Molecular Biology of Bhlh PAS Genes Involved in Dipteran Juvenile Hormone Signaling

Molecular Biology of bHLH PAS Genes Involved in Dipteran Juvenile Hormone Signaling

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Aaron A. Baumann. B.S.

Graduate Program in Entomology

The Ohio State University

2010

Dissertation Committee:

Thomas G. Wilson, Advisor

David Denlinger

H. Lisle Gibbs

Amanda Simcox

Aaron A. Baumann

2010

Abstract

Methoprene tolerant (Met), a member of the bHLH-PAS family of transcriptional

regulators, has been implicated in juvenile hormone (JH) signaling in Drosophila melanogaster. Met mutants are resistant to the toxic and morphogenetic defects of exogenous JH application. A paralogous gene in D. melanogaster, germ cell expressed

(gce), forms JH-sensitive heterodimers with MET, but a function for gce has not been reported.

DmMet orthologs from three mosquito species are characterized and, based on sequence analysis and intron position, are shown to have higher sequence identity to

Dmgce than to DmMet. An evolutionary scheme for the origin of Met from a gce-like

ancestor gene in lower Diptera is proposed. RNAi-driven underexpression of Met in the

Yellow Fever mosquito, Aedes aegypti, results in the concomitant reduction of putative

JH-inducible genes, suggesting involvement in JH signaling.

The viability of D. melanogaster Met mutants is thought to result from functional

redundancy conferred by gce. Therefore, genetic manipulation of gce expression was

used to probe the function of this gene. Overexpression of gce was shown to alleviate

preadult, but not adult Met phenotypes. RNAi-driven underexpression of gce resulted in

preadult lethality in both Met+ and Met mutant backgrounds. Therefore, unlike Met, gce is a vital gene.

Evolutionary analysis of 12 Met and gce orthologs showed that these genes are conserved across the genus Drosophila. Additionally, distinct signatures of selective pressure were identified in Met and gce via dN/dS analysis. The paucity of introns in Met relative to gce supports the notion of a retrotransposition mechanism of duplication, through which Met arose from a gce-like ancestor following the divergence of higher and lower Diptera. Furthermore, RT-PCR analysis revealed discrete embryonic expression profiles for Met and gce. Together, these results show a degree of post-duplication subfunctionalization.

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Dedication

To my parents and grandfather for their love and encouragement.

To my wife, Rachelle, for her patience during my pursuit of this endeavor.

Acknowledgements

I am indebted to my major advisor, Dr. Thomas Wilson, for his guidance and support throughout my graduate studies. When my confidence was shaken during bouts of frustrating results, a brief consultation with Tom allowed me to regain my poise, to try again, and ultimately to succeed. His mentorship afforded me valuable opportunities to explore and grow within the laboratory, and encouraged development of a self-motivated attitude that will no doubt serve me in the future.

I thank the members of my dissertation committee, Drs. Dave Denlinger, Amanda

Simcox, and Lisle Gibbs, for their comments and suggestions during committee meetings. Additionally, I extend thanks to Dr. Woodbridge Foster for guiding me through the process of rearing mosquitoes and for allowing me to maintain A. aegypti colonies in his laboratory space.

I am extremely grateful for Dr. Shaoli Wang’s instruction in various molecular techniques that were instrumental to my ability to design and perform much of the work presented herein.

Finally, I would like to thank Dr. Joshua Benoit for his assistance and encouragement, and my good friend Chris Herman for helping me with various manual tasks.

Vita

March 30, 1982……………………………………. Born-West Allis, Wisconsin

2000………………………………………………... Centerville High School

2003-2004………………………………….. ……... Student Research Assistant The Ohio State University

2004……………………………………………….. B.S., The Ohio State University

2005-2010…………………………………………. Graduate Teaching / Research Associate The Ohio State University

Publications

1. Baumann, A., Wilson, T.G., Barry, J., Wang, S. Juvenile hormone action requires paralogous genes in Drosophila melanogaster. Genetics. 185:1327-1336.

2. Baumann, A., Fujiwara, Y., Wilson, T.G. 2010. Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila. Journal of Insect Physiology. 56:1445-1455.

3. Wang, S., Baumann, A., Wilson, T.G. Drosophila melanogaster Methoprene-tolerant (Met) gene homologs from three mosquito species: members of PAS transcriptional factor family. Journal of Insect Physiology. 2007; 53:246–253.

Field of Study

Major field: Entomology

Table of Contents

Abstract…………………………………………………………………….. ……... ii

Dedication………………………………………………………………….. ……... iv

Acknowledgements………………………………………………………………… v

Vita…………………………………………………………………………………. vi

List of Tables………………………………………………………………………. vii

List of Figures……………………………………………………………………… ix

Chapters

1. Introduction………………………………………………………… ……... 1 Juvenile hormone in insect growth and development……………… 1 Juvenile hormone in insect reproduction…………………………... 3 Juvenile hormone structure………………………………………… 4 Synthesis and metabolism………………………………………….. 6 JH agonists: insecticidal use……………………………………….. 7 Molecular mechanisms of JH signal transduction…………………. 9 Molecular crosstalk between 20E and JH signaling pathways…….. 13 Directions………………………………………………………….. 16 Research goals……………………………………………………... 16 References………………………………………………………….. 18 Illustrations………………………………………………………… 24

2. Drosophila melanogaster Methoprene-tolerant (Met) gene homologs from three mosquito species: Members of PAS transcriptional factor family………………………………………………………………. ……....26 Abstract…………………………………………………………….. 26 Introduction………………………………………………………… 27 Materials and Methods…………………………………………….. 30 Results……………………………………………………………… 35 Discussion………………………………………………………….. 39 References………………………………………………………….. 43

vii

Illustrations………………………………………………………… 47

3. Paralogous Genes Involved in Juvenile Hormone Action in Drosophila melanogaster……………………………………………………………….. 57 Abstract…………………………………………………………….. 57 Introduction………………………………………………………… 58 Materials and Methods……………………………………………... 60 Results……………………………………………………………… 69 Discussion………………………………………………………….. 77 References………………………………………………………….. 83 Illustrations………………………………………………………….87

4. Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila………………. 98 Abstract…………………………………………………………….. 98 Introduction………………………………………………………… 99 Materials and Methods……………………………………………... 102 Results……………………………………………………………… 109 Discussion………………………………………………………….. 117 References………………………………………………………….. 122 Illustrations………………………………………………………….128

Conclusions………………………………………………………………………… 140

Appendix A: Revision of the genomic sequence of D. melanogaster gce………….147

Bibliography……………………………………………………………………….. 154

viii

List of Tables

Table Page

1.1 Representative table of some JH inducible genes in Drosophila melanogaster………………………………………………………………. 24

2.1 Gene-specific primers used for 5’ and 3’ RACE PCR amplification of mosquito Met homologs……………………………………………………………… 47

2.2 Amino acid identities among mosquito Met and Drosophila Met and gce genes…………………………………………………………… ……... 48

2.3 Primers used for RT-PCR analysis of dsMet and dsß-gal-injected Aedes aegypti…………………………………………….. 49

3.1 Number of defective eye facets in gce overexpressing and underexpressing strains of D. melanogaster………………………………………………………. 87

3.2 Larval and pupal survival rates of gce-dsRNA strains of D. melanogaster………………………………………………….……... 88

3.3 Pupal survival (%) of gce-dsRNA strains of D. melanogaster…………….. 89

4.1 Flybase (http://www.FlyBase.org) annotation symbols for Met and gce orthologs of 12 species of Drosophila……………………………………... 128

4.2 Primer sequences used for RT-PCR of selected gce orthologs……………. 129

4.3 Sequence identity matrices for gce and CG15032 orthologs in 12 species of Drosophila………………………………………………………………….130

4.4 Sequence identity matrices calculated from the amino acid sequences of functional domain and open reading frames of Met and gce orthologs from D. melanogaster, three mosquito species, and Tribolium castaneum….…….. 131

List of Figures

Figure Page

1.1 Representative juvenile hormones and juvenile hormone analogs………… 25

2.1. Multiple alignment of mosquito Met and D. melanogaster Met and gce amino acid sequences………………………………………………………. 50

2.2 RT-PCR products amplified from D. melanogaster and Aedes aegypti using degenerate or D. melanogaster Met-specific primers……………….. 53

2.3 Phylogenetic tree produced using Met and gce amino acid sequences from members of Culicidae and Drosophilidae………………………………….. 54

2.4 Relative expression of putative JH-inducible genes in dsMet and dsß-gal-injected A. aegypti………………………………………………… 55

2.5 Developmental expression profile of AaMet………………………………. 56

3.1 Northern blot analysis of the developmental expression of gce during D. melanogaster development………………………………………………… 90

3.2 RT-PCR analysis of Met and gce in selected tissues of D. melanogaster………………………………………………….…………91

3.3 Expression of gce in UAS-gce-overexpressing flies………………………. 92

3.4 Enhanced methoprene pupal toxicity of gce-overexpressing flies………… 93

3.5 Rescue of resistance to methoprene-induced male genitalia malrotation during pupal development in gce-overexpressing flies …………………… 94

3.6 Rescue of eye phenotype in Metw3 flies overexpressing gce………………. 95

3.7 Fertility of adult D. melnoagster overexpressing gce…………………….. 96

3.8 RT-PCR analysis of gce expression in UAS-dsRNA-gce larvae …………..97

4.1 Multiple sequence alignments of the functional domains in Met-like genes in Drosophila, mosquitoes, and Tribiolium……………………………….. 132

4.2 Intron position of Drosophila Met and gce orthologs as superimposed on the accepted phylogeny of the represented species………………………… 133

4.3 The revised genomic structure of D. melanogaster gce…………………….134

4.4 Multiple sequence alignment showing continuity of CG15032 and the bHLH domain of D. grimshawi and D. willistoni gce orthologs…………………...135

4.5 Microsynteny of CG15032 and gce orthologs within the genus Drosophila…………………………………………………………………. 136

4.6 RT-PCR analysis of embryonic Met and gce expression in D. melanogaster………………………………………………………………. 137

4.7 Plots of dN/dS ratios across the Met and gce coding sequences of 12 Drosophila species………………………………………………………… 138

4.8 Phylogenetic tree of available holometabolous Met and gce genes………...139

A.1 RACE PCR product obtained for D. melanogaster gce, including the 5’ region formerly annotated as CG15032…………………………………………… 148

Introduction

Juvenile hormone in insect growth and development

Insect development takes place through a series of discrete growth intervals termed instars. At the beginning of each instar, the deposition of a sclerotized, chitinous

exoskeleton establishes a physical boundary to limit growth during that developmental

stage. Developmental cues at the end of an instar cause the shedding of and ecdysis from

the previously deposited cuticle, and the deposition of a new, larger cuticle that defines

the maximal growth of the following instar. The events that occur during the

developmental transition from one instar to the next are referred to as molting.

In hemimetabolous insects, the procession of larval molts produces a series of

smaller but morphologically similar iterations of the adult. This direct, isomorphic

development defines incomplete metamorphosis. In contrast, holometabolous insects

undergo complete metamorphosis; holometabolous larvae progress through sequentially

larger, generally vermiform (i.e., maggots, caterpillars, and grubs), immature stages that

are morphologically distinct from the adult. In both groups, a hormonally controlled

developmental switch in the final instar initiates the commitment to adult development.

In the Holometabola, this decision precipitates the metamorphic molt to a transitory stage

called the pupa, from which the adult will ultimately eclose. Holometabolous

development is proposed to have allowed the exploitation of distinct ecological niches by

adults and immatures, facilitating the enormous evolutionary success of the

Holometabola (Truman and Riddiford, 1999). As an illustration of this success, an

estimated 75% of animals on Earth are beetles.

Insect molts are initiated by 20-hydroxy ecdysone (20-E), the so-called molting

hormone. Ecdysone is a steroid hormone produced by the prothoracic gland in response

to the release of prothoracicotropic hormone from the lateral neurosecretory cells in the

brain. It has long been known that ecdysteroids (ecdysone and structurally related

derivatives) exert their activity at the level of transcription, since pulses of ecdysone

induce transcriptional puffs in the polytene chromosomes of Drosophila melanogaster

larval salivary glands (Ashburner, 1972).

While the initiation of a molt is always triggered by ecdysone, the nature of the

molt is mediated by the interaction of ecdysone with juvenile hormone (JH), a product of

the corpus allatum (CA). When 5th instar Rhodnius prolixus hemipteran nymphs are

either parabiosed (surgically fused; the insects share a common hemolymph pool) to

earlier instar nymphs, or implanted with active CA, the 5th instar animals produce another set of immature, rather than adult, phenotypic characteristics (Wigglesworth, 1934).

Therefore, in the presence of JH, ecdysone directs the transition to a subsequent immature instar.

Conversely, surgical ablation of the CA results in a dramatic decrease in JH titer

and can elicit precocious metamorphosis. The metamorphic molt is achieved when

ecdysone, in the absence of JH, induces expression of genes whose products direct the

production of adult rather than larval structures (Zhou et al., 1998). Thus, it was

proposed that JH can modulate the expression of ecdysone-inducible genes (Hiruma et

al., 1999). Table 1.1 provides a representative list of some JH-inducible genes.

Juvenile hormone in insect reproduction

Following pupal development, the interaction of ecdysone and JH plays a pivotal

role in insect reproductive biology. In female insects, the activity of these counteracting

hormones is critical for oocyte development and maturation. Development of the D.

melanogaster oocyte is under the control of JH through previtellogenic stages 8-9. D.

melanogaster females with the apterous4 mutation are both sterile and JH-deficient

owing to reduced levels of JH synthesis (Bownes, 1989). These females synthesize yolk

proteins (vitellogenins; Vg), but Vg deposition in the oocyte is impaired (Gavin and

Williamson, 1976). JH treatment can rescue vitellogenic oocyte development in ap

females (Postlethwait and Weiser, 1973). Previtellogenic growth in Aedes aegypti is

likewise under the control of JH (Clements, 1992), whose primary role seems to be to

induce 20-E competence in the fat body, the site of post-blood meal Vg synthesis. In

contrast, vitellogenesis is retarded by JH treatment in the gypsy moth, Lymantria dyspar

(Davis et al., 1990), showing variation in hormonal control in insects.

In males, various aspects of accessory gland (MAG) function and courtship are under the control of JH. JH was first isolated in large quantities from the MAG of

Hyalophora cecropia (Williams, 1956), suggesting a role in male reproductive biology.

When adult male Tribolium castaneum are fed hydroprene, a JH agonist, an increase in mRNA and protein accumulation is observed in the MAG, with concomitant increase in

MAG size (Parthasarathy, et al., 2009). RNAi-driven reduction of JHAMT (JH acid methyl transferase, an enzyme in the JH biosynthetic pathway) expression and the

resultant reduction in JH inhibit male courtship and expression of accessory gland

proteins in T. castaneum. Similarly, in D. melanogaster, JH controls MAG protein

accumulation (Yamamoto et al., 1988) and male ap mutants court females less vigorously

than wild-type flies (Tompkins, 1990).

Juvenile Hormone Structure

Chemical analysis first resolved the sesquiterpenoid structure of endogenous JH

(Röller et al., 1967). Several homologs exist, each bearing opposing, terminal epoxide

and methyl ester functions (Figure 1.1). Variation in the degree and identity of alkyl group substitution at C3, C7, and C11 along the carbon skeleton defines the originally

identified homologs. The convention established for naming JH homologs reflects the number of carbon atoms; homologs of higher carbon count are given smaller numerical identifiers. JH 0, I, and II, are characterized by the presence of ethyl side chains, thus increasing the total carbon count, whereas methyl side chains characterize the 16-carbon

JH III. The major JHs are presented in Figure 1.1.

The evolutionary importance of multiple JH homologs is unclear. JH 0, I, II, and

III have all been isolated from members of the Lepidoptera. JH III is common throughout Hexapoda, including the most primitive, and is presumed to be the evolutionary precursor to the higher homologs. Dipteran insects, upon which this dissertation focuses, primarily produce JH III. JH bisepoxide (JHB3) has been identified as a product of the CA in higher Diptera including D. melanogaster (Richards et al.,

1989) and S. bullata (Bylemans et al., 1998) and of the CA and MAG of a lower dipteran, Aedes aegypti (Borovsky et al., 1994). Almost identical in structure to JH III,

JHB3 is distinguished by an additional epoxide group spanning C6-C7. JHB3 has only been isolated in vitro as a product of cultured CA and may be an oxidation product of JH

III.

Synthesis and Metabolism

Schooley et al., (1976) first elucidated the biosynthetic scheme for the production of the JH carbon skeleton. The biosynthesis of JH according to Klowden (2007) follows:

JH synthesis in the CA begins when three molecules of acetate are combined to form mevalonic acid, from which isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are formed. IPP and DMAPP are combined in a ratio of 2:1 to form farnesyl pyrophosphate (FPP). In a series of steps, FPP is converted to farnesol and then to the penultimate precursor, farnesoic acid. JH III is produced by the esterification and epoxidation of the opposing ends of farnesoic acid. The ethyl side chains found in higher homologs originate from the initial incorporation of homomevalonic acid precursors.

In a generalized insect body plan, the CA exist as paired glands posterior to and directly innervated by the brain. In higher Diptera, the fusion of the paired CA, corpora cardiaca, and prothoracic gland, forms a circular structure called the ring gland. JH biosynthesis in the CA is the primary determining factor of an insect’s JH titer. Once released into the hemolymph, lipophilic JH molecules are transported to target tissues via

JH binding proteins that additionally protect the hormone from specific and nonspecific esterases (Whitmore and Gilbert, 1972).

At certain times during insect development, when JH must be cleared from the

hemolymph, specific enzymes target the characteristic epoxide and methyl ester moieties.

JH esterase (JHE) converts JH to JH acid via hydrolysis of the methyl ester function,

while JH epoxide hydrolase attacks the epoxide function at C10-C11. The combined

activity of these enzymes produces JH acid diol, which is excreted (Klowden, 2007).

Experimental procedures have been developed to manipulate JH titer, including genetic ablation of the CA using targeted expression of the cell death genes (Liu et al., 2009;

Riddiford et al., 2010), ectopic overexpression of JH esterase (Tan et al., 2005), and

RNA interference-based knockdown of JH biosynthetic enzymes (Jones et al., in press;

Parthsarathy et al., 2009).

JH Agonists: Insecticidal Use

The physiology and chemistry of JH prompted intense research into the synthesis and commercial-scale production of JH analogs (JHAs), or juvenoids, for agricultural use. The allure of these compounds was at least twofold. First, juvenoids exhibit extremely low non-target (in particular, mammalian) toxicity. Second, it was originally thought that insect resistance to JHAs was unlikely, since an insect was not likely to become refractory to an endogenous hormone (Williams, 1967).

Dozens of JHAs show activity in Tenebrio molitor and Galleria mellonella bioassays (Henrick, et al., 1976). Intriguingly, JHAs need not mimic the chemical structure of endogenous JH, as exemplified by the pyridine-based pyriproxyfen (see

Figure 1.1), whose activity exceeds JH by two orders of magnitude in white puparial and larval assays (Riddiford and Ashburner, 1991). Methoprene, a juvenoid structurally similar to endogenous JH, is widely used in soluble formulations to manage populations of larval mosquitoes (e.g. Cornel et al., 2002). Owing to its extremely low vertebrate toxicity, many commercially available flea and tick medications for domestic animals utilize methoprene.

The dipteran response to JHA treatment is somewhat atypical. In Diptera, adult structures arise from imaginal discs. When flies are challenged with JHAs, adult structures that differentiate from imaginal discs remain unaffected (Postlethwait, 1974).

Diagnostic (sub lethal) doses of methoprene cause disruption of abdominal bristle formation in female D. melanogaster (Madhavan, 1973), and malrotation of the male genital disc (Postlethwait, 1974). The latter phenotype has reproductive consequences, since affected males are unable to mate (Bouchard and Wilson, 1987). Similarly, methoprene application results in a delay of male genital rotation in A. aegypti

(O’Donnell and Klowden, 1997).

JHA application at the onset of dipteran metamorphosis results in dose-dependent lethality at the pharate adult stage. This is in striking contrast to members of Lepidoptera and Coleoptera (Srivastava and Srivastava, 1983; Konopova and Jindra, 2007), in which the initiation of the pupal program is delayed until after the development of one or more supernumerary larval instars. In these insects, pupal JHA application can result in the deposition of a second pupal cuticle (Zhou and Riddiford, 2002).

Molecular mechanism of JH signal transduction

The Methoprene-tolerant (Met) locus was identified by Wilson and Fabian (1986) by screening progeny of ethyl methanesulfonate (EMS)-mutagenized D. melanogaster for resistance to methoprene. Met mutants displayed dramatically enhanced (~100 fold) resistance to methoprene exposure, but not to other classes of insecticides. Met mutants showed resistance to the previously mentioned morphogenetic defects caused by methoprene (Wilson and Fabian, 1986) and a more potent JHA, pyriproxyfen (Riddiford and Ashburner, 1991), suggesting that Met could function as a JH receptor. MET was shown to bind JH with nanomolar affinity (Shemshedini and Wilson, 1990) and MET product was identified in the nuclei of several known JH target tissues, including ovary,

MAG, and larval fat body (Pursley et al., 2000), further satisfying criteria of a hormone

receptor. Additionally, recombinant MET was able to drive the expression of a reporter

gene in a JH-sensitive manner (Miura et al., 2005).

Cloning and sequence analysis identified Met as a member of the anciently

conserved basic Helix-Loop-Helix Period Ahr Sim (bHLH PAS) family of transcriptional

regulators (Ashok et al., 1998). bHLH PAS proteins are manifest in a variety of

organisms, serving a diverse array of functions in development, xenobiotic binding, and

detection of environmental signals (Crews, 1993). bHLH PAS genes function as homo-

or heterodimers to affect gene regulation. Dimerization is facilitated by both the HLH

domain and PAS repeats (PAS A and PAS B), the latter functioning in both protein-

protein interaction and ligand binding (Crews, 2003). Dimerized PAS proteins bind

DNA sequences in the promoter region of target genes via the basic (bHLH) residues that

precede the HLH motif. Each dimerization partner recognizes and binds one half of a

palindromic E-box consensus sequence CANNTG.

Analysis of the null Met27 allele provided the first demonstration of insecticide resistance due to the absence of a target macromolecule (Wilson and Ashok, 1998). This

EMS-induced null allele contains a lesion in the 5’ untranslated region. Met27 flies are viable, but females experience substantially reduced oogenesis, consistent with the role for JH in this physiology. That Met27 flies survive to adulthood suggests that 1) MET

does not act alone as the JH receptor, or 2) there exists an alternative mechanism(s)

through which JH signaling is coordinated, since absence of the true JH receptor should preclude normal development. Indeed, it has been proposed that other, Met-independent

(but uncharacterized) mechanisms of JH signal transduction exist (Flatt et al., 2008;

Riddiford, 2010). However, the results of Konopova and Jindra (2007) clearly implicate a homolog of Met in the metamorphic activity of holometabolous insects.

To identify potential binding partners for MET, Godlewski et al., (2006)

employed column pulldown assays in Drosophila S2 cells, demonstrating that 1) MET

forms homodimers and 2) MET can bind the product of germ cell expressed (gce), a

bHLH PAS gene with high sequence identity to Met. Furthermore, the formation of

MET:MET or MET:GCE dimers is JH-dependent; addition of JH or either of two JHAs

substantially reduces interaction between MET and GCE. Structure-function analyses

using site-directed mutagenesis identified regions of MET that are necessary for dimer

formation. Point mutations in the HLH and PAS A domains (Met1 and Met3 alleles,

respectively) had no effect on partner binding. N- and C-terminal truncations, deletions in the HLH or PAS A domains, and a point mutation in the PAS B domain (Met128 allele) each inhibited dimerization (Godlewski, et al., 2006).

Gce, originally identified by Moore et al., (2000), was named for its expression in a subset of embryonic germ cells. The function of GCE has not been determined, nor is it known whether this bHLH PAS protein forms homodimers, like MET. Godlewski et

al., (2006) suggest that changes in the JH titer during development could influence the

stoichiometric equivalents of MET:MET and MET:GCE complexes. Stage-specific formation of these alternative dimers could have unique regulatory consequences on distinct suites of target genes. GAL4/UAS-driven (Brand and Perrimon, 1993) overexpression of Met+ from actin or tubulin promoters results in larval lethality in the absence of methoprene (Barry et al., 2008), perhaps by upsetting this stoichiometry and

favoring MET:MET formation at inappropriate times. MET can repress expression of a

reporter gene in the absence of JH (Miura et al., 2005), a phenomenon previously

reported in other PAS proteins (Dolwick et al., 1993). Recently, JH was shown to

counteract MET and GCE to inhibit the caspase-induced autolysis of larval fat body that

occurs during metamorphosis (Liu et al., 2009).

In D. melanogaster, USP binds JH III with micromolar affinity; the amount of ligand necessary for Kd saturation exceeds physiological levels by two orders of

magnitude (Jones et al., 2001). Therefore, it was proposed that JH III may

nonspecifically bind USP after saturating its true receptor (Jones et al., in press). In vitro

production of methyl farnesoate (MF) by cultured CA of A. aegypti binds Ultraspiracle

(USP; the invertebrate homolog of the retinoic acid receptor, RXR) with nanomolar affinity (Jones et al., in press) and MF exhibits increased JHA activity relative to JH III in larval assays (Harshman, et al., in press). However, no role for MF, the crustacean

juvenile hormone, is known for Diptera. Nevertheless, the proposal that alternative farnesol derivatives act at different times in development through distinct sets of specific receptors is intriguing. In particular, it is unknown whether GCE serves as a receptor for any farnesoids.

Molecular crosstalk between JH and E20 signaling pathways

Ecdysone is converted to the active form, 20-hydroxy-ecdysone (20-E) in target tissues. The lipophilic steroid hormone penetrates cell membranes to regulate gene transcription through a heterodimeric receptor complex comprised of two nuclear receptors: the ecdysone receptor (EcR), to which 20-E physically binds, and USP, the ortholog of the vertebrate retinoic acid receptor (RXR). When bound with 20-E, the

USP:ECR complex recognizes and binds ecdysone response elements (ERE) located in the promoter region of target genes, inducing a transcriptional cascade comprised of hierarchical network of early and late genes. The early genes either repress their own expression in the absence of 20-E, or induce expression of late genes (Ashburner et al.,

1974). Thus, the transcriptional events following E-20 induction are tightly regulated.

One of the early 20-E induced genes, Broad Complex (broad or BR-C), encodes a family of four alternatively spliced zinc finger transcription factors Z1-Z4. During larval development in D. melanogaster, JH represses the action of broad. However, JH

application at pupariation induces a second wave of broad expression, resulting in deposition of a second pupal cuticle (Minakuchi et al., 2008).

Restifo and Wilson (1998) demonstrated that certain broad alleles show phenotypes similar to those seen with methoprene exposure. Furthermore, phenotypic synergism is evident in the Met and broad double mutants produced from crosses of Met and broad homozygous flies (Wilson et al., 2006), indicating that MET and BROAD proteins interact, and providing a molecular bridge between JH and 20-E mediated signaling pathways.

In T. castaneum, broad is downstream of Met in the JH signaling pathway during metamorphosis (Konopova and Jindra, 2008). broad expression directs the progressive development of the hemimetabolous Oncopeltus fasciatus; changes in the broad expression profile may have been a determinant in the divergence between hemi- and holometabolous insects (Erezyilmaz et al., 2006). In the latter, broad expression is confined to the prepupal stage, where it acts as a pupal specifier (Zhou and Riddiford,

2002). Loss of broad expression, characteristic of the npr1 mutant (non-pupariating; one of several broad complementation groups), is a failure to enter the pupal program.

The molecular networks that link JH and E20 signaling pathways form the foundation of multiple aspects of insect physiology, as evidenced by the criticality of both hormones in development, reproduction, and diapause (Zhou and Riddiford, 2002;

Soller et al., 1999; Denlinger, 1985). Bitra and Palli (2009) demonstrated physical

interaction of MET with both components of the ecdysone receptor complex, ECR and

USP, using both two hybrid assays in yeast and Drosophila L57 cells, as well as column pulldown assays. However, in this system, neither E20 or JH addition enhanced MET interaction with USP or ECR.

Li et al., (2007) compared microarray data from D. melanogaster and A. mellifera to identify a subset of conserved, JH-inducible genes. In the promoter region of 16 of the

D. melanogaster orthologs, a conserved JH response element (JHRE) was identified.

RNAi-driven reduction of the expression of two JHRE binding proteins, FKBP39 and

Chd64, inhibits JHIII-induced expression of a reporter construct. Column pulldown assays showed FKBP39 and CHD64 as binding partners of D. melanogaster ECR, USP,

and MET. FKBP39, which is present at the onset of metamorphosis (Riddiford, 2008), is

an inhibitor of autophagy in D. melanogaster; FKBP39 overexpression precludes the

developmental autolysis of larval fat body cells in wandering third instar larvae (Juhász et al., 2007). However, Chd64 is expressed during larval molts, but not in the third instar or during metamorphosis (Riddiford, 2008). Therefore, the developmental expression profiles of Chd64 and FKBP39 need to be accommodated in this model.

Directions

An explanation for the viability of Met mutants suggests that the paralog, gce, offers functional redundancy. However, studies regarding the crosstalk of JH and 20-E have largely ignored GCE, so it remains unclear whether GCE functions as a homodimer or if it can act to bind any of the farnesoid products of the CA. Work presented here and supported by independent research (Konopova and Jindra, 2007) shows that the Met-like genes in more primitive holometabolous insects more closely resemble gce at the genomic level. Therefore, an analysis of the evolution of Met and gce and the subsequent functional divergence of these proteins in higher Diptera will help elucidate the molecular mechanisms of JH activity.

Research goals

Incidence of insecticide resistance can result from chronic exposure of an insect population to a particular toxicant. Indeed, resistant populations of Ochlerotatus nigromaculis mosquitoes under methoprene treatment were identified in Fresno,

California (Cornel et al., 2002), prompting our investigation of the Met orthologs in lower Diptera, particularly Aedes aegypti, a close relative of O. nigromaculis that is amenable to laboratory rearing and experimental manipulation.

The present body of work aims to answer questions regarding the evolution and molecular biology of “Met-like” genes within Diptera, and to identify conserved functions for Met, gce, and their lower dipteran orthologs. Emphasis is placed on the families Drosophilidae and Culicidae. The former family is represented by D. melanogaster, a model organism upon which much of modern genetics is based, and 11 other members of the genus Drosophila, whose genomes were recently elucidated

(Ashburner, 2007). Culicidae is a family of evolutionarily primitive Diptera, represented by numerous medically important disease vectors, including Anopheles gambiae, a primary vector of the malaria parasite, and Aedes aegypti, the yellow fever mosquito.

The primary goals of this dissertation research are:

1. Identify and characterize the Met orthologs in several medically important

mosquito species and propose a scheme for the evolutionary history of Met and

Met-like genes within the order Diptera.

2. Identify a role for gce product via over- and under-expression of gce in Met+ and

Met mutant backgrounds in D. melanogaster.

3. Identify and describe the Met and gce orthologs among members of the genus

Drosophila and examine the signatures of selection present across Met and gce

coding sequences.

A comparative genomics approach is used to investigate the evolutionary history

of Met and gce since the divergence of higher and lower Diptera. The results of this work

prompted an investigation into the molecular function of gce. Consequences of the

genetic manipulation of the expression of D. melanogaster gce and its putative ancestor

in A. aegypti are used to elucidate evolutionarily conserved or divergent molecular

mechanisms that form the foundation of JH signal transduction in Diptera.

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Symbol Gene name Molecular function Reference JhI-1 Endoribonuclease JH inducible protein 1 Dubrovsky et al., Unknown 2000 JhI-26 JH inducible protein 26

BTB POZ zinc finger Zhou and Riddiford, Br Broad-Complex (BR-C) transcription factor 2002

Amino acid transmembrane mnd Minidisks transporter Dubrovsky et al., Amino acid 2002 transmembrane JhI-21 JH inducible protein 21 transporter

JH-specific esterase JHE JH esterase Kethidi et al.., 2005

Ecdysone-induced Heme binding Dubrovsky et al., E75A protein 75B 2004 RNA polymerase II E74B Ecdysone-induced transcription factor protein 74EF activity

Beckstead et al., Phosphoenolpyruvate 2007 Phosphoenolpyruvate carboxykinase (GTP) pepck carboxykinase activity

Unknown CG14949 CG14949

Table 1.1. Representative table of some D. melanogaster JH-inducible genes. The molecular function for each gene is according to FlyBase (http://www.FlyBase.org).

Figure 1.1. Molecular structure of the major JH homologs and some representative JHAs. From Klowden, 2007.

Chapter 2

Drosophila melanogaster Methoprene-tolerant (Met) gene homologs from three mosquito species: Members of PAS transcriptional factor family

Abstract

The Methoprene-tolerant (Met) gene in Drosophila melanogaster has been shown

to function in juvenile hormone (JH) action. Met homologs were isolated from three

mosquito species, Culex pipiens, Aedes aegypti and Anopheles gambiae. Sequence

similarity was found to be high among conserved domains, and the majority of the 7–9

introns in AaMet and AgMet are located in either identical or similar positions, indicating evolutionary relatedness. Sequence comparison with Met and the similar germ cell

expressed (gce) gene in D. melanogaster showed that the mosquito genes are more

similar to gce than to Met. Moreover, the multiple introns in AgMet and AaMet are more

similar in number with the 7 introns in Dmgce than to the single intron in DmMet; in fact,

six intron positions in AaMet and AgMet are similar to those in Dmgce. Efforts to

identify a second homologous gene in mosquitoes were unsuccessful, suggesting a single

gene in lower Diptera, consistent with the single gene uncovered in genomic sequencing

of A. aegypti and A. gambiae. Additionally, RNAi-driven underexpression of Met in A.

aegypti resulted in reduced expression of some putative JH-inducible genes, indicating

that AaMet may function in mosquito JH signaling. These results suggest that a gene

duplication occurred during the evolution of higher Diptera, resulting in Met and gce.

Introduction

Juvenile hormone (JH) is involved in a variety of important functions in insects, including development, reproduction, caste determination, and behavior (Riddiford, 1985

and Riddiford, 1994; Nijhout and Wheeler, 1982). Although JH is a necessary molecule

at certain time in insect development, it is toxic when present during early metamorphosis in certain insects, especially Diptera (Wilson, 2004). Several chemical companies have taken advantage of this characteristic and synthesized chemical analogs of JH (JHA) having insecticidal activity for certain insects (Staal, 1975; Mulla, 1995; Ritchie and

Broadsmith, 1997). Perhaps the most successful JH analog is methoprene (isopropyl 11- methoxy-3, 7, 11-trimethyl-2, 4-dodecadienoate) (Henrick et al., 1973), which is very effective against dipteran insects (Staal, 1975; O’Donnell and Klowden, 1997).

However, the wide use of methoprene for mosquito control in recent years is perhaps exacting a toll: resistance evolution. Methoprene resistance has been shown in two

Florida populations of Aedes taeniorhynchus (Dame et al., 1998) and in California

populations of Ochlerotatus nigromaculis (formerly Aedes nigromaculis; Cornel et al.,

2000 and Cornel et al., 2002).

The Methoprene-tolerant (Met) gene was identified due to its involvement in

methoprene resistance in Drosophila melanogaster. Examination of Met allele

phenotypes and rescue by Met+ transgenes clearly showed Met to be directly responsible for JHA resistance as well as its involvement in a JH-mediated physiology, that of oogenesis (Wilson and Fabian, 1986; Wilson et al., 2006a; 2006b). Isolation and sequence analysis of Met showed this gene to be a member of the bHLH-PAS family of transcriptional regulators (Ashok et al., 1998). Met is proposed to be a component of the elusive JH receptor, based on the mutant phenotype (Wilson and Fabian, 1986), the binding of JH III by MET, its ability for transcriptional activation (Miura et al., 2005),

and its genetic interaction with the 20-hydroxyecdysone primary response gene, Broad-

Complex (Wilson et al., 2006a; 2006b). Another D. melanogaster bHLH-PAS gene, germ

cell expressed (gce), shows more than 50% sequence identity to Met (Moore et al., 2000);

however, a function for gce has not been reported. Both genes are also present in the

recently sequenced and annotated Drosophila pseudoobscura genome and have high

identity with those genes in D. melanogaster.

We wanted to determine if Met and/or gce homologs exist in mosquitoes, which

might allow identification of the responsible gene (s) for resistance to methoprene in

mosquitoes. Using degenerate primers from known Met genes combined with RACE-

PCR, we isolated and sequenced Met cDNAs in three different mosquito species. The mosquito Met genes have higher similarity to gce than to DmMet, leading to our proposal of an evolutionary scheme for these genes in Diptera. Finally, we used RNA interference to knock down Met transcript in A. aegypti. Concomitant reduction in the levels of some putative JH-inducible genes suggests that AaMet may share JH-related functional conservation with DmMet.

Materials and methods

Mosquito species

Mosquitoes used in this study were C. pipiens (provided by Dr. Rebecca M

Robich), A. aegypti, and A. gambiae (both provided by Dr. Woodbridge A Foster). The

rearing conditions for C. pipiens, A. aegypti, and A. gambiae can be found from Robich

and Denlinger (2005), Gary and Foster (2001), and Mostowy and Foster (2004), respectively. A partial sequence obtained from a fourth mosquito, Ochlerotatus

nigromaculis (field-caught specimens provided by Dr. Anthony Cornel), was included in

the phylogenetic analysis. Fourth instar larvae of each mosquito species were used for

PCR.

Degenerate primer design and RT-PCR

The amino acid sequences of Met (GenBank protein accession no. T09462),

CG1705-PA (NP_511126), gce CG6211-PA (NP_511160), and A. gambiae homolog

ENSANGP00000010697 (XP_316059) were compared to identify their conserved

regions. The degenerate primers were designed using the Consensus-Degenerate Hybrid

Oligonucleotide (CODEHOP) program (Rose et al., 1998). The forward primer 5′-CGC

GAT AAG CTG AACGGC WSN ATH CAR GA-3′ and the reverse primer 5′-CGC ACC

TCA TCC GTC ATG TAN CCN GCN AC-3′ were chosen to amplify the Met homologs from each mosquito species.

Total RNA was extracted from fourth instar larvae using Trizol Reagent

(Invitrogen). Five μg of total RNA were used for first strand cDNA synthesis at 50 °C in a 20 μl volume containing SuperScript III reverse transcriptase and oligo(dT) primer

(Invitrogen). RT-PCR amplification was performed with 1 μl first-strand cDNA obtained from the RT reaction as template, 1 μl (10 μM) of each primer, 0.1 μM of each dNTP, and 1.25 U High Fidelity Taq DNA polymerase (Invitrogen) in a final volume of 25 μl.

PCR amplification was carried out in an Eppendorf cycler with an initial denaturation step at 94 °C for 3 minutes. Amplifications were achieved through 35 cycles at 94 °C for

30 s, 56 °C for 1 min, and 72 °C for 1 min. A final extension step was carried out for

10 min at 72 °C. PCR products were purified, subcloned into the pCR 2.1 TOPO TA cloning vector (Invitrogen), and transformed into TOP 10 competent cells. Inserts from positive clones were sequenced, and the sequencing results were used to design specific primers for 5′ and 3′ rapid amplification of cDNA ends (RACE).

We additionally examined the expression of Met throughout development of A. aegypti. Total RNA was isolated from embryos, each larval instar, and male and female adults. Reverse transcription and PCR amplification were performed as above.

RACE

Full-length Met gene cDNAs were synthesized by 5′ and 3′ RACE (Invitrogen).

One gene-specific primer for 3′RACE-PCR and 3 gene-specific primers for 5′ RACE-

PCR were designed for each mosquito species (Table 2.1) using the Gene Fisher software

(http://bibiserv.techfak.uni-bielefeld.de/genefisher/). After separation on 0.8% agarose stained with ethidium bromide, the RACE-PCR products were excised, purified, and sequenced.

Genomic DNA was extracted using the method of Qiao and Raymond (1995).

Genomic DNA amplifications of open reading frames (ORFs) were carried out using primers designed from the cDNA sequences obtained from RACE-PCR. cDNA sequences from A. aegypti and A. gambiae (AaMet and AgMet, respectively) were arbitrarily divided into two fragments and two pairs of gene-specific primers were designed to amplify the separate fragments. We obtained the latter half, containing four introns, of the sequences for AaMet and AgMet and the remaining intron sequences were obtained when genomic data for A. aegypti

(http://www.broad.mit.edu/annotation/disease_vector/aedes_aegypti/) and A. gambiae

(http://www.ncbi.nlm.nih.gov) became available.

Phylogenetic analysis

Multiple alignments were performed using Clustal X 1.83 (Thompson et al., 1997).

Poorly aligning amino acids at the N- and C- termini were removed, providing a multiple sequence alignment spanning the HLH domain through the majority of the PASB domain. Portions of the alignment containing multiple gaps were manually removed.

Phylogenetic tree construction was performed with PAUP* 4.0 for Windows (Swofford,

2002) under the parsimony criterion. The eight in-group sequences include Met homologs of four mosquito species and Met and gce sequences from D. melanogaster and

D. pseudoobscura. Three bHLH-PAS genes obtained via BLAST searches, Dmcycle

(bHLH-PAS gene from D. melanogaster), XlARNT2, and DrARNT2 (bHLH-PAS genes from Xenopus laevis and Danio rerio, respectively), were assigned as outgroups. An exhaustive search produced a single tree, which was visualized with the TreeView program (Page, 1996).

Generation of double stranded RNA constructs

PCR products appended with 5’ T7 promoter sequences were generated for

AaMet and Aaß-galactosidase (Aaß-gal) under the following reaction conditions: 94º C for 2 minutes, followed by 30 cycles of 94º C for 30 seconds, 59º C for 30 seconds, and

72º C for 1 minute and a final elongation step for 10 minutes at 72º C. PCR products

were confirmed via electrophoresis on 2.0% agarose stained with ethidium bromide,

excised, and purified. The purified, T7-appended PCR products were used to generate

double stranded RNA complementary to AaMet or Aaß-gal using the HiScribe T7 in vitro

transcription kit (New England BioLabs).

Double stranded RNA Injection and RT-PCR analysis

Newly eclosed A. aegypti were collected every 2-4 hours. Mosquitoes were briefly cold anesthetized at -20º C and transferred to a glass dish kept on ice. Females were injected intrathoracically with approximately 1 μL of dsRNA suspension containing

0.5-1.0 ng/μL of either dsMet or dsß-gal. We observed enhanced Met knockdown with the simultaneous injection of multiple dsRNA constructs. Therefore, dsMet injections were performed using a mixture of three dsMet constructs: dsMet2, dsMet3, and dsMet5, targeting nt 1655-2207, 748-1221, and 394-1221 of AaMet, respectively. Primer sequences for each of the knockdown constructs are given in table 2.3. Following injection, mosquitoes were transferred cages and allowed access to 10% sucrose for four days.

In order to examine the effects of Met knockdown on JH inducible genes, we identified the A. aegypti homologs of D. melanogaster JH-inducible proteins 1 and 26

(Jh-I1, Jh-I26; Dubrovsky et al., 2000) and JH esterase (JHE) using tBLASTx searches

against both FlyBase (http://www.FlyBase.org) and the NCBI (http://ncbi.nih.gov) databases. Accession numbers are given for target genes according to VectorBase

(http://www.VectorBase.org).

Surviving females were collected and used in pairs for RNA isolation and RT-

PCR analysis as above. Expression data were generated using the ImageQuant 400

hardware and ImageQuant TL analysis software (GE Healthcare; Amersham

Biosciences) and subject to paired T-tests.

Results

Isolation of Met full-length cDNAs in mosquitoes

Based on the conserved amino acid sequences among DmMet, Dmgce, and a

partial homologous sequence from A. gambiae, a pair of degenerate primers were

designed and used for RT-PCR amplification of a fragment including the bHLH through

PASB domains. A single gel band of 900 bp was obtained from each mosquito species, whose translated sequence showed similarity to DmMet and Dmgce.

We used each of these sequences to design primers for 3′ and 5′ RACE PCR.

After sequence alignment of the products, the full-length cDNA of each species was constructed. The C. pipiens (CpMet) cDNA (AY895165) is 3109 bp in length and

contains an ORF that encodes a protein of 854 amino acid residues with a predicted MW

of 93.3 kDa. Preceding the ATG start codon are 94 nucleotides of 5′ untranslated region

(5′UTR). A TAG stop codon is followed by 453 nucleotides of 3′ untranslated sequence,

which includes the canonical AATAAA polyadenylation signal upstream from an 18 bp

poly (A) tail.

The AaMet cDNA (AY902310) is 3135 bp in length and contains an ORF of

2727 bp encoding a protein of 909 amino acids with MW of 98.4 kDa. There are 123 bp

of 5′UTR and 285 bp of 3′UTR sequence containing 20 bp of poly (A) tail. The AgMet

cDNA (DQ303468) is 4782 bp in length and includes an ORF of 3348 bp encoding a

protein of 1116 amino acids of MW 119.4 kDa. There are 189 bp of 5′UTR and 1245 bp

of 3′UTR sequence, including 28 bp of poly (A) tail. The developmental expression of

AaMet is shown in Figure 2.5.

Sequence analysis of Met genes in mosquitoes

All three mosquito Met genes showed (1) primary domains characteristic of

bHLH-PAS proteins: bHLH, PASA, and PASB and (2) sequence identity with each

other, the highest (64%) being between CpMet and AaMet (Fig. 1). Sequence identity

between each mosquito gene and DmMet ranges from 43–45%, from 48–57% with

Dmgce. The bHLH conserved domains share substantially higher sequence identitiy (70–

97%), favoring gce (84–97%) over Met (70–83%) (Table 2.2).

PCR amplification with degenerate primers consistently produced a single

amplification product in each mosquito species (Fig. 2). To determine if a second Met

homolog, sharing higher sequence identity with DmMet than Dmgce exists, two pairs of

Met-specific primers were designed to specifically target a putative Met homolog from A. aegypti. Two forward primers, FP1 (5′ CCC AGT CTS CAT CTR ACG GAC 3′) and

FP2 (5′ ATM GAG ACS CTG TTC TAT CAR CA 3′), were paired with one reverse

primer, RP (5′ CAG CTG RTA SGG TTC YGG CTG 3′), and used for amplification. No product was amplified from A. aegypti (Fig. 2, lanes 6 and 7), although the expected Met

sequence was amplified from D. melanogaster (Fig. 2, lanes 4 and 5).

Comparison of intron positions among AaMet, AgMet, and gce

We identified and examined the introns of AaMet and AgMet. In DmMet, there is

a single intron located in the PASB domain (Ashok et al., 1998). In Dmgce, 7 introns

(none in the PASB domain) are present. The intron numbers and locations in AaMet and

AgMet are more similar to gce than to Met in D. melanogaster: there are 9 and 7 introns

in AaMet and AgMet, respectively. Four introns exist in the same positions in AaMet and

AgMet, and 6 introns are located in similar positions in Dmgce, AaMet, and AgMet (Fig.

1). One intron in the PAS A region of AaMet and AgMet is absent in Dmgce.

Phylogenetic analysis

Sequences used in this analysis spanned the bHLH region through the PAS B domain, a total of 286 amino acids. The six genes examined (OnMet, AaMet, CpMet,

AgMet, Dmgce, Dpgce) were found to form a sister clade to the Met sequences from

Drosophila (DmMet, DpMet), a result consistent with our initial observations from the amino acid identities among these sequences. An exhaustive search using the parsimony criterion produced a single tree (Fig. 3), whose topology suggests that Dmgce and the mosquito Met homologs share gce-like common ancestor.

RNAi reduction of AaMet

Like DmMet, AaMet is expressed throughout development (Figure 2.5). RNAi- directed knockdown of AaMet was performed on adult female mosquitoes. RT-PCR analysis of AaMet showed that Met transcript abundance (relative to Rp49 expression) was reduced by nearly 90% in dsMet-injected females compared to dsß-gal-injected controls (Figure 2.4). In order to examine the effects of Met reduction on a subset of putative JH-inducible genes, cDNA isolated from dsMet and dsß-gal-injected survivors

was used for RT-PCR analysis of AAEL012886, AAEL006600, and AAEL000516, the

genes discovered in tBLASTx homology searches as the A. aegypti homologs of DmJHE,

DmJhI-1, and DmJhI-26, respectively. The expression of all three genes was significantly (p=0.013, 0.0002, and 0.0005, respectively) reduced in DsMet-injected females relative to dsß-gal controls (Figure 2.4).

Discussion

Mosquitoes transmit diseases affecting millions of humans worldwide. Vector control by insecticides is still important for disease control. Methoprene has been widely employed in the management of mosquito populations over the past 25 years (Schaefer et al., 1975), and resistance to methoprene has appeared in some mosquito species, including A. taeniorhynchus (Dame et al., 1998) and O. nigromaculis (Cornel et al.,

2002). However, the resistance mechanism is unknown. Identifying the mechanisms of methoprene resistance in mosquitoes will aid in efforts to manage mosquito populations.

In order to identify potential methoprene resistance genes in some medically important mosquito species, we isolated the Met homologs from A. aegypti, A. gambiae, and C. pipiens using degenerate primers designed in CODEHOP (Rose et al., 1998). The degenerate primers used in this study were designed based on conserved bHLH and PAS

B domain sequences to facilitate the simultaneous amplification of both DmMet and

Dmgce (Fig. 2, lane 2). However, in each mosquito species, only one PCR product was

obtained (Fig. 2, lane 3), suggesting the presence of a single Met-like gene in each

mosquito species, a result corroborated by the genomic sequencing data presently

available for A. aegypti and A. gambiae.

High sequence identity between Met and gce suggests that a gene duplication

occurred in higher Diptera, producing Met from a gce template. The similarities in the

intron positions among gce and the putative mosquito Met homologs lend support to this

hypothesis. It is also possible (though less likely) that the duplication occurred before the

divergence of higher and lower Diptera, and one copy was subsequently lost in

mosquitoes. Future study of homologous sequences from other dipteran species will

serve to further elucidate the evolutionary history of these genes.

Although the function of gce in D. melanogaster is unknown, GCE can dimerize

with MET in the absence of JH or JH agonist (Godlewski et al., 2006), suggesting that

gce and Met may share a common function. Since methoprene applied to A. aegypti

results in toxicity and morphogenetic defects (Braga et al., 2005) similar to those seen for

D. melanogaster (Postlethwait, 1974), we believe that these Met homologs function similarly in JH signaling and possibly resistance in mosquitoes.

We examined the transcriptional consequences of RNAi-driven AaMet

underexpression on a subset of putative JH-inducible genes. AaMet underexpression

resulted in the concomitant reduction of each gene compared to dsß-gal controls,

supporting the notion that AaMet functions similarly to DmMet to mediate some aspects of JH signaling. Additionally, we detected AaMet expression in the ovary and adult female fat body (not shown), two target tissues in the JH-regulated physiology of previtellogenic ovarian development in A. aegypti (Clements, 1992). Since AaMet was shown to possess high identity with Dmgce, an elucidation of the function of Dmgce may aid in our understanding of mosquito Met function.

The vertebrate gene with the highest sequence identity to either Met or gce is the aryl hydrocarbon receptor (ahr). Neither Met nor gce appears to be a homolog of this bHLH-PAS gene, however. AHR binds various xenobiotic compounds, such as dioxin, and subsequently transcriptionally activates certain genes, especially cytochrome P450s, involved in the degradation of these compounds (Hahn, 1998). Although the property of ligand binding by MET might suggest an evolutionary relationship of Met with ahr, the

resistance studies have shown Met interaction with JHAs, not with a variety of

chemically dissimilar insecticides (Wilson and Fabian, 1986) as might be expected for an

ahr homolog. Based on gene homology, the spineless (ss) gene of Drosophila has been

proposed as an invertebrate ahr (Duncan et al., 1998). Future ligand-binding studies with

SS (and GCE) may help our understanding of the evolution of these genes.

In conclusion, the Met homologous genes isolated from these mosquito species should facilitate studies of the mechanism of methoprene resistance seen in field populations as well as roles for JH in mosquitoes.

References

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Riddiford, 1994 L.M. Riddiford, Cellular and molecular actions of juvenile hormone I. General considerations and premetamorphic actions, Advances in Insect Physiology 24:213–274.

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Wilson, T.G., Wang, S., Beno, M., Farkas, R. 2006a. Wide mutational spectrum of a gene involved in hormone action and insecticide resistance in Drosophila melanogaster, Molecular Genetics and Genomics 276:294–303.

Wilson, T.G., Yerushalmi, Y., Donnell, D.M., Restifo, L.L. 2006b. Interaction between hormonal signaling pathways in Drosophila melanogaster as revealed by genetic interaction between Methoprene-tolerant and Broad-Complex, Genetics 172:253–264.

Table 2.1. Gene-specific primers used in 5′ and 3′ RACE-PCR in three mosquito species. a The anchor primers are provided in the RACE Kit (Invitrogen). AP: adapter primer; AAP: abridged anchor primer; AUAP:abridged universal amplification primers.

Table 2.2. Amino acid identities among DmMet, Dmgce, and Met homologs from three mosquito species. Amino acid identities in the entire ORF and in the bHLH conserved domain are shown above and below the diagonal, respectively, for each species pair. DmMet: (GenBank nucleotide accession no. AF034859); Dmgce: (AE003498); CpMet: (AY895165); AaMet: (AY902310); AgMet: (DQ303468).

Primer name Target gene Primer sequence 5-T7-Met-3 AaMet TAATACGACTCACTATAGGGCTGGAGCTAGTCCCAGATCG 3-T7-Met-3 TAATACGACTCACTATAGGGGACTGATCGACTGCGTGAAA 5-T7-Met-4 TAATACGACTCACTATAGGGCGATCTGAGCCAACAGCATA 3-T7-Met-4 TAATACGACTCACTATAGGGTACACAAAGTCGCCCGTTC 5-T7-Met-5 TAATACGACTCACTATAGGGCGGATGCTGAATGGATTCTT 3-T7-Met-5 TAATACGACTCACTATAGGGATACACAAAGTCGCCCGTTC 5-T7-B-gal AaB-gal TAATACGACTCACTATAGGGGCAATCAGCGTAGACCTGCT 3-T7-B-gal TAATACGACTCACTATAGGGTAGTCAGCTGGCTGGGATCT 5JHE AAEL012886 CCACTGTCGAAGGGATTGTT 3JHE GCCTTCGTAACGTTCTCAGC 5JhI-1 AAEL006600 CCAGCGTCCGTTTATTTGTT 3JhI-1 TTTGGCTTAGGTGCTGCTTT 5JhI-26 AAEL000516 TGCAAAAACTGGCAAAACTG 3JhI-26 ATTCCAAATCGCTGGTGAAG

Table 2.3. Primer sequences used to RNAi reduction of AaMet, AaB-gal, and for RT- PCR analysis of putative JH-inducible genes identified in BLAST searches against DmJHE, DmJhI-1, and DmJhI-26. T7 promoter sequence: TAATACGACTCACTATAGGG.

Figure 2.1 (continued)

Fig. 1. Amino acid sequences comparison among AaMet, AgMet, Dmgce, DmMet, and CpMet and intron positions among AaMet, AgMet, and Dmgce using Clustal W program (http://www.ebi.ac.uk/clustalw.html).

Asterisks and dots denote perfect and similar identities in the alignment positions, respectively; bHLH, PASA, PASB domains are indicated by underline, bold underline, and double underline, respectively, in the AaMet gene.

Six conserved intron positions among AaMet, AgMet, and Dmgce are indicated as bold and underlined. The other intron positions are all shown in bold. One intron in AaMet is located in the PASA region and is the same position (162Q) as AgMet; the second intron location (AaMet, 243R) is the same as Dmgce, but not in AgMet; the third one (34 K) is unique in AaMet.

Figure 2.1 (continued)

Figure 1 (continued)

Figure 2.1 (continued)

Fig. 2.2. RT-PCR results using Met and gce degenerate primers or two pairs of Met- specific primers.Lane 1: 1 KB ladder (Biolabs).Lane 2: Amplification using Met and gce degenerate primers in D. melanogaster. Top band: DmMet; bottom band: Dmgce.Lane 3: Amplification using Met and gce degenerate primers in A. aegypti. The single band shown is AaMet.Lane 4, 5: Amplification in D. melanogaster using each of two different pairs of Met-specific primers, showing the expected DmMet band in each lane.Lane 6, 7: Amplification in A. aegypti using the same primer pairs as in lanes 4 and 5, showing failure of amplification.

Fig. 2.3. Phylogenetic tree of DmMet and DpMet, Dmgce and Dpgce, and four mosquito Met homologs. An exhaustive search using amino acid sequences spanning the bHLH through PASB domain produced a single most parsimonious tree. Three additional bHLH-PAS family members serve as outgroups. Bootstrap values from 2000 replications are displayed on the appropriate branches. The Ochlerotatus nigromaculis (formerly Aedes nigromaculis) formed a clade with Aedes Met, as expected.

1.8

1.6

1.4

1.2

0.8

0.6

0.4

0.2

0 Met JHE (AAEL012886) JhI-1 (AAEL006600) JhI-26 (AAEL000516)

dsB-gal dsMet

Figure 2.4. Expression of three putative JH-inducible genes in dsMet treatments or dsB- gal controls. Bars: mean + SEM relative to Rp49 expression. RNAi reduction of AaMet expression indicates substantial knockdown. AaMet reduction (Met; shown above) results in the reduced expression of the putative A. aegypti homologs of DmJHE, DmJhI- 1, and DmJhI-26.

Figure 2.5. Developmental expression profile of AaMet relative to Rp49 in A. aegypti. E: embryo; L1-L4: larval instars 1-4; Pa: early pupa; Pb: late pupa.

Chapter 3

Paralogous Genes Involved in Juvenile Hormone Action in Drosophila melanogaster

Abstract

Juvenile hormone (JH) is critical for multiple aspects of insect development and physiology. Although roles for the hormone have received considerable study, an understanding of the molecules necessary for JH action in insects has been frustratingly

slow. Methoprene-tolerant (Met) in Drosophila melanogaster fulfills many of the

requirements for a hormone receptor gene. A paralogous gene with high sequence

identity, germ-cell expressed (gce), is a candidate as a Met partner in JH action.

Expression of gce was detected at multiple times and in multiple tissues during

development, similar to that previously found for Met. To probe roles of this gene in JH

action, we carried out in vivo gce over- and underexpression studies. We show by

overexpression studies that gce can substitute in vivo for Met to rescue preadult but not

adult phenotypic characters. We also demonstrate that RNAi-driven reduction of gce

expression in transgenic flies results in preadult lethality in the absence of MET. These

results show that (1) unlike Met, gce is a vital gene and shows functional flexibility and

(2) both gene products appear to promote JH action in preadult but not adult

development.

Introduction

The sesquiterpenoid juvenile hormones (JH) regulate numerous insect functions,

including molting, morphology and caste determination, and reproduction (Wheeler and

Nijhout, 2003). JH has been shown to regulate gene expression in carrying out many of

these functions (Beckstead et al., 2007; Dubrovsky et al., 2000; Li et al., 2007;

Minakuchi et al., 2008), but a crucial understanding of JH action is lacking, in part because elucidation of the hormone receptor has been difficult (Gilbert et al., 2000;

Willis, 2007). A likely prospect is the Methoprene-tolerant (Met) gene, originally discovered in a Drosophila melanogaster screen for mutants resistant to the JH insecticidal agonist methoprene (Wilson and Fabian, 1986). JH or JH agonists applied to dipteran insects at the onset of metamorphosis result in lethality and morphogenetic defects, such as failure of rotation of the male genitalia that normally occurs during pupal

development (Madhavan, 1973; Postlethwait, 1974), and Met mutants show resistance to

these JH effects (Wilson and Fabian, 1986). The Met gene product has been shown to

possess characteristics of a hormone receptor, such as high affinity JH binding (Miura et

al., 2005; Shemshedini and Wilson, 1990), expression in JH target tissues (Liu et al.,

2009; Pursley et al., 2000), and JH-dependent transcriptional activity (Miura et al., 2005).

Met is a member of the bHLH-PAS transcription factor gene family (Ashok et al., 1998).

Recently, a Met -like homolog of the beetle Tribolium castaneum was identified and

shown by RNAi experiments to be necessary for proper larval-larval molts (Konopova

and Jindra, 2007), a key role for JH in a variety of insects, thus strengthening the

likelihood of MET involvement in JH action.

This MET-JH action hypothesis was weakened when a null allele, Met 27, was

found to be homozygous viable ( Wilson and Ashok, 1998). Since JH is involved in

molting in many insects, a Met null allele might be expected to result in a lethal

phenotype (Riddiford, 2008). Perhaps another gene rescues the lethality of Met 27 flies.

The most likely candidate is a paralogous D. melanogaster gene, germ-cell expressed

(gce), identified as a bHLH-PAS gene upon analysis of the sequenced genome (Moore et al., 2000). The level of sequence similarity to Met is about 60%, highest in the conserved bHLH and PAS domains involved in DNA binding and protein-protein interaction

(Dolwick et al., 1993; Huang et al., l993). The function of gce is poorly understood.

Recently, however, GCE and MET were shown to induce programmed cell death in D. melanogaster larval fat body tissue during metamorphosis, a result that could be suppressed by methoprene application (Liu et al., 2009). Furthermore, attempts to isolate

Met homologous genes from three mosquito species and the beetle T. castaneum

(Konopova and Jindra, 2007) revealed only a single homolog in each species with higher

sequence and intron-exon structure similarity to gce than to Met (Wang et al., 2007).

Together, these results suggest that an ancestor gene duplication resulting in gce and Met occurred after the lower-higher dipteran split during evolutionary time. Unlike Met, no gce mutants have been reported, so the consequences of mutated gce are unknown.

To probe the role of gce in JH action, we carried out two sets of experiments: (1) overexpression of gce both in Met + flies to detect any new phenotype and in Met mutants to measure possible rescue of Met phenotypic characters and (2) RNAi-driven reduction of gce expression to determine the consequences of insufficient GCE. To overexpress gce and produce gce-RNAi in flies, we employed the GAL4-UAS system (Brand and

Perrimon l993), which uses a D. melanogaster gene promoter ligated to yeast GAL4, resulting in GAL4 protein product to drive expression of gce ligated to a UAS response element in transgenic flies.

Materials and Methods

gce overexpression construct

The GAL4-UAS system (Brand and Perrimon, l993) was used to overexpress gce in transgenic flies. A gce-bearing transgenic stock, UAS-gce, was constructed from gce cDNA following RT-PCR amplification. First, total RNA was isolated from adult flies

using TRIzol reagent, followed by treatment with RNase-free DNase-I (Invitrogen) and

bromochloropropane (Molecular Research Center, Inc) reagents. Two microgram

aliquots were reverse transcribed with 200 U/μl of MMLV reverse transcriptase

(Invitrogen) plus 500 ng of random hexamers (Ambion) in a 25 μl reaction mixture to

synthesize first-strand cDNA. The cDNA was amplified by PCR in a reaction mixture

consisting of 2.5 μl of 10X PCR buffer, 1 μl Taq DNA polymerase (Invitrogen), 1 μl of

10 mM dNTP mix, 1μl of 50 mM MgCl2, 1 ml each of 10 mM primer, 1 μl of cDNA, and

16 μl of H20. The primers used were forward 5’-

ATAGGTACCACGATTGCGAAATGTTATGC-3’ and reverse 5’-

ATATCTAGAGAAACCCTTCAGTCGAGACC-3’. The product was subcloned into the

TOPO vector (Invitrogen), sequenced, and inserted into the pUAST transformation vector

(Brand and Perrimon, 1993). Proper orientation of the fragment in the vector was verified

by restriction site analysis and sequencing.

gce RNAi construct

Expression of gce was also subjected to RNAi-mediated knockdown. A gce

RNAi-generating transgene was engineered from a fusion of genomic DNA and reverse

complement cDNA, which has been shown to give higher knockdown of the gene of

interest (Kalidas and Smith, 2002). First, genomic DNA was isolated as follows: 15 flies

were homogenized in 200 μL of extraction buffer (1% SDS, 50 mM Tris HCl (pH 8.0),

25 mM NaCl, 25 mM EDTA) and placed in a water bath at 65° for 1 hr. Then 100 μL of

3 M potassium acetate was added, and the sample was placed on ice for 1 hr. The sample

was centrifuged, and DNA was precipitated from the supernatant with two volumes of

EtOH and 0.1 volume of 3 M sodium acetate overnight at -20°. The pellet was rinsed in

70% EtOH, air-dried for 5-10 min, and then resuspended in 100 mM Tris HCl (pH 8.0), 1

mM EDTA.

A gce fragment was amplified from the genomic DNA by PCR as described above using forward primer 5’-TATAAGATCTCCGCAGTCGACCATCGATGT-3’

preceded by a BglII adapter site and reverse primer 5’-

AGATCTCGAGCTGCCGGCAAATAGTTCAAT-3’ preceded by a XhoI adapter site

(for ease of subcloning). The product, which spanned part of exon 6 and intron 6, was

subcloned into the TOPO vector (Invitrogen), sequenced, and inserted into the pUAST

transformation vector.

To create the cDNA fragment, total RNA was isolated from adult flies, and cDNA

was synthesized and amplified as described above using forward primer 5’

AGATTCTAGACACACAGATACCGCAGTCGA 3’ preceded by XbaI adapter site and

reverse primer 5’ TATACTCGAGCAGTGCTGGGTATAAAACGC 3’ preceded by

XhoI adapter site. The product was subcloned into the TOPO vector, sequenced, and

inserted into the pUAST transformation vector immediately downstream of the inserted

genomic fragment. Proper orientation of the fragments in the vector were verified by

restriction site analysis and sequencing.

D. melanogaster transformation

Germline transformation of either w or w MetW3 embryos was performed by

injecting dechorionated embryos with purified UAS-gce or UAS-gce-dsRNA plasmid together with “wings clipped” helper plasmid pπ 25.7 (Rubin and Spradling, 1982) in a ratio of 2-3:1. Transformant flies were recognized by partial restoration of eye color resulting from the mini-w+ gene included in the pUAST vector. Multiple UAS-gce and

UAS-gce-dsRNA transformant lines were isolated and made homozygous for the

transgene by selecting on w+ eye color. Each of these lines represents an independent

insertion of the transgene into the genome, and lines having the transgene inserted into

the third chromosome were selected and used in these studies. Other genes or alleles

were introduced into transformant lines by genetic crosses.

gce overexpression and knockdown in GAL4-UAS transgenic strains

Simple genetic crosses served to bring the UAS-linked and GAL4-linked

transgenes together to control expression. Each UAS line was crossed with flies carrying

either an actin-GAL4 or tubulin-GAL4 transgene heterozygous to a TM6 balancer

chromosome carrying a Tubby (Tb) dominant mutation, allowing ready identification of

larvae/pupae/adults carrying the balancer chromosome in the F1 generation. Similar

crosses with tubulin-GAL4/TM6, Lsp2-GAL4, and GawB}dan[AC116]-GAL4 fathers

allows tubulin, larval serum protein2, and compound eye promoters, respectively, to

drive UAS-gce expression in progeny. The Lsp2 promoter is expressed in third-instar fat

body, a JH target tissue (Liu et al., 2009), and GawB}dan[AC116] in the compound eye

(Flybase), thus allowing tissue-specific expression. F1 progeny carrying the TM6

balancer chromosome lack actin-GAL4 and therefore serve as control flies. Similar

27 crosses using w Met /Y; actin-GAL4 fathers resulted in F1 females that were

27 homozygous for Met , and using w/w; UAS-gce/UAS-gce mothers resulted in F1 that were homozygous/hemizygous for w Met+; both of these crosses were used in some

experiments.

Analogous crosses with either w Met27; UAS-gce-dsRNA or w Met+; UAS-gce-

dsRNA females allowed either tubulin or actin-driven expression of the dsRNA transgene

27 + in F1 individuals homozygous for either Met or Met .

RT-PCR

Overexpression or knockdown of gce was measured using RT-PCR. RNA extraction, reverse transcription, and PCR amplification was carried out as described above, except that PCR primers used for gce were forward primer

5’GGATGCCATCGATCGCAAGT 3’ and reverse primer 5’

GCTTCGTCACTACGCCGAAA 3’. The primers for Rp49 were forward primer

5’CCGCTTCAAGGGACAGTATC 3’ and reverse primer

5’ATCTCGCCGCAGTAAACG 3’. Ten µl of each PCR product was electrophoresed on a 1% agarose gel and stained with ethidium bromide. An image of the gel was captured with the ImageQuant 400 (GE Healthcare) and then analyzed using the ImageQuant TL software (Amersham Biosciences). PCR products were cloned into TOPO vector

(Invitrogen) and sequenced with M13 forward primer.

Northern analysis

Total RNA was isolated from D. melanogaster Oregon R strain using TRizol reagent (Invitrogen). The RNA was separated by electrophoresis on a 0.8% formaldehyde-agarose gel and then transferred to positively charged nylon membranes

(Roche Diagnostics, Indianapolis, IN). RNA probes were prepared with a DIG RNA labeling kit (Roche) using gce, Met, Rp49 and rRNA sequences as templates. The cDNA

used to prepare the probes were as follows: gce cDNA 5’ untranslated region that include

nucleotide +1 to +1683; gce cDNA 5’ coding region +1338 to +2381; gce cDNA 3’

coding region +3340 to +4216; Gce cDNA 3’ untranslated region +3989 to +5530; a Met cDNA +151 to +2151; rp49 cDNA +1 to +458; and 18S rRNA gene +500 to +1377. The membranes were hybridized with DIG labeled RNA probes for 12h at 65 oC with DIG

easy hyb (Roche). DIG labeled RNA was detected with an alkaline phosphatase-

conjugated anti-DIG antibody using CDP star (Roche).

Methoprene resistance

Cultures were assayed for methoprene sensitivity on each of 3-5 doses of

methoprene (isopropyl-(2E,4E)-11-methoxy-3,7,11-tri-methyl-2,4-dodecadienonate,

ChemService, PA), applied as ethanolic solutions (25 ml) to the surface of each culture

(food surface area 3.8 cm2) in doses ranging from of 0.27 to 5.4 mg/vial. Mortality

occurred predominately during the late pupal (pharate adult) stage; survivors were

examined for the methoprene-induced morphogenetic defect of malrotated male genitalia

or posterior sternal bristle defects (Madhavan, 1973; Wilson and Fabian, 1986).

Eye phenotype

The Met eye defect phenotype was quantitated by counting defective facets

(appearing black) in either pharate adult (nonsurvivors) or adult (survivors) males under

40X magnification. Preliminary experiments showed that the number of defective facets

does not increase between these two stages, thus allowing direct comparison of

individuals between the two developmental stages.

Oviposition

Females were isolated within 8 h after eclosion and allowed to age for 6 d in the

presence of wild-type (Oregon-RC) males and baker’s yeast sprinkled on the food surface

to maximize oogenesis. They were then placed individually in 28 X 95-mm plastic food

vials (Capitol, Fonda, NY) in the continued presence of yeasted food and males at 25º.

Eggs were counted during the next 4 d, a time period during development when the

ovipositional rate reaches a maximal steady-state value and allows strain comparison

(Wilson and Ashok, 1998). Egg fertility was noted, and vials having unfertilized eggs were discarded because unmated females are less fecund.

In some experiments oogenesis was examined by dissecting females in Ringer’s solution and censusing vitellogenic oocytes in each ovary using the staging described previously ( Wilson and Ashok, 1998).

Male courtship

Males of the appropriate genotypes were taken from cultures at age 3-5 d after eclosion following light etherization. They were allowed to recover for 16-24 h, then placed individually on food in 25 X 95 mm plastic food vials at 22º with two 4-6 day post-eclosion Oregon-RC virgin females. Courtship was noted for 1-2 h, then males were removed at designated times up to 18 h, and female fertility noted by F1 larvae appearing

3 d later, showing that mating had occurred.

Results

Expression of gce

The gce gene was first detected in embryonic germ cells, giving rise to its name

(Moore et al., 2000). We first examined the expression patterns of gce in wild-type to detect any differences from those found for Met (Pursley et al., 2000). A Northern analysis showed expression at multiple times during development, with periods of low- to-absent expression in early embryonic and pupal stages (Figure 3.1). The expression of

Met was found to be similarly widespread (Pursley et al., 2000), but the pattern was not identical to that of gce.

Expression of gce was also examined by RT-PCR in selected tissues, including two known JH target tissues, ovary and male accessory glands. Expression levels were

generally lower than that found for Met, especially in larval fat body, where it was

present in trace levels (Figure 3.2). Another study has shown expression of gce and Met

in a variety of tissues and expression levels (Chintapalli et al., 2007). Clearly, expression

of gce is not limited to its name-sake tissue in the embryo.

Overexpression of gce rescues preadult loss of Met function

The UAS-gce transgene was found to be overexpressed by either tubulin-GAL4 or

actin-GAL4, but more strongly by the tubulin promoter (Figure 3.3). Flies having gce overexpressed in a Met+ background showed no obvious novel phenotype—such as

lethality or change in development—evident at any stage of development. However, when gce was overexpressed in a Met 27 background, we found rescue of preadult Met27

phenotypic characters. These included both methoprene-conditional and nonconditional

characters. The conditional phenotype includes resistance to the well-documented lethal

and morphogenetic effects of methoprene either topically applied to Met+ prepupae

(Riddiford and Ashburner, 1991) or incorporated into the diet of Met+ larval cultures

(Wilson and Ashok, 1998). Met mutants survive similar treatments with methoprene,

other JH agonists (Riddiford and Ashburner, 1991), or JH III itself (Wilson and Fabian,

1986). When challenged with methoprene, both Met27; UAS-gce/tubulin-GAL4 and

Met27; UAS-gce/actin-GAL4 flies were less tolerant than balancer chromosome sibling

controls to the toxic effects at each of four doses (Figure 3.4), showing blockage of the name-sake Methoprene-tolerant phenotype of Met27. Lethality occurred at the end of

pupation, at the pharate adult stage, which is characteristic of the effect of methoprene on

Met+ flies (and other Diptera). In contrast, Met27; UAS-gce/TM6 control flies, having a

TM6 balancer chromosome substituted for the GAL4 driver chromosome, showed good

survival due to Met27 at all four doses tested (Figure 3.4).

Similarly, treatment of D. melanogaster Met+ prepupae with JH analogs resulted

in failure of complete rotation of the male genital disk that normally occurs during pupal

development (Adam et al., 2003), and Met mutants are completely resistant to this effect

(Wilson and Fabian 1986). In our experiments, resistance seen in Met27 to this

methoprene-induced effect was blocked in Met27; UAS-gce/ tubulin-GAL4 flies, resulting in abnormal genital rotation, a response similar to that found for Met+ (Figure 3.5). The

effect was widespread: 91% (N= 100) of males from cultures treated with 0.27 µg/vial of methoprene showed varying degrees of abnormal rotation, compared with 0% (N= 100) of treated Met27/Y; UAS-gce/TM6 control sibs that showed abnormal rotation, as expected

due to the Met27mutation. Therefore, the presence of overexpressed gce resulted in striking enhancement of both methoprene toxicity and a morphogenetic response to methoprene, showing that GCE can substitute for MET in the flies to restore methoprene sensitivity approaching Met+ levels.

Additionally, gce overexpression rescued a non-conditional Met phenotypic

character, that of defective posterior facets in the compound eye (Figure 3.6), previously

seen as grossly malformed facets in scanning electron microscopy (Wilson et al., 2006).

This phenotype has not been reported for other eye mutants and appears to be specific for

Met mutants, most strongly expressed in the MetW3 allele (Wilson et al., 2006).

Quantification of the phenotype and its rescue was carried out by counting darkened

facets on affected adults under light microscopy (Table 3.1). The eye phenotype was

strongly rescued in MetW3; UAS-gce flies driven by either GAL4 promoter.

Clearly, overexpression of GCE by a global GAL4 promoter such as actin or

tubulin can result in GCE substitution for MET to block the Met phenotype (Figure 3.4).

To examine the blockage effect using a tissue-specific promoter, we drove expression of

GCE using a larval fat body promoter in the Lsp2-GAL4 strain. Examination of 50 F1 males showed 100% morphologically normal male genitalia, characteristic of typical

Met-induced resistance, showing no substitution by GCE for MET to block the resistance.

However, resistance to either the eye defect (Table 3.1) or methoprene-induced pupal death (Figure 3.4) in MetW3 or Met27 was either essentially absent (eye phenotype) or

partially found (pupal death phenotype), respectively, in the UAS-gce/ Lsp2-GAL4

transgenic flies, showing at least partial substitution by GCE for MET. Therefore, GCE

overexpression in a specific tissue can partially substitute for MET to block Met-

generated resistance to pupal death and to the Met-generated eye defect, but not the male

genitalia defect. We also examined GCE driven by another GAL4 promoter,

GawB}dan[AC116]-GAL4, that is specific for the compound eye. Unlike GCE driven by

the Lsp2-GAL4 promoter, the F1 showed complete rescue of the Met eye phenotype,

resulting in morphologically normal eyes (Table 3.1). Therefore, GCE substitution for

MET can show tissue specificity.

While rescue of preadult Met phenotypic characters was clear and consistent, the

adult phenotype of Met27, consisting of deficient oogenesis/oviposition and male

courtship behavior (Wilson and Ashok, 1998; Wilson et al., 2003) was not appreciably

changed in Met27; UAS-gce/tubulin-GAL4 adults compared to Met27 balancer sibs (Figure

3.7). Both GAL4 drivers result in gce overexpression in adults, although to a lesser extent

than in larvae (Figure 3.3). Either this level of overexpression is insufficient for rescue,

or GCE is incapable of substituting for MET in adults.

Underexpression of gce results in lethality

Additional insight into the role of gce came to light when we examined the consequences of gce underexpression by using either actin-GAL4 or tubulin-GAL4 to drive expression of gce dsRNA, which results in RNAi directed to gce. Alone, neither of the two GAL4 constructs nor the UAS-gce dsRNA construct alone has a noticeable effect on survival or fertility in either a Met+ or Met27 background. However, the level of gce

transcript in late larvae expressing gce-RNAi was found to be considerably lowered

relative to that in balancer siblings (Figure 3.8), showing effectiveness of the dsRNA

transgene in reducing gce mRNA. Initally, we examined the phenotype of Met+/ Met+;

UAS-gce-dsRNA/ tubulin-GAL4 animals to understand the consequences of gce reduction

in a Met+ background. Embryos were seemingly unaffected, judging from good hatch

rates after egg fertilization at 25º, but no role for JH has been reported during D.

melanogaster embryogenesis, so this result is perhaps not surprising.

However, the consequences to postembryonic viability were severe: Met+; UAS-

gce-dsRNA/ tubulin-GAL4 homozygotes/hemizygotes, but not TM6 siblings, failed to

survive to adulthood, dying as mid-to-late stage larvae or, more commonly, pupae (Table

3.2). Visual examination of these pupae revealed that death typically occurred in pharate adults. When driven by the actin-GAL4 construct, gce RNAi expressed in Met+

homozygous progeny resulted in survival in reasonable numbers with many eclosing as adults (Table 3.2). However, these adults were affected by gce underexpression, since a majority of these females (but not the TM6 sibling adults) died within 2-3 days following eclosion.

To understand the consequences of gce reduction in a Met background, we

generated Met27; UAS-gce-dsRNA/ tubulin-GAL4 animals. Again, prepupal death

occurred, but lethality was shifted from pharate adults to earlier pupae (0-2 days),

showing more severe consequences of gce reduction in a Met27 than in a Met+

background. When driven by the actin-GAL4 construct, gce-dsRNA resulted in lethality,

primarily pharate adult (Table 3.2).

Therefore, reduction of gce is lethal in Met27 or Met+ preadults when driven by a

tubulin promoter but is lethal primarily only in Met27 preadults when driven by the

“weaker” (Barry et al., 2008) actin promoter. Another possibility is that the slightly

larger amount of GCE produced in the actin-driven strain (Figure 3.8) is sufficient to partially rescue the lethality, resulting in the higher survival seen relative to that with the tubulin-driven promoter. Similar results (not shown) were seen when MetW3 replaced

Met27 in these strains, showing the results not to be allele specific.

Methoprene effect during gce underexpression

It is clear that gce overexpression enhances methoprene toxicity (Figure 3.4). To

determine if methoprene could either rescue or exacerbate the preadult lethality

phenotype due to gce underexpression, we examined the consequences of methoprene

application when gce is underexpressed. Methoprene clearly acts as a juvenile hormone at both the organismal and molecular level in a variety of insects (Wilson, 2004). Since

Met27; UAS-gce-dsRNA cultures are preadult lethal (Table 3.2), we examined Met+; UAS- gce-dsRNA cultures exposed to a range of methoprene doses, including milder, generally sublethal doses (0.27 and 0.54 mg/vial) and stronger doses (2.7 and 5.4 mg/vial). Little difference on the toxicity to gce-dsRNA/actin or TM6 controls was found (Table 3.3).

Methoprene/JH III application to Met27 adults

JH is required for oogenesis and oocyte production in a variety of insects (Wyatt

and Davey, 1996), including D. melanogaster (Postlethwait and Handler, 1978; Wilson,

1982). If Met is involved in JH action, then the poor oogenesis and oviposition

previously seen in Met27 females (Wilson and Ashok, 1998) might reflect either

insufficient JH or some defect in either the JH receptor or an associated component in JH action in these flies. We examined this question by exposing newly eclosed Met27 females to methoprene or JH III applied to the food surface, a valid method of application

(Wilson et al., 2003). Daily oviposition was measured during a 5-day window of steady hormone exposure and oviposition. The mean daily oviposition by 8 Met27 females (5

replicates) exposed to methoprene was 59.6 eggs (2.2 SEM); to JH III was 57.1 (2.0

SEM); to ethanol was 52.6 (2.2 SEM). Oviposition by methoprene- or JH III-treated

adults was higher, but not significantly different from that of ethanol-treated siblings

(P=0.066, one-way ANOVA). Therefore, the poor oviposition in Met27 is not due to

significant JH deficiency and is consistent with a phenotype expected to result from an

abnormal component of JH reception.

Underexpression of gce magnifies Met eye phenotype

Since overexpression of gce blocked the preadult Met phenotypic characters

(Figures 3.4-3.6), we examined the effect of gce underexpression on the MetW3 eye

phenotype. The eye defect phenotype could be examined in moribund MetW3/Y; UAS-

gce-dsRNA / actin-GAL4 male pharate adults because eye development in the pharate

adult stage appears morphologically normal (except for the facet phenotype). When the

Met eye phenotype was compared between actin-driven and TM6-balancer siblings, a

significant (but not dramatic) enhancement of the facet defect abundance was evident

(Table 3.1). Met must be lesioned to produce the eye phenotype; if Met+ is substituted

W3 + for Met in the mother, the eye remains wild-type in the F1 Met /Y; gce-RNAi males,

showing that a low GCE level alone does not result in the eye phenotype. Therefore, reduction of gce in MetW3 hemizygous flies results in a mild enhancement of the

nonconditional eye phenotype.

Discussion

Our previous work established Met involvement in JH action in D. melanogaster,

a conclusion that has been supported by subsequent independent work demonstrating

high affinity JH binding to MET, JH-driven transcriptional activity (Miura et al., 2005),

and involvement of a Met homolog in control of metamorphosis in the beetle T.

castaneum (Konopova and Jindra, 2007). We now show by under- and overexpression

studies that the paralogous gene gce in D. melanogaster not only plays a vital role during

pupal development but also can substitute for Met, suggesting involvement in the action

of this hormone.

Is the rescue due to an abundance of GCE or to expression in tissue not normally expressing endogenous gce? To address the issue of tissue specificity of Met/gce expression, we turned to recent results demonstrating that larval fat body catabolism, required for completion of metamorphosis, is initiated by ecdysone, MET, and GCE and can be blocked by JH application (Liu et al., 2009). Perhaps high pupal survival of Met mutants following methoprene application results from an absence of methoprene- induced blockage of catabolism, and the lowered pupal survival in Met27; gce transgenic flies (Figure 3.4) results from substitution of GCE for the absent MET. Overexpression of GCE specifically in larvae fat body was carried out using a larval fat body GAL4 driver, and resistance to methoprene-induced mortality and male genitalia malrotation was examined. Methoprene-treated Met27; UAS-gce /lfb-GAL4 were found to be completely (50/50 examined) resistant to the male genitalia defect, indicating no blockage of the Met27mutation. However, the progeny were only partially resistant to pupal death, showing that GCE can partially substitute for MET in this tissue to block the

Met27mutation and suggesting that tissue specific, not widespread gce overexpression, may underlie the basis of the GCE substitution effect. Since the larval fat body shows little or no gce (Figure 3.1), then supplying GCE to this tissue can explain the tissue- specific effect seen on the pupal death phenotype. The eye phenotype was not rescued by larval fat body promoter-driven GCE, but it was completely rescued by compound eye

promoter-driven GCE (Table 3.1). Therefore, GCE expressed in the larval fat body can

partially substitute for MET in the tissue(s) responsible for pharate adult death, but not

eye or male genitalia, demonstrating tissue specificity of expression or utilization of

GCE/MET.

We could find little effect of gce overexpression on adult reproductive

phenotypes of Met27. JH plays roles in both male and female reproduction in D.

melanogaster as well as in many other insects (Wyatt and Davey, 1996). Clearly, overexpressed GCE can substitute for MET in Met preadults, but since the adult transgenic fly reproductive phenotypes were similar to those in adults not overexpressing

the gce transgene (Figure 3.7), this substitution appears to be unproductive in adults.

This result suggests that MET may be the major player in adults. The presence of only a single Met/gce homolog in three mosquito species (Wang et al., 2007) and in the beetle T.

castaneum (Konopova and Jindra, 2007) with higher similarity to gce than to Met

suggests that this reproductive role for MET evolved following the gene duplication seen

in higher Diptera. Both Met and gce are present in the 12 species of D. melanogaster

whose genomes have been sequenced (http://flybase.org); therefore, this duplication

occurred earlier than the evolutionary divergence of these species that occurred as much

as 60 million years ago.

Clearly, gce underexpression can be lethal to either larvae or pupae, especially

pupae, especially in the absence of Met+ (Table 3.2). The presence of background Met+

allowed greater pupal development, shifting pupal death in RNAi individuals from the

early pupal stage to the pharate adult/adult stage, depending on the promoter used and

thus presumably the level of RNAi produced. Underexpression of gce in the absence of

Met+, in a Met27background, is more severe and results in a total loss of preadult viability

(Table 3.2). This suggests that both GCE and MET can interact to promote some vital

aspect of larval/pupal development. Our previous work using D. melanogaster S-2 cells has shown that MET and GCE can heterodimerize (Godlewski et al., 2006), suggesting a mechanistic basis for MET-GCE interaction. However, underexpression driven by the stronger tubulin promoter results in pupal death in a Met+ background (Table 3.2), so the

absence of Met+ is not a prerequisite for this phenotype. If both MET and GCE are

necessary for JH action, then underexpression of both genes could result in death due to a

failure of some JH-controlled developmental event, for example. The greater phenotypic

severity of gce underexpression in a mutant Met background (Table 3.2) argues for the

JH action role scenario.

Tissue-specific expression levels of Met and gce are given for 24 larval and adult

tissues in FlyAtlas database, determined by microarray analysis. The data shows robust

expression (at least 2/4 “present” calls in the 4 microarray replicates) for gce in 12 tissues

and for Met in 10 tissues. Seven tissues showed robust expression of both genes;

interestingly, none are demonstrated JH target tissues. This could mean that (1) the presence of both GCE and MET are not required for JH action, (2) only one is required, or (3) few JH target tissues have been identified (perhaps more likely explanation).

However, there are some surprises in the FlyAtlas dataset; for example, neither gene showed good expression in ovary or larval fat body, and only gce showed strong expression in male accessory glands, all demonstrated JH target tissues. Possibly, additional regulatory roles, independent of JH, for either of these transcription factors exists and is reflected in the FlyAtlas dataset.

Does this work show MET and GCE involvement in a JH receptor complex?

Although the phenotypic characteristics of Met suggest involvement in JH reception, there is no direct evidence for involvement in a bona fide receptor. The disparate levels of transcript found for Met and gce in certain tissues (Figure 3.2) might suggest separate roles for either or both of the gene products in certain tissues, not formation of a mandatory heterodimer that might be predicted for a JH receptor. Indeed, one of the roles might involve eye development, which can be disrupted when either Met is mutated

(Figure 3.6; Table 3.1) or JH titer is low (Riddiford et al., 2010). Likewise, the lack of substantial resistance to methoprene in the Met+; UAS-gce dsRNA/actin-GAL4 flies

(Table 3.3) might seem perplexing, considering the high resistance seen in Met mutants, but this conundrum might simply reflect a lack of strong JH binding by GCE, a possible

requirement for resistance to the hormone and insecticide. Ligand binding might be the sole property of MET in a MET/GCE heterodimer, and loss of GCE affects JH action, but not due to failure of JH binding.

Future studies focusing on the role of gce may lead to more rapid progress defining a JH receptor.

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Table 3.1. Defective eye facets in gce overexpressed and underexpressed strains. Defects were counted in both eyes of 25 males of the indicated genotype. lfb=larval fat body; eye=compound eye. Standard error of mean values given in parentheses. NA=Not Applicable.

Table 3.2. Larval and pupal survival values of gce-dsRNA strains. Each value is the mean of 50 larvae or pupae, 5 replications. A larval survivor pupariated; a pupal survivor eclosed. actin refers to actin-GAL4; tubulin to tubulin- GAL4

% Survivor % Survivor

Genotype larvae (SEM) pupae (SEM)

27 80 (2.9) 84 (2.0) Met /Y; UAS-gce-dsRNA/TM6

27 89 (2.0) 84 (2.1) Met /+; UAS-gce-dsRNA/TM6

27 60 (1.8) 0 Met /Y; UAS-gce-dsRNA/actin

27 78 (2.3) 50 (3.4) Met /+; UAS-gce-dsRNA/actin

+/+; UAS-gce-dsRNA/actin 82 (2.5) 83 (2.9)

27 34 (4.1) 0 Met /Y; UAS-gce-dsRNA/tubulin

27 57 (2.2) 0 Met /+; UAS-gce-dsRNA/tubulin

+/+; UAS-gce-dsRNA/tubulin 64 (3.3) 0

Table 3.3. Survival of gce RNAi transgenic pupae on methoprene. Each value is the mean of 30 pupae, 5 replications. Pupal survival was defined by eclosion.

% Pupal Survival (SEM)

Methoprene dose (μg/vial) +/+; UAS-gce dsRNA/ actin-GAL4 +/+; UAS-gce dsRNA/ TM6

0 89 (1.7) 85 (1.7)

0.27 84 (2.4) 72 (1.7)

0.54 82 (2.9) 71 (3.5)

2.7 57 (3.5) 43 (3.0)

5.4 27 (4.9) 21 (4.2)

Figure 3.1. Northern hybridization analysis of gce transcripts. RNA was isolated from whole animals at various developmental ages. 5 µg total RNA was electrophoresed and transferred to nylon membranes. The membranes were hybridized with DIG labeled gce, Rp49 and rRNA probes. The identity of the 7.5 kb minor band is undetermined.

Figure 3.2. RT-PCR analysis of Met and gce transcripts from selected tissues. The first strand cDNA was synthesized, and PCR was performed with primer pairs for gce, Met, and Rp49 genes in adult brain, ovary, male accessory gland (MAG) and third-instar larval fat body. Values are means (SEM) of three separate determinations expressed relative to those of Rp49. The Met PCR primer sequences are given in (Barry et al., 2008).

Figure 3.3. Expression of gce in A UAS-gce overexpressing strains. Expression was measured by RT-PCR in both larvae and adults. B Expression values for gce and rp49 in gce-overexpressed larvae and adults. Values are normalized to those of +; UAS- gce/TM6 sibs. Each value is the mean (SEM error bar) of three determinations of larvae or adults of the indicated genotype.

Figure 3.4. Enhanced methoprene pupal toxicity of gce-overexpressing strains. gce refers to UAS-gce driven by GAL4-actin, GAL4-tubulin, or GAL4-LFB (larval fat body) promoters. Each value is the mean (SEM error bar) of 30 pupae, three determinations. Pupal survival was defined as eclosion. Methoprene doses in µg/vial.

Figure 3.5. Rescue of resistance to methoprene-induced failure of male genitalia rotation during pupal development. A. Normal-appearing genitalia phenotype in w Met27; UAS- gce /TM6 male exposed to 5.4 µg/vial methoprene during larval development. The absence of Met+ protects the fly from methoprene-induced malrotation, and the appearance is normal, having the anal plates posterior and the penis apparatus anterior. B. Abnormal-appearing genitalia in Met27; UAS-gce/tubulin-GAL4 adult exposed to 0.027µg/vial methoprene. The genitalia has failed to complete the normal 360º rotation by approximately 100 degrees, an effect expected in methoprene-treated flies carrying Met+.

Figure 3.6. Rescue of eye phenotype in MetW3 flies. A. Nonconditional eye phenotype of disrupted posterior facets (evidenced by darkening) in w MetW3; UAS-gce /TM6 adult. B. Complete rescue of eye phenotype in w MetW3; UAS-gce /tubulin-GAL4 adult. The light red eye color in A results from the w+-carrying UAS-gce transgene, and the darker red color in B from the additional w+-carrying tubulin-GAL4 transgene.

Figure 3.7. Fertility of adults overexpressing gce. A Eggs laid/female during a 4-d window of optimal oviposition. Each value is the mean of 30 females of the indicated genotype. B Males fertilizing newly encountered wild-type females during the indicated time periods, cumulative values. Met+/Y; UAS-gce/tubulin-GAL4 males were tested only at the 0-1 hr period. Each value is the mean of 20 males, 5 replicates. 96

Figure 3.8. Expression of gce in A UAS-dsRNA gce larvae having the designated GAL4 promoter or TM6 balancer chromosome as measured by RT-PCR. O-RC refers to the Oregon-RC wild-type strain. B Expression values for gce and rp49 in Met27; UAS-gce- dsRNA larvae. Values are normalized to those in Oregon-RC wild-type larvae. Values are means (SEM error bars) of three determinations of larvae of the indicated genotype.

Chapter 4

Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila

Abstract

Juvenile hormone (JH) signaling underpins both regulatory and developmental pathways in insects. However, the JH receptor is poorly understood.

Methoprene tolerant (Met) and germ cell expressed (gce) have been implicated in JH signaling in Drosophila. We investigated the evolution of Met and gce across 12

Drosophila species and found that these paralogs are conserved across at least 63 million years of dipteran evolution. Distinct patterns of selection found using estimates of dN/dS ratios across Drosophila Met and gce coding sequences, along with their incongruent temporal expression profiles in embryonic D. melanogaster, illustrate avenues through which these genes have diverged within the Diptera. Additionally, we demonstrate that the annotated gene CG15032 is the 5’ terminus of gce.

In mosquitoes and beetles, a single Met-like homolog displays structural similarity to both Met and gce, and the intron locations are conserved with those of gce.

We found that Tribolium and mosquito Met orthologs are assembled from Met- and gce- specific domains in a modular fashion. Our results suggest that Drosophila Met and gce

experienced divergent evolutionary pressures following the duplication of an ancestral gce-like gene found in less derived holometabolous insects.

Introduction

The juvenile hormones (JH) are sesquiterpenoid products of the insect corpus allatum. JH endocrinology underpins molting and metamorphosis (Riddiford, 1994), reproduction (Bownes, 1989; Dubrovsky, 2002), diapause (Denlinger, 1985), and various aspects of behavior and physiology, some unique to social insects (Robinson and Vargo,

1997).

Molting and metamorphosis reflect the interplay of JH with 20-OH-ecdysone (20-

E), the steroid molting hormone. In the presence of JH, 20-E directs molting to the next larval instar. In the final larval instar, a substantial depression of JH in the hemolymph facilitates 20-E directed metamorphosis; exogenous JH applied at this time disrupts the normal developmental program (Truman and Riddiford, 2007; Liu et al., 2009). In

Lepidoptera and Coleoptera, JH application elicits a classical antimetamorphic effect, and larvae undergo supernumerary larval molts. The dipteran response to exogenous JH is incongruent with other Holometabola. When challenged with sublethal doses of JH, metamorphosis proceeds, but adult flies emerge with various developmental defects,

including malrotation of the male genitalia or female sternite bristle disruption

(O’Donnell and Klowden, 1997; Madhavan, 1973). Death occurs at the pharate adult stage in a dose-dependent manner (Liu et al., 2009).

The JH analog (JHA) methoprene can disrupt development of certain insects

(Staal, 1975). However, documented methoprene-resistant populations of aedine

mosquitoes (Cornell et al., 2002; Dame et al., 1998) has demonstrated that, contrary to

initial expectations (Williams, 1967), certain insects can become refractory to treatment

with compounds chemically derived from one of their endogenous hormones.

An understanding of the molecular mechanism of methoprene resistance and JH

activity in general has been hindered by the inability to identify and characterize a bona

fide JH receptor. A prime candidate gene for a JH receptor component, Methoprene

tolerant (Met), was identified in a screen for Drosophila mutants resistant to methoprene

(Wilson and Fabian, 1986). Met is a member of the bHLH-PAS family of transcriptional

regulators (Ashok et al., 1998), genes involved in various developmental and regulatory

networks as well as response to environmental signals and ligand binding (Crews, 2003).

MET was shown to bind JH III with nanomolar affinity and to regulate transcription in a

JH-dependent manner (Miura et al., 2005), suggesting that Met can play a central role in

JH signaling in Drosophila. However, loss of MET function is not lethal, suggesting that

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another gene affords functional redundancy, or that alterative JH signaling mechanisms exist (Wilson and Ashok, 1998; Wilson et al., 2003; Riddiford et al., 2010).

A genome-wide survey of D. melanogaster bHLH PAS proteins revealed germ

cell expressed (gce), a gene with high sequence identity to Met (Moore et al., 2000).

Pulldown assays showed GCE as a JH-sensitive MET binding partner (Godlewski et al.,

2006). Recently, Liu et al., (2009) showed that JH influences the ability of MET and

GCE to transcriptionally regulate the caspase genes that direct programmed cell death

(PCD) that occurs during metamorphosis (Liu et al., 2009).

The red flour beetle, Tribolium castaneum, has a single Met-like ortholog. RNAi gene silencing experiments have shown that TcMet is involved in the JH response

(Konopova and Jindra, 2007; Parthasarathy et al., 2008). TcMet-deficient beetles undergo precocious larval-pupal metamorphosis (Konopova and Jindra, 2007) and display resistance to JHAs (Parthasarathy et al., 2008). Aedes aegypti, Culex pipiens, and

Anopheles gambiae mosquitoes each contain a single gce-like ortholog (Wang et al.,

2007). Presumably, these proteins mediate JH signaling similarly to the single gene in coleopteran insects.

The existence of two Met-like genes in Drosophila versus a single ortholog in

Culicidae led to the proposal that Met and gce are paralogs derived from an ancestral gene (Wang et al., 2007). The conservation of several intron positions among the

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mosquito orthologs, TcMet, and Dmgce supports the notion that the intron-sparse Met

arose after the duplication of a gce-like ancestor sometime during early dipteran evolution.

In this work, we took advantage of the recently elucidated 12 Drosophila

genomes (Ashburner, 2007) and examined the evolution of Met and gce across this

genus. We then compared various functional domains of Met and gce orthologs across

Holometabola, finding conservation of Met-specific PAS A and PAC domains in mosquitoes and beetles. We used RACE PCR to obtain the entire D. melanogaster gce mRNA, and found that the 5’ terminus of this message is the annotated sequence

CG15032, conserved across Drosophila but absent in mosquitoes and beetles. Since gene duplication events often result in relaxed selective constraint on the parental sequence

(MacCarthy and Bergman, 2007), we examined the signatures of selection across the Met and gce paralogs. Using maximum likelihood estimates of dN/dS ratios, we found that

Met and gce have been subject to distinct patterns of selection. Finally, we show that unlike Met, gce is not contributed as a maternal mRNA in D. melanogaster.

Materials and Methods

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Drosophila stocks

D. virilis was a gift from Amanda Simcox, Ohio State University. D. persimilis

and D. willistoni were obtained from the UC San Diego Drosophila Stock Center

(http://stockcenter.ucsd.edu). D. melanogaster w1118 and O-RC wild-type flies were

obtained from the Bloomington Drosophila Stock Center, Indiana University

(http://flystocks.bio.indiana.edu). All flies were cultured at 25º C on a 12:12 L:D

photoperiod on a standard yeast-molasses-cornmeal medium augmented with propionic acid to retard mold growth.

Sequences

Coding sequences (CDS) for the putative Met, gce, and CG15032 orthologs from

the 12 available Drosophila genomes were identified using the GBrowse function of

Flybase (Tweedie et al., 2009; http://www.Flybase.net) or the BLink function of the

NCBI web server (http://www.NCBI.nih.gov). Flybase annotation tags for each

identified Met and gce ortholog are listed in Table 4.1. GBrowse generates a list of

annotated orthologs from a user-specified query and provides the microsyntenic relationships for each predicted ortholog so that flanking genes can be compared to the orthologous regions in D. melanogaster. Using this output, we inferred the spatial relationships of gce and CG15032 orthologs across Drosophila. Unclear annotations were subject to RT-PCR analyses in order to confirm or revise database predictions.

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Conservation of intron positions in Met and gce across taxa was examined using multiple

pair-wise alignments of each species’ genomic sequence (decorated FASTA format) with

the corresponding CDS.

RT-PCR Analysis

Adult O-RC wild-type flies and w1118 D. melanogaster embryos were used for RT-

PCR analyses. For analysis of gce and CG15032 orthologs, total RNA was isolated from adult D. melanogaster (O-RC strain), D. persimilis, D. willistoni, and D. virilis using

TriZol reagent (Invitrogen, Carlsbad, CA). Reverse transcription was performed using the SuperScript II reverse transcription kit from Invitrogen (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

PCR amplification to confirm physical separation of GK17951 and D. willistoni

gce was performed with primers GK17951F and Wil-gce-R. PCR amplification to

confirm the cDNA sequence for D. persimilis Met was performed with primers

GL13106F and GL13107R. Reaction conditions were: initial incubation for 3 min at

94ºC, 30 cycles of 30 sec at 94ºC, 30 sec at 60ºC, and 30 sec at 72ºC, followed by a final

10 minute elongation step at 72ºC. D. virilis gce was amplified from adult cDNA using

primers VGce05 and VGce02, which were designed from D. virilis genomic sequences.

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A 3.1 kb cDNA sequence including the full coding region was TOPO TA-cloned using the PCR 4.0 vector (Invitrogen, Carlsbad, CA) and sequenced.

For analysis of embryonic Met and gce expression in D. melanogaster, total RNA was isolated from adult w1118 flies as described above. cDNA synthesis and RACE PCR

were performed with the SMART RACE cDNA amplification kit (Clontech Laboratory,

Mountain View, CA) according to the manufacturer’s instructions. 5’ RACE was

performed using primer Gce14, which was designed from a gce cDNA sequence deduced

from the genomic sequence (GenBank accession no. NC004354). A second round of 5’

RACE analysis was performed using primer Gce28, which was designed from the

sequence obtained by the first 5’ RACE analysis. 3’ RACE was performed using primer

Gce7. Finally, the entire coding region of the gce cDNA was amplified using primers

Gce13 and Gce02Xho to confirm integrity of the sequences. Sequences of all primers used in this study are summarized in Table 4.2.

RT-PCR analysis of embryonic transcripts

RT-PCR was performed to analyze gce and Met expression during embryonic development in D. melanogaster. Total RNA from D. melanogaster w1118 embryos was isolated as above, and approximately 5 µg was reverse transcribed using MMLV reverse

transcriptase (Invitrogen). PCR amplification was carried out using primer pairs Gce33

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and Gce12 or Gce1245 and Gce1371 for gce (Baumann et al., unpublished), primer pairs

Met1594 and Met1739 for Met, and RP49-138 and RP49-293 for rp49 (Barry et al.,

2008). Amplification was performed in a 20-µl volume containing 0.4 pmol of each

primer, 0.5U of Taq polymerase (Qiagen), 4.0 pmol of each dNTP and 1X accessory

buffer. Reaction conditions were: initial incubation for 3 minutes at 94º C followed by

25 cycles of 30 sec at 94º C, 30 sec at 57º C (Gce1245 and Rp49) or 62o C (Gce33 and

Met), and 30 sec at 72º C, with a final elongation step at 72º C for 2 minutes.

Functional domain analysis

All multiple sequence alignments (MSA) were performed in ClustalW

(Thompson et al., 1994) using the default settings for gap penalties. After alignment,

sequences were edited by hand in BioEdit (Hall, 1999) in order to adjust or remove

poorly aligning sequence. Under the justification that sequence identity implies

functional conservation, we quantified sequence identity among conserved domains in

Met- or gce-like sequences across several holometabolous insects. In particular, we sought to address what structural features are shared between Met, gce, and the putative antecedents of these genes. Met and gce display functional redundancy in D. melanogaster; certain phenotypic characters can be rescued by gce overexpression

(Baumann et al., unpublished). We expected that the single ortholog in Tribolium and

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each of the culicids might display high identity with both Met and gce functional domains, based on our assumption that they maintain the functions of both Drosophila paralogs as a single, composite unit. Sequence identity matrices using the amino acid sequences for the bHLH, PAS A, PAS B, and PAC domains from TcMet, DmMet,

DpseMet, AaMet, AgMet, and Dmgce were constructed with BioEdit (Hall, 1999), using the previously defined domain boundaries (Ashok et al., 1998), MSAs for which are presented in Figure 4.1. These domains were chosen based on previous work demonstrating their functional importance in the dimerization of MET and GCE

(Godlewski et al., 2006). A phylogenetic tree of all available Met and gce amino acid sequences (truncated D. persimilis sequences were omitted in order to optimize alignment) was constructed in MEGA 4.0 (Tamura et al., 2007) using the neighbor joining method in order to provide context for the evolutionary history of these genes across the holometabola (Figure 4.8).

In order to identify functional domains in D. melanogaster CG15032, we performed searches using MOTIF (http://motif.genome.jp), ExPasy

(http://www.expasy.org), and the conserved domain database (CDD; Marchler-Bauer et al., 2009) available through the NCBI web server

(http://www.ncbi.nlm.gov/structure/cdd/cdd.shtml). CDD imports several external databases including Pfram, SMART, COG, PRK, and TIGRFAM. PAC domain

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sequences for D. melanogaster Met and gce were obtained from the SMART protein

domain database (SMART accession no. SM00086).

dN/dS analysis

In order to infer the relative selective pressures operating across the CDS of Met

and gce, we obtained separate estimates of nonsynonymous to synonymous substitution

ratios (dN/dS) for the each of 12 drosophilid Met and gce orthologs. Sequences were translated, aligned, and reverse-translated in BioEdit (Hall, 1999) to prevent reading frame disruption during alignment. Datasets were analyzed with DataMonkey

(Kosakovsky Pond and Frost, 2005), a web-based implementation of the HyPhy

(hypothesis testing using phylogenies; Kosakovsky Pond and Muse, 2005a) package.

Codon substitution models 000010 and 010020 (TrN) were chosen via the DataMonkey

automatic selection function for Met and gce, respectively, and each MSA was evaluated using the PARRIS (Scheffler et al., 2006) and REL (random effects likelihood) methods

(Kosakovsky Pond and Muse, 2005b). The PARRIS algorithm evaluates an entire coding sequence to infer positive selection (dN/dS > 1), while the REL algorithm estimates a site-based dN/dS ratio at each codon position. Given the small size of the dataset and the susceptibility of the REL method to Type 1 statistical error, we used the REL method to identify regions of Met or gce where the relative dN/dS is elevated or depressed across

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the CDS (Kosakovsky Pond, personal communication), based on the a priori hypothesis that Met and gce are subject to differential post-duplication selective pressures.

Results

Identification of Met and gce orthologs in 12 Drosophila species

The phylogeny of the 12 species for which complete genome sequence data are available can be split into two basal lineages, the Sophophora and Drosophila, which are believed to have diverged from a common ancestor approximately 63 million years ago

(Tamura et al., 2004). Both Met and gce orthologs were identified in each of these 12 genomes, indicating that these two genes predate the origin of the genus.

The relative numbers and positions of introns across duplicate genes may imply the duplication mechanism. In the case of exon shuffling, recombination between introns results in the modular transfer of exons between genes (Patthy, 1999). In retrotransposition, an mRNA copy of the parental gene is incorporated into the genome.

The intron positions of all Met and gce orthologs identified in this study are presented in a phylogenetic tree (Figure 4.2) of the accepted taxonomic relationships among these flies (Powell, 1997). The annotation state of several species precluded exact

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prediction of the locations of novel, species-specific introns. Problematic sequences

include D. mojavensis Met, D. willistoni gce, and D. grimshawi gce.

The location of a single intron in the PAS B region of Met is conserved across the

genus. Within the Sophophora, one intron, immediately C-terminal to PAS A, was

gained in the lineage leading to D. simulans. In the lineage leading to D. willistoni, a

single intron was gained C-terminal to the PAS B domain. The D. mojavensis Met

ortholog harbors a second intron, but the annotation state of this species’ genome

prevented determination of its exact location.

The positions of six introns are common to all gce orthologs. D. melanogaster, D.

pseudoobscura, and D. mojavensis have an additional, seventh intron in the bHLH

domain. D. persimilis gce contains only three introns, one in the bHLH domain and two

C-terminal to the PAC domain. A large deletion spanning the C-terminal portion of

bHLH to the PAC domain exists in this gene (Figure 4.2), accounting for the missing

introns.

CG15032 is in the N-terminus of gce

To obtain full-length gce cDNA, we performed 5’ and 3’RACE, obtaining approximately 800 bp of newly identified coding sequence at the 5’ terminus. RT-PCR analysis confirmed the integrity of this coding region in both D. melanogaster and D.

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virilis. The newly identified region had been previously annotated as a neighboring gene

(GenBank accession no. CG15032) in the database. However, our analysis indicates that this sequence is not a separate gene, but is part of gce. While this manuscript was under

revision, an annotation update removed CG15032 from the database, elongating the D.

melanogaster gce transcripts and translation products. We obtained a cDNA sequence of

5.5 kb with 1337 bp 5’ and 1344 bp 3’ untranslated region. Figure 4.3 shows a modified

representation of the genomic structure of gce, consisting of 10 exons and 9 introns and

consistent with the current FlyBase annotation (Figure A.1).

No functional domains were identified in CDD searches using the annotated D.

melanogaster CG15032 protein sequence. MOTIF analysis of CG15032

(http://motif.genome.jp/) indicated the presence of a glycine-rich region spanning

residues 182-220 and a histidine-rich region spanning residues 229-265. The region

spanning residues 229-265 in CG15032 contains His-Pro repeats (Fig 4a). These His-Pro

repeats are not unique; they are present in the products of transcription factor genes

Bicoid, Paired, and Tango, the Drosophila bHLH PAS ortholog of the human Aryl

hydrocarbon nuclear translocator (Arnt) (Crews, 2003).

BLAST searches with CG15032 against the Tribolium, Aedes, and Anopheles

genomes produced no significant hits, indicating that feature of gce evolved after the

divergence of Culicidae and Drosophilidae. Sequence identity among CG15032

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orthologs is substantially lower (24-98%) than that among annotated gce orthologs (68-

97%, excluding D. persimilis gce). Sequence identity matrices, showing the relative conservation among CG15032 and gce orthologs, are presented in Table 4.3.

Microsynteny of CG15032 and gce orthologs

Analysis of the microsynteny, the spatial arrangement of loci in a particular chromosomal region, illustrates how genetic elements are maintained and rearranged through evolutionary time. In particular, microsyntenic analysis allows visualization of genomic localization of duplicate genes (Sztal et al., 2007).

CG15032 orthologs are conserved across Drosophila, and the syntenic organization of CG15032 and gce orthologs within the genus is generally conserved.

However, a comparison between D. melanogaster and D. pseudoobscura reveals that the

D. pseudoobscura CG15032 ortholog and its 5’ neighbor (the ortholog of D. melanogaster CG5877) reside on chromosome 2R, while D. pseudoobscura gce remains on the X chromosome. In D. persimilis, the CG15032 and gce orthologs retain the melanogaster arrangement, indicating that the rearrangement of these elements occurred independently in D. pseudoobscura.

The CG15032 ortholog prediction for D. willistoni, GK17951, encodes a 34 amino acid protein that exists downstream of the D. willistoni MED8 ortholog, a gene

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located on chromosome 2R in D. melanogaster (data not shown). RT-PCR analysis using

primers designed to span GK17951 and D. willistoni gce confirmed the discontinuity of

these sequences. In both this species and in D. grimshawi, the N-terminal portion of the gce translation aligns to D. melanogaster CG15032 sequence. In the annotated proteins for both of these orthologs, the bHLH domain immediately follows the CG15032 orthologous region (Figure 4.4b). The microsynteny of CG15032 and gce orthologs

across the genus Drosophila is provided in Figure 4.5. For simplicity, all orthologs have been named according to their D. melanogaster counterparts.

Embryonic expression profiles

We investigated the expression profiles of Met and gce in D. melanogaster embryos. Using RT-PCR, we detected no gce transcript from 0 to 8 hours after oviposition. This suggests that embryos begin to synthesize endogenous gce 8h after egg fertilization. This is in striking contrast with Met transcript, which is found in 0-8h embryos as maternal mRNA (Figure 4.6). We have found that Met transcript in Aedes

aegypti embryos (data not shown), and both Met and gce transcripts in D. virilis embryos

are maternally deposited (data not shown), indicating that this shift in embryonic gce

expression may be novel to the Sophophora, or to D. melanogaster alone.

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Conservation of functional domains

As expected (Wang et al., 2007) sequence comparisons of the entire mosquito

Met open reading frames from A. aegypti, A gambiae, and C pipiens (ORF) demonstrate

higher overall sequence identity to Dmgce than to DmMet (Table 4.4). We observed that the bHLH and PAS B functional domains of TcMet, AaMet, and AgMet share higher

sequence identity with the corresponding domains from Dmgce, but the PAS A and PAC domains of these genes show higher identity with DmMet, an observation consistent with

previous reports for TcMet (Konopova and Jindra, 2007). Contrary to expectation based

on genomic structural similarities shared by TcMet and Dmgce, identity matrices

produced from our MSAs indicate that the TcMet ORF shares higher sequence identity

with DmMet than with Dmgce.

MSAs of all Drosophila gce orthologs indicate that D. persimilis gce lacks the

PAS (A and B) domains, while the PAC domain is conserved. The D persimilis Met

ortholog identified by GBrowse, GL13016, harbors a complete PAS A domain but the

PAS B is truncated. GL13107, the downstream neighbor of GL13106 also contains

sequence that aligns to D. melanogaster Met. When the GL13106 and GL13107

sequences were combined, the chimeric construct aligned with other Drosophila Met

sequences, suggesting that D. persimilis Met may be a composite of these annotations,

each of which has an ATG start and TAG stop codon. We performed RT-PCR with

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primers spanning GL13106 and GL13107, but failed to amplify a hybrid GL13107-

GL13107 product, confirming that these Met-aligning sequences are discrete entities.

Neighbors of the truncated D. persimilis gce, which lacks both PAS A and PAS B

domains, are the orthologs of D. melanogaster CG15032 and CG8173, respectively,

neither of which contain the missing PAS domains. The 5’ and 3’ termini of D.

persimilis gce align to the corresponding regions of D. melanogaster gce, illustrating that

a substantial deletion has removed the region spanning the 3’ end of the bHLH domain through the PAS B domain.

Each Met and gce ortholog retains a strongly conserved PAC domain. This

domain, immediately C-terminal to the PAS B domain, is thought to play a role in PAS

domain folding (Zhulin et al., 1997). The gce PAC domain shows 87% sequence identity across Drosophila whereas the Met PAC domain shows 82% identity. The single

exception is D. persimilis Met, which apparently lacks a PAC domain in either GL13106

or GL13107.

dN/dS in Met and gce

The relative selective pressure across a CDS can be examined by estimating the

nonsynonymous to synonymous substitution ratio (dN/dS). This method has been used to

scan genomes for conserved protein coding sequences, based on the expectation that

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regions where synonymous substitutions are greatly favored relative to nonsynonymous

substitutions (i.e. dN/dS << 1) are likely to be evolutionarily conserved, functional units.

An extension of this approach employs dN/dS ratios at individual sites along a CDS in

order to infer codons subject to positive or negative selection. When dN/dS = 1, the effects of nonsynonymous and synonymous substitutions are effectively equal, implying neutral evolution. When dN/dS > 1, adaptive evolution (positive selection) is inferred, whereas dN/dS < 1 implies purifying selection. Sites under positive selection have been found to include active surface residues of venom toxins (Gibbs and Rossiter, 2008) and salivary proteins of arthropod vectors (Schwalie and Schultz, 2009).

Unfortunately, statistical support for site-based dN/dS estimates of selection is hindered by our small dataset of 12 orthologs. First, analysis of the entire CDS for both

Met and gce was performed using the PARRIS algorithm (Scheffler et al., 2006) in order to infer positive selection in either gene. We found no statistically supported (p ≤ 0.05) instances of positive selection in either gce or Met. We then used the REL method to infer regional trends in the dN/dS ratio across each coding sequence rather than to identify particular residues under the influence of positive or negative selection.

Analysis using the REL method demonstrated striking dissimilarity in the relative distributions and locations of selectively constrained regions in Met vs. gce. Notably, the

N-terminal half of gce has markedly depressed dN/dS ratios compared to the C-terminal

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half. The region of gce spanning codon positions 400-650 appears to be subject to relaxed evolutionary constraint as indicated by dN/dS values close to 1 (Figure 4.7b).

This scenario is in stark contrast with the dN/dS trend seen across Met orthologs, where dN/dS ratios are largely depressed, indicating that the majority of the coding sequence is subject to strong selective constraint (Fig 7a). As expected, the bHLH, PAS A, PAS B, and PAC domains reside in regions of depressed dN/dS, in accordance with an evolutionarily conserved function.

Discussion

In this study, we have investigated the evolutionary history of paralogous genes involved in juvenile hormone signaling across the genus Drosophila. Distinct signatures of selective pressure and differential embryonic expression patterns suggest that Met and gce remain functionally conserved. In addition, we have shown that the Flybase annotation CG15032 is the 5’terminal sequence of the gce transcript and that this sequence is conserved across the genus. The genomic structure and functionally important features of the Met and gce orthologs are largely conserved. Conservation of a single intron gained in the PAS B domain of all Drosophila Met sequences indicates that the duplication that produced Met and gce predates the family Drosophilidae.

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The strikingly fewer number of introns in Met vs. gce across Drosophila suggest that Met arose via retrotransposition from the parental gce ancestor. A duplicate gene that arises by retrotransposition, if retained, often diverges from the parental gene.

Functional redundancy afforded by an increase in copy number of the parental gene subjects one or both paralogs to relaxed selective constraint, leading to sub- or neo- functionalization. Based on our results and recent work demonstrating that overexpressed gce can rescue some Met phenotypic characters (Baumann et al., unpublished), we propose that Met has co-opted some JH-related physiological function(s) in adult flies.

The viability of Met mutants (Wilson and Ashok, 1998; Wilson et al., 2003) has been suggested to argue against a role for MET in JH signaling as a receptor component

(Riddiford, 2007; Gilbert, 2000). However, GCE might play a redundant role to rescue survival when MET is absent or nonfunctional. Mosquitoes exhibit a morphogenetic response to exogenous JH that mimics higher Diptera (O’Donnel and Klowden, 1997), presumably through the action of a single Met-like ortholog. The mosquito orthologs retain higher sequence identity to the entire gce ORF but have a strongly conserved Met- like PAS A. These genes may function through the cooperation of alternatively spliced dimerization partners, and act as JH-dependent transcriptional regulators in a manner reflective of the MET:GCE heterodimers (Godlewski et al., 2006) of Drosophila.

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Alternatively, other unidentified proteins may participate in forming the putative JH-

receptor complex in lower Diptera. Studies addressing the cross talk between JH and

ecdysteroid signaling systems have shown that the multipartite machinery that directs the

molting process is complex (Li et al., 2007).

We used estimates of dN/dS ratios across the CDS of Met and gce to demonstrate

that the relative intensities and localizations of selective constraint are distinct for each paralog. We expected elevated dN/dS estimates in gce relative to those in Met based on

our a priori interpretation that Met has at least partially replaced gce in JH signaling

(Wang et al., 2007), leading to relaxed selective constraint across the gce CDS.

Interestingly, this phenomenon was only observed in a subset of C-terminal residues in gce. Substantially depressed dN/dS ratios in the N-terminal half of gce imply a conserved function subject to purifying selection. C-terminal degeneracy was found to confer differential ligand-specificity and downstream regulatory targets of human and mouse aryl hydrocarbon receptors (Ramadoss and Perdew, 2005; Flaveny et al., 2010),

suggesting that C-terminal differentiation between Met and gce may serve to regulate the

transcription of distinct sets of downstream effector genes.

With few exceptions, each identified Met and gce ortholog harbors the primary functional domains (bHLH, PAS A and B) and a PAC domain immediately C- terminal to PAS B. PAC conservation is substantial across the genus Drosophila (84-

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100% in Met, 93-100% in gce). Functionally, PAC domains have been reported to be

involved in PAS domain folding (Crews, 2003). While no studies have addressed the

functional importance of PAC in Met or gce, Wilson et al., (2006) identified the mutation

sites in Met alleles, and demonstrated that a PAC domain mutation (E478K) results in the

weak Met31 allele that confers a modest level of methoprene resistance and refractoriness

to methoprene-induced adult morphogenetic phenotypic characters. Features of a

transactivation domain (TAD), whose sequences are broadly defined but include regions

rich in glutamine and aspartic acid residues, are evident in the C-terminal regions of both

Met and gce across Drosophila (Figure 4.2).

Our RACE PCR results show that CG15032 is the previously unidentified 5’

terminus of the gce message. Girard et al., (2006) reported the recovery of a chimeric

fragment consisting of CG15032 and gce in a search for SoxNeuro target genes, a result

that corroborates our finding. A CG15032 ortholog does not appear in BLAST searches

against Culicid or Tribolium genomes, indicating that the incorporation of this sequence

into gce occurred following the origination of Met and gce.

We recently (Baumann et al., unpublished) examined adult JH target tissues and found that Met and gce are often co-expressed. While Met mRNA appears in embryos as

a maternal transcript, gce is only transcribed after the first 8 hours of embryonic

development (Figure 4.6). Roles for JH in the Drosophila embryo have not been

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reported, but certain juvenoids applied to early embryos cause death (Smith and Arking,

1975). Overexpression of Met+ in combination with exogenous JH application has been

shown to cause early larval mortality, shifting the sensitive and critical periods ahead

several instars (Barry et al., 2008) presumably by altering the stoichiometric equilibrium

between MET and GCE and perhaps favoring MET homodimerization that might alter

the expression profiles of downstream targets. Similarly, it is possible that in the early embryo, where MET is present in the absence of gce, MET homodimers perform a JH- sensitive regulatory role distinct from MET:GCE dimers.

In this paper we have identified all Met and gce orthologs in 12 members of the genus Drosophila whose evolutionary history spans nearly 63 million years (Tamura et

al., 2002). Functional domain identity between PAS A and PAC domains in mosquitoes,

beetles, and Drosophila Met offer potential targets for the functional dissection of GCE in

Drosophila. The modular architecture of the culicid ortholog, comprised of Met- and

gce-specific functional domains, suggests that the mosquito genes may function similarly

to MET:GCE complexes, but as individual entities, perhaps through homodimerization.

The differences in selective constraint along Met and gce and the demonstration of

differences in temporal expression in embryonic D. melanogaster provide a platform for

further investigations of the evolution of this bipartite system from a single component

transcriptional regulator.

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Table 4.1. The Flybase annotation symbols for each of the Drosophila Met and gce orthologs. A * indicates that the sequence is the identified ortholog via BLASTp searches with a D. melanogaster CG15032 query.

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Species Primer Sequence D. melanogaster Gce14 CTGTTTGAGCATGTTCTGGTCCTCGGGATG Gce28 CGCTTGTGCAGTTTGTTTACGCCTCTATCC Gce7 TGTGGCCAGGAATTTGCGGGAACAGAGTCA Gce13 GAGAACATGGAGGGTGCCAGTCGCAG Gce02Xho AATCTCGAGCTAGTCCTGGTCGTCCTC D. persimilis GL13106F GGCACACAGAATCCCAGTCT GL13107R CAGATGGAGGTGGAGAGGAG D. willistoni GK17951F ATGGATCGCGAATCCCTTAG Wil-gce-R TATTGGCATCAACCAGACGA D. virilis DvGce05 AAAATGAGCAGCCTCGATGCCTATCTG DGcev02 CGCCTTTGCCGTTGGCTGCCACATT

Table 4.2. List of primer sequences used in RT-PCR analyses.

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GCE mel sim sec yak ere ana pse per gri wil vir sim 0.973 sec 0.923 0.944 yak 0.951 0.956 0.907 ere 0.869 0.869 0.891 0.888 ana 0.817 0.815 0.774 0.82 0.751 pse 0.821 0.821 0.775 0.83 0.76 0.805 per 0.435 0.438 0.445 0.443 0.432 0.425 0.572 gri 0.732 0.734 0.697 0.741 0.681 0.73 0.744 0.37 wil 0.79 0.791 0.748 0.795 0.731 0.779 0.803 0.417 0.77 vir 0.73 0.733 0.692 0.739 0.681 0.724 0.755 0.372 0.807 0.771 moj 0.741 0.746 0.707 0.755 0.701 0.741 0.767 0.38 0.836 0.779 0.861

CG15032 mel sim sec yak ere ana pse per gri wil vir sim 0.981 sec 0.949 0.96 yak 0.919 0.926 0.894 ere 0.858 0.865 0.83 0.828 ana 0.55 0.553 0.537 0.529 0.51 pse 0.412 0.415 0.396 0.415 0.378 0.391 per 0.408 0.41 0.392 0.41 0.374 0.395 0.959 gri 0.28 0.287 0.274 0.271 0.258 0.252 0.246 0.239 wil 0.294 0.294 0.28 0.291 0.288 0.291 0.257 0.254 0.341 vir 0.265 0.265 0.262 0.264 0.263 0.262 0.241 0.239 0.381 0.315 moj 0.265 0.268 0.271 0.261 0.267 0.262 0.23 0.228 0.372 0.306 0.63

Table 4.3. (A) Sequence identity matrix for gce orthologs (omitting CG15032 orthologous sequence) across Drosophila. Low sequence identity in D. persimilis gce is a result of truncated gce CDS. (B) Sequence identity matrix for CG15032 orthologs identified in the genus Drosophila.

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bHLH DmMet TcMet CpMet AgMet AaMet TcMet 0.492 CpMet 0.692 0.538 0.738 0.507 0.861 AgMet AaMet 0.692 0.492 0.83 0.815 Dmgce 0.723 0.523 0.769 0.8 0.83

PAS A DmMet TcMet CpMet AgMet AaMet TcMet 0.482 CpMet 0.637 0.465 AgMet 0.689 0.5 0.862 AaMet 0.637 0.517 0.862 0.896 Dmgce 0.689 0.413 0.603 0.603 0.586

PAS B DmMet TcMet CpMet AgMet AaMet TcMet 0.537 CpMet 0.58 0.567 AgMet 0.586 0.592 0.851 AaMet 0.58 0.567 0.858 0.82 Dmgce 0.709 0.549 0.629 0.641 0.629

PAC DmMet TcMet CpMet AgMet AaMet TcMet 0.659 CpMet 0.568 0.59 AgMet 0.636 0.659 0.863 AaMet 0.613 0.568 0.886 0.84 Dmgce 0.681 0.568 0.568 0.59 0.568

Entire ORF DmMet TcMet CpMet AgMet AaMet TcMet 0.401 CpMet 0.486 0.401 AgMet 0.505 0.403 0.765 AaMet 0.49 0.395 0.79 0.744 Dmgce 0.596 0.386 0.539 0.547 0.545

Table 4.4. Sequence identity matrices for bHLH, PAS A, PAS B, and ORF amino acid sequences for the Met and gce orthologs from D. melanogaster, three mosquitoes, and Tribolium.

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bHLH

PAS A

PAS B

PAC

Figure 4.1. Multiple sequence alignment of Drosophila, mosquito, and beetle Met/gce bHLH, PAS A, PAS B, and PAC domains.

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Figure 4.2. Intron position in Drosophila Met (a) and gce (b) orthologs presented as superimposed on the Drosophila phylogeny. Instances where intron position could not be determined are indicated by question marks. Functional domains and identified motifs are indicated in boxes across each CDS.

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Figure 4.3. The revised genomic organization of gce consists of 10 exons and 9 introns. Shaded regions represent coding sequence. White regions indicate 3’ and 5’ UTR.

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A 10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| [melanogaster] QAAYPGHSAHMHALHHQYP------QPHPHAHHPQHPHHS------P [simulans] QAAYPGHSAHMHALHHQYP------QPHPHAHHPQHPHHS------P [sechellia] QAAYPGHSAHMHALHHEYL------QPHPHAHHPQHPHHS------P [yakuba] QAAYPGHSAHMHALHHQYP------QPHPHAHHPQHPHHS------P [erecta] QAAYPGHSAHMHALHHQYP------QPHPHAHHPQHPHHS------P [ananassae] HPAGHMHPAMHHQYPPSHP------HGHPHPHQHPHLHHQ------Q [persimilis] FGAHTAHRHPLHHQYPHMP------VPHPHPHPHQHTHHHPQLQHQHQ [pseudoobscura] FGAHTAHRHPLHHQYPHMP------VPHPHPH--QHTHHHPQLQHQHQ [grimshawi] GHVLHPHPHHHHPHSHP------HPHSHHPHTHSHP------[willistoni] LHPHHPQHHPHHPQHHPSAHHLHHHGPPPPPTALHSHHSHPHGHHPH-HTHP------[virilis] -MLPHGQHQ-HAPLHH------PHPHPHAHPHPHHQ------[mojavensis] -MLPHGHHHPHAPLHHSHTHPLPH------PHPHPHALPHPHHQ------

B 70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| [melanogaster] HP------HHPHPHETMMEMFQLSNR------[simulans] HP------HHPHPHETMMEMFQLSNR------[sechellia] HP------HHPHPHETMMEMFQLSNR------[yakuba] HP------HHPHPHETMMEMFQLSNR------[erecta] HP------HHPHPHETMMEMFQLSNR------[ananassae] HP------HHQHPHDTMMEMFQLSNR------[persimilis] HPHPHPHQHPHPHPHETMMEMFQLSNR------[pseudoobscura] HP--HAHQHPHPHPHETMMEMFQLSNSYVRSRSTPQSPHSPQASAHILGRAAHCQSPASP [grimshawi] HH------QHPHPHETMMEMFQLSNSGREARNRAEKNRRDKLNGSIQELSTMVPHVAES [willistoni] HP------HGHHPHETMMEMFQLSNSGREARNRAEKNRRDKLNGSIQELSTMVPHVAES [virilis] HT------HHPHPHDSMMEMFQLSNR------

Figure 4.4. A portion of the multiple alignment of identified CG15032 orthologs. (A) The region of CG15032 orthologs containing His-Pro paired repeats is immediately N- terminal to the continuation of the annotated D. willistoni and D. grimshawi gce proteins. (B) The beginning of the bHLH domain in these proteins is underlined in black.

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Figure 4.5. Microsynteny of gce and CG15032 orthologs across 12 Drosophila species inferred from GBrowse. Genes are color-coded according to D. melanogaster orthologs. Colorless blocks indicate genes with no identified orthologs in D. melanogaster or genes that correspond to D. melanogaster orthologs on other chromosomes. The double shading in D. willistoni GK16199 and D. grimshawi GH17161 indicate annotations comprised of sequences homologous to both D. melanogaster gce and CG15032.

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Figure 4.6. Embryonic expression profiles of Met and gce in D. melanogaster relative to Rp49 expression.

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Figure 4.7. dN/dS plots from REL analysis of 12 Drosophila Met (a) and 11 Drosophila (D. persimilis excluded) gce (b) orthologs. Estimated dN/dS ratios are plotted against codon position.

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Figure 4.8. Neighbor joining phylogenetic tree of all available Met or gce orthologs (excluding D. persimilis). The Homo sapiens aryl hydrocarbon nuclear translocator (ahrnt) bHLH PAS gene serves as an outgroup sequence.

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Conclusions

This study examined the molecular biology and evolution of the paralogs Met and gce, genes involved in JH signaling, in evolutionarily distant members of the order

Diptera. In mosquitoes (lower Diptera), a single Met-like gene exists and shares higher identity with gce from D. melanogaster (higher Diptera) rather than with Met. This suggests that, during Dipteran evolution, Met arose from the duplication of a gce-like ancestor sequence. In D. melanogaster, overexpression of gce can rescue some Met phenotypes in a tissue-specific manner. Additionally, gce is critical for survival to adulthood; gce underexpression in either Met+ or especially Met genetic backgrounds results in preadult lethality. Met and gce are conserved throughout the genus Drosophila, and dN/dS analysis indicates that distinct selective pressures have acted on each paralog during their evolutionary history.

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I. Drosophila melanogaster Methoprene-tolerant (Met) gene homologs from three mosquito species, members of PAS transcriptional factor family

1. RT-PCR analysis with degenerate primers identified a single Met-like

homolog in the genome of each of the three mosquito species, Aedes

aegypti, Anopheles gambiae, and Culex pipiens.

2. Phylogenetic analysis, as well as comparison of intron number and

position in each of the identified mosquito genes, indicated that the

mosquito Met orthologs share higher sequence identity with Dmgce than

DmMet. This result indicates that DmMet arose from the duplication of an

ancestral, gce-like gene in lower Diptera.

3. Aedes aegypti Met is expressed throughout development, congruent with

previous reports showing widespread expression of DmMet during the life

cycle of Drosophila melanogaster. AaMet expression was detected in

whole body extracts of both adult males and females, and in the ovary and

fat body of the adult female, two known JH target tissues.

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4. RNAi-driven reduction of AaMet results in concomitant reduction in two

JH-inducible genes, Juvenile hormone inducible protein (AAEL006600)

and JH esterase (AAEL012886) in A. aegypti, implicating AaMet in the

involvement of adult JH signaling in this mosquito.

II. Juvenile hormone action requires paralogous genes in Drosophila melanogaster.

1. gce expression, determined by Northern analysis, was shown to be

widespread throughout development in D. melanogaster, with minimal

levels of whole body expression found during embryonic and pupal

development.

2. RT-PCR analysis of selected tissues finds gce expressed in known JH

target tissues, including the ovary and male accessory gland.

Additionally, gce is generally co-expressed with Met, except for late third

instar fat body, where its expression is substantially reduced relative to

Met.

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3. Overexpression of gce in a Met mutant background results in a dramatic

enhancement of methoprene-conditional toxic and morphogenetic defects,

resulting in phenotypes similar to those seen in wild type (Met+) flies. Met

mutant flies overexpressing gce show rescue of a non-conditional adult

phenotype, that of defective development of posterior facets in the

compound eye.

4. While gce overexpression could rescue preadult phenotypic characters in a

Met mutant background, its overexpression failed to rescue the phenotypes

of deficient oogenesis or reduced male courtship characteristic of Met

adults, showing that Met has co-opted the role of mediating JH-regulated

reproductive functions in Drosophila.

5. RNAi-driven reduction of gce expression from either an actin or tubulin

promoter demonstrates that gce is a vital gene; gce underexpression in

both Met+ and Met genetic backgrounds results in lethality. Met+ ; UAS-

gce-dsRNA / tubulin-GAL4 homo-/hemizygotes do not survive to

adulthood, and die primarily at the pharate adult stage. The same gce

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RNAi construct expressed in a Met mutant background shifts lethality to

early pupae.

III. Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila

1. Met and gce are conserved within the genus Drosophila, dating the

duplication that gave rise to these paralogs at least 63 million years ago,

based on the phylogeny of the genus. The paucity of introns in Met

orthologs relative to gce suggests a retrotransposition mechanism of

duplication.

2. The genomic structure of gce was revised. Using RACE PCR, we show

that the gene CG15032 is actually the 5’ terminus of gce, instead of a

separate gene.

3. The newly identified region of gce was identified in each represented

member of the genus Drosophila, but conservation of this region of gce is

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poor. In D. pseudoobscura, this portion of gce resides on a separate

chromosome. These data, along with the apparent absence of functional

domains in this region, suggest that this sequence likely plays no

functional role in gce.

4. Met and gce show different temporal expression profiles in D.

melanogaster embryos. Unlike Met, gce is not a maternal message; gce is

expressed in the embryo after 8 hours.

5. D. persimilis Met and gce have both experienced substantial deletions or

truncations. In this species, the Met ortholog appears to be represented by

two separate genes, each of which aligns with a distinct region of DmMet.

The PAC domain, present in all Met or gce genes examined, is missing

from either of the two putative D. persimilis Met sequences.

6. Analysis of the nonsynonymous-to-synonymous substitution ratios

(dN/dS) of Met and gce orthologs within the genus Drosophila indicates a

substantial relaxation of selective pressure on the C-terminal half of gce,

downstream of the functional domains. Conversely, nonsynonymous

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substitutions in the N-terminal half are stringently selected against.

Depressed dN/dS values across the Met coding sequence indicate strong selective constraint over the entire open reading frame.

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Appendices

Appendix A:

Revision of the genomic sequence of D. melanogaster gce.

5’ and 3’ RACE PCR were used to obtain the full length cDNA of D.

melanogaster gce. Our results indicated that the gene previously annotated as CG15032

by FlyBase (http://www.FlyBase.org) is the 5’ terminus of gce, not a separate gene.

While the manuscript that comprises Chapter 4 of this dissertation was under review, the annotation state of gce was revised in FlyBase to reflect the physical continuity of

CG15032 and gce. Presented in Figure A.1 is a sequence alignment of our RACE product with the updated annotation for D. melanogaster gce. This figure was published as supplemental material in Baumann, et al., 2010.

147 850 860 870 880 890 900 ....|....|....|....|....|....|....|....|....|....|....|....| gce AAGTGTGTTTTAAAAATGTTTGAACAAATACCATAGAGAATACCGTTAGGTGGCGGTAGT gce-RD_FBgn0261703 AAGTGTGTTTTAAAAATGTTTGAACAAATACCATAGAGAATACCGTTAGGTGGCGGTAGT 910 920 930 940 950 960 ....|....|....|....|....|....|....|....|....|....|....|....| gce GTAACTGTTAC-ACTGTAAAAATACCAAGTGAATTGAATTAGGTGCAAGAAAATACTGAA gce-RD_FBgn0261703 GTAACTGTTAC-ACTGTAAAAATACCAAGTGAATTGAATTAGGTGCAAGAAAATACTGAA 970 980 990 1000 1010 1020 ....|....|....|....|....|....|....|....|....|....|....|....| gce ATTTAGTTACTGGTTGTTAATATTTTCGTTTATTTTTTTTTTTTCAAAATAAAAAACAAA gce-RD_FBgn0261703 ATTTAGTTACTGGTTGTTAATATTTTCGTTTATTTTTTTTTTT-CAAAATAAAAAACAAA 1030 1040 1050 1060 1070 1080 ....|....|....|....|....|....|....|....|....|....|....|....| gce GGTACAAAGAATCCCGAAGAAATATTAAAGAAACACAAATAACGGGGAAGATAATAAAGC gce-RD_FBgn0261703 GGTACAAAGAATCCCGAAGAAATATTAAAGAAACACAAATAACGGGGAAGATAATAAAGC 1090 1100 1110 1120 1130 1140 ....|....|....|....|....|....|....|....|....|....|....|....| gce GAATTGCAACTGCAGGACAGTAACAACAACAAAGCAAAACAAAATAATACAAAATATATA gce-RD_FBgn0261703 GAATTGCAACTGCAGGACAGTAACAACAACAAAGCAAAACAAAATAATACAAAATATATA 1150 1160 1170 1180 1190 1200 ....|....|....|....|....|....|....|....|....|....|....|....| gce ATTCACCAATATAACACCAACAACCAGCAGACAGAGCGAGACGGATAGAGGCGTAAACAA gce-RD_FBgn0261703 ATTCACCAATATAACACCAACAACCAGCAGACAGAGCGAGACGGATAGAGGCGTAAACTA 1210 1220 1230 1240 1250 1260 ....|....|....|....|....|....|....|....|....|....|....|....| gce ACTGCACAAGCGAGTGCAAGCGAGAGGGCGTGCGTGAGAGCTTGCCGAAAGAGCGCAAGA gce-RD_FBgn0261703 ACTGCACAAGCGAGTGCAAGCGAGAGGGCGTGCGTGAGAGCTTGCCGAAAGAGCGCAAGA 1270 1280 1290 1300 1310 1320 ....|....|....|....|....|....|....|....|....|....|....|....| gce AAGAGAGAAGGGGGGCGAGAGAAAGAGAGAGCGAAAGCTTCATGCAGAATAACAAGAAAC gce-RD_FBgn0261703 AAGAGAGAAGGGGGGCGAGAGAAAGAGAGAGCGAAAGCTTCATGCAGAATAACAAGAAAC 1330 1340 1350 1360 1370 1380 ....|....|....|....|....|....|....|....|....|....|....|....| gce AGCGCAAAACATCTGGAGAACATGGAGGGTGCCAGTCGCAGCAGGAACTCCTCCACGAGC gce-RD_FBgn0261703 AGCGAAAAACATCTGGAGAACATGGAGGGTGCCAGTCGCAGCAGGAACTCCTCCACGAGC 1390 1400 1410 1420 1430 1440 ....|....|....|....|....|....|....|....|....|....|....|....| gce CACAGTCAAGGTCGCGGCCAGGACATTGAGGATCTCAAGCAAGACATTCCCTACTTTGAC gce-RD_FBgn0261703 CACAGTCAGGGTCGCGGCCAGGACATTGAGGATCTCAAGCAAGACATTCCCTACTTTGAC 1450 1460 1470 1480 1490 1500 ....|....|....|....|....|....|....|....|....|....|....|....| gce GAACCGCCGGCGCTCGATGCCGATCTGCTTGTTCTGGGCAAGAGCGAATGCCAGCTGGAC gce-RD_FBgn0261703 GAACCGCCGGCGCTCGATGCCGATCTGCTTGTTCTGGGCAAGAGCGAATGCCAGCTGGAC 1510 1520 1530 1540 1550 1560 ....|....|....|....|....|....|....|....|....|....|....|....| gce GAGCTGGCCTGGGATCGTGATGCGGATGGGGATGCGGATGCGCCGCTAGAGACGGCACCC gce-RD_FBgn0261703 GAGCTGGCCTGGGATCGTGATGCGGATGGGGATGCGGATGCGCCGCTAGAGACGGCACCC 1570 1580 1590 1600 1610 1620 ....|....|....|....|....|....|....|....|....|....|....|....| gce GCCGTCGATCTCGAGGAGGATAACTATCCGGATGAGAACGAGAGCTCGGTGCTGGGCAGC gce-RD_FBgn0261703 GCCGTCGATCTCGAGGAGGATAACTATCCGGATGAGAACGAGAGCTCGGTGCTGGGCAGC

1630 1640 1650 1660 1670 1680 ....|....|....|....|....|....|....|....|....|....|....|....| gce GACTATGCGCCCAGTGGGAGTGGGAGTGGTGCGAACAGTTTCTACCAATCGCCGACGCCC gce-RD_FBgn0261703 GACTATGCGCCCAGTGGGAGTGGGAGTGGTGCGAACAGTTTCTACCAATCGCCGACGCCC 1690 1700 1710 1720 1730 1740 ....|....|....|....|....|....|....|....|....|....|....|....| gce TCTGCCACCGGATCTGGTTGCGATCTGATGCTCCGACCGCCATCGAATTCCATGTACCAT gce-RD_FBgn0261703 TCTGCCACCGGATCTGGTTGCGATCTGATGCTCCGACCGCCATCGAATTCCATGTACCAT

Figure A.1 (continued)

Figure A.1. Sequence alignment of D. melanogaster gce RACE PCR and the corresponding FlyBase annotation.

148

Figure A.1 (continued)

1750 1760 1770 1780 1790 1800 ....|....|....|....|....|....|....|....|....|....|....|....| gce TTCAATTACCGATCGCCGGGTAGTCCAATGCCTGTGGCTCCAGGAGTGACCAACTCACGA gce-RD_FBgn0261703 TTCAATTACCGATCGCCGGGTAGTCCAATGCCTGTGGCTCCAGGAGTGACCAACTCACGA 1810 1820 1830 1840 1850 1860 ....|....|....|....|....|....|....|....|....|....|....|....| gce GGTCTGCATCCGTATGCACATTCCCCGGCGCATGGTAATCCGCCTGGTTTCTATCCGAAT gce-RD_FBgn0261703 GGTCTGCATCCGTATGCACATTCCCCGGCGCATGGTAATCCGCCTGGTTTCTATCCGAAT 1870 1880 1890 1900 1910 1920 ....|....|....|....|....|....|....|....|....|....|....|....| gce ATGTGGTATCCGAATGCACCATACGGATCGGCAGGAGCGGCAGGATCCGCCGGAGGAGCC gce-RD_FBgn0261703 ATGTGGTATCCGAATGCACCATACGGATCGGCAGGAGCGGCAGGATCCGCCGGAGGAGCC 1930 1940 1950 1960 1970 1980 ....|....|....|....|....|....|....|....|....|....|....|....| gce GTTTCCGGTGGCAGGTACATGGGCTATGGACCGGGCGGAGTGCCAGGAGGAACCAATTCG gce-RD_FBgn0261703 GTTTCCGGTGGCAGGTACATGGGCTATGGACCGGGCGGAGTGCCAGGAGGAACCAATTCG 1990 2000 2010 2020 2030 2040 ....|....|....|....|....|....|....|....|....|....|....|....| gce GGTCCGGGTGCGGGTCCTGGAGCAATGCAAGCTGCGTATCCTGGACACTCGGCTCACATG gce-RD_FBgn0261703 GGTCCGGGTGCGGGTCCTGGAGCAATGCAAGCTGCGTATCCTGGACACTCGGCTCACATG 2050 2060 2070 2080 2090 2100 ....|....|....|....|....|....|....|....|....|....|....|....| gce CATGCGCTGCATCATCAATATCCGCAGCCACATCCGCACGCCCACCACCCGCAGCATCCG gce-RD_FBgn0261703 CATGCGCTGCATCATCAATATCCGCAGCCACATCCGCACGCCCACCACCCGCAGCATCCG 2110 2120 2130 2140 2150 2160 ....|....|....|....|....|....|....|....|....|....|....|....| gce CATCATTCACCACATCCGCACCACCCGCATCCGCACGAGACCATGATGGAGATGTTTCAG gce-RD_FBgn0261703 CATCATTCACCACATCCGCACCACCCGCATCCGCACGAGACCATGATGGAGATGTTTCAG 2170 2180 2190 2200 2210 2220 ....|....|....|....|....|....|....|....|....|....|....|....| gce CTCTCGAATAGTGGTCGAGAAGCGCGAAACCGAGCGGAAAAGAATCGGCGGGACAAATTG gce-RD_FBgn0261703 CTCTCGAATAGTGGTCGAGAGGCGCGAAACCGAGCGGAAAAGAATCGGCGGGACAAACTG

2230 2240 2250 2260 2270 2280 ....|....|....|....|....|....|....|....|....|....|....|....| gce AACGGATCCATCCAGGAACTATCCACAATGGTGCCCCACGTGGCGGAGTCGCCACGTCGA gce-RD_FBgn0261703 AACGGATCCATCCAGGAACTATCCACAATGGTGCCCCACGTGGCGGAGTCGCCACGTCGA 2290 2300 2310 2320 2330 2340 ....|....|....|....|....|....|....|....|....|....|....|....| gce GTGGACAAAACAGCCGTTCTGCGCTTCGCCGCCCATGCATTGCGATTGAAGCATGCCTTT gce-RD_FBgn0261703 GTGGACAAAACAGCCGTTCTGCGCTTTGCCGCCCATGCATTGCGATTGAAGCATGCCTTT 2350 2360 2370 2380 2390 2400 ....|....|....|....|....|....|....|....|....|....|....|....| gce GGCAACAGTCTGATGCAGCAGCGACCACAGATCACGGACACCCTGATGGACATGCTGGAC gce-RD_FBgn0261703 GGCAACAGTCTGATGCAGCAGCGACCACAGATCACGGACACCCTGATGGACATGCTGGAC 2410 2420 2430 2440 2450 2460 ....|....|....|....|....|....|....|....|....|....|....|....| gce AGCTTCTTTCTCACACTCACCTGCCATGGCCACATACTGCTGATCTCGGCCAGCATTGAA gce-RD_FBgn0261703 AGCTTCTTTCTCACACTCACCTGCCATGGCCACATACTGCTGATCTCGGCCAGCATTGAA 2470 2480 2490 2500 2510 2520 ....|....|....|....|....|....|....|....|....|....|....|....| gce CAGCATTTGGGCCACTGTCAGTCGGATTTGTATGGTCAGAGCATTATGCAGATCACGCAT gce-RD_FBgn0261703 CAGCATTTGGGCCACTGTCAGTCGGATTTGTATGGTCAGAGCATTATGCAGATCACGCAT 2530 2540 2550 2560 2570 2580 ....|....|....|....|....|....|....|....|....|....|....|....| gce CCCGAGGACCAGAACATGCTCAAACAGCAGCTGATCCCCACCGAGTTGGAGAACCTCTTC gce-RD_FBgn0261703 CCCGAGGACCAGAACATGCTCAAACAGCAGCTGATCCCCACCGAGTTGGAGAACCTCTTC 2590 2600 2610 2620 2630 2640 ....|....|....|....|....|....|....|....|....|....|....|....| gce GATGCCCATGGCGATTCGGATGCGGAAGGTGAGCCCCGCCAGCGGAGCAAAGCCGAGGAG gce-RD_FBgn0261703 GATGCCCATGGCGATTCGGATGCGGAAGGTGAGCCCCGCCAGCGGAGCAAAGCCGAGGAG

Figure A.1 (continued)

149

Figure A.1 (continued)

2650 2660 2670 2680 2690 2700 ....|....|....|....|....|....|....|....|....|....|....|....| gce GATGCCATCGATCGCAAGTTGCGCGAGGATAGACGCAGTTTTCGCGTGAGATTGGCTCGT gce-RD_FBgn0261703 GATGCCATCGATCGCAAGTTGCGCGAGGATAGACGCAGTTTTCGCGTGAGATTGGCTCGT 2710 2720 2730 2740 2750 2760 ....|....|....|....|....|....|....|....|....|....|....|....| gce GCTGGTCCCAGATCGGAACCCACCGCCTACGAAGTGGTCAAGATCGATGGCTGCTTTCGG gce-RD_FBgn0261703 GCTGGTCCCAGATCGGAACCCACCGCCTACGAAGTGGTCAAGATCGATGGCTGCTTTCGG 2770 2780 2790 2800 2810 2820 ....|....|....|....|....|....|....|....|....|....|....|....| gce CGTAGTGACGAAGCGCCACGCGGCGTACGCTCCAATCATTTCAGCTCCAATCTGCAGTTA gce-RD_FBgn0261703 CGTAGTGACGAAGCGCCACGCGGCGTACGCTCCAATCATTTCAGCTCCAATCTGCAGTTA 2830 2840 2850 2860 2870 2880 ....|....|....|....|....|....|....|....|....|....|....|....| gce ATCAGGCGGACACGCGGTCGTGACGATGTCATTCCACTGCACACCATTAGCGGCAATGAT gce-RD_FBgn0261703 ATCAGGCGGACACGCGGTCGTGACGATGTCATTCCACTGCACACCATTAGCGGCAATGAT 2890 2900 2910 2920 2930 2940 ....|....|....|....|....|....|....|....|....|....|....|....| gce ATCATTCTGACTGGCTGCGCCCGGATAATCCGTCCGCCGAAGATCGCCAGTCGGCTGATC gce-RD_FBgn0261703 ATCATTCTGACTGGCTGCGCCCGGATAATCCGTCCGCCGAAGATCGCCAGTCGGCTGATC 2950 2960 2970 2980 2990 3000 ....|....|....|....|....|....|....|....|....|....|....|....| gce GATGCCAATACACTGGAGTACAAAACCCGCCACTTGATTGACGGCAGGATCATCGACTGC gce-RD_FBgn0261703 GATGCCAATACACTGGAGTACAAAACCCGCCACTTGATTGACGGCAGGATCATCGACTGC 3010 3020 3030 3040 3050 3060 ....|....|....|....|....|....|....|....|....|....|....|....| gce GACCAGAGAATTGGTATCGTGGCCGGCTACATGACGGATGAGGTGCGCAATCTCAGTCCC gce-RD_FBgn0261703 GACCAGAGAATTGGTATCGTGGCCGGCTACATGACGGATGAGGTGCGCAATCTCAGTCCC 3070 3080 3090 3100 3110 3120 ....|....|....|....|....|....|....|....|....|....|....|....| gce TTCACCTTCATGCACAACGACGATGTGCGCTGGGTGATCGTGGCCCTGCGTCAAATGTAC gce-RD_FBgn0261703 TTCACCTTCATGCACAACGACGATGTGCGCTGGGTGATCGTGGCCCTGCGTCAAATGTAC 3130 3140 3150 3160 3170 3180 ....|....|....|....|....|....|....|....|....|....|....|....| gce GACTGCAATAGTTCATATGGCGAGTCCACTTACCGGCTATTCACCCGCAACGGGAACATC gce-RD_FBgn0261703 GACTGCAATAGTTCATATGGCGAGTCCACTTACCGGCTATTCACCCGCAACGGGAACATC 3190 3200 3210 3220 3230 3240 ....|....|....|....|....|....|....|....|....|....|....|....| gce ATCTACCTGCAATCGAAGGGCTATCTGGAGATCGACAAGGAGACGAACAAGGTGCACTCC gce-RD_FBgn0261703 ATCTACCTGCAATCGAAGGGCTATCTGGAGATCGACAAGGAGACGAACAAGGTGCACTCC 3250 3260 3270 3280 3290 3300 ....|....|....|....|....|....|....|....|....|....|....|....| gce TTTGTCTGTGTGAACACGCTGCTTGGCGAGGAGGAGGGCAAGCGGCGGGTGCAGGAGATG gce-RD_FBgn0261703 TTTGTCTGTGTGAACACGCTGCTTGGCGAGGAGGAGGGCAAGCGGCGGGTGCAGGAGATG 3310 3320 3330 3340 3350 3360 ....|....|....|....|....|....|....|....|....|....|....|....| gce AAGAAGAAGTTCTCGGTAATTATCAACACACAGATACCGCAGTCGACCATCGATGTGCCC gce-RD_FBgn0261703 AAGAAGAAGTTCTCGGTAATTATCAACACACAGATACCGCAGTCGACCATCGATGTGCCC 3370 3380 3390 3400 3410 3420 ....|....|....|....|....|....|....|....|....|....|....|....| gce GCCTCCGAGCATCCGGCTCTGCTGGAGAAGGCCGTGCTGCGGCTCATCCAGAATCTGCAG gce-RD_FBgn0261703 GCCTCCGAGCATCCGGCTCTGCTGGAGAAGGCCGTGCTGCGGCTCATCCAGAATCTGCAG 3430 3440 3450 3460 3470 3480 ....|....|....|....|....|....|....|....|....|....|....|....| gce AAGTCTGGCGAGAATGGTGGCCACGATGACGGCGACGAGGACGATGATGCGCAGGATGGC gce-RD_FBgn0261703 AAGTCTGGCGAGAATGGTGGCCACGATGACGGCGACGAGGACGATGATGCGCAGGATGGC 3490 3500 3510 3520 3530 3540 ....|....|....|....|....|....|....|....|....|....|....|....| gce GACGACGACGAGGAGGATGATGATGACGATCAGGACGATGGCGCGCGCAGCATGTCCGAA gce-RD_FBgn0261703 GACGACGACGAGGAGGATGATGATGACGATCAGGACGATGGCGCGCGCAGCATGTCCGAA

Figure A.1 (continued)

150

Figure A.1 (continued)

3550 3560 3570 3580 3590 3600 ....|....|....|....|....|....|....|....|....|....|....|....| gce TTCGGTGATCCCTATGGCAGTCATCACGGCCGATCGCATCATGGTTCCTCCGCTCTATCC gce-RD_FBgn0261703 TTCGGTGATCCCTATGGCAGTCATCACGGCCGATCGCATCATGGTTCCTCCGCTCTATCC 3610 3620 3630 3640 3650 3660 ....|....|....|....|....|....|....|....|....|....|....|....| gce TCACACGGGCATGGTAACGCCAAAACCCCACCCCTGGCACTGGTTCCGCCGGAAGCGTCC gce-RD_FBgn0261703 TCACACGGGCATGGTAACGCCAAAACCCCACCCCTGGCACTGGTTCCGCCGGAAGCGTCC 3670 3680 3690 3700 3710 3720 ....|....|....|....|....|....|....|....|....|....|....|....| gce TCTGTCAAATCGGCGATTACGAAGAGTATCAGTGTGGTCAACGTTACGGCGGCCAAACAT gce-RD_FBgn0261703 TCTGTCAAATCGGCGATTACGAAGAGTATCAGTGTGGTCAACGTTACGGCGGCCAAACAT 3730 3740 3750 3760 3770 3780 ....|....|....|....|....|....|....|....|....|....|....|....| gce TTGCGTGGTATCCACGCATCGACGGCAGTCAAATCGCCCAGCTCCTTGGGCAGCTGCACC gce-RD_FBgn0261703 TTGCGTGGTATCCACGCATCGACGGCAGTCAAATCGCCCAGCTCCTTGGGCAGCTGCACC 3790 3800 3810 3820 3830 3840 ....|....|....|....|....|....|....|....|....|....|....|....| gce TGCAGCGATTCGCATTCGCCTTGCGACTTCTGCCAGGGTGCACCCACGACCGATCTCCAG gce-RD_FBgn0261703 TGCAGCGATTCGCATTCGCCTTGCGACTTCTGCCAGGGTGCACCCACGACCGATCTCCAG 3850 3860 3870 3880 3890 3900 ....|....|....|....|....|....|....|....|....|....|....|....| gce GCGGTTGGGTCCAATCTGAAGAGGGGCAGCACTGCTCACGTGGAAACCGAAGAGAAGCTG gce-RD_FBgn0261703 GCGGTTGGGTCCAATCTGAAGAGGGGCAGCACTGCTCACGTGGAAACCGAAGAGAAGCTG 3910 3920 3930 3940 3950 3960 ....|....|....|....|....|....|....|....|....|....|....|....| gce TCCAAGCGGCGTTTTATACCCAGCACTGAAATCGAGCATGTGCTGCACACATCTTTGGAC gce-RD_FBgn0261703 TCCAAGCGGCGTTTTATACCCAGCACTGAAATCGAGCATGTGCTGCACACATCTTTGGAC 3970 3980 3990 4000 4010 4020 ....|....|....|....|....|....|....|....|....|....|....|....| gce CAAATCGGGCGTAATCTTACCCAGCAGCTCAATGTGGCCAGGAATTTGCGGGAACAGAGT gce-RD_FBgn0261703 CAAATCGGGCGTAATCTTACCCAGCAGCTCAATGTGGCCAGGAATTTGCGGGAACAGAGT 4030 4040 4050 4060 4070 4080 ....|....|....|....|....|....|....|....|....|....|....|....| gce CAGCGATACGAGTTGCCCCATGCCAATCAGCGATTCGATGAGATCATGCAGGAGCACCAG gce-RD_FBgn0261703 CAGCGATACGAGTTGCCCCATGCCAATCAGCGATTCGATGAGATCATGCAGGAGCACCAG 4090 4100 4110 4120 4130 4140 ....|....|....|....|....|....|....|....|....|....|....|....| gce AAGCAGAGTGAACTGTATGTGAACATCAAGAGCGAGTACGAGGTGCAGCTGCAGCATAAG gce-RD_FBgn0261703 AAGCAGAGTGAACTGTATGTGAACATCAAGAGCGAGTACGAGGTGCAGCTGCAGCATAAG 4150 4160 4170 4180 4190 4200 ....|....|....|....|....|....|....|....|....|....|....|....| gce GCAAGCACCCGGAAGTCATCGGATTCGGATCGGAATCAGGAGCAGCCGCCGCCGCCGCTT gce-RD_FBgn0261703 GCAAGCACCCGGAAGTCATCGGATTCGGATCGGAATCAGGAGCAGCCGCCGCCGCCGCTT 4210 4220 4230 4240 4250 4260 ....|....|....|....|....|....|....|....|....|....|....|....| gce CAGGAGGACGACCAGGACTAGAGTGATGGAGAGCGCCCGATCGGGTCTCGACTGAAGGGT gce-RD_FBgn0261703 CAGGAGGACGACCAGGACTAGAGTGATGGAGAGCGCCCGATCGGGTCTCGACTGAAGGGT 4270 4280 4290 4300 4310 4320 ....|....|....|....|....|....|....|....|....|....|....|....| gce TTCATCGATGTGATATGTAACTACTGTCCTGGCAGTCAGCTCAAGCCCCAACATACAAAG gce-RD_FBgn0261703 TTCATCGATGTGATATGTAACTACTGTCCTGGCAGTCAGCTCAAGCCCCAACATACAAAG 4330 4340 4350 4360 4370 4380 ....|....|....|....|....|....|....|....|....|....|....|....| gce TGTCCCTAATTCGTACCAACCAATAGAACCAATAATTCAGGGTTACTTCTCGTTCGGTCA gce-RD_FBgn0261703 TGTCCCTAATTCGTACCAACCAATAGAACCAATAATTCAGGGTTACTTCTCGTTCGGTCA 4390 4400 4410 4420 4430 4440 ....|....|....|....|....|....|....|....|....|....|....|....| gce ACTGATGGGTAACTGCTGCGGTACAGTTTAGGTACAGATACAGATACGGATAGATAACCT gce-RD_FBgn0261703 ACTGATGGGTAACTGCTGCGGTACAGTTTAGGTACAGATACAGATACGGATAGATAACCT

Figure A.1 (continued)

151

Figure A.1 (continued)

4450 4460 4470 4480 4490 4500 ....|....|....|....|....|....|....|....|....|....|....|....| gce TTAACCCCAACTTATGAGTGAGCCTGAGACAAGCATACTTTAGATACCCTGCCCACATGC gce-RD_FBgn0261703 TTAACCCCAACTTATGAGTGAGCCTGAGACAAGCATACTTTAGATACCCTGCCCACATGC 4510 4520 4530 4540 4550 4560 ....|....|....|....|....|....|....|....|....|....|....|....| gce ACACACTCACATATATATATAGATAT------ATATGTACATATATACACTGTTTGTT gce-RD_FBgn0261703 ACACACTCACATATATATATATATATATATATATATATGTACATATATACACTGTTTGTT 4570 4580 4590 4600 4610 4620 ....|....|....|....|....|....|....|....|....|....|....|....| gce CAGTTTGTTGTTTTTAACTTCCTGTCCCACGTAGAAGGACTTTTACATGCATGTAGAACT gce-RD_FBgn0261703 CAGTTTGTTGTTTTTAACTTCCTGTCCCACGTAGAAGGACTTTTACATGCATGTAGAACT 4630 4640 4650 4660 4670 4680 ....|....|....|....|....|....|....|....|....|....|....|....| gce AGTTACCGTTACCGTTTCCGTTTCCGATTTCGTTTAGAATGTTAGACACATACATATTTA gce-RD_FBgn0261703 AGTTACCGTTACCGTTTCCGTTTCCGATT------4690 4700 4710 4720 4730 4740 ....|....|....|....|....|....|....|....|....|....|....|....| gce GTAGCTTAATCCAATCACAGCTGATTCTTTTGAACACAACTTGTTATTTGCTGATCCCAA gce-RD_FBgn0261703 ------4750 4760 4770 4780 4790 4800 ....|....|....|....|....|....|....|....|....|....|....|....| gce TTTTCTAAAACAAAAAGATGGTAGAACAAATATGAATAGAAAAGGGTCACGTCAATTTGC gce-RD_FBgn0261703 ------4810 4820 4830 4840 4850 4860 ....|....|....|....|....|....|....|....|....|....|....|....| gce ATCGAAAGCGGAAACGGCAACAAACACCCACAAAGGATTAAGATAATGATATAAGTAATG gce-RD_FBgn0261703 ------4870 4880 4890 4900 4910 4920 ....|....|....|....|....|....|....|....|....|....|....|....| gce ATTAGCGTAAATGGGAGGATTTAAACAATTATTATGCTAAATGAAATATACTATTAAAAA gce-RD_FBgn0261703 ------4930 4940 4950 4960 4970 4980 ....|....|....|....|....|....|....|....|....|....|....|....| gce CCTATGTACCATACGTTGTGCGTAATGCCCAAACTAAACGTGATAAAAGACTGTTACCAA gce-RD_FBgn0261703 ------4990 5000 5010 5020 5030 5040 ....|....|....|....|....|....|....|....|....|....|....|....| gce CTACATACACACATACATAATACCGAGATGGAAACAACCGTAAATCTAAAACTAAAACCT gce-RD_FBgn0261703 ------5050 5060 5070 5080 5090 5100 ....|....|....|....|....|....|....|....|....|....|....|....| gce AAACTTAAATCGGCATAGTTTCGCATGAAAAGGTAATATATACATACATACATAAATATA gce-RD_FBgn0261703 ------5110 5120 5130 5140 5150 5160 ....|....|....|....|....|....|....|....|....|....|....|....| gce ACAAATATATTCATACGCGCACTAAAAGGAAGCGTTGCATAGTTTAATTACATTTACCGT gce-RD_FBgn0261703 ------5170 5180 5190 5200 5210 5220 ....|....|....|....|....|....|....|....|....|....|....|....| gce ACGAACACTTTTTATAGATGTTTAATGTTGTTATGGTTAAGATAAATCCGGACGAATTGA gce-RD_FBgn0261703 ------5230 5240 5250 5260 5270 5280 ....|....|....|....|....|....|....|....|....|....|....|....| gce TGGATTTTGGCCAAGCACGGTGCACTGCATATATGTAGCAAGTGAGCCAAGTACAATTTA gce-RD_FBgn0261703 ------5290 5300 5310 5320 5330 5340 ....|....|....|....|....|....|....|....|....|....|....|....| gce CACAAATGTATACATAATTAGATGCATATATGGTTCGACGTAAACTAATAATAAATCGAT gce-RD_FBgn0261703 ------

Figure A.1 (continued)

152

Figure A.1 (continued)

5350 5360 5370 5380 5390 5400 ....|....|....|....|....|....|....|....|....|....|....|....| gce GTAAAAGCGTAAAAACGGAAGTTAAGAAACCACACACACACACAAACACACACACCCACA gce-RD_FBgn0261703 ------5410 5420 5430 5440 5450 5460 ....|....|....|....|....|....|....|....|....|....|....|....| gce AAAACCAGAAAAAGCTGAGGCGACAAAAATATGAGAACTCTGTTAAATGTTTGTTATGCA gce-RD_FBgn0261703 ------5470 5480 5490 5500 5510 5520 ....|....|....|....|....|....|....|....|....|....|....|....| gce TTTGTTTTTTTTTTTATCTAAATTAATATTAAACTAATGTATTAGTGAATAAACTTGTGT gce-RD_FBgn0261703 ------5530 5540 5550 5560 5570 ....|....|....|....|....|....|....|....|....|....|... gce ACCTACATACAAAACAATGATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA gce-RD_FBgn0261703 ------

References

Baumann, A., Fujiwara, Y., Wilson, T.G. 2010. Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila. Journal of Insect Physiology. 56:1445-1455.

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