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.     

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.     

The Drosophila Planar Polarity Proteins Inturned and Multiple Wing Hairs Interact Physically and Function Together

The conserved frizzled (fz) pathway regulates planar cell polarity in both vertebrate and invertebrate animals. This pathway has been most intensively studied in the wing of Drosophila, where the proteins encoded by pathway genes all accumulate asymmetrically. Upstream members of the pathway accumulate on the proximal, distal, or both cell edges in the vicinity of the adherens junction. More downstream components including Inturned and Multiple Wing Hairs accumulate on the proximal side of wing cells prior to hair initiation. The Mwh protein differs from other members of the pathway in also accumulating in growing hairs. Here we show that the two Mwh accumulation patterns are under different genetic control with the early proximal accumulation being regulated by the fz pathway and the latter hair accumulation being largely independent of the pathway. We also establish recruitment by proximally localized Inturned to be a putative mechanism for the localization of Mwh to the proximal side of wing cells. Genetically inturned (in) acts upstream of mwh (mwh) and is required for the proximal localization of Mwh. We show that Mwh can bind to and co-immunoprecipitate with Inturned. We also show that these two proteins can function in close juxtaposition in vivo. An In∷Mwh fusion protein provided complete rescue activity for both in and mwh mutations. The fusion protein localized to the proximal side of wing cells prior to hair formation and in growing hairs as expected if protein localization is a key for the function of these proteins.THE frizzled (fz) signaling pathway regulates tissue planar cell polarity (PCP) in the epidermis of both vertebrate and invertebrate animals (Lawrence et al. 2007; Montcouquiol 2007; Wang and Nathans 2007; Zallen 2007). PCP is dramatic in the cuticle of insects such as Drosophila, which is decorated with arrays of hairs and sensory bristles.The genetic basis for tissue polarity has been most extensively studied in the fly wing (Wong and Adler 1993). The Planar Polarity (PCP) genes of the fz pathway (also known as the core PCP genes), the planar polarity effector (PPE) genes and the multiple wing hairs (mwh) gene encode key components that regulate planar polarity in the wing. fz, disheveled (dsh), prickle/spiny leg (pk/sple), Van Gogh (Vang) (aka strabismus), starry night (stan) (aka flamingo) and diego (dgo) are members of the PCP group (Vinson and Adler 1987; Wong and Adler 1993; Taylor et al. 1998; Wolff and Rubin 1998; Chae et al. 1999; Gubb et al. 1999; Usui et al. 1999). A distinctive feature of these genes is that their protein products accumulate asymmetrically on the distal (Fz, Dsh, and Dgo) (Axelrod 2001; Feiguin et al. 2001; Shimada et al. 2001; Strutt 2001), proximal (Vang, Pk)(Tree et al. 2002; Bastock et al. 2003), or both distal and proximal (Stan) (Usui et al. 1999) sides of wing cells. These genes/proteins act as a functional group and are corequirements for the asymmetric accumulation of the others.The PPE includes inturned (in), fuzzy (fy), and fritz (frtz) (Park et al. 1996; Collier and Gubb 1997; Collier et al. 2005). These genes are thought to function downstream of the PCP genes and the proteins encoded by these genes also accumulate asymmetrically in wing cells (Adler et al. 2004; Strutt and Warrington 2008). As is the case for the PCP genes, the PPE genes/proteins also appear to be a functional group and to be corequirements for the asymmetric accumulation of the others. Several observations support the hypothesis that the PPE genes are essential downstream effectors of the PCP genes. The earliest appreciation of this came from careful observations of the mutant phenotypes. A common feature of mutations in all of these genes is that they do not result in a randomization of hair polarity, but rather in a similar complicated and abnormal stereotypic pattern (Gubb and Garcia-Bellido 1982; Adler et al. 2000). That the abnormal patterns were so similar suggested that these genes all functioned in the same process (Wong and Adler 1993). The mutant phenotypes differed in that the vast majority of PCP mutant wing cells form a single hair, while many PPE mutant wing cells form two or three hairs. Mutations in PPE genes are epistatic to both loss- and gain-of-function mutations in PCP genes (Wong and Adler 1993; Lee and Adler 2002). Further evidence that the PPE genes function downstream of the PCP genes comes from the analysis of protein localization. PPE gene function is not needed for the proper asymmetric localization of PCP proteins (Usui et al. 1999; Strutt 2001; Tree et al. 2002; Collier et al. 2005) but in contrast PCP gene function is essential for the asymmetric accumulation of PPE proteins (Adler et al. 2004; Strutt and Warrington 2008). Further, the PCP genes/proteins instruct the localization of the PPE proteins (Adler et al. 2004).The multiple-wing-hairs (mwh) gene is thought to function downstream of both the PCP and PPE genes (Wong and Adler 1993). This conclusion comes from analyses that are similar to those that established that the PPE genes function downstream of the PCP genes. The overall hair polarity pattern of mwh mutant wings shares the same complicated and abnormal stereotypic hair polarity pattern seen in PCP and PPE mutants. However, mwh cells differ by producing a larger number of hairs (typically three to four hairs) (Wong and Adler 1993). mwh mutations are epistatic to mutations in both the PCP and PPE genes and mwh is not required for the asymmetric accumulation of either PCP or PPE proteins (Usui et al. 1999; Strutt 2001; Adler et al. 2004; Strutt and Warrington 2008).The mwh gene was recently determined to encode a novel G protein binding–formin homology 3 (GBD-FH3) protein with a complex accumulation pattern in wing cells (Strutt and Warrington 2008; Yan et al. 2008). Prior to hair initiation Mwh accumulates along the proximal side of wing cells and during hair growth Mwh accumulates in the growing hair. Temperature-shift experiments with a temperature-sensitive allele provided evidence for two temporally separate mwh functions and it was proposed that the two accumulation patterns were associated with the two temporal functions (Yan et al. 2008). Here we show that the early proximal accumulation of Mwh requires the function of the PCP and PPE genes (a result also seen previously in Strutt and Warrington 2008), while the hair accumulation of Mwh is largely independent of these two groups of genes providing further genetic evidence for Mwh having two independent functions.How does the Mwh protein accumulate proximally? An obvious possibility is that Mwh interacts directly with one or more of the upstream proteins and in this way is recruited to the proximal side. The PPE proteins are strong candidates to interact directly with Mwh, as they function genetically in between the PCP gene and Mwh (Wong and Adler 1993). Consistent with this possibility we found that In and Mwh interacted in the yeast two-hybrid system and that these two proteins co-immunoprecipated from wing cells. This interaction was found not to be dependent on the function of the PCP genes consistent with the data from genetic studies that both in and mwh retain at least partial function in a fz mutant wing (Wong and Adler 1993). The hypothesis that Mwh is recruited to the proximal side by interacting with In predicts that these two proteins function in close proximity to one another. Consistent with these expectations we found that an In∷Mwh fusion protein provided both In and Mwh function.     

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.     

Profiling Sex-Specific piRNAs in Zebrafish

Piwi proteins and their partner small RNAs play an essential role in fertility, germ-line stem cell development, and the basic control and evolution of animal genomes. However, little knowledge exists regarding piRNA biogenesis. Utilizing microfluidic chip analysis, we present a quantitative profile of zebrafish piRNAs expressed differentially between testis and ovary. The sex-specific piRNAs are derived from separate loci of repeat elements in the genome. Ovarian piRNAs can be categorized into groups that reach up to 92 members, indicating a sex-specific arrangement of piRNA genes in the genome. Furthermore, precursor piRNAs preferentially form a hairpin structure at the 3′end, which seem to favor the generation of mature sex-specific piRNAs. In addition, the mature piRNAs from both the testis and the ovary are 2′-O-methylated at their 3′ ends.SMALL RNAs, ranging from 19 to 30 nucleotides (nt) in length, constitute a large family of regulatory molecules with diverse functions in invertebrates, vertebrates, plants, and fungi (Bartel 2004; Nakayashiki 2005). Two major classes of small RNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs). The functions of small RNAs have been conserved through evolution; they have been shown to inhibit gene expression at the levels of mRNA degradation, translational repression, chromatin modification, heterochromatin formation, and DNA elimination (Mochizuki et al. 2002; Bartel 2004; Kim et al. 2005; Brodersen and Voinnet 2006; Lee and Collins 2006; Vaucheret 2006).Over the past few years, focus on the genetics of small RNAs has helped clarify the mechanisms behind the regulation of these molecules. While hundreds of small RNAs have been identified from mammalian somatic tissues, relatively little is known about small RNAs in germ cells. A recent breakthrough has been the identification of small RNAs that associate with Piwi proteins (piRNAs) from Drosophila and mammalian gonads (Aravin et al. 2001, 2006; Girard et al. 2006; Grivna et al. 2006; Vagin et al. 2006; Watanabe et al. 2006). piRNAs and their interacting proteins Ziwi/Zili have also been identified in zebrafish (Houwing et al. 2007, 2008). Increasing evidence indicates that piRNAs play roles mainly in germ cell differentiation and genomic stability (Carthew 2006; Lau et al. 2006; Vagin et al. 2006; Brennecke et al. 2007; Chambeyron et al. 2008; Klattenhoff and Theurkauf 2008; Kuramochi-Miyagawa et al. 2008; Kim et al. 2009; Lim et al. 2009; Unhavaithaya et al. 2009). Moreover, although piRNAs are mostly expressed in germ line cells, recent studies showed piRNA expression in nongerm cells, for example, T-cell lines (Jurkat cells and MT4) (Azuma-Mukai et al. 2008; Yeung et al. 2009), indicating other functions such as in the immune system. piRNAs do not appear to be derived from double-stranded RNA precursors, and their biogenesis mechanisms, although unclear, may be distinct from those of siRNA and miRNA. Recently, two distinct piRNA production pathways were further proposed: the “ping-pong” model (Brennecke et al. 2007; Gunawardane et al. 2007) and the Ago3-independent piRNA pathway centered on Piwi in somatic cells (Li et al. 2009; Malone et al. 2009). However, the mechanistic pathways of piRNA activity and their biogenesis are still largely unknown.Teleost fishes comprise >24,000 species, accounting for more than half of extant vertebrate species, displaying remarkable variation in morphological and physiological adaptations (see review in Zhou et al. 2001). Recently, Houwing et al. (2007, 2008) reported findings on Ziwi/Zili and associated piRNAs, implicating roles in germ cell differentiation, meiosis, and transposon silencing in the germline of the zebrafish. However, some of the identified zebrafish piRNAs are nonrepetitive and nontransposon-related piRNAs, suggesting that piRNAs may have additional unknown roles. In this study, we show that for males and females, piRNAs are specifically derived from separate loci of the repeat elements, and that ovarian piRNAs are far more often associated in groups. Genomic analysis of piRNAs indicates a tendency to folding at the 3′ end of the piRNA precursor, which may favor cleavage of the piRNA precursor to generate mature sex-specific piRNAs. Furthermore, methylation modification occurs at the 2′-O-hydroxyl group on the ribose of the final 3′ nucleotide in both the testis and the ovary.     

The Population Genomics of Trans-Specific Inversion Polymorphisms in Anopheles gambiae

In the malaria mosquito Anopheles gambiae polymorphic chromosomal inversions may play an important role in adaptation to environmental variation. Recently, we used microarray-based divergence mapping combined with targeted resequencing to map nucleotide differentiation between alternative arrangements of the 2La inversion. Here, we applied the same technique to four different polymorphic inversions on the 2R chromosome of An. gambiae. Surprisingly, divergence was much lower between alternative arrangements for all 2R inversions when compared to the 2La inversion. For one of the rearrangements, 2Ru, we successfully mapped a very small region (∼100 kb) of elevated divergence. For the other three rearrangements, we did not identify any regions of significantly high divergence, despite ample independent evidence from natural populations of geographic clines and seasonal cycling, and stable heterotic polymorphisms in laboratory populations. If these inversions are the targets of selection as hypothesized, we suggest that divergence between rearrangements may have escaped detection due to retained ancestral polymorphism in the case of the youngest 2R rearrangements and to extensive gene flux in the older 2R inversion systems that segregate in both An. gambiae and its sibling species An. arabiensis.MORE than 70 years ago Dobzhansky and Sturtevant (1938) first discovered polymorphic inversion arrangements carried by various Drosophila pseudoobscura populations. After observing correlations between environmental conditions and inversion frequencies, Dobzhansky proposed that inversions are under strong selection due to their role in promoting local adaptation to the heterogeneous conditions a species encounters both spatially and temporally (Dobzhansky 1944, 1948; Powell 1997). More recent studies have implicated chromosomal inversions in the adaptation of a diversity of eukaryotes including humans (Coluzzi et al. 1979; Feder et al. 2003; Hoffmann et al. 2004; Stefansson et al. 2005). Long known to be common in dipteran insects, more recent HapMap data suggest that polymorphic inversions may be numerous in human populations and by extension other mammals (Bansal et al. 2007). Given their potential importance in facilitating adaptation, surprisingly little is known about the mechanism(s) or the genes responsible for maintaining inversion polymorphisms in natural populations.Gene exchange between inverted and standard arrangements, although reduced, can still occur through gene flux: the action of gene conversion and multiple crossovers in inversion heterozygotes (heterokaryotypes) (Chovnick 1973; Navarro et al. 1997; Schaeffer and Anderson 2005). Over time allelic variation unrelated to ecological adaptation should become homogenized between arrangements, while alleles which are under divergent selection pressures should remain in linkage disequilibrium with each other and with the inversion itself, leading to heightened differentiation between standard and inverted arrangements at and near the target loci. In principle, this process allows the identification of specific loci involved in adaptive divergence (Schaeffer et al. 2003; Schaeffer and Anderson 2005; Storz 2005). Consistent with this model, previous low-resolution studies of Drosophila inversions revealed heterogeneous patterns of nucleotide diversity relative to divergence, as well as the interspersion of regions of high and low genetic association potentially due to the interaction of selection and gene flux (Schaeffer et al. 2003; Kennington et al. 2006; but see Munte et al. 2005). The application of high-resolution tools flowing from completely sequenced genomes will facilitate the mapping of genes that are the targets of divergent natural selection within gene arrangements.Although Drosophila has been the favored model, the African malaria vector Anopheles gambiae sensu stricto also provides an excellent system for studying the maintenance of inversion polymorphisms, not only within a species but across speciation events of different ages in the An. gambiae sibling species complex. The nominal species An. gambiae s.s. (hereafter, An. gambiae) is synanthropic: almost exclusively biting humans, resting indoors, and exploiting anthropogenic larval habitats (Coluzzi 1999). This close association with humans, vital to making An. gambiae one of the most proficient vectors of malaria, is likely to have been facilitated by chromosomal inversions thought to confer adaptive benefits in heterogeneous climatic and ecological settings in Africa. Seven common polymorphic inversions exist on the second chromosome. Six of these are located on the right arm (2R): j, b, c, u, d, and k, while 2La is the only inversion on the left arm (Coluzzi et al. 2002). Facilitated by the sequenced reference genome (Holt et al. 2002), some of the breakpoints for these polymorphic inversions have been localized to small genomic regions (Sharakhov et al. 2006; Coulibaly et al. 2007; Sangare 2007). Most of these inversions appear to be the targets of strong selection. Five of the inversions (2La and 2Rb, -c, -d, and -u) are nonrandomly associated with degree of aridity; each cycles seasonally with rainfall, and all except 2Ru form stable geographic clines in frequency from mesic forest to xeric regions bordering the Sahara (Coluzzi et al. 1979; Toure et al. 1994, 1998; Powell et al. 1999). Inversion 2Rj is not clinal, but its distribution in Mali is consistent with adaptation to novel rockpool niches (Coluzzi et al. 1985; Manoukis et al. 2008).In the An. gambiae species complex, inversion polymorphisms can be maintained across the boundaries of emerging and even full species. An. gambiae and its sibling An. arabiensis, strictly sympatric throughout most of their extensive ranges in sub-Saharan Africa, differ by multiple fixed chromosomal rearrangements on the X but share three chromosome 2 inversions: 2La, fixed in An. arabiensis and polymorphic in An. gambiae; and 2Rb and -c, polymorphic in both species (Coluzzi et al. 1979, 2002). Moreover, these same inversions and all other common An. gambiae inversions with the exception of 2Rj are shared and polymorphic in two lineages apparently undergoing ecological speciation within An. gambiae—the assortatively mating M and S molecular forms (della Torre et al. 2002, 2005). Inversion frequencies are correlated with climatic and ecological conditions in parallel in both lineages (Costantini et al. 2009; Simard et al. 2009). Unlike the full species, the M and S incipient species are not distinguished by any fixed inversion differences. Indeed, genomewide divergence mapping between the M and S forms revealed that significant differentiation was confined to two small low-recombination regions adjacent to the centromeres of 2L and X which are distant from any inversions (Turner et al. 2005). Thus, in distinction to models of speciation invoking inversions as facilitating the persistence of hybridizing species (Noor et al. 2001; Rieseberg 2001; Ortiz-Barrientos et al. 2002; Navarro and Barton 2003), the An. gambiae data suggest that chromosome 2 inversions are not directly responsible for reproductive isolation. Instead, the same chromosome 2 inversion polymorphisms appear to confer similar ecological benefits, within and across species boundaries. A long-term research goal is to identify the mechanisms and the genes controlling these processes.Previously we conducted the first high-density genomic scan of divergence across a chromosomal inversion (2La) in An. gambiae (White et al. 2007). By hybridizing genomic DNA from S form mosquitoes homokaryotypic for alternate gene arrangements on chromosome 2L (2La or 2L+^a) to oligonucleotide microarrays we were able to measure divergence across the 22-Mb inversion at nearly 14,000 markers. Differentiation in the rearranged region was significantly higher than in collinear portions of chromosome 2L. Between breakpoints the pattern of differentiation was heterogeneous: two genomic clusters of significantly higher divergence were identified near but not adjacent to the breakpoints. Directed resequencing within the S form confirmed these results and suggested that both clusters contained genes targeted by selection. Observed levels of linkage disequilibrium between the 2La breakpoints and markers in the clusters are highly unlikely under a neutral scenario, in light of known recombination rates and plausible estimates of the age of the inversion.The present study characterizes the patterns of genetic variation in polymorphic rearrangements on the opposite (right) arm of chromosome 2: 2Rj, -b, -c, and -u. With the goal of identifying candidate genes maintaining these inversions in natural populations, we applied microarray-based divergence mapping to measure differentiation between alternative 2R arrangements. Because three of four inversions have taxonomic distributions that span incipient and/or completed speciation events, we validated the microarray findings by targeted sequencing in multiple taxa: sympatric Malian populations of An. gambiae M and S forms, and the sibling species An. arabiensis.     

Mating-System Variation,Demographic History and Patterns of Nucleotide Diversity in the Tristylous Plant Eichhornia paniculata

Inbreeding in highly selfing populations reduces effective size and, combined with demographic conditions associated with selfing, this can erode genetic diversity and increase population differentiation. Here we investigate the role that variation in mating patterns and demographic history play in shaping the distribution of nucleotide variation within and among populations of the annual neotropical colonizing plant Eichhornia paniculata, a species with wide variation in selfing rates. We sequenced 10 EST-derived nuclear loci in 225 individuals from 25 populations sampled from much of the geographic range and used coalescent simulations to investigate demographic history. Highly selfing populations exhibited moderate reductions in diversity but there was no significant difference in variation between outcrossing and mixed mating populations. Population size interacted strongly with mating system and explained more of the variation in diversity within populations. Bayesian structure analysis revealed strong regional clustering and selfing populations were highly differentiated on the basis of an analysis of F_st. There was no evidence for a significant loss of within-locus linkage disequilibrium within populations, but regional samples revealed greater breakdown in Brazil than in selfing populations from the Caribbean. Coalescent simulations indicate a moderate bottleneck associated with colonization of the Caribbean from Brazil ∼125,000 years before the present. Our results suggest that the recent multiple origins of selfing in E. paniculata from diverse outcrossing populations result in higher diversity than expected under long-term equilibrium.THE rate of self-fertilization in hermaphrodite organisms is expected to affect a number of important features of population genetic structure and diversity. Most directly, homozygosity increases as a function of the selfing rate and thus reduces the effective population size (N_e), up to twofold with complete selfing (Pollak 1987; Charlesworth et al. 1993; Nordborg 2000). Further, because of increased homozygosity, crossing over rarely occurs between heterozygous sites, thus increasing linkage disequilibrium (LD). Higher LD causes stronger hitchhiking effects such as selective sweeps, background selection, and Hill–Robertson interference, all of which are expected to further reduce the amount of neutral genetic variation within populations (reviewed in Charlesworth and Wright 2001).Population genetic processes resulting from inbreeding may be further augmented by demographic and life-history characteristics associated with the selfing habit. In particular, selfing populations can be founded by single individuals, resulting in striking reductions in diversity as a result of genetic bottlenecks and reproductive isolation. The capacity for uniparental reproduction gives many selfers prolific colonizing ability and the capacity to establish after long-distance dispersal, especially in comparison with obligate outcrossers (Baker 1955; Pannell and Barrett 1998). The colonization–extinction dynamics typical of many selfing species and limited pollen-mediated gene flow also increase differentiation among populations, resulting in considerable population subdivision (Hamrick and Godt 1990, 1996; Schoen and Brown 1991). Although the total amounts of among-population variation may be less affected by these processes (Pannell and Charlesworth 1999; Ingvarsson 2002), the demographic and life-history characteristics of many selfing species are likely to result in nonequilibrium conditions occurring in selfing populations.In many taxa where selfing has evolved it may be of relatively recent origin (Schoen et al. 1997; Takebayashi and Morrell 2001; Foxe et al. 2009; Guo et al. 2009). Where selfing has recently established, demographic forces associated with colonization may be as important as the mating system per se in structuring patterns of diversity. For example, if selfing originates through the establishment of a small number of founders, we would expect a sharp reduction in diversity relative to the outcrossing progenitor and a strong signature of a genetic bottleneck. In contrast, if selfing has evolved recently through the spread of genetic modifiers of small effect, newly established populations may retain significant amounts of ancestral polymorphism from their outcrossing progenitors. In this latter case populations may retain considerably more variation than expected under long-term equilibrium predictions.Molecular evidence for reduced nucleotide diversity and greater differentiation among populations of selfing taxa compared to populations of related outcrossing taxa has been reported from Leavenworthia (Liu et al. 1998, 1999), Arabidopsis (Savolainen et al. 2000; Wright et al. 2002), Solanum (Baudry et al. 2001), Mimulus (Sweigart and Willis 2003), Amsinckia (Perusse and Schoen 2004), and Caenorhabditis (Graustein et al. 2002; Cutter et al. 2006; Cutter 2008). In each case the reduction in diversity was more severe than the twofold reduction predicted for selfing populations at equilibrium. This indicates that factors in addition to the mating system are reducing diversity, but it has been difficult to uncouple the relative importance of genetic hitchhiking from the ecology and demographic history of selfing taxa. This challenge parallels similar difficulties in efforts to distinguish selective from demographic explanations in population genetic studies of Drosophila (Haddrill et al. 2005; Ometto et al. 2005; Thornton and Andolfatto 2006; Jensen et al. 2008). However, in many plant populations, especially those with annual life histories and small structured populations, demographic processes may play a more prominent role in causing reduced diversity than increased hitchhiking associated with selfing.Molecular population genetic studies of selfing in plants have generally focused on either small samples from a large number of populations (e.g., Sweigart and Willis 2003; Nordborg et al. 2005) or relatively large within-population samples from a small number of populations (e.g., Baudry et al. 2001). Ideally, a deeper sampling both within and among populations combined with independent ecological and historical information is required to improve understanding of the interplay of demographic and selective factors. Here we address these issues by examining patterns of nucleotide diversity within a large sample of populations of Eichhornia paniculata (Pontederiaceae), an annual species for which there is considerable ecological and demographic information (reviewed in Barrett and Husband 1997).E. paniculata occurs primarily in northeastern (N.E.) Brazil and the Caribbean islands of Cuba and Jamaica. Various lines of evidence suggest that Brazil is the original source region for Caribbean populations (reviewed in Barrett et al. 2009). Populations of E. paniculata exhibit striking mating-system diversity, ranging from predominantly outcrossing to those that are highly selfing (outcrossing rate, t = 0.002–0.96; n = 54 populations) (Barrett and Husband 1990; Barrett et al. 1992). Variation in mating system is associated with the evolutionary breakdown of the species'' tristylous genetic polymorphism and the spread and fixation of selfing variants capable of autonomous self-pollination (Barrett et al. 1989). Populations of E. paniculata are characterized by three morph structures: trimorphic with long-, mid-, and short-styled morphs (hereafter L-, M-, and S-morphs); dimorphic, with two floral morphs, most commonly the L- and M-morphs; and monomorphic, primarily composed of selfing variants of the M-morph. The morph structure and presence of selfing variants within populations explain ∼60% of the variation in outcrossing rates among populations (Barrett and Husband 1990). Trimorphic populations are largely outcrossing, dimorphic populations display mixed mating, and monomorphic populations are highly selfing. Patterns of allozyme variation indicate a reduction in diversity with increased selfing rates and greater among-population differentiation (Glover and Barrett 1987; Barrett and Husband 1990; Husband and Barrett 1993). Finally, studies of the inheritance of mating-system modifiers (Fenster and Barrett 1994; Vallejo-Marín and Barrett 2009) in combination with allozyme (Husband and Barrett 1993) and molecular evidence (Barrett et al. 2009) indicate that the transition from outcrossing to selfing in E. paniculata has occurred on multiple occasions.The goal of our study was to investigate the relation between mating-system variation and neutral molecular diversity for a large sample of E. paniculata populations encompassing most of the geographical range. This was accomplished by collecting multilocus nucleotide sequence data from 225 individuals sampled from 25 populations including trimorphic, dimorphic, and monomorphic populations. Because it has been previously demonstrated that this sequence of morph structures is strongly associated with increasing rates of self-fertilization (see Barrett and Husband 1990), we predicted a decrease in neutral diversity and increases in F_st and linkage disequilibrium from floral trimorphism to monomorphism. This extensive population-level sampling across a wide range of selfing rates allowed us to investigate the relative importance of mating system, geography, and current population size in structuring genetic variation. We also applied the approaches of Bayesian clustering (Pritchard et al. 2000; Falush et al. 2003; Gao et al. 2007) and divergence population genetics (Wakeley and Hey 1997; Hey and Nielsen 2004; Becquet and Przeworski 2007) to investigate the demographic history of E. paniculata and to provide a framework for understanding island colonization and the transition from outcrossing to selfing.     

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.     

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.