3p heterodimer requires both subunits to bind ATP but only one to efficiently hydrolyze it. Additionally, two different amino acid substitutions to the same residue (G97) in Mlh3p affect the minor mismatch repair function of Mlh3p while only one of them compromises its ability to promote crossing over. These studies thus reveal different functional requirements among the heterodimers formed by Mlh1p.CROSSING over during meiosis not only generates variation but is also important for providing the necessary interactions between homologous chromosomes that ensure correct segregation at division I of meiosis. Recombination is initiated by the production of programmed double-strand breaks (DSBs), catalyzed by the covalently attached Spo11p (Bergerat et al. 1997; Keeney et al. 1997), aided by a number of proteins (reviewed in Keeney and Neale 2006). DSBs are made at a much higher frequency than crossovers, and designation of only a subset to yield crossovers is thought to occur during early stages of DSB repair (Borner et al. 2004). At least two distinct pathways contribute to the production of crossover events in Saccharomyces cerevisiae. The major pathway is dependent on Msh4p/Msh5p and the mismatch repair proteins Mlh1p and Mlh3p (Ross-MacDonald and Roeder 1994; Hollingsworth et al. 1995; Hunter and Borts 1997; Wang et al. 1999; Abdullah et al. 2004) and the second pathway is dependent on Mus81p/Mms4p endonuclease (de los Santos et al. 2001, 2003).Mitotic mismatch repair (MMR) is the process by which mutations that arise during DNA replication and recombination are recognized and removed (reviewed in Kolodner 1996; Harfe and Jinks-Robertson 2000). Msh2p forms a heterodimer with Msh6p (MutSα) to repair base–base mismatches and small insertions and/or deletions and with Msh3p (MutSβ) to repair large insertions and/or deletions (reviewed in Jiricny 2006). Mlh1p forms heterodimers with Pms1p, Mlh2p, and Mlh3p to coordinate the removal of these mismatches (Prolla et al. 1994; Wang et al. 1999). Mlh1p/Pms1p (MutLα) are involved in the repair of all types of mismatches in combination with MutSα and MutSβ, and in the absence of either protein a mutator phenotype is observed (Habraken et al. 1997, 1998). Mlh1p/Mlh2p (MutLβ) and Mlh1p/Mlh3p (MutLγ) are involved in the MutSβ pathway only, which repairs frameshift mutations caused by insertions or deletions. Consequently mlh3Δ mutants only exhibit a weak mutator phenotype, due to a lesser involvement in mismatch repair and a partial overlap in function with Pms1p (Flores-Rozas and Kolodner 1998; Harfe et al. 2000).Although the MutL homologs interact primarily through their C-terminal domains (Pang et al. 1997; Ban and Yang 1998), it is thought that the N-terminal domains must also interact for the complex to be fully functional (Ban and Yang 1998). Binding of ATP causes the proteins to undergo conformational changes, which are essential for the interaction between the N termini (Ban et al. 1999; Tran and Liskay 2000; Sacho et al. 2008). ATP hydrolysis and subsequent release of ADP is required to allow the protein complex to return to its initial state, completing the cycle so that the subunits are ready to bind ATP again if required. Using mutants of MLH1 and PMS1 that are presumed to be defective for ATP binding and/or ATP hydrolysis, it has been shown that both of these functions are essential for fully effective mismatch repair (Tran and Liskay 2000). However, the ATP binding and ATP hydrolysis mutants of PMS1 exhibited lower mitotic mutation rates than the corresponding MLH1 ATPase mutants, suggesting that there is functional asymmetry within the Mlh1p/Pms1p heterodimer (Tran and Liskay 2000; Hall et al. 2002). Another example of the asymmetry in the contributions of these subunits to function can be seen in assays that measure recombination between diverged sequences (homeologous recombination). The Mlh1p ATPase activity has been shown to be more important for the suppression of homeologous recombination than Pms1p ATPase activity (Welz-Voegele et al. 2002). This functional asymmetry is supported by in vitro biochemical analysis that demonstrated Pms1p has a lower ATP binding affinity than Mlh1p (Hall et al. 2002).As mentioned above, Mlh1p/Mlh3p function in the Msh4p/Msh5p pathway for meiotic recombination (Hunter and Borts 1997; Santucci-Darmanin et al. 2000). The Msh4p/Msh5p complex is thought to act in the stabilization of Holliday junction intermediates to allow their resolution in a crossover configuration (Snowden et al. 2004). The Mlh1p/Mlh3p complex has been suggested to act in the resolution of these structures, either directly or indirectly. Human Pms2 and its yeast homolog, Pms1p, have been shown to possess a latent endonuclease activity, conferred by a motif that is conserved among some of the MutL homologs, including Mlh3p (Kadyrov et al. 2006, 2007). Mutations in the DHQA(X)₂E(X)₄E motif in yeast MLH3 cause defects in both mismatch repair and meiotic recombination equivalent to mlh3Δ, suggesting that Mlh3p may also possess an endonuclease activity that is important for the generation of crossovers (Nishant et al. 2008).ATP binding by Mlh1p has been shown to be important for both of its meiotic functions (crossing over and repair of heteroduplex DNA) (Pang et al. 1997; Tran and Liskay 2000; Hoffmann et al. 2003). In contrast, the ATP hydrolysis mutant mlh1-E31A/mlh1-E31A appears to have no effect on meiotic recombination (Tran and Liskay 2000; Hoffmann et al. 2003). This may partly be explained by in vitro studies demonstrating that this mutant exhibits a low level of ATPase activity (Hall et al. 2002).The meiotic functions of MLH1 can be functionally separated as shown by mutating the same residue, G98, to different amino acids (Hoffmann et al. 2003). The residue G98 is situated in the ATPase motif in the GFRGEAL box (GYRGDAL in Mlh3p), which forms the lid of the ATP binding pocket. Mutations in this motif are predicted to affect ATP binding and/or heterodimerization with Pms1p (Ban and Yang 1998; Ban et al. 1999). Mutating the residue G98 in the ATP binding lid to alanine resulted in defective repair of heteroduplex DNA while crossing over was unaffected, but when the same residue was mutated to valine both mismatch repair and crossover functions were defective (Hoffmann et al. 2003). The mlh1-G98V mutant disrupts the interaction of Mlh1p with Pms1p, while mlh1-G98A does not (Pang et al. 1997). This may contribute to the difference observed in the effect on crossing over as Mlh1p is thought to interact with Pms1p and Mlh3p through the same residues (Wang et al. 1999; Kondo et al. 2001). Consequently if the interaction with Pms1p is affected then it is likely that the interaction with Mlh3p is also disrupted.We constructed mlh3 mutants corresponding to the ATP binding and ATP hydrolysis mutants of mlh1 to explore the role of Mlh3p in meiotic recombination. We also constructed mlh3-G97A and mlh3-G97V mutants, equivalent to the mlh1-G98A/V pair that has been shown to differentially affect the mitotic and meiotic functions of Mlh1p. All mutants were assayed for mitotic mismatch repair, meiotic heteroduplex repair, crossing over, and chromosome segregation.