Phenogenetics of triploid intersexes in Drosophila melanogaster

Triploid intersexes homozygous for a mutant (msl-2) known to impede the hyperactivation of the X chromosome in diploid males differentiate into adults, sexually indistinguishable from their heterozygous sibs. A shift toward female sexual differentiation mediated by manipulating the rearing temperature is accompanied by an apparent increase in the level of an X-linked gene product. This unexpected result is rationalized in terms of differential lethality of individuals at the two extremities of the distribution of X-activity levels in intersexes raised at a particular temperature. No evidence of a mosaicism comparable to the sexual mosaicism exhibited could be found with respect to an X-linked gene product in triploid intersexes.     

Studies on the Sex-Specific Lethals of DROSOPHILA MELANOGASTER. IV. Gynandromorph Analysis of Three Male-Specific Lethals, mle, msl-227 and mle(3)132

Mutants at three male-specific lethal loci of Drosophila melanogaster (mle, msl-2²⁷ and mle(3)132) were examined by gynandromorph analysis. In all cases only a very few gynandromorphs with small X/O patches appeared. Most of these small X/O patches were in the abdomen, and the structures in these X/O regions were reduced in size. These results indicate that the primary effects of these mutants are not on any particular organs or tissues, but rather on individual cells. mle and msl-2 have been shown by Belote and Lucchesi (1980a) to be defective in dosage compensation in X/Y males. We suggest that this dosage-compensation defect results in the expression of Minute-like phenotypes in X/O cells, and hence results in the death of X/O males and flies with large X/O tissue areas.     

Male-Specific Lethal Mutations of DROSOPHILA MELANOGASTER

A total of 7,416 ethyl methanesulfonate (EMS)-treated second chromosomes and 6,212 EMS-treated third chromosomes were screened for sex-specific lethals. Four new recessive male-specific lethal mutations were recovered. When in homozygous condition, each of these mutations kills males during the late larval or early pupal stages, but has no detectable effect in females. One mutant, mle^ts, is a temperature sensitive allele of maleless, mle (Fukunaga, Tanaka and Oishi 1975), while the other three mutants identify two new loci: male-specific lethal-1 (msl-1) (two alleles) at map position 2-53.3 and male-specific lethal-2 (msl-2) at 2-9.0.——The male-specific lethality associated with these mutants is not related to the sex per se of the mutant flies, since sex-transforming genes fail to interact with these mutations. Moreover, the presence or absence of a Y chromosome in males or females has no influence on the male-specific lethal action of these mutations. Finally, no single region of the X chromosome, when present as a duplication, is sufficient to rescue males from the lethal effects of msl-1 or msl-2. These results suggest that the number of complete X chromosomes determines whether a fly homozygous for a male-specific lethal mutation lives or dies.     

Identification of a New Gene Family Expressed during the Onset of Sexual Reproduction in the Centric Diatom Thalassiosira weissflogii

An intriguing feature of the diatom life cycle is that sexual reproduction and the generation of genetic diversity are coupled to the control of cell size. A PCR-based cDNA subtraction technique was used to identify genes that are expressed as small cells of the centric diatom Thalassiosira weissflogii initiate gametogenesis. Ten genes that are up-regulated during the early stages of sexual reproduction have been identified thus far. Three of the sexually induced genes, Sig1, Sig2, and Sig3, were sequenced to completion and are members of a novel gene family. The three polypeptides encoded by these genes possess different molecular masses and charges but display many features in common: they share five highly conserved domains; they each contain three or more cysteine-rich epithelial growth factor (EGF)-like repeats; and they each display homology to the EGF-like region of the vertebrate extracellular matrix glycoprotein tenascin X. Interestingly, the five conserved domains appear in the same order in each polypeptide but are separated by variable numbers of nonconserved amino acids. SIG1 and SIG2 display putative regulatory domains within the nonconserved regions. A calcium-binding, EF-hand motif is found in SIG1, and an ATP/GTP binding motif is present in SIG2. The striking similarity between the SIG polypeptides and extracellular matrix components commonly involved in cell-cell interactions suggests that the SIG polypeptides may play a role in sperm-egg recognition. The SIG polypeptides are thus important molecular targets for determining when and where sexual reproduction occurs in the field.     

Studies on the Sex-Specific Lethals of DROSOPHILA MELANOGASTER. V. Sex Transformation Caused by Interactions between a Female-Specific Lethal, Sxlf#1, and the Male-Specific Lethals mle(3)132, msl-227, and mle

Interactions between a female-specific lethal mutant, Sxl^f#1, and each of three male-specific lethal mutants, mle(3)132, msl-2²⁷ and mle, of Drosophila melanogaster were observed to produce morphological changes in various sexually dimorphic external characters. Chromosomal females heterozygous for Sxl^f#1 and homozygous for any one of the male-specific lethals (and to a lesser degree heterozygous for male-specific lethals) sometimes had sex combs, male-type tergites, male-type sternites, male-type anal plates or male-type external genitalia. Penetrance was not high and expression was often incomplete; single individuals never had all the sexually dimorphic structures transformed. When mothers were homozygous for male-specific lethals, higher proportions of female progeny were affected than when mothers were heterozygous, suggesting a maternal effect.     

Sex-specific Trans-regulatory Variation on the Drosophila melanogaster X Chromosome

The X chromosome constitutes a unique genomic environment because it is present in one copy in males, but two copies in females. This simple fact has motivated several theoretical predictions with respect to how standing genetic variation on the X chromosome should differ from the autosomes. Unmasked expression of deleterious mutations in males and a lower census size are expected to reduce variation, while allelic variants with sexually antagonistic effects, and potentially those with a sex-specific effect, could accumulate on the X chromosome and contribute to increased genetic variation. In addition, incomplete dosage compensation of the X chromosome could potentially dampen the male-specific effects of random mutations, and promote the accumulation of X-linked alleles with sexually dimorphic phenotypic effects. Here we test both the amount and the type of genetic variation on the X chromosome within a population of Drosophila melanogaster, by comparing the proportion of X linked and autosomal trans-regulatory SNPs with a sexually concordant and discordant effect on gene expression. We find that the X chromosome is depleted for SNPs with a sexually concordant effect, but hosts comparatively more SNPs with a sexually discordant effect. Interestingly, the contrasting results for SNPs with sexually concordant and discordant effects are driven by SNPs with a larger influence on expression in females than expression in males. Furthermore, the distribution of these SNPs is shifted towards regions where dosage compensation is predicted to be less complete. These results suggest that intrinsic properties of dosage compensation influence either the accumulation of different types of trans-factors and/or their propensity to accumulate mutations. Our findings document a potential mechanistic basis for sex-specific genetic variation, and identify the X as a reservoir for sexually dimorphic phenotypic variation. These results have general implications for X chromosome evolution, as well as the genetic basis of sex-specific evolutionary change.     

Exploring a Local Genetic Interaction Network Using Evolutionary Replay Experiments

Understanding how genes interact is a central challenge in biology. Experimental evolution provides a useful, but underutilized, tool for identifying genetic interactions, particularly those that involve non-loss-of-function mutations or mutations in essential genes. We previously identified a strong positive genetic interaction between specific mutations in KEL1 (P344T) and HSL7 (A695fs) that arose in an experimentally evolved Saccharomyces cerevisiae population. Because this genetic interaction is not phenocopied by gene deletion, it was previously unknown. Using “evolutionary replay” experiments, we identified additional mutations that have positive genetic interactions with the kel1-P344T mutation. We replayed the evolution of this population 672 times from six timepoints. We identified 30 populations where the kel1-P344T mutation reached high frequency. We performed whole-genome sequencing on these populations to identify genes in which mutations arose specifically in the kel1-P344T background. We reconstructed mutations in the ancestral and kel1-P344T backgrounds to validate positive genetic interactions. We identify several genetic interactors with KEL1, we validate these interactions by reconstruction experiments, and we show these interactions are not recapitulated by loss-of-function mutations. Our results demonstrate the power of experimental evolution to identify genetic interactions that are positive, allele specific, and not readily detected by other methods, shedding light on an underexplored region of the yeast genetic interaction network.     

Homing endonuclease mediated gene targeting in Anopheles gambiae cells and embryos

Homing endonuclease genes (HEGs) are ‘selfish’ genetic elements that combine the capability to selectively disrupt specific gene sequences with the ability to rapidly spread from a few individuals to an entire population through homologous recombination repair events. Because of these properties, HEGs are regarded as promising candidates to transfer genetic modifications from engineered laboratory mosquitoes to wild-type populations including Anopheles gambiae the vector of human malaria. Here we show that I-SceI and I-PpoI homing endonucleases cleave their recognition sites with high efficiency in A. gambiae cells and embryos and we demonstrate HEG-induced homologous and non-homologous repair events in a variety of functional assays. We also propose a gene drive system for mosquitoes that is based on our finding that I-PpoI cuts genomic rDNA located on the X chromosome in A. gambiae, which could be used to selectively incapacitate X-carrying spermatozoa thereby imposing a severe male-biased sex ratio.     

Genetic interactions between chromosomes 11 and 18 contribute to airway hyperresponsiveness in mice

We used two-dimensional quantitative trait locus analysis to identify interacting genetic loci that contribute to the native airway constrictor hyperresponsiveness to methacholine that characterizes A/J mice, relative to C57BL/6J mice. We quantified airway responsiveness to intravenous methacholine boluses in eighty-eight (C57BL/6J X A/J) F₂ and twenty-seven (A/J X C57BL/6J) F₂ mice as well as ten A/J mice and six C57BL/6J mice; all studies were performed in male mice. Mice were genotyped at 384 SNP markers, and from these data two-QTL analyses disclosed one pair of interacting loci on chromosomes 11 and 18; the homozygous A/J genotype at each locus constituted the genetic interaction linked to the hyperresponsive A/J phenotype. Bioinformatic network analysis of potential interactions among proteins encoded by genes in the linked regions disclosed two high priority subnetworks - Myl7, Rock1, Limk2; and Npc1, Npc1l1. Evidence in the literature supports the possibility that either or both networks could contribute to the regulation of airway constrictor responsiveness. Together, these results should stimulate evaluation of the genetic contribution of these networks in the regulation of airway responsiveness in humans.     

The translation initiation factor eIF4E regulates the sex-specific expression of the master switch gene Sxl in Drosophila melanogaster

In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors--the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d--that have been directly implicated in Sxl splicing regulation.     

Phenogenetics of triploid intersexes in Drosophila melanogaster

Triploid intersexes homozygous for a mutant (msl-2) known to impede the hyperactivation of the X chromosome in diploid males differentiate into adults, sexually indistinguishable from their heterozygous sibs. A shift toward female sexual differentiation mediated by manipulating the rearing temperature is accompanied by an apparent increase in the level of an X-linked gene product. This unexpected result is rationalized in terms of differential lethality of individuals at the two extremities of the distribution of X-activity levels in intersexes raised at a particular temperature. No evidence of a mosaicism comparable to the sexual mosaicism exhibited could be found with respect to an X-linked gene product in triploid intersexes.     

Testing for the Footprint of Sexually Antagonistic Polymorphisms in the Pseudoautosomal Region of a Plant Sex Chromosome Pair

The existence of sexually antagonistic (SA) polymorphism is widely considered the most likely explanation for the evolution of suppressed recombination of sex chromosome pairs. This explanation is largely untested empirically, and no such polymorphisms have been identified, other than in fish, where no evidence directly implicates these genes in events causing loss of recombination. We tested for the presence of loci with SA polymorphism in the plant Silene latifolia, which is dioecious (with separate male and female individuals) and has a pair of highly heteromorphic sex chromosomes, with XY males. Suppressed recombination between much of the Y and X sex chromosomes evolved in several steps, and the results in Bergero et al. (2013) show that it is still ongoing in the recombining or pseudoautosomal, regions (PARs) of these chromosomes. We used molecular evolutionary approaches to test for the footprints of SA polymorphisms, based on sequence diversity levels in S. latifolia PAR genes identified by genetic mapping. Nucleotide diversity is high for at least four of six PAR genes identified, and our data suggest the existence of polymorphisms maintained by balancing selection in this genome region, since molecular evolutionary (HKA) tests exclude an elevated mutation rate, and other tests also suggest balancing selection. The presence of sexually antagonistic alleles at a locus or loci in the PAR is suggested by the very different X and Y chromosome allele frequencies for at least one PAR gene.     

A vitamin D pathway gene–gene interaction affects low-density lipoprotein cholesterol levels

Much evidence suggests an association between vitamin D deficiency and chronic diseases such as obesity and dyslipidemia. Although genetic factors play an important role in the etiology of these diseases, only a few studies have investigated the relationship between vitamin D-related genes and anthropometric and lipid profiles. The aim of this study was to investigate the association of three vitamin D-related genes with anthropometric and lipid parameters in 542 adult individuals. We analyzed the rs2228570 polymorphism in the vitamin D receptor gene (VDR), rs2134095 in the retinoid X receptor gamma gene (RXRG) and rs7041 in the vitamin D-binding protein gene (GC). Polymorphisms were genotyped by TaqMan allelic discrimination. Gene–gene interactions were evaluated by the general linear model. The functionality of the polymorphisms was investigated using the following predictors and databases: SIFT (Sorting Intolerant from Tolerant), PolyPhen-2 (Polymorphism Phenotyping v2) and Human Splicing Finder 3. We identified a significant effect of the interaction between RXRG (rs2134095) and GC (rs7041) on low-density lipoprotein cholesterol (LDL-c) levels (P=.005). Furthermore, our in silico analysis suggested a functional role for both variants in the regulation of the gene products. Our results suggest that the vitamin D-related genes RXRG and GC affect LDL-c levels. These findings are in agreement with other studies that consistently associate vitamin D and lipid profile. Together, our results corroborate the idea that analyzing gene–gene interaction would be helpful to clarify the genetic component of lipid profile.     

Genetic Basis of Self-Incompatibility in the Lichen-Forming Fungus Lobaria pulmonaria and Skewed Frequency Distribution of Mating-Type Idiomorphs: Implications for Conservation

Fungal populations that reproduce sexually are likely to be genetically more diverse and have a higher adaptive potential than asexually reproducing populations. Mating systems of fungal species can be self-incompatible, requiring the presence of isolates of different mating-type genes for sexual reproduction to occur, or self-compatible, requiring only one. Understanding the distribution of mating-type genes in populations can help to assess the potential of self-incompatible species to reproduce sexually. In the locally threatened epiphytic lichen-forming fungus Lobaria pulmonaria (L.) Hoffm., low frequency of sexual reproduction is likely to limit the potential of populations to adapt to changing environmental conditions. Our study provides direct evidence of self-incompatibility (heterothallism) in L. pulmonaria. It can thus be hypothesized that sexual reproduction in small populations might be limited by an unbalanced distribution of mating-type genes. We therefore assessed neutral genetic diversity (using microsatellites) and mating-type ratio in 27 lichen populations (933 individuals). We found significant differences in the frequency of the two mating types in 13 populations, indicating a lower likelihood of sexual reproduction in these populations. This suggests that conservation translocation activities aiming at maximizing genetic heterogeneity in threatened and declining populations should take into account not only presence of fruiting bodies in transplanted individuals, but also the identity and balanced representation of mating-type genes.