High-resolution, high-throughput SNP mapping in Drosophila melanogaster

Single nucleotide polymorphisms (SNPs) are useful markers for genetic mapping experiments in model organisms. Here we report the establishment of a high-density SNP map and high-throughput genotyping assays for Drosophila melanogaster. Our map comprises 27,367 SNPs in common laboratory Drosophila stocks. These SNPs were clustered within 2,238 amplifiable markers at an average density of 1 marker every 50.3 kb, or 6.3 genes. We have also constructed a set of 62 Drosophila stocks, each of which facilitates the generation of recombinants within a defined genetic interval of 1-2 Mb. For flexible, high-throughput SNP genotyping, we used fluorescent tag-array mini-sequencing (TAMS) assays. We designed and validated TAMS assays for 293 SNPs at an average resolution of 391.3 kb, and demonstrated the utility of these tools by rapidly mapping 14 mutations that disrupt embryonic muscle patterning. These resources enable high-resolution high-throughput genetic mapping in Drosophila.     

Haplotype reconstruction from SNP alignment.

In this paper, we describe a method for statistical reconstruction of haplotypes from a set of aligned SNP fragments. We consider the case of a pair of homologous human chromosomes, one from the mother and the other from the father. After fragment assembly, we wish to reconstruct the two haplotypes of the parents. Given a set of potential SNP sites inferred from the assembly alignment, we wish to divide the fragment set into two subsets, each of which represents one chromosome. Our method is based on a statistical model of sequencing errors, compositional information, and haplotype memberships. We calculate probabilities of different haplotypes conditional on the alignment. Due to computational complexity, we first determine phases for neighboring SNPs. Then we connect them and construct haplotype segments. Also, we compute the accuracy or confidence of the reconstructed haplotypes. We discuss other issues, such as alternative methods, parameter estimation, computational efficiency, and relaxation of assumptions.     

SNP Haplotype Mapping in a Small ALS Family

The identification of genes for monogenic disorders has proven to be highly effective for understanding disease mechanisms, pathways and gene function in humans. Nevertheless, while thousands of Mendelian disorders have not yet been mapped there has been a trend away from studying single-gene disorders. In part, this is due to the fact that many of the remaining single-gene families are not large enough to map the disease locus to a single site in the genome. New tools and approaches are needed to allow researchers to effectively tap into this genetic gold-mine. Towards this goal, we have used haploid cell lines to experimentally validate the use of high-density single nucleotide polymorphism (SNP) arrays to define genome-wide haplotypes and candidate regions, using a small amyotrophic lateral sclerosis (ALS) family as a prototype. Specifically, we used haploid-cell lines to determine if high-density SNP arrays accurately predict haplotypes across entire chromosomes and show that haplotype information significantly enhances the genetic information in small families. Panels of haploid-cell lines were generated and a 5 centimorgan (cM) short tandem repeat polymorphism (STRP) genome scan was performed. Experimentally derived haplotypes for entire chromosomes were used to directly identify regions of the genome identical-by-descent in 5 affected individuals. Comparisons between experimentally determined and in silico haplotypes predicted from SNP arrays demonstrate that SNP analysis of diploid DNA accurately predicted chromosomal haplotypes. These methods precisely identified 12 candidate intervals, which are shared by all 5 affected individuals. Our study illustrates how genetic information can be maximized using readily available tools as a first step in mapping single-gene disorders in small families.     

Haplotype reconstruction from SNP fragments by minimum error correction

MOTIVATION: Haplotype reconstruction based on aligned single nucleotide polymorphism (SNP) fragments is to infer a pair of haplotypes from localized polymorphism data gathered through short genome fragment assembly. An important computational model of this problem is the minimum error correction (MEC) model, which has been mentioned in several literatures. The model retrieves a pair of haplotypes by correcting minimum number of SNPs in given genome fragments coming from an individual's DNA. RESULTS: In the first part of this paper, an exact algorithm for the MEC model is presented. Owing to the NP-hardness of the MEC model, we also design a genetic algorithm (GA). The designed GA is intended to solve large size problems and has very good performance. The strength and weakness of the MEC model are shown using experimental results on real data and simulation data. In the second part of this paper, to improve the MEC model for haplotype reconstruction, a new computational model is proposed, which simultaneously employs genotype information of an individual in the process of SNP correction, and is called MEC with genotype information (shortly, MEC/GI). Computational results on extensive datasets show that the new model has much higher accuracy in haplotype reconstruction than the pure MEC model.     

Evolution of sexual dimorphism of wing shape in the Drosophila melanogaster subgroup

Background  

Sexual dimorphism of body size has been the subject of numerous studies, but few have examined sexual shape dimorphism (SShD) and its evolution. Allometry, the shape change associated with size variation, has been suggested to be a main component of SShD. Yet little is known about the relative importance of the allometric and non-allometric components for the evolution of SShD.     

Cytoplasmic myosin from Drosophila melanogaster

Myosin is identified and purified from three different established Drosophila melanogaster cell lines (Schneider's lines 2 and 3 and Kc). Purification entails lysis in a low salt, sucrose buffer that contains ATP, chromatography on DEAE-cellulose, precipitation with actin in the absence of ATP, gel filtration in a discontinuous KI-KCl buffer system, and hydroxylapatite chromatography. Yield of pure cytoplasmic myosin is 5-10%. This protein is identified as myosin by its cross-reactivity with two monoclonal antibodies against human platelet myosin, the molecular weight of its heavy chain, its two light chains, its behavior on gel filtration, its ATP-dependent affinity for actin, its characteristic ATPase activity, its molecular morphology as demonstrated by platinum shadowing, and its ability to form bipolar filaments. The molecular weight of the cytoplasmic myosin's light chains and peptide mapping and immunochemical analysis of its heavy chains demonstrate that this myosin, purified from Drosophila cell lines, is distinct from Drosophila muscle myosin. Two-dimensional thin layer maps of complete proteolytic digests of iodinated muscle and cytoplasmic myosin heavy chains demonstrate that, while the two myosins have some tryptic and alpha-chymotryptic peptides in common, most peptides migrate with unique mobility. One-dimensional peptide maps of SDS PAGE purified myosin heavy chain confirm these structural data. Polyclonal antiserum raised and reacted against Drosophila myosin isolated from cell lines cross-reacts only weakly with Drosophila muscle myosin isolated from the thoraces of adult Drosophila. Polyclonal antiserum raised against Drosophila muscle myosin behaves in a reciprocal fashion. Taken together our data suggest that the myosin purified from Drosophila cell lines is a bona fide cytoplasmic myosin and is very likely the product of a different myosin gene than the muscle myosin heavy chain gene that has been previously identified and characterized.     

The Zuker collection: a resource for the analysis of autosomal gene function in Drosophila melanogaster

The majority of genes of multicellular organisms encode proteins with functions that are not required for viability but contribute to important physiological functions such as behavior and reproduction. It is estimated that 75% of the genes of Drosophila melanogaster are nonessential. Here we report on a strategy used to establish a large collection of stocks that is suitable for the recovery of mutations in such genes. From approximately 72,000 F(3) cultures segregating for autosomes heavily treated with ethyl methanesulfonate (EMS), approximately 12,000 lines in which the treated second or third chromosome survived in homozygous condition were selected. The dose of EMS induced an estimated rate of 1.2-1.5 x 10(-3) mutations/gene and predicts five to six nonessential gene mutations per chromosome and seven to nine alleles per locus in the samples of 6000 second chromosomes and 6000 third chromosomes. Due to mosaic mutations induced in the initial exposure to the mutagen, many of the lines are segregating or are now fixed for lethal mutations on the mutagenized chromosome. The features of this collection, known as the Zuker collection, make it a valuable resource for forward and reverse genetic screens for mutations affecting a wide array of biological functions.     

Fast egg collection method greatly improves randomness of egg sampling in Drosophila melanogaster

When obtaining samples for population genetic studies, it is essential that the sampling is random. For Drosophila, one of the crucial steps in sampling experimental flies is the collection of eggs. Here an egg collection method is presented, which randomizes the eggs in a water column and diminishes environmental variance. This method was compared with a traditional egg collection method where eggs are collected directly from the medium. Within each method the observed and expected standard deviations of egg-to-adult viability were compared, whereby the difference in the randomness of the samples between the two methods was assessed. The method presented here was superior to the traditional method. Only 14% of the samples had a standard deviation higher than expected, as compared with 58% in the traditional method. To reduce bias in the estimation of the variance and the mean of a trait and to obtain a representative collection of genotypes, the method presented here is strongly recommended when collecting eggs from Drosophila.     

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster

Summary Previous studies have demonstrated that the expression of the -amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the -amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.Offprint requests to: D.A. Hickey     

HnRNP particles from Drosophila melanogaster cells

The isolation and characterization of HnRNP from cultured Drosophila melanogaster cells is described. HnRNP particles were extracted from the purified nuclei by sonication in the presence of rat liver cytosol RNAase inhibitor. The nuclear extract was centrifuged on a 15–30% sucrose gradient. The main part of the heterogeneous HnRNP material was localized in the 30 to 80S region of the sucrose gradient. According to the results of re-sedimentation studies the monomer particle was 45S. The buoyant density of HnRNP particles from different regions of the sucrose gradient were equal to 1.4. The protein composition of the particles was analyzed by urea-SDS-polyacrylamide gel electrophoresis. There are five main and a few minor bands. Only the main polypeptides have a slightly higher molecular weight than those of the major polypeptides of 30S subparticles from rat liver nuclei. According to electron = microscopic studies the particles are heterogeneous and the average diameter was found to be 24–26 nm both on the basis of negative contrast and platinum-palladium shadowed pictures.     

Purification of a lysosomal DNase from Drosophila melanogaster.

An acid DNase was purified from Drosophila melanogaster till apparent homogeneity by six consecutive chromatographic steps. The enzyme is a lysosomal DNase, because it is glycosylated and carries 1.8-2.4 mol of mannose-6-phosphate/mol of enzyme. The enzyme is fully active without any divalent cation and introduces single stranded nicks into a supercoiled DNA.     

Genetic analysis of modifiers affecting sexual dimorphism and temperature sensitivity of bx1 in Drosophila melanogaster

Two modifiers of bithorax1 phenotypic expression are described. An X-chromosome region is associated with sexual dimorphism in bx1 penetrance. It is hypothesized that sexual dimorphism is in part due to a lack of dosage compensation of the modifier, in males. A third chromosome region that segregates with the pink peach allele is implicated in mediating temperature sensitivity. By appropriate combinations of modifiers, both sexual dimorphism and temperature sensitivity can be greatly reduced.     

Effect of genomic deficiencies on sexual size dimorphism through modification of developmental time in Drosophila melanogaster

Sexual size dimorphism (SSD), a difference in body size between sexes, is common in many taxa. In insects, females are larger than males in >70% of all taxa in most orders. The fruit fly, Drosophila melanogaster is one prominent model organism to investigate SSD since its clear and representative female-biased SSD and its growth regulation are well studied. Elucidating the number and nature of genetic elements that can potentially influence SSD would be helpful in understanding the evolutionary potential of SSD. Here, we investigated the SSD pattern caused by artificially introduced genetic variation in D. melanogaster, and examined whether variation in SSD was mediated by the sex-specific modification of developmental time. To map the genomic regions that had effects on sexual wing size and/or developmental time differences (SDtD), we reanalyzed previously published genome-wide deficiency mapping data to evaluate the effects of 376 isogenic deficiencies covering a total of ~67% of the genomic regions of the second and third chromosomes of D. melanogaster. We found genetic variation in SSD and SDtD generated by genomic deficiencies, and a negative genetic correlation between size and development time. We also found SSD and SDtD allometries that are not qualitatively congruent, which however overall at best only partly help in explaining the patterns found. We identified several genomic deficiencies with the tendency to either exaggerate or suppress SSD, in agreement with quantitative genetic null expectations of many loci with small effects. These novel findings contribute to a better understanding of the evolutionary potential of sexual dimorphism.