Whole-Genome Sequencing and iPLEX MassARRAY Genotyping Map an EMS-Induced Mutation Affecting Cell Competition in Drosophila melanogaster.
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ABSTRACT:
SUBMITTER: Lee CH
PROVIDER: S-EPMC5068942 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Next-generation methods for rapid whole-genome sequencing enable the identification of single-base-pair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mutant chromosomes, we obtained high-quality reads for base pairs composing approximately 70% of the third chromosome of both DNA samples. We verified 103 single-base-pair changes between the two chromosomes. Nine changes were nonsynonymous mutations and two were nonsense mutations. One nonsense mutation was in a gene, encore, whose mutations produce an egg shell phenotype also observed in progeny of homozygous mutant mothers. Complementation analysis revealed that the chromosome carried a new functional allele of encore, demonstrating that one round of next-generation sequencing can identify the causative lesion for a phenotype of interest. This new method of whole-genome sequencing represents great promise for mutant mapping in flies, potentially replacing conventional methods.
| S-EPMC2674820 | biostudies-literature
Project description:Epigenetic research has been focused on cell-type-specific regulation; less is known about common features of epigenetic programming shared by diverse cell types within an organism. Here, we report a modified method for chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and its use to construct a high-resolution map of the Drosophila melanogaster key histone marks, heterochromatin protein 1a (HP1a) and RNA polymerase II (polII). These factors are mapped at 50-bp resolution genome-wide and at 5-bp resolution for regulatory sequences of genes, which reveals fundamental features of chromatin modification landscape shared by major adult Drosophila cell types: the enrichment of both heterochromatic and euchromatic marks in transposons and repetitive sequences, the accumulation of HP1a at transcription start sites with stalled polII, the signatures of histone code and polII level/position around the transcriptional start sites that predict both the mRNA level and functionality of genes, and the enrichment of elongating polII within exons at splicing junctions. These features, likely conserved among diverse epigenomes, reveal general strategies for chromatin modifications.
| S-EPMC3240582 | biostudies-literature
Project description:BackgroundRecent advances in molecular genetics have enabled to determine the genetic causes of non-syndromic hearing loss, and more than 100 genes have been related to the phenotype. Due to this extraordinary genetic heterogeneity, a large percentage of patients remain without any molecular diagnosis. This condition imply the need for new methodological strategies in order to detect a greater number of mutations in multiple genes. In this work, we optimized and tested a panel of 86 mutations in 17 different genes screened using a high-throughput genotyping technology to determine the molecular etiology of hearing loss.MethodsThe technology used in this work was the MassARRAY iPLEX® platform. This technology uses silicon chips and DNA amplification products for accurate genotyping by mass spectrometry of previous reported mutations. The generated results were validated using conventional techniques, as direct sequencing, multiplex PCR and RFLP-PCR.ResultsAn initial genotyping of control subjects, showed failures in 20 % of the selected alterations. To optimize these results, the failed tests were re-designed and new primers were synthesized. Then, the specificity and sensitivity of the panel demonstrated values above 97 %. Additionally, a group of 180 individuals with NSHL without a molecular diagnosis was screened to test the diagnostic value of our panel, and mutations were identified in 30 % of the cases. In 20 % of the individuals, it was possible to explain the etiology of the HL. Mutations in GJB2 gene were the most prevalent, followed by other mutations in in SLC26A4, CDH23, MT-RNR1, MYO15A, and OTOF genes.ConclusionsThe MassARRAY technology has the potential for high-throughput identification of genetic variations. However, we demonstrated that optimization is required to increase the genotyping success and accuracy. The developed panel proved to be efficient and cost-effective, being suitable for applications involving the molecular diagnosis of hearing loss.
| S-EPMC4581412 | biostudies-literature
Project description:In many species, sperm can remain viable in the reproductive tract of a female well beyond the typical interval to remating. This creates an opportunity for sperm from different males to compete for oocyte fertilization inside the female's reproductive tract. In Drosophila melanogaster, sperm characteristics and seminal fluid content affect male success in sperm competition. On the other hand, although genome-wide association studies (GWAS) have demonstrated that female genotype plays a role in sperm competition outcome as well, the biochemical, sensory, and physiological processes by which females detect and selectively use sperm from different males remain elusive. Here, we functionally tested 26 candidate genes implicated via a GWAS for their contribution to the female's role in sperm competition, measured as changes in the relative success of the first male to mate (P1). Of these 26 candidates, we identified eight genes that affect P1 when knocked down in females, and showed that five of them do so when knocked down in the female nervous system. In particular, Rim knockdown in sensory pickpocket (ppk)+ neurons lowered P1, confirming previously published results, and a novel candidate, caup, lowered P1 when knocked down in octopaminergic Tdc2+ neurons. These results demonstrate that specific neurons in the female's nervous system play a functional role in sperm competition and expand our understanding of the genetic, neuronal, and mechanistic basis of female responses to multiple matings. We propose that these neurons in females are used to sense, and integrate, signals from courtship or ejaculates, to modulate sperm competition outcome accordingly.
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Project description:Dwindling male fertility due to xenobiotics is of global concern. Accordingly, male reproductive toxicity assessment of xenobiotics through semen quality analysis in exposed males, and examining progeny production of their mates is critical. These assays, in part, are biased towards monogamy. Females soliciting multiple male partners (polyandry) is the norm in many species. Polyandry incites sperm competition and allows females to bias sperm use. However, consequences of xenobiotic exposure to the sperm in the light of sperm competition remain to be understood. Therefore, we exposed Drosophila melanogaster males to endosulfan, and evaluated their progeny production as well as the ability of their sperm to counter rival control sperm in the storage organs of females sequentially mated to control/exposed males. Endosulfan (2 μg/ml) had no significant effect on progeny production and on the expression of certain genes associated with reproduction. However, exposed males performed worse in sperm competition, both as 1(st) and 2(nd) male competitors. These findings indicate that simple non-competitive measures of reproductive ability may fail to demonstrate the harmful effects of low-level exposure to xenobiotics on reproduction and advocate consideration of sperm competition, as a parameter, in the reproductive toxicity assessment of xenobiotics to mimic situations prevailing in the nature.
| S-EPMC4262826 | biostudies-literature
Project description:Males of many species use social cues to predict sperm competition (SC) and tailor their reproductive strategies, such as ejaculate or behavioural investment, accordingly. While these plastic strategies are widespread, the underlying mechanisms remain largely unknown. Plastic behaviour requires individuals to learn and memorize cues associated with environmental change before using this experience to modify behaviour. Drosophila melanogaster respond to an increase in SC threat by extending mating duration after exposure to a rival male. This behaviour shows lag times between environmental change and behavioural response suggestive of acquisition and loss of memory. Considering olfaction is important for a male's ability to assess the SC environment, we hypothesized that an olfactory learning and memory pathway may play a key role in controlling this plastic behaviour. We assessed the role of genes and brain structures known to be involved in learning and memory. We show that SC responses depend on anaesthesia-sensitive memory, specifically the genes rut and amn We also show that the γ lobes of the mushroom bodies are integral to the control of plastic mating behaviour. These results reveal the genetic and neural properties required for reacting to changes in the SC environment.
| S-EPMC5998109 | biostudies-literature
Project description:Phenotypic plasticity can allow animals to adapt their behavior, such as their mating effort, to their social and sexual environment. However, this relies on the individual receiving accurate and reliable cues of the environmental conditions. This can be achieved via the receipt of multimodal cues, which may provide redundancy and robustness. Male Drosophila melanogaster detect presence of rivals via combinations of any two or more redundant cue components (sound, smell, and touch) and respond by extending their subsequent mating duration, which is associated with higher reproductive success. Although alternative combinations of cues of rival presence have previously been found to elicit equivalent increases in mating duration and offspring production, their redundancy in securing success under sperm competition has not previously been tested. Here, we explicitly test this by exposing male D. melanogaster to alternative combinations of rival cues, and examine reproductive success in both the presence and absence of sperm competition. The results supported previous findings of redundancy of cues in terms of behavioral responses. However, there was no evidence of reproductive benefits accrued by extending mating duration in response to rivals. The lack of identifiable fitness benefits of longer mating under these conditions, both in the presence and absence of sperm competition, contrasted with some previous results, but could be explained by (a) damage sustained from aggressive interactions with rivals leading to reduced ability to increase ejaculate investment, (b) presence of features of the social environment, such as male and female mating status, that obscured the fitness benefits of longer mating, and (c) decoupling of behavioral investment with fitness benefits.
| S-EPMC7319233 | biostudies-literature
Project description:Although traditional genetic assays have characterized the pattern of crossing over across the genome in Drosophila melanogaster, these assays could not precisely define the location of crossovers. Even less is known about the frequency and distribution of noncrossover gene conversion events. To assess the specific number and positions of both meiotic gene conversion and crossover events, we sequenced the genomes of male progeny from females heterozygous for 93,538 X chromosomal single-nucleotide and InDel polymorphisms. From the analysis of the 30 F1 hemizygous X chromosomes, we detected 15 crossover and 5 noncrossover gene conversion events. Taking into account the nonuniform distribution of polymorphism along the chromosome arm, we estimate that most oocytes experience 1 crossover event and 1.6 gene conversion events per X chromosome pair per meiosis. An extrapolation to the entire genome would predict approximately 5 crossover events and 8.6 conversion events per meiosis. Mean gene conversion tract lengths were estimated to be 476 base pairs, yielding a per nucleotide conversion rate of 0.86 × 10(-5) per meiosis. Both of these values are consistent with estimates of conversion frequency and tract length obtained from studies of rosy, the only gene for which gene conversion has been studied extensively in Drosophila. Motif-enrichment analysis revealed a GTGGAAA motif that was enriched near crossovers but not near gene conversions. The low-complexity and frequent occurrence of this motif may in part explain why, in contrast to mammalian systems, no meiotic crossover hotspots have been found in Drosophila.
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Project description:The rosy locus in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase (XDH). Previous studies defined a "control element" near the 5' end of the gene, where variant sites affected the amount of rosy mRNA and protein produced. We have determined the DNA sequence of this region from both genomic and cDNA clones, and from the ry+10 underproducer strain. This variant strain had many sequence differences, so that the site of the regulatory change could not be fixed. A mutagenesis was also undertaken to isolate new regulatory mutations. We induced 376 new mutations with 1-ethyl-1-nitrosourea (ENU) and screened them to isolate those that reduced the amount of XDH protein produced, but did not change the properties of the enzyme. Genetic mapping was used to find mutations located near the 5' end of the gene. DNA from each of seven mutants was cloned and sequenced through the 5' region. Mutant base changes were identified in all seven; they appear to affect splicing and translation of the rosy mRNA. In a related study (T. P. Keith et al. 1987), the genomic and cDNA sequences are extended through the 3' end of the gene; the combined sequences define the processing pattern of the rosy transcript and predict the amino acid sequence of XDH.
| S-EPMC1203121 | biostudies-other
Project description:The ability of female insects to retain and use sperm for days, months, or even years after mating requires specialized storage organs in the reproductive tract. In most orders, these organs include a pair of sclerotized capsules known as spermathecae. Here, we report that some Drosophila melanogaster females exhibit previously uncharacterized structures within the distal portion of the muscular duct that links a spermatheca to the uterus. We find that these 'spermathecal duct presences' (SDPs) may form in either or both ducts and can extend from the duct into the sperm-storing capsule itself. We further find that the incidence of SDPs varies significantly between genotypes, but does not change significantly with the age or mating status of females, the latter indicating that SDPs are not composed of or stimulated by sperm or male seminal proteins. We show that SDPs affect neither the number of first male sperm held in a spermatheca nor the number of offspring produced after a single mating. However, we find evidence that SDPs are associated with a lack of second male sperm in the spermathecae after females remate. This raises the possibility that SDPs provide a mechanism for variation in sperm competition outcome among females.
| S-EPMC7137968 | biostudies-literature