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Victoria's Masters Thesis A Generative Cell Specific 1 Ortholog in Drosophila melanogaster Victoria Elisabeth Garcia A thesis Submitted in partial fulfillment Of the requirements for the degree of Master of Science University of Washington 2012 Committee: Barbara Wakimoto Martha Bosma Jeffrey Riffell Program authorized to offer degree: Biology INTRODUCTION AND BACKGROUND Sexual reproduction is the dominant mode of propagation in eukarya.1 It has been shown to facilitate genomic complexity and robustness, and to speed adaptation while aiding in the suppression of harmful mutations.2 Though the process is profoundly widespread, the cellular mechanics of the fertilization process are not tightly conserved, and can vary tremendously from species to species:3 The extending pollen tubes and double fertilizations of angiosperms, for example, differ vastly from flagellum-mediated clinches and aggregations seen with the plus and minus gametes of Chlamydomonas. Variation is the rule even at the protein level: factors that mediate gamete-gamete interaction tend to be highly species-specific.4 Indeed, genes encoding many fertilization proteins show signatures of positive selection, and have been shown to have roles in speciation.5 The Generative Cell Specific 1 (GCS1) protein is exceptional in this regard:6 First identified in Arabidopsis thaliana in 2004,7 its orthologs have been recognized in a wide range of eukaryotic taxa, including plants, protists, and invertebrates.8 This uniquely broad distribution has led researchers to suggest that it may have had a fundamental role in the evolution of sexual reproduction. 6 Despite its intriguing phylogenetic distribution, the protein’s mode of action remains largely uncharacterized. In several species, the protein has been demonstrated to have an essential role in mediating the coalescence of gametes: Functional studies of the protein in Chlamydomonas 9 demonstrated a role in gamete-gamete plasma membrane fusion, and the work to date in Plasmodium9,10 and Arabidopsis11 is consistent with a similar role. The exact mechanism by which the protein does this, however, has not been characterized. Few clues can be gleaned from a superficial examination of the amino acid sequence: other than a transmembrane domain (TMD) and a putative signal sequence, the protein has no known functional motifs. It also has no known interaction partners. GCS1’s lack of recognizable functional motifs has led to speculation that it may penetrate the opposing gamete’s plasma membrane directly, like a viral FAST protein,6 but this has not yet been explored experimentally. A number of promising avenues for elucidating the GCS1 mystery exist, however. One important route is functional study of the protein in a broader variety of species. To date, GCS1 has been studied in only a fraction of the model systems where its putative orthologs have been - 1 - discerned. The vast majority of published GCS1 studies have focused on angiosperms, malarial parasites, and green algae. Though potential orthologs have recently been recognized in a Mycetozoa, Cilliatea, and Euglenazoa,12 it has never been studied functionally in any of those taxa. In animals, putative orthologs have been identified in Hydra and Nematostella,8 as well as in several species of arthropods;13 yet a single whole-organism in situ hybridization study constitutes the whole of the published, experimental attention its expression has there received.14 Study of GCS1s in these systems has the potential to reveal both new fusion mechanisms and new gamete interaction pathways. Additionally, studying the protein’s evolved functions across many taxa has the potential to help illuminate the workings of the common ancestral form, and thus has the potential to illuminate the evolution of sexual reproduction itself.6 Exploration of the protein’s mode of operation in new species is therefore an important avenue for research. Very recently, a large-scale computational genomics project identified potential GCS1 orthologs in several Drosophilid species.13 For several reasons, the D. melanogaster system is a particularly apt one for study of male-specific membrane-bound gamete proteins: Firstly, the stages of spermiogenesis in D. melanogaster are well characterized.15 Secondly, because tail elongation and cytokinesis are extremely membrane-intensive, the endomembrane system in developing sperm is highly sensitized, allowing interrogation of mechanisms that would otherwise be shrouded by redundancy.16 Along with ease of husbandry, and quick generation time17, there is a wealth of genetic and cytological tools available. Most importantly, the Wakimoto lab has isolated the largest collection of male sterile mutant strains existing for any organism to date. The availability of these powerful experimental tools predict that functional studies of GCS1 in D. melanogaster are likely to be highly profitable. The confirmed presence of a Drosophila GCS1 ortholog would open up a number of promising lines of inquiry. The purpose of this project, therefore, is to resolve the question of whether a GCS1 ortholog is involved in male fertility in Drosophila. The literature regarding GCS1 is here reviewed, and a promising male sterile mutant from the Wakimoto lab is analyzed. Evidence regarding the mutant’s genotype and phenotype is then evaluated in light of current knowledge of GCS1 and Drosophila spermiogenesis, and predictions are made about how a GCS1 protein might function in Drosophila. - 2 - RESULTS A. Transgenic rescue of pskl- by the putative Drosophila melanogaster GCS1. The mutations under study in this work were identified and partially characterized prior to this project’s beginning. Originally recovered in a large-scale screen of EMS-induced male sterile mutations,18,19 psklZ3119, psklZ 3059, and psklZ0070 males were sterile despite producing motile sperm that could enter eggs, and had been shown to be allelic via complementation analyses. Meiotic recombination and deficiency mapping limited the pskl gene to a 66.55 kb region on chromosome 3R that included 17 genes annotated genes. One of these was CG34027, an uncharacterized gene that had recently been identified by OrthoDB as a potential ortholog of GCS1.13 The similarities between the pskl- phenotype and the GCS1 mutant phenotypes observed in Arabidposis11, Chlamydomonas9, and Plasmodium9 and were striking. In all of these, mutants produced male gametes that matured normally, and that could reach and make contact with female gametes (albeit inefficiently in Arabidopsis).20 Despite contact between the gametes, in all of these, a complete or nearly complete block to fertilization was observed. The mechanisms underlying the pskl and GCS1 mutant phenotypes furthermore also appeared to have certain commonalities: Strong evidence from Chlamydomonas9 had demonstrated a role in gamete plasma membrane interaction for GCS1, and studies in Arabidopsis11 and Plasmodium9,10 suggested that the same was true in these systems. Activity at the gamete plasma membrane seemed likely to be affected by the Pskl protein as well. The pskl mutations had been classified as one of five members of the snky class of male sterile mutations. The founding member, snky, was found to permit the formation of motile sperm that are capable of entering the egg but result in failure in sperm plasma membrane breakdown (PMBD), an essential step that occurs in the egg cytoplasm in Drosophila fertilization.21,22 Similar dynamics were also observed with mutations in the misfire 23,24, aghino, and kugi genes. CG34027 also showed a promising expression profile. A genome-wide microarray study showed that expression of CG34027 is highly enriched in the testis.25 None of the other 16 genes within the 66.55 kb region-of-interest showed similar patterns of enrichment. - 3 - Given these parallels, we decided to test whether lesions in CG34027 were responsible for the pskl- phenotype. To do this, we performed a transgenic rescue using a 10.323 kb KpnI genomic fragment containing CG34027. An insertion of the transgene was obtained via P- element germ line transformation. This transgene was able to rescue fertility in pskl/Df males completely or nearly completely (Table 1). We therefore concluded that the 10.323 kb fragment contains all of the sequences necessary for pskl expression and function. Since the fragment contains only one predicted gene, CG34027, we can further conclude that pskl and CG34027 are one in the same. B. CG34027/pskl as an ortholog of GCS1. The suggestion that CG34027 could be a D. melanogaster ortholog of GCS1 comes from OrthoDB,13 which identified it as being a member of a protein family that included previously- identified GCS1s from the louse Pediculus 14 and the beetle Tribolium.9 This proposal conflicted with the findings of several recently published phylogenetic studies of GCS1,9,14 which had expressly concluded that GCS1 did not exist in Drosophila. To resolve the conflict, we created - 4 - an alignment of CG34027 with other identified arthropod orthologs of GCS1 (Fig. 1). We also surveyed the extant GCS1 literature to identify common traits regarding subcellular localization, amino acid sequence, and potential functional domains, which we compared with the predicted amino acid sequence and functional domains for CG34027. Finally, we sequenced CG34027 in the three pskl strains to determine whether the locations of the lesions were consistent with a GCS1 identity (Fig. 2). 1. CELLULAR AND SUBCELLULAR LOCALIZATION a. In Arabidopsis, Chlamydomonas, and Plasmodium, GCS1 is a sex-specific,
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