Evolution of Neo-Sex Chromosomes in Silene diclinis

A small cluster of dioecious species in the plant genus Silene has evolved chromosomal sex determination and sex chromosomes relatively recently, within the last 10 million years (MY). Five dioecious Silene species (section Elisanthe) are very closely related (1–2 MY of divergence) and it was previously thought that all five have similar sex chromosomes. Here we demonstrate that in one of these species, Silene diclinis, the sex chromosomes have been significantly rearranged, resulting in the formation of neo-sex chromosomes. Fluorescence in situ hybridization with genic and repetitive probes revealed that in S. diclinis a reciprocal translocation has occurred between the ancestral Y chromosome and an autosome, resulting in chromosomes designated Y1 and Y2. Both Y1 and Y2 chromosomes are male specific. Y1 pairs with the X chromosome and with the autosome (the neo-X), which cosegregates with X. Y2 pairs only with the neo-X, forming a chain X-Y1-neo-X-Y2 in male meiosis. Despite very recent formation of the neo-sex chromosomes in S. diclinis, they are present in all surveyed individuals throughout the species range. Evolution of neo-sex chromosomes may be the cause of partial reproductive isolation of this species and could have been the isolating mechanism that drove speciation of S. diclinis.PAIRING of homologous chromosomes during meiosis, in the majority of diploid plants and animals, leads to the formation of bivalents at first metaphase and subsequently the correct segregation of the chromosomes. Chromosomal translocations that produce multivalents usually result in unbalanced segregation, which consequently affects fertility. However, chain or ring configurations appear to be stably inherited in some species. An extreme example is found in the plant genus Oenothera, where many species display a ring involving all 14 chromosomes (Cleland 1972). In animals these configurations may include sex chromosomes, resulting in the formation of multiple X and Y chromosomes. For example, the monotreme platypus possesses five X and five Y chromosomes that form a chain of alternating X and Y chromosomes in male meiosis (Bick and Sharman 1975; Gruetzner et al. 2006). Such chains are formed due to several interchromosomal translocation events, including sex chromosome–autosome translocations (Gruetzner et al. 2006). Since sex chromosomes are rare in plants, examples of plant sex-linked chromosome multiples have been reported on only a few occasions. A chain of four X and five Y has been identified in an East African mistletoe Viscum fischeri (Wiens and Barlow 1975) and a chain of two X and two Y has been found in Humulus lupulus ssp. cordifolius (Shephard et al. 2000). Trivalent formation comprising Y1 X Y2 has been observed both in H. japonicus (Shephard et al. 2000) and in a number of dioecious species in the genus Rumex (Cunado et al. 2007; Navajas-Perez et al. 2009). Here we report that the plant species Silene diclinis has multiple sex chromosomes that form a chain of four during meiosis metaphase I.S. diclinis is a member of a small group of dioecious species (having separate male and female plants) in section Elisanthe in the plant genus Silene (Caryophyllaceae). The other members of this group are S. latifolia, S. dioica, S. heuffelii, and S. marizii (Prentice 1978). The presence of large heteromorphic sex chromosomes in S. latifolia and S. dioica has been known for many years (Westergaard 1958). Due to the ease of cytogenetic identification of the sex chromosomes, the clear morphological difference between the sexes and the short generation time, S. latifolia was used in early genetic research concerning sex determination in plants. The male was shown to be the heterogametic sex (XY) with the larger Y chromosome having a decisive role in sex determination (Westergaard 1958). Since then, S. latifolia has become a species of choice for studies in plant genetics, ecology, and evolution (Bernasconi et al. 2009). It is particularly useful for studies of sex chromosome evolution because the sex chromosomes in Silene are of relatively recent origin compared to those of mammals (Charlesworth 2002; Ming and Moore 2007; Marais et al. 2008).Experimental crosses involving all five dioecious species in Silene section Elisanthe in various pairwise combinations have produced viable hybrids and, although some combinations were less successful than others, the formation of these hybrids suggests a close relationship within this group (Prentice 1978). This close relationship is also illustrated by DNA sequence comparisons that show that interspecific silent divergence between these species does not exceed 2%, which is comparable to intraspecific polymorphism in S. latifolia (Ironside and Filatov 2005). S. diclinis is a rare and restricted endemic, found only in Southern Valencia, Spain in an area smaller than 18 × 9 km (Prentice 1976; Montesinos et al. 2006). Of the other four Elisanthe species, only S. latifolia occurs in this region, and experimental crosses between these two species are the least successful (Prentice 1978). Hybrids between S. latifolia and S. dioica occur naturally in regions where their populations coincide (Baker 1948) but no natural hybrids of S. diclinis and S. latifolia have been reported.Cytogenetic analysis of S. diclinis has been limited. Examination of mitotic metaphase spreads in root tip squash preparations from adult male and female plants indicated that the male had one X and one Y chromosome. Both chromosomes were large but the difference between them was slight (van Nigtevecht and Prentice 1985). Regular pairing of chromosomes with 12 bivalents at metaphase I in pollen mother cells has been reported (Morisset and Bozman 1969). However, these observations were made without the benefit of a marker for the Y chromosome. Recently, sequences with homology to an Ogre retrotransposon have been isolated from S. latifolia and used as probes in fluorescence in situ hybridization (FISH) experiments on mitotic (Cermak et al. 2008) and both mitotic and meiotic (Filatov et al. 2009) chromosome spreads. The pattern of hybridization showed that these sequences are widespread over the X chromosome and all of the autosomes but are mainly confined to a small section at the pairing region of the Y chromosome in S. latifolia. Therefore, these probes “paint” all the chromosomes apart from the Y, providing a “negative paint” for the Y chromosome. By using one of these probes (clone 4.2) on meiotic spreads of S. dioica and S. marizii, we confirmed that these species have sex chromosomes similar to those of S. latifolia (Filatov et al. 2009). The X and Y formed a rod bivalent and the Y chromosome was larger than both the X and autosomes.In this article we report our FISH experiments with S. diclinis using the negative paint probe together with probes containing S. latifolia sex-linked gene sequences. We demonstrate that S. diclinis males have two Y chromosomes that differ in the distribution of the paint signal and these gene sequences. In meiotic metaphase I, one Y pairs with the X and an autosome while the second Y pairs with the other arm of this autosome, forming a chain of four chromosomes. We suggest that an autosome–Y reciprocal translocation was involved in the evolution of neo-sex chromosomes in this species.     

Interlock Formation and Coiling of Meiotic Chromosome Axes During Synapsis

The meiotic prophase chromosome has a unique architecture. At the onset of leptotene, the replicated sister chromatids are organized along an axial element. During zygotene, as homologous chromosomes pair and synapse, a synaptonemal complex forms via the assembly of a transverse element between the two axial elements. However, due to the limitations of light and electron microscopy, little is known about chromatin organization with respect to the chromosome axes and about the spatial progression of synapsis in three dimensions. Three-dimensional structured illumination microscopy (3D-SIM) is a new method of superresolution optical microscopy that overcomes the 200-nm diffraction limit of conventional light microscopy and reaches a lateral resolution of at least 100 nm. Using 3D-SIM and antibodies against a cohesin protein (AFD1/REC8), we resolved clearly the two axes that form the lateral elements of the synaptonemal complex. The axes are coiled around each other as a left-handed helix, and AFD1 showed a bilaterally symmetrical pattern on the paired axes. Using the immunostaining of the axial element component (ASY1/HOP1) to find unsynapsed regions, entangled chromosomes can be easily detected. At the late zygotene/early pachytene transition, about one-third of the nuclei retained unsynapsed regions and 78% of these unsynapsed axes were associated with interlocks. By late pachytene, no interlocks remain, suggesting that interlock resolution may be an important and rate-limiting step to complete synapsis. Since interlocks are potentially deleterious if left unresolved, possible mechanisms for their resolution are discussed in this article.MEIOSIS is a specialized cell division found in all organisms with a sexual life cycle. It requires the intricate coordination and precise timing of a series of cellular processes to ensure proper chromosome segregation and reduction in ploidy level. Meiotic prophase is initiated by the formation of cytologically characteristic leptotene chromosomes, which requires the installation of axial elements (AEs) onto the chromosomes. Cohesin complexes, required for sister chromatid cohesion during mitosis and meiosis, are an essential component of AE formation or maintenance (Klein et al. 1999). After the formation of double-strand breaks to initiate recombination, there is a global reorganization of chromosomes at the leptotene–zygotene transition as telomeres cluster on the nuclear envelope in the bouquet configuration (Harper et al. 2004). As zygotene proceeds, the close association of the paired homologs is stabilized by formation of the synaptonemal complex (SC). During synapsis, a transverse element is installed between the AEs, now called the lateral elements (LEs), to assemble the tripartite ladder-like SC (Page and Hawley 2003). On the basis of electron microscopy (EM) surveys, synapsis typically starts from the ends of chromosomes and works its way inward, although interstitial sites of synapsis initiation are also found, especially in organisms that have long chromosomes such as maize (Burnham et al. 1972; Zickler and Kleckner 1999). As homologous chromosomes synapse, their axes coil around each other. This feature has been called “relational coiling/twisting” of homologs (Moens 1972, 1974; Zickler and Kleckner 1999). The cause or possible function of this coiling is not known.As first described by Gelei, during zygotene chromosomes can become entangled within other synapsing pairs, forming interlocks (Gelei 1921). Either a bivalent (type I interlock) or one unsynapsed chromosome (type II interlock) can become trapped between unsynapsed AEs and caught by the formation of the SC on both sides of the loop. In one example described in maize, multiple chromosomes are trapped, forming a “complex interlock” (Gillies 1981). Since interlocks can be deleterious if left unresolved, mechanisms should be present in meiotic nuclei to prevent or resolve their occurrence (von Wettstein et al. 1984). To date, no mechanism has yet been identified. Interlocks could be resolved by coordinated breakage and rejoining of chromosomes (Holm et al. 1982; Rasmussen 1986; Moens 1990) or by chromosome movement and SC disassembly during zygotene and pachytene (Conrad et al. 2008; Koszul et al. 2008). For chromosomes to escape an interlock by this latter mechanism, one or more telomeres of the interlocking chromosomes either have to separate from each other on the nuclear envelope or be released from the nuclear envelope so that one interlocking chromosome can be pulled away from the other (Rasmussen and Holm 1980).Meiotic chromosomes are large, complex structures that can be tens of microns in length, yet the size of many structural elements of interest such as recombination nodules (50–200 nm diameter) and the LEs of the SC (spaced 100–200 nm apart) is just beyond the resolution of conventional wide-field microscopy (von Wettstein et al. 1984). In favorable light microscope preparations, DNA is organized into chromomeres of condensed chromatin; however, their organization with respect to AEs cannot be easily resolved by conventional light microscopy. Although analysis of spatial organization of AEs at a high resolution can be accomplished by three-dimensional (3D) EM reconstructions of entire nuclei (e.g., Gillies 1973; Zickler and Olson 1975; Goldstein and Moens 1976), 3D EM analysis of even one cell is arduous. It has been difficult to describe the spatial behavior of AEs during synapsis, which constrains our understanding of the kinetics of this dynamic process. Three-dimensional structured illumination microscopy (3D-SIM), a new method of superresolution light microscopy, can achieve a resolution of better than 100 nm in the lateral (xy) plane and 250 nm along the z-axis (Gustafsson 2008; Gustafsson et al. 2008; Schermelleh et al. 2008). The advantages of 3D-SIM over EM are the ability to use FISH and immunolocalizations with fluorescently labeled proteins to specifically and simultaneously detect DNA, RNA, and proteins on chromosomes and the ability to obtain three-dimensional information from thick specimens.Maize, a diploid (2n = 20) monocot grass of the Poaceae, is one of the few organisms with a large genome in which chromatin structure, homologous pairing, and synapsis are amenable to analysis by a combination of cytological, genetic, molecular, and biochemical techniques (Cande et al. 2009). Here, we use 3D-SIM to study chromomere and AE/LE organization of maize pachytene chromosomes. In maize, the cohesin REC8 alpha-kleisin homolog, encoded by absence of first division1 (afd1), is required for maintenance of AEs and proper leptotene chromosome structure (Golubovskaya et al. 2006). We used antibodies against AFD1/REC8 and ASYNAPTIC1 (ASY1), the Arabidopsis homolog of the yeast HOP1 gene (Armstrong et al. 2002), to delineate the prophase chromosome axis and show it changes during synapsis. This is the first exploration of chromosome behavior and synapsis at such a high resolution by light microscopy in any organism, and our findings allow us to quantitatively analyze key features of SC axial element behavior during zygotene and pachytene.     

A Neo-Sex Chromosome That Drives Postzygotic Sex Determination in the Hessian Fly (Mayetiola destructor)

Two nonoverlapping autosomal inversions defined unusual neo-sex chromosomes in the Hessian fly (Mayetiola destructor). Like other neo-sex chromosomes, these were normally heterozygous, present only in one sex, and suppressed recombination around a sex-determining master switch. Their unusual properties originated from the anomalous Hessian fly sex determination system in which postzygotic chromosome elimination is used to establish the sex-determining karyotypes. This system permitted the evolution of a master switch (Chromosome maintenance, Cm) that acts maternally. All of the offspring of females that carry Cm-associated neo-sex chromosomes attain a female-determining somatic karyotype and develop as females. Thus, the chromosomes act as maternal effect neo-W''s, or W-prime (W′) chromosomes, where ZW′ females mate with ZZ males to engender female-producing (ZW′) and male-producing (ZZ) females in equal numbers. Genetic mapping and physical mapping identified the inversions. Their distribution was determined in nine populations. Experimental matings established the association of the inversions with Cm and measured their recombination suppression. The inversions are the functional equivalent of the sciarid X-prime chromosomes. We speculate that W′ chromosomes exist in a variety of species that produce unisexual broods.SEX chromosomes are usually classified as X, Y, Z, or W on the basis of their pattern of segregation and the gender of the heterogametic sex (Ohno 1967). However, when chromosome-based sex determination occurs postzygotically, the same nomenclature confounds important distinctions and may hide interesting evolutionary phenomena. The Hessian fly (Mayetiola destructor), a gall midge (Diptera: Cecidomyiidae) and an important insect pest of wheat, presents an excellent example (Stuart and Hatchett 1988, 1991). In this insect, all of the female gametes and all of the male gametes have the same number of X chromosomes (Figure 1A); no heterogametic sex exists. Nevertheless, Hessian fly sex determination is chromosome based; postzygotic chromosome elimination produces different X chromosome to autosome ratios in somatic cells (male A1A2X1X2/A1A2OO and female A1A2X1X2/A1A2X1X2, where A1 and A2 are the autosomes, X1 and X2 are the X chromosomes, and the paternally derived chromosomes follow the slash) (Stuart and Hatchett 1991; Marin and Baker 1998). Thus, Hessian fly “X” chromosomes are defined by their haploid condition in males, rather than by their segregation in the gametes.Open in a separate windowFigure 1.—Chromosome behavior and sex determination in the Hessian fly. (A) Syngamy (1) establishes the germ-line chromosome constitution: ∼32 maternally derived E chromosomes (represented as a single white chromosome) and both maternally derived (black) and paternally derived (gray) autosomes and X chromosomes. During embryogenesis, while the E chromosomes are eliminated, the paternally derived X chromosomes are either retained (2) or excluded (3) from the presumptive somatic cells. When the paternally derived X chromosomes are retained (2), a female-determining karyotype is established. When they are eliminated (3), a male-determining karyotype is established. Thelygenic mothers carry Cm (white arrow), which conditions all of their offspring to retain the X chromosomes. Recombination occurs during oogenesis (4). All ova contain a full complement of E chromosomes and a haploid complement of autosomes and X chromosomes. Chromosome elimination occurs during spermatogenesis (5). Sperm contain only the maternally derived autosomes and X chromosomes. (B) The segregation of Cm (white dot) on a Hessian fly autosome among monogenic families. Thelygenic females produce broods composed of equal numbers of thelygenic (Cm/−) and arrhenogenic (−/−) females (box 1). Arrhenogenic females produce males (box 2). (C) Matings between monogenic and amphigenic families. Cm (white dot) is dominant to the amphigenic-derived chromosomes (gray dot) and generates all-female offspring (box 3). Amphigenic-derived chromosomes are dominant to the arrhenogenic-derived chromosomes (no dot) and generate offspring of both sexes (box 4).An autosomal, dominant, genetic factor called Chromosome maintenance (Cm) complicates Hessian fly sex determination further (Stuart and Hatchett 1991). Cm has a maternal effect that acts upstream of X chromosome elimination during embryogenesis (Figure 1A). It prevents X chromosome elimination so that all of the offspring of Cm-bearing mothers obtain a female-determining karyotype. Cm-bearing females produce only female offspring and are therefore thelygenic. The absence of Cm usually has the opposite effect; all of the offspring of most Cm-lacking females obtain a male-determining karyotype. These Cm-lacking females produce only male offspring and are therefore arrhenogenic. Like a sex-determining master switch, Cm is usually heterozygous and present in only one sex (Figure 1B). Thus, thelygenic females (Cm/−) are “heterogametic,” as their Cm-containing gametes and Cm-lacking gametes produce thelygenic (Cm/−) and arrhenogenic (−/−) females in a 1:1 ratio. Collectively, thelygenic and arrhenogenic females are called monogenic because they produce unisexual families. However, some Hessian fly females produce broods of both sexes and are called amphigenic. No mating barrier between monogenic and amphigenic families exists (Figure 1C), but amphigenic females have always been found in lower abundance (Painter 1930; Gallun et al. 1961; Stuart and Hatchett 1991). In experimental matings, the inheritance of maternal phenotype was consistent with the segregation of three Cm alleles (Figure 1C): a dominant thelygenic allele, a hypomorphic amphigenic allele, and a null arrhenogenic allele (Stuart and Hatchett 1991).Here we report the genetic and physical mapping of Cm on Hessian fly autosome 1 (A1). Two nonoverlapping inversions were identified that segregated perfectly with Cm. The most distal inversion was present in all thelygenic females examined. The more proximal inversion extended recombination suppression. These observations suggested that successive inversions evolved to suppress recombination around Cm after it arose. The inversions therefore appear to have evolved in response to the forces that shaped vertebrate Y and W chromosomes (Charlesworth 1996; Graves and Shetty 2001; Rice and Chippindale 2001; Carvalho and Clark 2005). We therefore believe the inversion-bearing chromosomes may be classified as maternal effect neo-W''s.     

Anecdotal,Historical and Critical Commentaries on Genetics: Personal Reflections on the Origins and Emergence of Recombinant DNA Technology

The ability to identify genetic markers in nonmodel systems has allowed geneticists to construct linkage maps for a diversity of species, and the sex-determining locus is often among the first to be mapped. Sex determination is an important area of study in developmental and evolutionary biology, as well as ecology. Its importance for organisms might suggest that sex determination is highly conserved. However, genetic studies have shown that sex determination mechanisms, and the genes involved, are surprisingly labile. We review studies using genetic mapping and phylogenetic inferences, which can help reveal evolutionary pattern within this lability and potentially identify the changes that have occurred among different sex determination systems. We define some of the terminology, particularly where confusion arises in writing about such a diverse range of organisms, and highlight some major differences between plants and animals, and some important similarities. We stress the importance of studying taxa suitable for testing hypotheses, and the need for phylogenetic studies directed to taxa where the patterns of changes can be most reliably inferred, if the ultimate goal of testing hypotheses regarding the selective forces that have led to changes in such an essential trait is to become feasible.THE ever-increasing accessibility of genetic markers is allowing sex-determining regions to be genetically mapped in a growing number of nonmodel organisms. There are several reasons for studying sex determination. In animals, gonadal differences are often accompanied by striking somatic secondary sexual dimorphisms, which are interesting in an evolutionary context (Shine 1989; Badyaev 2002). In plants, females and males often differ in flower morphology and abundance (Dawson and Geber 1998), and, although sex differences are often minor outside the flowers (or inflorescences), they do exist (Dawson and Geber 1998; Eppley and Wenk 2001). The genetic control of these phenotypes is a fundamental biological process, and studying sex determination pathways is important in animal developmental biology (Adams and McLaren 2002; Pinyopich et al. 2003), including genetic pathway evolution (Wilkins 1995; Williams and Carroll 2009).Until recently, sex determination was generally studied by testing for genetic control vs. partial or complete environmental influences. Genetic systems were examined cytologically to determine the level of heteromorphism between the sex chromosomes and to identify whether females or males are heterogametic (see the comprehensive review in Bull 1983). Male heterogametic systems, referred to as XY, were also tested to identify whether the Y chromosome carries a male-determining gene, as in almost all therian mammals and most dioecious plants so far studied, or whether sex is determined through X–autosome balance, as in Drosophila and Caenorhabditis elegans (Haag 2005). For female heterogametic (ZW) species, analogous tests were used to identify how femaleness is determined.Until recently, sex-determining genes and regions could be genetically mapped in only a few model species, but now that molecular genetic markers can be developed in nonmodel species, new information is becoming available about how genetic sex determination (GSD) mechanisms have changed during evolutionary history. It has long been known from genetic mapping in model systems, including mammals, and (more recently) birds, that sex chromosomes often have large nonrecombining regions (Bull 1983; Charlesworth 1991; Charlesworth et al. 2005). However, in other organisms, nonrecombining regions are not always large and may sometimes be absent. The evolution of sexual reproduction and recombination have been the focus of many years of discussion in evolutionary biology (Otto 2009), and studies of sex chromosomes are important for understanding why recombination is often lost, and elucidating the evolutionary consequences of recombination suppression (Charlesworth 1996; Otto and Barton 1997; Barton and Charlesworth 1998). The adaptation of genes on the sex chromosomes is also interesting, because this location affects the outcome of sex-specific selection pressures (Rice 1984; Charlesworth et al. 1987; Vicoso and Charlesworth 2006; Mank 2009a). Finally, the mechanism of sex determination can affect sex ratios (West and Sheldon 2002; Dorken and Pannell 2008; West 2009) and is therefore significant in evolutionary ecology.Sex determination is also relevant in applied biology. In many domesticated animals, one sex may be of greatest economic interest to farmers and breeders. Modern meat production is largely based on males, including industrial production of chicken, cattle, and many fish, whereas females are the sex required for milk (cattle) and egg (chicken) production. Similarly, a few crop plants are dioecious, and, in some of these, the crop is produced by females (e.g., grapes, dates, and papaya), while in other species the sexes differ in characteristics such as fiber or chemical content. Because immature birds, fish, and plants have no obvious phenotypic sex differences, maximizing agricultural returns often requires genetically sexing juveniles. Mapping sex determination is an important first step toward identifying the sex-determining genes or finding other sex-specific markers to develop molecular sexing methods.In this review, we first summarize recent developments in genetic mapping of sex determination, concentrating on nonmodel plants and animals with genetic sex determination. We show how this information can be useful for understanding the evolution of sex determination and sex chromosomes and identify some important unanswered questions.     

Mek1 Suppression of Meiotic Double-Strand Break Repair Is Specific to Sister Chromatids,Chromosome Autonomous and Independent of Rec8 Cohesin Complexes

During meiosis, recombination is directed to occur between homologous chromosomes to create connections necessary for proper segregation at meiosis I. Partner choice is determined at the time of strand invasion and is mediated by two recombinases: Rad51 and the meiosis-specific Dmc1. In budding yeast, interhomolog bias is created in part by the activity of a meiosis-specific kinase, Mek1, which is localized to the protein cores of condensed sister chromatids. Analysis of meiotic double-strand break (DSB) repair in haploid and disomic haploid strains reveals that Mek1 suppresses meiotic intersister DSB repair by working directly on sister chromatids. Rec8 cohesin complexes are not required, however, either for suppression of intersister DSB repair or for the repair itself. Regulation of DSB repair in meiosis is chromosome autonomous such that unrepaired breaks on haploid chromosomes do not prevent interhomolog repair between disomic homologs. The pattern of DSB repair in haploids containing Dmc1 and/or Rad51 indicates that Mek1 acts on Rad51-specific recombination processes.IN eukaryotes, meiosis is a specialized type of cell division that produces the gametes required for sexual reproduction. In meiosis, one round of DNA replication is followed by two rounds of chromosome segregation, termed meiosis I and II. As a result of the two divisions, four haploid cells are produced, each containing half the number of chromosomes as the diploid parent. Proper segregation at meiosis I requires connections between homologous chromosomes that are created by a combination of sister chromatid cohesion and recombination (Petronczki et al. 2003). In vegetative cells, cohesion is mediated by multisubunit ring-shaped complexes that are removed by proteolysis of the kleisin subunit, Mcd1/Scc1 (Onn et al. 2008). In meiotic cells, introduction of a meiosis-specific kleisin subunit, Rec8, allows for a two-step removal of cohesion with loss of arm cohesion at anaphase I and centromere cohesion at anaphase II (Klein et al. 1999). Missegregation of chromosomes during meiosis causes abnormal chromosome numbers in gametes that may lead to infertility and genetic disorders such as trisomy 21 or Down''s syndrome.In mitotically dividing budding yeast cells, recombination is mediated by an evolutionarily conserved RecA-like recombinase, Rad51, and occurs preferentially between sister chromatids (Kadyk and Hartwell 1992). In contrast, recombination during meiosis is initiated by the deliberate formation of double-strand breaks (DSBs) by an evolutionarily conserved, topoisomerase-like protein, Spo11, and occurs preferentially between homologous chromosomes (Jackson and Fink 1985; Schwacha and Kleckner 1997; Keeney 2001). After DSB formation, the 5′ ends on either side of the breaks are resected, resulting in 3′ single stranded (ss) tails. Rad51, and the meiosis-specific recombinase Dmc1, bind to the 3′ ssDNA tails to form protein/DNA filaments that promote strand invasion of homologous chromosomes. DNA synthesis and ligation result in the formation of double Holliday junctions, which are then preferentially resolved into crossovers (Allers and Lichten 2001; Hunter 2007).The precise roles that the Rad51 and Dmc1 recombinase activities play in meiotic recombination have been unclear because experiments have indicated both overlapping and distinct functions for the two proteins (Sheridan and Bishop 2006; Hunter 2007). While both rad51Δ and dmc1Δ mutants reduce interhomolog recombination, other studies suggest that Rad51, in complex with the accessory protein Rad54, is involved primarily in intersister DSB repair. In contrast, Dmc1, in conjunction with the accessory protein Rdh54/Tid1 (a paralog of Rad54), effects DSB repair in meiotic cells by invasion of nonsister chromatids (Dresser et al. 1997; Schwacha and Kleckner 1997; Shinohara et al. 1997a,b; Arbel et al. 1999; Bishop et al. 1999; Hayase et al. 2004; Sheridan and Bishop 2006).The preference for recombination to occur between homologous chromosomes during meiosis is created in part by Dmc1. DSBs accumulate in dmc1Δ diploids due to a failure in strand invasion (Bishop et al. 1992; Hunter and Kleckner 2001). In the efficiently sporulating SK1 strain background, these unrepaired breaks trigger the meiotic recombination checkpoint, resulting in prophase arrest (Lydall et al. 1996; Roeder and Bailis 2000). In dmc1Δ mutants, Rad51 is present at DSBs, yet there is no strand invasion of sister chromatids (Bishop 1994; Shinohara et al. 1997a). These results suggest that in addition to Dmc1 promoting interhomolog strand invasion, Rad51 activity must also be suppressed.Recent studies have shown that during meiosis Rad51 recombinase activity is inhibited by two different mechanisms that decrease the formation of Rad51/Rad54 complexes: (1) binding of the meiosis-specific Hed1 protein to Rad51, thereby excluding interaction with Rad54, and (2) reduction in the affinity of Rad54 for Rad51 due to phosphorylation of Rad54 by Mek1 (Tsubouchi and Roeder 2006; Busygina et al. 2008; Niu et al. 2009). Mek1 is a meiosis-specific kinase that is activated in response to DSBs (Niu et al. 2005, 2007; Carballo et al. 2008). In addition to phosphorylating Rad54, Mek1 phosphorylation of an as yet undetermined substrate is required to suppress Rad51/Rad54-mediated strand invasion of sister chromatids (Niu et al. 2009).To dissect the mechanism by which Mek1 suppresses meiotic intersister DSB repair, we took advantage of the ability of yeast cells to undergo haploid meiosis. The lack of homologous chromosomes in haploid cells makes it possible to examine sister-chromatid-specific events in the absence of interhomolog recombination. De Massy et al. (1994) previously observed a delay in DSB repair in haploid cells and proposed that this delay was due to a constraint in using sister chromatids. We have shown that this delay is dependent on MEK1 and utilized the haploid system to determine various biological parameters required to suppress meiotic intersister DSB repair. Our results indicate that Rad51 and Dmc1 recombinase activities have distinct roles during meiosis and that interhomolog bias is established specifically on sister chromatids through regulation of Rad51, not Dmc1. rec8Δ diploids exhibit defects in meiotic DSB repair (Klein et al. 1999; Brar et al. 2009). Given that cohesin complexes are specific for sister chromatids, we investigated the role of REC8 in intersister DSB repair and found it is required neither for suppressing intersister DSB repair during meiosis nor for the repair itself.     

Gorilla Mothers Also Matter! New Insights on Social Transmission in Gorillas (Gorilla gorilla gorilla) in Captivity

The present paper describes two distinct behaviors relating to food processing and communication that were observed in a community of five separately housed groups of lowland gorillas (Gorilla gorilla gorilla) in captivity during two study periods one decade apart: (1) a food processing technique to separate wheat from chaff, the so-called puff-blowing technique; and (2) a male display used to attract the attention of visitors, the so-called throw-kiss-display. We investigated (a) whether the behaviors were transmitted within the respective groups; and if yes, (b) their possible mode of transmission. Our results showed that only the food processing technique spread from three to twenty-one individuals during the ten-year period, whereas the communicative display died out completely. The main transmission mode of the puff-blowing technique was the mother-offspring dyad: offspring of puff-blowing mothers showed the behavior, while the offspring of non- puff-blowing mothers did not. These results strongly support the role mothers play in the acquisition of novel skills and vertical social transmission. Furthermore, they suggest that behaviors, which provide a direct benefit to individuals, have a high chance of social transmission while the loss of benefits can result in the extinction of behaviors.     

Identification of the Major Sex-Determining Region of Turbot (Scophthalmus maximus)

Sex determination in fish is a labile character in evolutionary terms. The sex-determining (SD) master gene can differ even between closely related fish species. This group is an interesting model for studying the evolution of the SD region and the gonadal differentiation pathway. The turbot (Scophthalmus maximus) is a flatfish of great commercial value, where a strong sexual dimorphism exists for growth rate. Following a QTL and marker association approach in five families and a natural population, we identified the main SD region of turbot at the proximal end of linkage group (LG) 5, close to the SmaUSC-E30 marker. The refined map of this region suggested that this marker would be 2.6 cM and 1.4 Mb from the putative SD gene. This region appeared mostly undifferentiated between males and females, and no relevant recombination frequency differences were detected between sexes. Comparative genomics of LG5 marker sequences against five model species showed no similarity of this chromosome to the sex chromosomes of medaka, stickleback, and fugu, but suggested a similarity to a sex-associated QTL from Oreochromis spp. The segregation analysis of the closest markers to the SD region demonstrated a ZW/ZZ model of sex determination in turbot. A small proportion of families did not fit perfectly with this model, which suggests that other minor genetic and/or environmental factors are involved in sex determination in this species.SEX ratio is a central demographic parameter directly related to the reproductive potential of individuals and populations (Penman and Piferrer 2008). The phenotypic sex depends on the processes of both sex determination and sex differentiation. Exogenous factors, such as temperature, hormones, or social behavior, can modify the gonad development pathway in fish (Baroiller and D''Cotta 2001; Piferrer and Guiguen 2008). Both genetic (GSD) and environmental sex determination has been reported in this group (Devlin and Nagahama 2002; Penman and Piferrer 2008), although primary sex determination is genetic in most species (Valenzuela et al. 2003). Among GSD, single, multiple, or polygenic sex-determining (SD) gene systems have been documented (Kallman 1984; Matsuda et al. 2002; Lee et al. 2004; Vandeputte et al. 2007).Sex determination in fish can evolve very rapidly (Woram et al. 2003; Peichel et al. 2004; Ross et al. 2009). Different sex determination mechanisms have been reported between congeneric species and even between populations of the same species (Almeida-Toledo and Foresti 2001; Lee et al. 2004; Mank et al. 2006). The evolution of sex chromosomes involves the suppression of recombination between homologous chromosomes probably to maintain sex-related coadapted gene blocks (Charlesworth et al. 2005; Tripathi et al. 2009). The sex determination pathway appears to be less conserved than other developmental processes (Penman and Piferrer 2008). However, differences are more related to the top of the hierarchy in the developmental pathway, while downstream genes are more conserved (Wilkins 1995; Marín and Baker 1998). As a consequence, the SD master gene in fish can vary among related species (Kondo et al. 2003; Tanaka et al. 2007; Alfaqih et al. 2009). In this sense, fish represent an attractive model for studying the evolution of SD mechanisms and sex chromosomes (Peichel et al. 2004; Kikuchi et al. 2007).A low proportion of fish species have demonstrated sex-associated chromosome heteromorphisms (Almeida-Toledo and Foresti 2001; Devlin and Nagahama 2002; Penman and Piferrer 2008). This is congruent with the rapid evolution of the SD region in fish, and thus in most species the male and female version of this chromosome region appears largely undifferentiated. In spite of this, indirect clues related to progenies of sex/chromosome-manipulated individuals or to segregation of morphologic/molecular sex-associated markers indicate that mechanisms of sex determination in fish are similar to other vertebrates (Penman and Piferrer 2008). With the arrival of genomics, large amounts of different genetic markers and genomic information are available for scanning genomes to look for their association with sex determination. Quantitative trait loci (QTL) (Cnaani et al. 2004; Peichel et al. 2004) or marker association (Felip et al. 2005; Chen et al. 2007) approaches have been used to identify the SD regions in some fish species. Also, microarrays constructed from gonadal ESTs have been applied to detect differentially expressed genes in the process of gonadal differentiation (Baron et al. 2005). Further, the increased genomic resources in model and aquaculture species have allowed the development of both comparative genomics (Woram et al. 2003; Kikuchi et al. 2007; Tripathi et al. 2009) and candidate gene (Shirak et al. 2006; Alfaqih et al. 2009) strategies to identify and characterize the SD region in fish. This has permitted the identification of the SD region in eight fish, including both model and aquaculture species (reviewed in Penman and Piferrer 2008).The turbot is a highly appreciated European aquaculture species, whose harvest is expected to increase from the current 9000 tons to >15,000 tons in 2012 (S. Cabaleiro, personal communication). Females of this species reach commercial size 4–6 months before males do, explaining the interest of the industry in obtaining all-female populations. Although some differences between families can be observed in the production process at farms, sex ratio is usually balanced at ∼1:1. Neither mitotic nor meiotic chromosomes have shown sex-associated heteromorphisms in turbot (Bouza et al. 1994; Cuñado et al. 2001). The proportion of sexes observed in triploid and especially gynogenetic progenies moved Cal et al. (2006a,b) to suggest an XX/XY mechanism in turbot with some additional, either environmental or genetic, factor involved. However, Haffray et al. (2009) have recently claimed a ZZ/ZW mechanism on the basis of the analysis of a large number of progenies from steroid-treated parents. These authors also suggested some (albeit low) influence of temperature in distorting sex proportions after the larval period. Finally, hybridizations between brill (Scophthalmus rhombus) and turbot render monosex progenies, depending on the direction of the cross performed, which suggests different SD mechanisms in these congeneric species (Purdom and ThaCker 1980).In this study, we used the turbot genetic map (Bouza et al. 2007, 2008; Martínez et al. 2008) to look for sex-associated QTL in this species. The identification of a major QTL in a specific linkage group (LG) in the five families analyzed prompted us to refine the genetic map at this LG and to perform a comparative genomics approach against model fish species for a precise location and characterization of the putative SD region. Also, sex-associated QTL markers were screened in a large natural population to provide additional support to our findings and to obtain population parameters at sex-related markers that could aid in interpreting the evolution of this genomic region.     

Different Aneuploidies Arise From the Same Bridge-Induced Chromosomal Translocation Event in Saccharomyces cerevisiae

Chromosome translocations are gross chromosomal rearrangements that have often been associated with cancer development in mammalian cells. The feasibility of drastically reshaping the genome with a single translocation event also gives this molecular event a powerful capacity to drive evolution. Despite these implications and their role in genome instability, very little is known about the molecular mechanisms that promote and accompany these events. Here, at the molecular level, we describe 10 morphologically and physiologically different translocants ensuing from the induction of the same bridge-induced translocation (BIT) event in the budding yeast Saccharomyces cerevisiae. We have demonstrated that, despite their common origin from the integration of the same linear DNA construct, all 10 translocation mutant strains have different phenotypes and the ability to sporulate and regulate gene expression and morphology. We also provide insights into how heterogeneous phenotypic variations originate from the same initial genomic event. Here we show eight different ways in which yeast cells have dealt with a single initial event inducing translocation. Our results are in agreement with the formation of complex rearrangements and abnormal karyotypes described in many leukemia patients, thus confirming the modellistic value of the yeast BIT system for mammalian cells.TRANSLOCATIONS between nonhomologous chromosomes are some of the most severe genomic aberrations, which in higher eukaryotes often lead to malignant transformation. In humans, they have been associated with hematological cancers such as myelogenous leukemia but also with lymphomas, solid tumors, and recently, with mesenchymal and epithelial cancers (Dalla-Favera et al. 1982; Nowell 1988; Rowley 2001, 2008; Taki and Taniwaki 2006; Gasparini et al. 2007; Nickoloff 2008). In the past few decades, molecular and cytological studies have demonstrated that different chromosomal abnormalities such as aneuploidy can be associated with a translocation event in cancer cells. For example, acute transformation of leukemia patients is often associated with the duplication of the Philadelphia chromosome, trisomy 8 and isochromosome 17q, or other complex chromosomal rearrangements (Muehleck et al. 1984; Bernstein 1988; De Braekeleer 2007). Furthermore, unbalanced translocations can be detected in older leukemia patients. Karyotypic analyses have revealed that unbalanced translocations can advance into further rearrangements of the chromosomes involved in the translocations. This can give rise to complex and abnormal karyotypes characterized by monosomy, disomy, and trisomy for different segments of the translocation participant chromosomes (Pedersen et al. 2000).Many molecular studies have been performed to better understand the formation of such severe chromosomal aberrations (Kanaar et al. 1998; Delneri et al. 2003; Aylon and Kupiec 2004; Egli et al. 2004; Motegi et al. 2006; Weinstock et al. 2006; Motegi and Myung 2007). Nevertheless, very little is known about the mechanisms by which chromosome translocations and secondary rearrangements arising from these events occur. The occurrence of two or more double-strand breaks (DSB) and an inappropriate use of the recombination machinery of the cells are supposed to be some of the main ways in which a DNA translocation is promoted (Rowley 2001, 2008; Agarwal et al. 2006). In humans, defects in many DSB repair mechanisms such as non-allelic homologous recombination, non-homologous end-joining, and fork stalling and template switching (FoSTeS) have been reported to be involved in translocation formation (Gu et al. 2008). In yeast cells, single-strand annealing and break-induced replication (BIR) pathways have been shown to be involved in the formation of chromosome rearrangements and translocations (Bosco and Haber 1998; Haber 2006).Recently, our group described a method that allows the generation and subsequent selection of chromosomal translocations between any two chromosomal loci in diploid yeast strains. This was done by transformation of whole yeast cells with a linear DNA construct, carrying the KAN^R selectable marker flanked by DNA sequences homologous to two different chromosomal loci (Tosato et al. 2005, 2009; Nikitin et al. 2008).We used this method to investigate the multiple molecular mechanisms and pathways that might be involved during a translocation event in Saccharomyces cerevisiae. To this end, we generated a collection of 10 different mutant strains harboring a translocation between chromosomes XVI and IX (Figure 1), followed by their comparative molecular and physiological analyses. Results obtained from our experiments suggest that the induction of the same chromosomal translocation can lead to a wide variety of secondary chromosomal rearrangements, generating aneuploidy derived from partial or complete chromosome duplication or loss of genetic material.Open in a separate windowFigure 1.—Schematic of BIT between chromosomes XVI and IX. The 777,694-bp aberrant chromosome together with the two hypothetic fragments originating after a non-reciprocal BIT translocation are shown. Genes analyzed by Southern hybridization are reported along the chromosomes together with the primers used to test the integration of the BIT construct at the level of the two targeted loci. SSU1Fw and SUc2RevNEW primers were used to perform the bridge-PCR to validate the presence of the translocant chromosome (see results). TBP, translocation breakpoint.Physiological analyses also revealed that the 10 translocants obtained with the bridge-induced translocation (BIT) system in this work have different mutant phenotypes, which are analogous to the high variation of phenotypes characteristic of cancer cells. We demonstrated that these mutant strains exhibit altered behavior and fitness in different carbon sources for growth, different sporulation efficiencies, and ability to flocculate. Expression analyses also show that they exhibit different expression profiles of various genes located along the translocated chromosome or involved in cellular processes such as apoptosis, cell cycle regulation, and oxidative stress response. Overall, our work shows that the single integration of a linear DNA cassette with homology on two different chromosomes not only can generate a translocation, but also might be responsible for other complex chromosomal rearrangements. Such complex genomic rearrangements seen in yeast may play a role as key evolutionary forces, reshaping and remodeling genomes, followed by selection and adaptation into specialized cells or even neoplastic transformation, as observed in mammalian cancer cells.     

Alternative Splicing Modulates Ubx Protein Function in Drosophila melanogaster

Polyploidy is an important aspect of the evolution of flowering plants. The potential of gene copies to diverge and evolve new functions is influenced by meiotic behavior of chromosomes leading to segregation as a single locus or duplicated loci. Switchgrass (Panicum virgatum) linkage maps were constructed using a full-sib population of 238 plants and SSR and STS markers to access the degree of preferential pairing and the structure of the tetraploid genome and as a step toward identification of loci underlying biomass feedstock quality and yield. The male and female framework map lengths were 1645 and 1376 cM with 97% of the genome estimated to be within 10 cM of a mapped marker in both maps. Each map coalesced into 18 linkage groups arranged into nine homeologous pairs. Comparative analysis of each homology group to the diploid sorghum genome identified clear syntenic relationships and collinear tracts. The number of markers with PCR amplicons that mapped across subgenomes was significantly fewer than expected, suggesting substantial subgenome divergence, while both the ratio of coupling to repulsion phase linkages and pattern of marker segregation indicated complete or near complete disomic inheritance. The proportion of transmission ratio distorted markers was relatively low, but the male map was more extensively affected by distorted transmission ratios and multilocus interactions, associated with spurious linkages.POLYPLOIDY is common among plants (Masterson 1994; Levin 2002) and is an important aspect of plant evolution. Widespread paleopolyploidy in flowering plant lineages suggests that ancient polyploidization events have contributed to the radiation of angiosperms (Soltis et al. 2009; Van de Peer et al. 2009a). Whole genome duplications are thought to be the sources of evolutionary novelty (Osborn et al. 2003; Freeling and Thomas 2006; Chen 2007; Hegarty and Hiscock 2008; Flagel and Wendel 2009; Leitch and Leitch 2008). Other attributes of polyploids considered to promote evolutionary success include increased vigor, masking of recessive alleles, and reproductive barriers arising from loss of one of the duplicate genes (Soltis and Soltis 2000; Comai 2005; Otto 2007; Van de Peer et al. 2009b). Among crop species, polyploidy likely contributed to trait improvement under artificial selection (Paterson 2005; Udall and Wendell 2006; Dubcovsky and Dvorak 2007; Hovav et al. 2008).Disomic inheritance in polyploids, in contrast to polysomic inheritance, presents opportunities for duplicated genes to diverge and evolve new functions. The relative age of whole genome duplications and the extent of homology between subgenomes greatly influence chromosomal pairing at meiosis (Soltis and Soltis 1995; Wolfe 2001; Ramsey and Schemske 2002). Polysomic inheritance resulting from random chromosome pairing is associated with doubling of a single set of chromosomes. Disomic inheritance resulting from preferential pairing is often associated with polyploidy arising from combinations of divergent genomes. The evolutionary process of diploidization leads to a shift from random to preferential pairing that is not well understood but is genetically defined in systems such as Ph1 of wheat (Triticum aestivum) and PrBn of Brassica napus (Riley and Chapman 1958; Vega and Feldman 1998; Jenczewski et al. 2003). The degree of preferential pairing also affects allelic diversity and the ability to detect linkage. Accurate information about chromosome pairing and whole or partial genome duplications is thus important for both evolutionary studies and in linkage analysis.Such information is extremely limited in the C4 panicoid species Panicum virgatum (switchgrass), which is now viewed as a promising energy crop in the United States and Europe (Lewandowski et al. 2003; McLaughlin and Kszos 2005) and is planted extensively for forage and soil conservation (Vogel and Jung 2001). Little is known about either its genome structure or inheritance. Much current bioenergy feedstock development is focused on tetraploid cytotypes (2n = 4x = 36) due to their higher yield potentials, and an initial segregation study indicated a high degree of preferential pairing in a single F1 mapping population (Missaoui et al. 2005). A once-dominant component of the tallgrass prairie in North America, switchgrass is largely self-incompatible (Martinez-Reyna and Vogel 2002) with predominantly tetraploid or octoploid cytotypes (Hultquist et al. 1997; Lu et al. 1998). Limited gene flow appears possible between different cytotypes suggested by DNA content variation within collection sites and seed lots (Nielsen 1944; Hultquist et al. 1997; Narasimhamoorthy et al. 2008). True diploids appear to be rare (Nielsen 1944; Young et al. 2010). Multivalents in meiosis have not been observed in tetraploids or F1 hybrids between upland and lowland tetraploids, although rare univalents occurred (Barnett and Carver 1967; Martinez-Reyna et al. 2001). However, polysomic inheritance may occur with random bivalent pairing (Howard and Swaminathan 1953).Sustainable production of switchgrass for bioenergy to meet the goal of reducing greenhouse gas emissions will require advances in feedstock production that include improvements in yield (Carroll and Somerville 2009). Switchgrass has extensive genetic diversity and potential for genetic improvements, but each cycle of phenotypic selection can take several years (McLaughlin and Kszos 2005; Parrish and Fike 2005; Bouton 2007). Detailed understanding of genome structure to enable efficient marker-assisted selection (MAS) can speed this process considerably. Complete linkage maps are therefore required to both understand chromosome pairing and allow MAS.We report the construction of the first complete linkage maps of two switchgrass genotypes. The linkage maps provide genetic evidence for disomic inheritance in lowland, tetraploid switchgrass. Gene-derived markers enabled a comparative analysis to sorghum, revealing syntenic relationships between the diploid sorghum genome and the tetraploid switchgrass subgenomes. Transmission ratio distortion and multilocus interactions were analyzed in detail to document their potential influence on map accuracy and map-based studies in switchgrass.     

Retrogenes Reveal the Direction of Sex-Chromosome Evolution in Mosquitoes

The mosquito Anopheles gambiae has heteromorphic sex chromosomes, while the mosquito Aedes aegypti has homomorphic sex chromosomes. We use retrotransposed gene duplicates to show an excess of movement off the An. gambiae X chromosome only after the split with Ae. aegypti, suggesting that their ancestor had homomorphic sex chromosomes.HETEROMORPHIC sex chromosomes, both XX/XY and ZZ/ZW systems, have evolved independently multiple times in both animals and plants (Bull 1983; Charlesworth 1996; Rice 1996). Sex chromosomes are thought to evolve from a pair of autosomes that acquire a new sex-determining locus. Theory suggests that natural selection will favor tight linkage between the newly arisen sex-determining locus and sexually antagonistic alleles (i.e., genes that are beneficial in one sex, but detrimental in the other), which favors the suppression of recombination near the sex-determining locus (Charlesworth et al. 2005). In some species, this nonrecombining region includes only a small portion of the sex chromosome (hereafter referred to as homomorphic sex chromosomes), whereas in other species, this region encompasses most of the sex chromosomes (heteromorphic sex chromosomes). In many species the nonrecombining region progressively expands from only the portion near the sex-determining locus to nearly the full extent of the sex chromosomes (Lahn and Page 1999; Lawson Handley et al. 2004; Nicolas et al. 2005). However, the broad phylogenetic distribution of homomorphic sex chromosomes suggests that this progression does not happen in every species (e.g., Matsubara et al. 2006; Tsuda et al. 2007), although why it should occur in some lineages and not in others is unknown. As noted by Gilchrist and Haldane (1947, p. 187): “It is a striking fact that this [the suppression of recombination across the sex chromosome] has not happened in many large and successful groups.”Within the order Diptera, there are a wide variety of sex chromosomes and sex-determination mechanisms, including XY, ZW, multiple-X, and homomorphic systems, often varying within the same family (Marin and Baker 1998; Schutt and Nothiger 2000; Sanchez 2008). The mosquito Anopheles gambiae (a species in the subfamily Anophelinae) has fully differentiated heteromorphic X and Y chromosomes that show no evidence of recombination (Krzywinski et al. 2004). The mosquito Aedes aegypti (subfamily Culicinae) has a nonrecombining sex-determining region that spans only a few megabases on chromosome 1; this chromosome is homologous to chromosomes X and 2R of An. gambiae (Nene et al. 2007). An. gambiae and Ae. aegypti diverged ∼150 million years ago (Krzywinski et al. 2006).Because of the rapid turnover of sex-chromosome systems among the Diptera, it is not clear if the common ancestor of Ae. aegypti and An. gambiae had only a sex-determining region (i.e., homomorphic sex chromosomes) or fully differentiated heteromorphic sex chromosomes (Rai and Black 1999). The generally accepted model of sex-chromosome evolution, in which homomorphic sex chromosomes progressively suppress recombination and become heteromorphic, predicts that the common ancestor of Ae. aegypti and An. gambiae had homomorphic sex chromosomes (Figure 1A). This implies that evolution of heteromorphic sex chromosomes in An. gambiae occurred in a short period of time after the split between these two lineages and before the radiation of the Anophelines and that the homomorphic sex chromosomes of Ae. aegypti have been nearly static over evolutionary time. Alternatively, the common ancestor may have had nearly or fully differentiated sex chromosomes, and Ae. aegypti evolved from heteromorphic sex chromosomes to having only a small sex-determining region (Figure 1B; Rao and Rai 1987). We imagine this transition may have occurred by one of two mechanisms: either the sex-determining locus was transposed from the ancestral sex chromosome to an autosome or, in an XO sex-determination system, one of the “numerator” genes located on the X chromosome sustained an inactivating mutation, effectively making a karyotypic XX individual into a genetically male XO individual. (The precise mechanism of sex determination in Ae. aegypti is not known.)Open in a separate windowFigure 1.—Hypotheses for sex-chromosome evolution in Anopheles gambiae and Aedes aegypti. (A) The ancestor of An. gambiae and Ae. aegypti had homomorphic sex chromosomes and heteromorphism evolved along the Anopheline lineage. (B) The ancestor of An. gambiae and Ae. aegypti had heteromorphic chromosomes and homomorphism evolved along the Culicine lineage.To determine the state of the mosquito common ancestor, we examined genes duplicated by retrotransposition in the An. gambiae genome. Several organisms with heteromorphic sex chromosomes, including mammals and Drosophila, have an excess of retrotransposed genes moving from the X chromosome to autosomes compared to genes moving between autosomes or from the autosomes to the X (Betran et al. 2002; Emerson et al. 2004; Vinckenbosch et al. 2006; Meisel et al. 2009). This pattern is further found to be strongly associated with the origin of new X chromosomes in both mammals and Drosophila (Potrzebowski et al. 2008; Meisel et al. 2009), although it continues long after X chromosomes arise. While there are many hypotheses for the evolutionary forces that drive gene movement off X chromosomes—including sexual antagonism and meiotic sex-chromosome inactivation (e.g., Hense et al. 2007)—it is likely that all of these forces also act in mosquitoes, implying excess movement off the heteromorphic X in this clade as well. We reasoned that if the common ancestor of Ae. aegypti and An. gambiae had homomorphic sex chromosomes (Figure 1A), there should be an excess of retrogene movement off the X chromosome in An. gambiae only after the divergence of the two lineages (i.e., since An. gambiae evolved a differentiated X chromosome). In contrast, if the common ancestor had fully heteromorphic chromosomes (Figure 1B), then our prediction is that there will be an excess of gene movement off the An. gambiae X on both the shared ancestral branch and the Anopheles-specific branch after the split with Aedes. (Note that the Ae. aegypti genome is largely not assembled onto chromosomes, precluding a similar analysis in this species.)We collected data on all functional, intact duplicates in the An. gambiae genome and all orthologs between An. gambiae and Ae. aegypti from Ensembl version 54. When genes are retrotransposed there will be introns in the parental copy, but no introns in the daughter copy, allowing us to polarize gene movement. Although introns may be lost—and more rarely gained—over time, the rate of such changes is quite low (Coulombe-Huntington and Majewski 2007). Nevertheless, unless a parental gene loses all of its introns and the daughter gene gains introns, such changes will merely cause us to miss events rather than to assign them to an incorrect chromosome. Using gene-tree/species-tree reconciliation (Goodman et al. 1979), we identified retrotransposition events in the An. gambiae genome that have occurred since the split with Drosophila melanogaster and assigned them to a branch on the basis of the timing of the inferred duplication event in the gene tree. Calculating the expected number of movements on the basis of the equations presented in Betran et al. (2002), we find that an excess of movement off the X chromosome has in fact occurred since the split with D. melanogaster (χ² = 23.83, d.f. = 2, P = 6.7 × 10⁻⁶). We then divided the retrotransposition events into those that occurred before the divergence of An. gambiae and Ae. aegypti and those that occurred only in An. gambiae since the split. We determined that there is a 400% excess of retrotransposition events off the X chromosome since the An. gambiae and Ae. aegypti split (Figure 2: χ² = 51.97, d.f. = 2, P = 5.2 × 10⁻¹²). However, there is no excess of retrotransposition off the X chromosome prior to the split between An. gambiae and Ae. aegypti (Figure 2: χ² = 1.51, d.f. = 2, P = 0.47). This strongly suggests a recent origin of fully differentiated heteromorphic sex chromosomes in An. gambiae.Open in a separate windowFigure 2.—Retroposition events off the X chromosome. There is an excess of genes moving off the X chromosome on the An. gambiae-specific lineage, but not on the branch leading to the common ancestor of An. gambiae and Ae. aegypti.The deepest split between species within the subfamily Anophelinae—all of which have fully differentiated sex chromosomes—occurs soon after the split with the Culicinae (Krzywinski et al. 2006). This implies that the evolution of heteromorphic sex chromosomes must have occurred very soon after the split with Ae. aegypti. To determine whether there was a burst of retrotransposition off the X following this split, we examined the amino acid sequence identity between X-to-autosome retrotransposed proteins and their parental paralogs. A comparison of these distributions indicates that there is no difference in the percentage of identity of genes retrotransposed off the An. gambiae X chromosome and one-to-one orthologs between An. gambiae and Ae. aegypti (71.1% vs. 70.7%, t-test, P = 0.92; JTT amino acid distances, 0.508 vs. 0.436, t-test, P = 0.57). Given the fact that functional retrotransposed genes have been found to evolve more rapidly than single-copy genes (Betran et al. 2002), these results support the idea that these duplication events occurred soon after the split between An. gambiae and Ae. aegypti.Our results have important implications for two further areas of research. First, a recent article (Moyle et al. 2010) proposed that X-to-autosome duplication events could be partly responsible for the large X-effect—the disproportionate effect of the X chromosome on reproductive isolation (Coyne and Orr 2004). This is because gene movement between chromosomes can itself cause reproductive isolation (e.g., Masly et al. 2006), and any excess movement involving the X will lead to an excess of reproductive isolation loci mapping to this chromosome. One prediction of this model is that species showing the large X-effect should also show an excess of X-to-autosome gene movement. As An. gambiae does in fact exhibit patterns consistent with the large X-effect (Slotman et al. 2005), our demonstration of an excess of movement off the X supports this model.Second, it has been proposed that the excess movement off the X in Drosophila is the cause of the deficit of male-biased genes on the X in the same species (e.g., Vibranovski et al. 2009), although the number of retrotransposed genes is much smaller than the number of missing male-biased genes (Betran et al. 2002; Parisi et al. 2003). We have previously shown that there is no deficit of male-biased genes on the An. gambiae X chromosome, at any significance level (Hahn and Lanzaro 2005). Given the observed excess of gene movement off the X presented here, we therefore find little support for a causal link between movement and genome-wide patterns of male-biased gene expression.Our results suggest that retrogene movement is a general feature of sex-chromosome evolution and support the hypothesis that the common ancestor of An. gambiae and Ae. aegypti had homomorphic sex chromosomes. It appears that the nonrecombining region around the sex-determining locus in An. gambiae expanded rapidly after the divergence with Ae. aegypti. Further investigation into the causes of the rapid expansion in the An. gambiae lineage and the long-term stasis in the Ae. aegypti lineage is clearly warranted.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

Evolution of a Distinct Genomic Domain in Drosophila: Comparative Analysis of the Dot Chromosome in Drosophila melanogaster and Drosophila virilis

The distal arm of the fourth (“dot”) chromosome of Drosophila melanogaster is unusual in that it exhibits an amalgamation of heterochromatic properties (e.g., dense packaging, late replication) and euchromatic properties (e.g., gene density similar to euchromatic domains, replication during polytenization). To examine the evolution of this unusual domain, we undertook a comparative study by generating high-quality sequence data and manually curating gene models for the dot chromosome of D. virilis (Tucson strain 15010–1051.88). Our analysis shows that the dot chromosomes of D. melanogaster and D. virilis have higher repeat density, larger gene size, lower codon bias, and a higher rate of gene rearrangement compared to a reference euchromatic domain. Analysis of eight “wanderer” genes (present in a euchromatic chromosome arm in one species and on the dot chromosome in the other) shows that their characteristics are similar to other genes in the same domain, which suggests that these characteristics are features of the domain and are not required for these genes to function. Comparison of this strain of D. virilis with the strain sequenced by the Drosophila 12 Genomes Consortium (Tucson strain 15010–1051.87) indicates that most genes on the dot are under weak purifying selection. Collectively, despite the heterochromatin-like properties of this domain, genes on the dot evolve to maintain function while being responsive to changes in their local environment.EUKARYOTIC genomes are packaged into two major types of chromatin: euchromatin is gene rich and has a diffuse appearance during interphase, while heterochromatin is gene poor and remains densely packaged throughout the cell cycle (Grewal and Elgin 2002). The distal 1.2 Mb of the fourth chromosome of Drosophila melanogaster, known as the dot chromosome or Muller F element, is unusual in exhibiting an amalgamation of heterochromatic and euchromatic properties. This domain has a gene density that is similar to the other autosomes (Bartolomé et al. 2002; Slawson et al. 2006). However, it appears heterochromatic by many criteria, including late replication and very low levels of meiotic recombination (Wang et al. 2002; Arguello et al. 2010). It exhibits high levels of association with heterochromatin protein 1 (HP1) and histone H3 di- and trimethylated at lysine 9 (H3K9me2/3), as shown by immunofluorescent staining of the polytene chromosomes (Riddle and Elgin 2006; Slawson et al. 2006). This association with heterochromatin marks has recently been confirmed by the modENCODE Project [N. C. Riddle, A. Minoda, P. V. Kharchenko, A. A. Alekseyenko, Y. B. Schwartz, M. Y. Tolstorukov, A. A. Gorchakov, C. Kennedy, D. Linder-Basso, J. D. Jaffe, G. Shanower, M. I. Kuroda, V. Pirrotta, P. J. Park, S. C. R. Elgin, G. H. Karpen, and the modENCODE Consortium (http://www.modencode.org), unpublished results]. To understand this unique domain and to examine the evolution of a region with very low levels of recombination, we have undertaken a comparative study using the dot chromosome of D. virilis, a species that diverged from D. melanogaster 40–60 million years ago (Powell and Desalle 1995). We sequenced and improved the assembly of the D. virilis dot chromosome and created a manually curated set of gene models to ensure that both the assembly and the gene annotations are at a quality comparable to those in D. melanogaster. We then compared the sequence organization and gene characteristics of the distal portion of the D. virilis dot chromosome with the corresponding region from the D. melanogaster dot chromosome.In addition to examining the long-term dot chromosome evolution, we also investigated the short-term dot chromosome evolution by comparing the genomic sequences from two different strains of D. virilis. Agencourt Biosciences (AB) has previously produced a whole genome shotgun assembly of Tucson strain 15010–1051.87, while we have sequenced Tucson strain 15010–1051.88 of D. virilis [the Genomics Education Partnership (GEP) assembly]. The AB assembly has been improved by the Drosophila 12 Genomes Consortium and released as part of the comparative analysis freeze 1 (CAF1) assembly (Drosophila 12 Genomes Consortium et al. 2007).Using the GEP and CAF1 assemblies from D. virilis, and the high-quality D. melanogaster assembly and its gene annotations from FlyBase (Crosby et al. 2007), we compared the gene properties and sequence organization of the dot chromosomes and reference euchromatic and heterochromatic domains. The dot chromosomes from D. melanogaster and D. virilis are distinct from the heterochromatic and euchromatic regions of the two genomes, both in organization (e.g., repeat density) and in characteristics of the genes (e.g., size, codon bias). The two dot chromosomes resemble each other by most criteria and differ only in the types of repetitive sequences present and in relative gene order and orientation. Despite the very low rate of meiotic recombination, comparison of the two D. virilis strains shows that dot chromosome genes are under weak purifying selection. Our analysis of genes that are present in a euchromatic chromosome arm in one species and on the dot chromosome in the other (the “wanderer” genes) shows that this set of genes evolves to maintain function while responding to the changes in the local chromosomal environment.     

Structural and Functional Characterization of VanG d-Ala:d-Ser Ligase Associated with Vancomycin Resistance in Enterococcus faecalis

d-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes.     

Deficiency in l-Serine Deaminase Interferes with One-Carbon Metabolism and Cell Wall Synthesis in Escherichia coli K-12

Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes l-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S l-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of l-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C₁ units and interferes with cell wall synthesis. We suggest that at high concentrations, l-serine decreases synthesis of UDP-N-acetylmuramate-l-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high l-serine is overcome in several ways: by a large concentration of l-alanine, by overproducing MurC together with a low concentration of l-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.The Escherichia coli genome contains three genes, sdaA, sdaB, and tdcG, specifying three very similar 4Fe4S l-serine deaminases. These enzymes are very specific for l-serine for which they have unusually high K_m values (3, 32). Expression of the three genes is regulated so that at least one of the gene products is synthesized under all common growth conditions (25). This suggests an important physiological role for the enzymes. However, why E. coli needs to deaminate l-serine has been a long-standing problem of E. coli physiology, the more so since it cannot use l-serine as the sole carbon source.We showed recently that an E. coli strain devoid of all three l-serine deaminases (l-SDs) loses control over its size, shape, and cell division when faced with complex amino acid mixtures containing l-serine (32). We attributed this to starvation for single-carbon (C₁) units and/or S-adenosylmethionine (SAM). C₁ units are usually made from serine via serine hydroxymethyl transferase (GlyA) or via glycine cleavage (GCV). The l-SD-deficient triple mutant strain is starved for C₁ in the presence of amino acids, because externally provided glycine inhibits GlyA and a very high internal l-serine concentration along with several other amino acids inhibits glycine cleavage. While the parent cell can defend itself by reducing the l-serine level by deamination, this crucial reaction is missing in the ΔsdaA ΔsdaB ΔtdcG triple mutant. We therefore consider these to be “defensive” serine deaminases.The fact that an inability to deaminate l-serine leads to a high concentration of l-serine and inhibition of GlyA is not surprising. However, it is not obvious why a high level of l-serine inhibits cell division and causes swelling, lysis, and filamentation. Serine toxicity due to inhibition of biosynthesis of isoleucine (11) and aromatic amino acids (21) has been reported but is not relevant here, since these amino acids are provided in Casamino Acids.We show here that at high internal concentrations, l-serine also causes problems with peptidoglycan synthesis, thus weakening the cell wall. Peptidoglycan is a polymer of long glycan chains made up of alternating N-acetylglucosamine and N-acetylmuramic acid residues, cross-linked by l-alanyl-γ-d-glutamyl-meso-diaminopimelyl-d-alanine tetrapeptides (1, 28). The glucosamine and muramate residues and the pentapeptide (from which the tetrapeptide is derived) are all synthesized in the cytoplasm and then are exported to be polymerized into extracellular peptidoglycan (2).In this paper, we show that lysis is caused by l-serine interfering with the first step of synthesis of the cross-linking peptide, the addition of l-alanine to uridine diphosphate-N-acetylmuramate. This interference is probably due to a competition between serine and l-alanine for the ligase, MurC, which adds the first l-alanine to UDP-N-acetylmuramate (7, 10, 15). As described here, the weakening of the cell wall by l-serine can be overcome by a variety of methods that reduce the endogenous l-serine pool or counteract the effects of high levels of l-serine.