Directionality of yeast mating-type interconversion

The mating-type a and α alleles of the yeast Saccharomyces cerevisiae interconvert by a transposition-substitution reaction where replicas of the silent mating loci, at HML and HMR, are transmitted to the expressed mating-type locus (MAT). HML is on the left arm and HMR on the right arm, while MAT is in the middle of chromosome III. Cells with the genotype HMLα HMRa switch mating type efficiently at a frequency of about 86%. Since well over 50% of the cells switch, it is thought that switches do not occur randomly, but are directed to occur to the opposite mating-type allele. In contrast, we report that strains possessing the reverse HMLa HMRα arrangement switch (phenotype) inefficiently at a maximum of about 6%. The basis for this apparent reduced frequency of switching is that these strains preferentially yield futile homologous MAT locus switches—that is, MATa to MATa and MATα to MATα—and consequently, most of these events are undetected. We used genetically marked HM loci to demonstrate that a cells preferentially choose HMR as donor and a cells preferentially choose HML as donor, irrespective of the genetic content of the silent loci. Because of this feature, HMLα HMRa strains generate predominantly heterologous while HMLa HMRα strains produce predominantly homologous MAT switches. The control for directionality of switching therefore is not at the level of transposing heterologous mating-type information, but only at the level of choosing HML versus HMR as the donor. In strains where the preferred donor locus is deleted, the Inefficient donor becomes capable of donating efficiently. Thus the preference seems to be mediated by competition between the HM loci for donating information to MAT.     

Illegal transposition of mating-type genes in yeast

Mating-type switching in the yeast Saccharomyces cerevisiae involves the transposition of a copy of a or α information from unexpressed “library” genes, HML or HMR, to replace the sequence at the mating type locus, MAT. In normal homothallic strains, where conversions of MAT may occur as often as every cell division, the switching of MAT alleles does not alter the alleles at HML or HMR. We have discovered that several mutations within or adjacent to MAT that impair the excision of the MAT allele permit conversions of the alleles at HML or HMR in more than 1% of the cells analyzed. The two mutations within the MAT locus (MATa-inc and MATα-inc) can transpose to HML or HMR without being lost at MAT. Thus a MATα-inc HMLα HMRa HO strain can switch to MATα-inc HMLα HMRα-inc HO. Even though the α-inc and a-inc alleles prevent their own replacement at MAT, these sequences are efficiently transposed back from HMLα-inc or HMLa-inc to replace normal MAT alleles. When these alleles reappear at MAT, they are again blocked in excision. Thus the sequences used to remove an allele from MAT must differ from those used to replicate and transpose it. Two cis-acting stk mutations adjacent to MAT that block switching of MATa to MATα also induce the conversion of HMLα to HMLa. However, we have previously shown that these events do not occur in strains carrying a recessive “switch” mutant (swi1) or in strains carrying a defective allele of the HO gene. In stk1 MATa HO strains, HMLα was converted to HMLa in approximately 4% of the subclones examined. In contrast, the HMLα-inc sequence was not converted in similar stk1 MATa HO strains. Thus the excision of the α-inc sequence seems to be prevented at both MAT and HML. These results suggest that the illegal conversions of HML and HMR occur by a mechanism similar to that used for normal conversions of MAT.     

Regulation of Nuclear Positioning and Dynamics of the Silent Mating Type Loci by the Yeast Ku70/Ku80 Complex

We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLα or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and α cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLα, unlike HMRa and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLα donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLα creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus.     

Diversity of Mating-Type Chromosome Structures in the Yeast Zygosaccharomyces rouxii Caused by Ectopic Exchanges between MAT-Like Loci

We investigated sex chromosome diversity in Zygosaccharomyces rouxii (Z. rouxii). In the current study, we show that the organization of the mating-type (MAT) locus is highly variable in the Z. rouxii population, indicating the MAT, HML, and HMR loci are translocation hotspots. Although NBRC1130 and CBS732 were originally two stocks of the type strain of the species, only NBRC1130 retains the original karyotype. A reciprocal translocation between the MAT and HMR loci appears to have occurred during the early passage culture of CBS732, which was used for genome sequencing. In NBRC1733, NBRC0686, NBRC0740 and NBRC1053, the terminal region of the chromosome containing the HMR locus was replaced with the chromosomal region to the left of the MAT or HML loci. The translocation events found in NBRC1733, NBRC0686, NBRC0740, and NBRC1053 were reconstructed under our experimental conditions using the DA2 background, and the reconstruction suggests that the frequency of this type of translocation is approximately 10⁻⁷. These results suggest that the MAT and MAT-like loci were the susceptible regions in the genome, and the diversity of mating-type chromosome structures in Z. rouxii was caused by ectopic exchanges between MAT-like loci.     

The trans action of HMRa in mating type interconversion

Summary HML and HMR are the sites of cryptic mating type genes in the yeast Saccharomyces cerevisiae. In the presence of the HO gene, the information from HML or HMR (an a or cassette) is transferred to the mating type locus (MAT). HML, HMR, and MAT are located on chromosome III, yet are widely separeted. Similarly, in other yeasts, at least some of the genes involved in mating type interconversion are linked to the mating type locus. We demonstrate here that a cassette donor (HMR) and the cassette target (MAT) need not be physically linked for successful mating type interconversion. In particular, we show that HMR a on one chromosome can donate an a cassette to the mating type locus on a homologous chromosome III.     

Efficient Mating-Type Switching in Candida glabrata Induces Cell Death

Candida glabrata is an apparently asexual haploid yeast that is phylogenetically closer to Saccharomyces cerevisiae than to Candida albicans. Its genome contains three MAT-like cassettes, MAT, which encodes either MATa or MATalpha information in different strains, and the additional loci, HML and HMR. The genome also contains an HO gene homolog, but this yeast has never been shown to switch mating-types spontaneously, as S. cerevisiae does. We have recently sequenced the genomes of the five species that, together with C. glabrata, make up the Nakaseomyces clade. All contain MAT-like cassettes and an HO gene homolog. In this work, we express the HO gene of all Nakaseomyces and of S. cerevisiae in C. glabrata. All can induce mating-type switching, but, despite the larger phylogenetic distance, the most efficient endonuclease is the one from S. cerevisiae. Efficient mating-type switching in C. glabrata is accompanied by a high cell mortality, and sometimes results in conversion of the additional cassette HML. Mortality probably results from the cutting of the HO recognition sites that are present, in HML and possibly HMR, contrary to what happens naturally in S. cerevisiae. This has implications in the life-cycle of C. glabrata, as we show that efficient MAT switching is lethal for most cells, induces chromosomal rearrangements in survivors, and that the endogenous HO is probably rarely active indeed.     

Interconversion of Yeast Cell Types by Transposable Genes

The a and α cell types of budding yeast Saccharomyces cerevisiae are controlled by alternate alleles of the mating-type locus (MAT), MATa and MATα. The cell types can be interconverted by switching alleles of MAT. The loci HMRa and HMLα, which are loosely linked to MAT, are involved in mating-type switching. Experimental evidence for their role in MAT interconversion is presented. As a result of switching, the homothallic and heterothallic strains containing the amber and ochre mutations within the HMRa locus yield corresponding amber and ochre mutant mata loci. Similarly, the hmlα mutant strain generates matα mutant alleles. That is, specific mutations from HMRa and HMLα are transmitted to MAT. A replica of the mating-type coding information originating from these loci is transposed to MAT, where it replaces the existing information. Furthermore, "Hawthorne deletions" in strains containing hmra-amber/ochre result in production of mata-amber/ochre alleles. Therefore, genetic information for MATa resides at HMRa. The switches occur in a defined set of clonally related cells. Thus, the efficient interconversion of yeast cell types is mediated by an unidirectional transfer of genetic information between nonallelic sites in a nonrandom and programmed fashion. The results are inconsistent with the "flip-flop" models, but satisfy a key prediction of the general controlling element and the specific cassette models proposed for mating-type interchange.     

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.     

Congenic Mapping and Allele-Specific Alteration Analysis of Stmm1 Locus Conferring Resistance to Early-Stage Chemically Induced Skin Papillomas

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to early-stage chemically induced skin papillomas on chromosome 7 with a large number of [(FVB/N×MSM/Ms)×FVB/N] F1 backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 7. We used linkage analysis and congenic mouse strains to refine the location of Stmm1 (Skin tumor modifier of MSM 1) locus within a genetic interval of about 3 cM on proximal chromosome 7. In addition, we used patterns of allele-specific imbalances in tumors from F1 backcross and N10 congenic mice to narrow down further the region of Stmm1 locus to a physical distance of about 5.4 Mb. To gain the insight into the function of Stmm1 locus, we carried out a long term BrdU labelling experiments with congenic mice containing Stmm1 locus. Interestingly, we observed a decrease of BrdU-LRCs (Label Retaining Cells) in a congenic strain heterozygous or homozygous for MSM allele of Stmm1. These results suggest that Stmm1 responsible genes may have an influence on papillomagenesis in the two-stage skin carcinogenesis by regulating epidermal quiescent stem cells.     

Identification of Receptor-Tyrosine-Kinase-Signaling Target Genes Reveals Receptor-Specific Activities and Pathway Branchpoints During Drosophila Development

Genetic maps provide a means to estimate the probability of the co-inheritance of linked loci as they are transmitted across generations in both experimental and natural populations. However, in the age of whole-genome sequences, physical distances measured in base pairs of DNA provide the standard coordinates for navigating the myriad features of genomes. Although genetic and physical maps are colinear, there are well-characterized and sometimes dramatic heterogeneities in the average frequency of meiotic recombination events that occur along the physical extent of chromosomes. There also are documented differences in the recombination landscape between the two sexes. We have revisited high-resolution genetic map data from a large heterogeneous mouse population and have constructed a revised genetic map of the mouse genome, incorporating 10,195 single nucleotide polymorphisms using a set of 47 families comprising 3546 meioses. The revised map provides a different picture of recombination in the mouse from that reported previously. We have further integrated the genetic and physical maps of the genome and incorporated SSLP markers from other genetic maps into this new framework. We demonstrate that utilization of the revised genetic map improves QTL mapping, partially due to the resolution of previously undetected errors in marker ordering along the chromosome.GENETIC maps exist for hundreds of different species, and genetic map construction continues to play an important role in the characterization of genomes (Tanksley et al. 1992; Kong et al. 2002; Chowdhary and Raudsepp 2006; Stapley et al. 2008). A genetic map defines the linear order and relative distances among a set of marker loci in units that correspond to the frequency of meiotic recombination between the loci. Until recently mouse genetic maps based on simple sequence length polymorphism (SSLP) markers (Lyon 1976) have been sufficient for most experimental purposes since, unlike the hundreds of thousands of markers required in human genetic association studies, a relatively small number of markers is needed to map crosses between inbred mouse strains. However, recent developments in whole-genome high-resolution mapping in the mouse (Churchill et al. 2004; Valdar et al. 2006) and interest in examining recombination rates at an ultra-fine scale (Myers et al. 2005) have reawakened the need to develop a high-resolution genetic map in the mouse.The current standard genetic map of the mouse has been compiled from a substantial body of historical data and maintained by the Mouse Genome Informatics (MGI) project at The Jackson Laboratory (Bult et al. 2008). We will refer to it as the MGI map. The primary sources of data used to construct the MGI map were two mapping panels, described here. However, the current map is based on a consensus developed by the 2000 Chromosome Committee using all available published data. The map has continued to be maintained by MGI with the addition of new genetic markers and data but, because the map is based on consensus, published errors may have been perpetuated.The Jackson Laboratory developed a genetic map based on two sets of 94 progeny obtained from reciprocal backcrosses (BSB and BSS) between the inbred strains C57BL/6J and SPRET/EiJ (Rowe et al. 1994). These strains represent two different species of mouse (Mus musculus and Mus spretus). The map provides a wealth of genetic information, but problems with male fertility restrict breeding options and thus the map is female specific. The problems with male fertility may have resulted in some multi-locus distortion in the mapping panel (Montagutelli et al. 1996). Currently, 1372 and 4913 markers have been typed on the BSB and BSS backcross panels, respectively (Broman et al. 2002). Researchers at The Whitehead Institute and the Massachusetts Institute of Technology (MIT) developed a map of 4006 SSLP markers using an intercross population of 46 mice derived from strains OB (C57BL/6J-Lep^ob/ob) and CAST (CAST/EiJ) (Dietrich et al. 1994). Both parental strains OB and CAST are derived from M. musculus, but CAST is from a distinct subspecies, M. m. castaneus. The intercross mating strategy produces observable recombination from both male and female parents, but the two cannot be distinguished. Thus the map is sex averaged and based on 92 meioses. The Whitehead/MIT map was expanded to include 7377 SSLP markers (Dietrich et al. 1996). These are denoted as, e.g., D7Mit54, where “7” indicates the chromosome to which the marker is mapped and “54” is an arbitrary index. They are commonly referred to as “Mit” markers.Map resolution is limited by the number of observable recombination events in each of these panels. With 94 meioses (in each backcross) and an average of 14 recombination events/haploid genome transmitted, limiting resolution is on the order of 1 cM. A much larger panel would be needed to achieve subcentimorgan resolution and to accurately position high-density sets of SNP markers.Here we propose a new standard genetic map of the laboratory mouse based on data from a large heterogeneous stock (HS) mouse population descended from eight inbred strains (DBA/2J, C3H/HeJ, AKR/J, A/J, BALB/cJ, CBA/J, C57BL/6J, and LP/J) representing a diverse sample of the classical inbred strains (Petkov et al. 2004). Shifman et al. (2006) calculated genetic maps based on 11,247 informative SNP markers in 2293 HS individuals. The marker set is dense with 99% of the SNP intervals <500 kb, 81.2% <250 kb, and an estimated allele inheritance-based accuracy of 99.98%. Map positions were calculated separately for male and female meioses using CRIMAP software (Green et al. 1990), and the total length of the sex-averaged map is 1630 cM, as defined by the most distal SNP markers in their panel. The MGI map is 1783 cM on the basis of the most distal available marker position for each chromosome. However, on the basis of the most distal shared markers, the original Shifman map at 1612 cM is substantially longer than the MGI map at 1445 cM. It was not immediately clear if this discrepancy was due to the nature of recombination in the HS population or to their method of map estimation.There are at least two methodological problems with the HS map reported in Shifman et al. (2006). First, the map was constructed using a sliding window of 5–15 SNPs to handle eight multi-generation families within the CRIMAP software. Ideally, one considers all markers on a chromosome simultaneously in constructing a genetic map, and we found that this could be accomplished by splitting the complex pedigrees into sibships. Although splitting the pedigree results in slightly less efficient estimates of intermarker distances, this approach should incur no bias. Maps based on the full set of markers but with the complex pedigrees split into sibships are thus arguably better than maps based on the full pedigrees but with a sliding window of 5–15 markers. Second, analysis of families with incomplete parental genotypes may have contributed to an inflated map size. Sixteen of the 72 families lack parental genotypes or have genotypes for just one parent (15 of the 72), and many of them are small (26 have six or fewer siblings). Sibships with no parental genotype data of their own can give no information about sex-specific recombination rates. In conjunction with other sibships for which parental genotypes are available, they can provide some information, but the CRIMAP software (last modified in 1990) makes some approximations that result in a large bias even in the sex-averaged genetic maps for small sibships lacking parental genotype data.For these reasons, we recomputed the mouse genetic map on the basis of the original data reported and discuss the differences between the original Shifman map and the revised Shifman map below. The revised Shifman map provides a markedly different picture of recombination in the mouse: the estimated sex-averaged chromosome lengths correspond more closely to those in the original MGI map; the sex difference in the overall recombination rate is greatly reduced; and numerous narrow regions of high recombination rate, apparent in the original Shifman map, have disappeared.We propose the revised Shifman map as a new standard genetic map for the mouse. The new genetic map represents a substantial improvement over the existing MGI map due to the large number of meioses and to the genetic diversity of strains in the HS population. We have generated male, female, and sex-averaged genetic maps with physical positions and updated locus identifiers. We have established the correspondence between physical and genetic positions of 7080 Mit markers and corrected inconsistencies in the MGI map. We provide a web-based tool for the interpolation of new marker loci into the genetic map and for converting genetic map positions to NCBI mouse build 37 coordinates. Finally, we examine the effect of changing to this revised genetic map on QTL mapping in five previously published data sets (Beamer et al. 1999, 2001; Ishimori et al. 2004, 2008; Wergedal et al. 2006).     

Essential Loci in Centromeric Heterochromatin of Drosophila melanogaster. I: The Right Arm of Chromosome 2

With the most recent releases of the Drosophila melanogaster genome sequences, much of the previously absent heterochromatic sequences have now been annotated. We undertook an extensive genetic analysis of existing lethal mutations, as well as molecular mapping and sequence analysis (using a candidate gene approach) to identify as many essential genes as possible in the centromeric heterochromatin on the right arm of the second chromosome (2Rh) of D. melanogaster. We also utilized available RNA interference lines to knock down the expression of genes in 2Rh as another approach to identifying essential genes. In total, we verified the existence of eight novel essential loci in 2Rh: CG17665, CG17683, CG17684, CG17883, CG40127, CG41265, CG42595, and Atf6. Two of these essential loci, CG41265 and CG42595, are synonymous with the previously characterized loci l(2)41Ab and unextended, respectively. The genetic and molecular analysis of the previously reported locus, l(2)41Ae, revealed that this is not a single locus, but rather it is a large region of 2Rh that extends from unextended (CG42595) to CG17665 and includes four of the novel loci uncovered here.THE term “heterochromatin” was introduced by Heitz (1928) to describe regions of mitotic chromosomes that remain condensed throughout the cell cycle, in contrast to regions of euchromatin, which condense only during cell division. Heterochromatin was later divided into two classes: constitutive and facultative heterochromatin (Brown 1966). Constitutive heterochromatin is found in large blocks near centromeres and telomeres, while facultative heterochromatin can be described as silenced euchromatin that undergoes heterochromatization at specific developmental stages. Other properties of constitutive heterochromatin include late replication in S phase, low gene density, strikingly reduced level of meiotic recombination, enrichment in transposable element sequences and highly repetitive satellite DNA sequences, and the ability to silence euchromatic gene expression in a phenomenon called position effect variegation.Approximately 30% of the Drosophila melanogaster genome consists of constitutive heterochromatin (Gatti and Pimpinelli 1992). Centromeric heterochromatin in D. melanogaster is composed of mainly middle-repeat satellite DNA sequences and clusters of transposable element sequences (Lohe et al. 1993; Pimpinelli et al. 1995). Genes that reside in the heterochromatin are scattered like islands between the satellites and clusters of transposable elements. On average, heterochromatic genes are larger than euchromatic genes, primarily due to the prevalent accumulation of transposable element sequences in their introns (Devlin et al. 1990; Biggs et al. 1994; Dimitri et al. 2003a,b; Hoskins et al. 2007). Heterochromatic genes also tend to be AT-rich compared to their euchromatic counterparts; there is some evidence suggesting that the coding sequences of heterochromatic genes evolve toward AT richness in response to being located in heterochromatin (Yasuhara et al. 2005; Díaz-Castillo and Golic 2007).Drosophila heterochromatin is vastly under-replicated in polytene chromosomes, so heterochromatic genes cannot easily be mapped through polytene analysis. However, by using Hoechst 33258 and N-chromosome banding techniques, Dimitri (1991) was successful in dividing heterochromatin in mitotic chromosomes into distinct cytological bands; this was an important step in mapping the precise location of heterochromatic genes because before this time heterochromatic genes could be mapped only relative to one another. Here we focus on further refining the previous mapping work on essential genes in the proximal heterochromatin of the right arm of the second chromosome (2Rh) in cytological region h41–h46 of D. melanogaster (Hilliker 1976; Hilliker et al. 1980; Coulthard et al. 2003; Myster et al. 2004).Early mapping studies in D. melanogaster putatively placed the light (lt) and rolled (rl) genes in, or near, chromosome 2 heterochromatin (Schultz 1936; Hannah 1951; Hessler 1958). The first large-scale mutagenesis specifically directed at finding vital loci in second chromosome heterochromatin was conducted by Hilliker (1976). Using heterochromatic deletions created by Hilliker and Holm (1975), Hilliker (1976) set out to map vital loci using the mutagen ethyl methanesulfonate (EMS). He identified seven individual lethal complementation groups in 2Rh that were interpreted as representing seven vital loci. One of these heterochromatic loci was identified as the previously described rl gene. Two of the remaining vital loci have since been identified: Nipped-A is synonymous with the l(2) 41Ah complementation group (Rollins et al. 1999) and RpL38 is synonymous with Minute(2)41A and Hilliker''s (1976) l(2)41Af complementation group (Marygold et al. 2005; also referred to as l(2)Ag in FlyBase). In addition, Rollins et al. (1999) found the Nipped-B gene to be located in 2Rh, but how this locus fit into the data from Hilliker (1976) was unclear.With the limited release of some of the more distal heterochromatic sequences (Hoskins et al. 2002), a more recent mutagenesis screen focusing on distal 2Rh was conducted by Myster et al. (2004). In the region defined by the overlap between Df(2R)41A8 and Df(2R)41A10 (the latter was previously shown to be deficient for most of 2Rh; Hilliker and Holm 1975), Myster et al. (2004) reported the existence of 15 vital loci, considerably more than the 4 essential loci predicted by Hilliker (1976). The discrepancy between these two studies was the catalyst for this current work. Each group used the same mutagen, EMS, yet each group came up with very different interpretations of the number of vital loci.Hilliker''s interpretation relied on earlier evidence that EMS preferentially produced point mutations and not large-scale aberrations (Lim and Snyder 1974). Assuming that the mutants isolated in his study were point mutations, or small aberrations limited to one locus, Hilliker found that some of the loci that he identified exhibited complex interallelic complementation; the most complex complementation pattern was observed with locus l(2)41Ae. On the other hand, the interpretation of Myster et al. (2004) was that heterochromatin was more sensitive to EMS and that EMS could produce large heterochromatic deletions; they proposed that the complex interallelic complementation in l(2)41Ae was due to the presence of deletions and that l(2)41Ae represented a region of 2Rh containing many genes, rather than being a single locus.To resolve these different interpretations of the genomic segment containing l(2)41Ae (i.e., is it a single locus or a region of 2Rh), we set out to map l(2)41Ae and the region surrounding the presumed location of l(2)41Ae (as in Myster et al. 2004) by performing a large-scale inter se complementation analysis between all available mutant lines that were previously mapped to l(2)41Ae (including Nipped-B). In addition, we undertook a molecular mapping and sequence analysis, using a candidate gene approach with the most recent annotation of 2Rh (Hoskins et al. 2007), to characterize the region and identify as many essential genes as possible. We also used these approaches to map l(2)41Ab and unextended [two of the more proximal complementation groups identified by Hilliker (1976)]. Finally, we also utilized available RNA interference (RNAi) lines to knock down the expression of 12 genes in 2Rh in an attempt to identify essential genes.     

Amino Acid and vitamin requirements of several bacteroides strains

Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required l-arginine hydrochloride, l-tryptophan, l-leucine, l-histidine hydrochloride, l-cysteine hydrochloride, dl-valine, dl-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, dl-lysine hydrochloride, dl-isoleucine, l-proline, l-glutamic acid, dl-alanine, dl-phenylalanine, dl-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium d-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% l-tryptophan, 0.09% l-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in l-proline, glycine, l-glutamic acid, dl-serine, l-histidine hydrochloride, dl-alanine, or l-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% dl-serine but without vitamins.     

Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long-Range Interactions

The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic sub-nuclear compartments. In Saccharomyces cerevisiae heterochromatic loci, including telomeres and silent mating type loci, form clusters at the nuclear periphery. We have employed live cell 3-D imaging and chromosome conformation capture (3C) to determine the contribution of nuclear positioning and heterochromatic factors in mediating associations of the silent mating type loci. We identify specific long-range interactions between HML and HMR that are dependent upon silencing proteins Sir2p, Sir3p, and Sir4p as well as Sir1p and Esc2p, two proteins involved in establishment of silencing. Although clustering of these loci frequently occurs near the nuclear periphery, colocalization can occur equally at more internal positions and is not affected in strains deleted for membrane anchoring proteins yKu70p and Esc1p. In addition, appropriate nucleosome assembly plays a role, as deletion of ASF1 or combined disruption of the CAF-1 and HIR complexes abolishes the HML-HMR interaction. Further, silencer proteins are required for clustering, but complete loss of clustering in asf1 and esc2 mutants had only minor effects on silencing. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin at the silent loci and, in addition, identify an Asf1p-, Esc2p-, and Sir1p-dependent step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions.     

A Hidden Markov Model Combining Linkage and Linkage Disequilibrium Information for Haplotype Reconstruction and Quantitative Trait Locus Fine Mapping

Faithful reconstruction of haplotypes from diploid marker data (phasing) is important for many kinds of genetic analyses, including mapping of trait loci, prediction of genomic breeding values, and identification of signatures of selection. In human genetics, phasing most often exploits population information (linkage disequilibrium), while in animal genetics the primary source of information is familial (Mendelian segregation and linkage). We herein develop and evaluate a method that simultaneously exploits both sources of information. It builds on hidden Markov models that were initially developed to exploit population information only. We demonstrate that the approach improves the accuracy of allele phasing as well as imputation of missing genotypes. Reconstructed haplotypes are assigned to hidden states that are shown to correspond to clusters of genealogically related chromosomes. We show that these cluster states can directly be used to fine map QTL. The method is computationally effective at handling large data sets based on high-density SNP panels.ARRAY technology now allows genotyping of large cohorts for thousands to millions of single nucleotide polymorphisms (SNPs), which are becoming available for a growing list of organisms including human and domestic animals. Among other applications, these advances permit systematic scanning of the genome to map trait loci by association (e.g., Wellcome Trust Case Control Consortium 2007; Charlier et al. 2008), to predict genomic breeding values for complex traits (Meuwissen et al. 2001; Goddard and Hayes 2009), or to identify signatures of selection (e.g., Voight et al. 2006).Present-day genotyping platforms do not directly provide information about linkage phase; i.e., co-inherited alleles at adjacent heterozygous markers (haplotypes) are not identified as such. As haplotype information may considerably empower genetic analyses, indirect phasing strategies have been devised: haplotypes can be reconstructed from unphased genotypes using either familial information (Mendelian segregation and linkage) and/or population information (linkage disequilibrium, LD, and surrogate parents) (e.g., Windig and Meuwissen 2004; Scheet and Stephens 2006; Kong et al. 2008).Haplotype-based approaches are routinely applied in animal genetics for combined linkage and LD mapping of QTL (e.g., Meuwissen and Goddard 2000; Blott et al. 2003). In these studies, phasing has so far relied on familial information provided by the extended pedigrees typical of livestock (e.g., Windig and Meuwissen 2004). This approach, however, leaves a nonnegligible proportion of genotypes unphased, especially for the less connected individuals. After phasing, identity-by-descent (IBD) probabilities conditional on haplotype data—needed for QTL mapping—are computed for all chromosome pairs, using familial as well as population information (hence combined linkage and LD mapping – L + LD) (e.g., Meuwissen and Goddard 2001). However, the use of high-density SNP chips and the analysis of ever larger cohorts render the computation of pairwise IBD probabilities a bottleneck.We herein propose a more efficient, heuristic approach based on hidden Markov models (HMM). It simultaneously phases and sorts haplotypes in clusters that can be used directly for mapping or other purposes. The proposed method exploits familial as well as population information, and imputes missing genotypes. We herein describe the accuracy of the proposed method and its use for L + LD mapping of QTL.     

Fine Mapping and Marker Development for the Crossability Gene SKr on Chromosome 5BS of Hexaploid Wheat (Triticum aestivum L.)

Most elite wheat varieties cannot be crossed with related species thereby restricting greatly the germplasm that can be used for alien introgression in breeding programs. Inhibition to crossability is controlled genetically and a number of QTL have been identified to date, including the major gene Kr1 on 5BL and SKr, a strong QTL affecting crossability between wheat and rye on chromosome 5BS. In this study, we used a recombinant SSD population originating from a cross between the poorly crossable cultivar Courtot (Ct) and the crossable line MP98 to characterize the major dominant effect of SKr and map the gene at the distal end of the chromosome near the 5B homeologous GSP locus. Colinearity with barley and rice was used to saturate the SKr region with new markers and establish orthologous relationships with a 54-kb region on rice chromosome 12. In total, five markers were mapped within a genetic interval of 0.3 cM and 400 kb of BAC contigs were established on both sides of the gene to lay the foundation for map-based cloning of SKr. Two SSR markers completely linked to SKr were used to evaluate a collection of crossable wheat progenies originating from primary triticale breeding programs. The results confirm the major effect of SKr on crossability and the usefulness of the two markers for the efficient introgression of crossability in elite wheat varieties.DURING domestication and selection of a number of important crop species, diversity has eroded resulting in increased vulnerability to biotic and abiotic stresses while also jeopardizing the potential for sustained genetic improvement of elite cultivars over the long term (Tanksley and McCouch 1997; Fu and Somers 2009). The reintroduction of the remarkable diversity present in the different gene pools into elite varieties through intra- and interspecific crosses (primary and secondary gene pools) and intergeneric crosses (tertiary gene pools) has been practiced for decades in cereals (Feuillet et al. 2008). Despite some highly significant successes, including the incorporation of dwarfing and disease-resistance genes that fueled the Green Revolution, introgression remains laborious and, for complex characters, largely unfulfilled. Wheat (Triticum aestivum L.) has been crossed with a wide range of related species from the Triticeae tribe (Jiang et al. 1994), such as Aegilops, Agropyron, Haynaldia, Secale, and Hordeum, which represent a reservoir of interesting alleles for improving wheat resistance to biotic (diseases, insects) and abiotic stresses (cold, salinity, and drought) as well as for quality traits such as grain protein content (Fedak 1985). Intergeneric crosses have resulted in the transfer of desirable rye (Secale cereale L.) characteristics into wheat (Florell 1931) with one of the best examples being the 1BL/1RS chromosomal translocation that provided novel race-specific resistance to rust diseases, improved adaptation and stress tolerance, superior aerial biomass, and higher kernel weight to wheat varieties (Zarco-Hernandez et al. 2005). However, most of the adapted wheat germplasm is not crossable with alien species thereby restricting the panel of lines that can be used for alien introgression in wheat breeding (Krolow 1970) or for the production of primary triticale, a man-made wheat–rye hybrid.Beginning in the early 1900s, researchers were producing experimental crosses between bread wheat, T. aestivum L. (2n = 6x = 42) as a recipient, and rye, S. cereale L. (2n =14) as the pollen donor (Backhouse 1916). Genetic studies conducted by Lein (1943) showed that dominant alleles of two genes, named Kr1 and Kr2, are responsible for the poor crossability between bread wheat and rye. Kr1 and Kr2 genes were localized roughly on chromosome 5B and 5A, respectively (Riley and Chapman 1967) and subsequently located more precisely on the long arms of these two chromosomes (Lange and Riley 1973; Sitch et al. 1985). Further studies indicated that the dominant alleles driving incompatibility of crossing wheat with rye act by actively inhibiting the production of intergeneric hybrids (Riley and Chapman 1967; Lange and Wojciechowska 1976; Jalani and Moss 1980, 1981; Cameron and Reger 1991 ). Other crossability genes, such as Kr3 on chromosome 5D (Krolow 1970) and Kr4 on chromosome 1A (Zheng et al. 1992), were identified later. Finally, a study elucidated that chromosome 1A, derived from the Chinese (Sichuan) tetraploid wheat T. turgidum L. cv. Ailanmai, carries a recessive allele for high crossability with rye (Liu et al. 1999). Genetic studies also indicated that Kr genes have different effects on wheat–rye crossability. For example, by testing the cultivars Chinese Spring (CS), Hope, and the substitution lines CS/Hope 5B and CS/Hope 5A, Riley and Chapman (1967) demonstrated that Kr1 has a stronger effect than Kr2, whereas Kr3 seemed weaker than the two other genes (Krolow 1970).To further explore the mechanisms controlling crossability in wheat, Snape et al. (1979) performed crosses between the wild barley Hordeum bulbosum and the wheat cultivars Chinese Spring and Hope as well as 21 substitution lines carrying individual chromosomes of Hope in the background of Chinese Spring. The results revealed that the Kr1 and Kr2 genes on chromosomes 5B and 5A that govern crossability between wheat and rye also are involved in controlling crossability between wheat and barley, although the percentage of crossability observed was significantly lower than with rye. Using crosses between Chinese Spring, Hope, and the entire series of substitution lines with the cultivated barley (H. vulgare L.) cv. Betzes, Fedak and Jui (1982) also suggested that the homeologous alleles of the Kr genes on chromosome group 5 of Chinese Spring (5A, 5B, and 5D) favor crossability with additive effects.In 1998, a new locus, named SKr, controlling crossability between wheat and rye was detected using a mapping population of 187 double haploid (DH) lines produced by anther culture from F₁ hybrids of a cross between the noncrossable (NC) French wheat cv. Courtot (Ct) and the Chinese crossable (C) cv. Chinese Spring (Tixier et al. 1998). SKr was identified as a major QTL located on the distal end of the short arm of chromosome 5B within a confidence interval ranging from 8.7 to 20.9 cM (Lamoureux et al. 2002). In this population, the effect of SKr was stronger (22.1% of heritability) than the one of a QTL identified on 5BL (supposedly Kr1, 5.5% of heritability), whereas no significant effect was detected on 5AL (for Kr2). Moreover, the results indicated a 95% crossability rate for cv. Chinese Spring and ∼10% for Courtot, suggesting that the Courtot genotype is Kr1Kr1/kr2kr2, whereas Chinese Spring would be kr1kr1/kr2kr2.Here, we report the construction of a high resolution genetic map at the SKr locus using a single seed descent (SSD) population derived from a cross between Courtot and a crossable DH line (MP98) that allowed us to assess the crossability phenotype as a single “Mendelian factor” and to demonstrate the major dominant effect of SKr on crossability. Synteny between wheat, barley, and rice was used to increase the density of markers and reduce the genetic interval around the SKr gene to 0.3 cM. BAC contigs from Chinese Spring were established with closely linked and cosegregating markers to lay the foundation for positional cloning of the gene. Finally, a SSR marker cosegregating with SKr was developed and its value for the exploitation of SKr in breeding was assessed in a collection of crossable lines used to produce primary triticale.     

Simple Y-Autosomal Incompatibilities Cause Hybrid Male Sterility in Reciprocal Crosses Between Drosophila virilis and D. americana

Postzygotic reproductive isolation evolves when hybrid incompatibilities accumulate between diverging populations. Here, I examine the genetic basis of hybrid male sterility between two species of Drosophila, Drosophila virilis and D. americana. From these analyses, I reach several conclusions. First, neither species carries any autosomal dominant hybrid male sterility alleles: reciprocal F₁ hybrid males are perfectly fertile. Second, later generation (backcross and F₂) hybrid male sterility between D. virilis and D. americana is not polygenic. In fact, I identified only three genetically independent incompatibilities that cause hybrid male sterility. Remarkably, each of these incompatibilities involves the Y chromosome. In one direction of the cross, the D. americana Y is incompatible with recessive D. virilis alleles at loci on chromosomes 2 and 5. In the other direction, the D. virilis Y chromosome causes hybrid male sterility in combination with recessive D. americana alleles at a single QTL on chromosome 5. Finally, in contrast with findings from other Drosophila species pairs, the X chromosome has only a modest effect on hybrid male sterility between D. virilis and D. americana.SPECIATION occurs when populations evolve one or more barriers to interbreeding (Dobzhansky 1937; Mayr 1963). One such barrier is intrinsic postzygotic isolation, which typically evolves when diverging populations accumulate different alleles at two or more loci that are incompatible when brought together in hybrid genomes; negative epistasis between these alleles renders hybrids inviable or sterile (Bateson 1909; Dobzhansky 1937; Muller 1942). Classical and recent studies in diverse animal taxa have provided support for two evolutionary patterns that often characterize the genetics of postzygotic isolation (Coyne and Orr 1989a). The first, Haldane''s rule, observes that when there is F₁ hybrid inviability or sterility that affects only one sex, it is almost always the heterogametic sex (Haldane 1922). Over the years, many researchers have tried to account for this pattern, but only two ideas are now thought to provide a general explanation: the “dominance theory,” which posits that incompatibility alleles are generally recessive in hybrids, and the “faster-male theory,” which posits that genes causing hybrid male sterility diverge more rapidly than those causing hybrid female sterility (Muller 1942; Wu and Davis 1993; Turelli and Orr 1995; reviewed in Coyne and Orr 2004). In some cases, however, additional factors might contribute to Haldane''s rule, including meiotic drive, a faster-evolving X chromosome, dosage compensation, and Y chromosome incompatibilities (reviewed in Laurie 1997; Turelli and Orr 2000; Coyne and Orr 2004).The second broad pattern affecting the evolution of postzygotic isolation is the disproportionately large effect of the X chromosome on heterogametic F₁ hybrid sterility (Coyne 1992). This “large X effect” has been documented in genetic analyses of backcross hybrid sterility (e.g., Dobzhansky 1936; Grula and Taylor 1980; Orr 1987; Masly and Presgraves 2007) and inferred from patterns of introgression across natural hybrid zones (e.g., Machado et al. 2002; Saetre et al. 2003; Payseur et al. 2004). However, in only one case has the cause of the large X effect been unambiguously determined: incompatibilities causing hybrid male sterility between Drosophila mauritiana and D. sechellia occur at a higher density on the X than on the autosomes (Masly and Presgraves 2007). Testing the generality of this pattern will require additional high-resolution genetic analyses in diverse taxa (Presgraves 2008). But whatever its causes, there is now general consensus that the X chromosome often plays a special role in the evolution of postzygotic isolation (Coyne and Orr 2004).The contribution of the Y chromosome to animal speciation is less clear. Y chromosomes have far fewer genes than the X or autosomes, and most of these genes are male specific (Lahn and Page 1997; Carvalho et al. 2009). In Drosophila species, the Y chromosome is typically required for male fertility, but not for viability (Voelker and Kojima 1971). How often, then, does the Y chromosome play a role in reproductive isolation? In crosses between Drosophila species, hybrid male sterility is frequently caused by incompatibilities between the X and Y chromosomes (Schafer 1978; Heikkinen and Lumme 1998; Mishra and Singh 2007) or between the Y and heterospecific autosomal alleles (Patterson and Stone 1952; Vigneault and Zouros 1986; Lamnissou et al. 1996). In crosses between D. yakuba and D. santomea, the Y chromosome causes F₁ hybrid male sterility, and accordingly, shows no evidence for recent introgression across a species hybrid zone (Coyne et al. 2004; Llopart et al. 2005). In mammals, reduced introgression of Y-linked loci (relative to autosomal loci) has been shown across natural hybrid zones of mice (Tucker et al. 1992) and rabbits (Geraldes et al. 2008), suggesting that the Y chromosome contributes to reproductive barriers.Here I examine the genetic basis of hybrid male sterility between two species of Drosophila, D. virilis and D. americana. These species show considerable genetic divergence (K_s ∼0.11, Morales-Hojas et al. 2008) and are currently allopatric: D. virilis is a human commensal worldwide with natural populations in Asia, and D. americana is found in riparian habitats throughout much of North America (Throckmorton 1982; McAllister 2002). Nearly 70 years ago, Patterson et al. (1942) showed that incompatibilities between the D. americana Y chromosome and the second and fifth chromosomes from D. virilis cause hybrid male sterility, a result that was confirmed in a more recent study (Lamnissou et al. 1996). Another study suggested that the X chromosome might play the predominant role in causing hybrid male sterility between D. virilis and D. americana (Orr and Coyne 1989). But because previous genetic analyses had to rely on only a few visible markers to map hybrid male sterility, they lacked the resolution to examine the genomic distribution of incompatibility loci.Using the D. virilis genome sequence, I have developed a dense set of molecular markers to investigate the genetic architecture of hybrid male sterility between D. virilis and D. americana. In this study, I perform a comprehensive set of crosses to address several key questions: What is the effect of the X chromosome on hybrid male sterility between D. virilis and D. americana? What is the effect of the Y chromosome? Approximately how many loci contribute to hybrid male sterility between these Drosophila species? Perhaps surprisingly, the answers to these questions differ dramatically from what has been found for other Drosophila species, including the well-studied D. melanogaster group.