Are all MTH-like genes involved in lifespan determination?

ARE ALL MTH-LIKE GENES INVOLVED IN LIFESPAN DETERMINATION? ANA RITA CARVALHO ARAÚJO Dissertação de Mestrado em Bioquímica Universidade do Porto Faculdade de Ciências Instituto de Ciências Biomédicas Abel Salazar 2012 ANA RITA CARVALHO ARAÚJO ARE ALL MTH-LIKE GENES INVOLVED IN LIFESPAN DETERMINATION? Dissertação de Candidatura ao grau de Mestre em Bioquímica da Universidade do Porto Orientador – Doutor Jorge Vieira Categoria – Investigador Afiliação – IBMC 2012 Are all mth-like genes involved in lifespan determination? i ACKNOWLEDGEMENTS To my supervisor, Professor Jorge Vieira, and to Professor Cristina Vieira for receiving me in the Molecular Evolution Lab at IBMC. I am very grateful for the guidance and advice that they gave me through this long journey. Thank you for making me a real scientist. To my tutor, Professor Pedro Alexandrino, for all the support and the availability to answer all my questions and for encouraging me to never give up. I would like to thank to my mother Olga and my father Manuel for always supporting my decisions and be by my side no matter what happened, and to my little sister Marta for all the conversations, arguments and comprehension and for always being there for me when I needed to explode. To my closest friends, Joana and Cátia, who always have a word to say when I lost my way, and for always being there to answer all my doubts and helping me to become a better person. To all my Master colleagues, especially, Elísio, Pedro, Andreia and Jéssica for being patient with me and for the friendship that grew up so fast within a year. To all my lab colleagues at IBMC, especially Ana Estefânia and Inês for all the coffee breaks, conservations and good mood that we shared. Finally, I would like to acknowledge FCT for funding this work. Are all mth-like genes involved in lifespan determination? ii Are all mth-like genes involved in lifespan determination? iii ABSTRACT The Drosophila methuselah (mth) gene was described for the first time in 1998 as being associated to increased lifespan. A mutant line of mth lived 35% longer than the wild type flies at both 25ºC and 29ºC. Moreover, mth flies were resistant to oxidative stress and starvation. The mth gene encodes a putative G protein-coupled receptor (GPCR), showing a recognizable Mth ectodomain containing ten conserved cysteine residues in the N-terminal and a seven-transmembrane (7tm) domain in the C-terminal, which are called Mth typical features. The function of this protein is not known yet, but despite of being implicated in lifespan, studies in D. melanogaster showed evidences that it might be involved in the control of the synaptic efficacy at glutamatergic neuromuscular junctions, enhancement of resistance to stress, higher wing beat frequency and coordinated visuomotor entrainment during simulated flight, as well as maintaining the rate of stem cell division with age. mth gene is only present in melanogaster subgroup of species and it has an estimated age of 10 million years. In D. melanogaster, mth belongs to a family comprising 16 genes: mth and its paralogs, mth-like genes. However, this family of genes only has two members in Bombyx mori and seven in Anopheles gambie. For that reason, we here establish the history of mth-like family through the Drosophila genus and concluded that the number of members of this family is not retained between Drosophila species. Hence, it is of interest to determine if other mth-like genes can influence lifespan too in species that are distantly related to D. melanogaster such as D. americana – since association between naturally occurring Mth amino acid polymorphisms and lifespan were showed in D. melanogaster. D. americana has been diverging from D. melanogaster for about 40 million years and does not have a mth orthologous gene copy. However, here we show that markers for mth-like genes with the typical Mth protein features are able to explain a considerable amount of lifespan differences observed in a F2 association study. Consequently, this suggests that mth-like genes might be involved in lifespan determination in a species distantly related to D. Are all mth-like genes involved in lifespan determination? iv melanogaster, and furthermore they could be associated with lifespan in all the insect orders where they were described. Are all mth-like genes involved in lifespan determination? v RESUMO O gene methuselah (mth) de Drosophila foi descrito pela primeira vez em 1998 como estando associado ao aumento da longevidade. Uma linha mutante deste gene vive cerca de 35% mais do que as moscas do tipo selvagem a 25ºC e a 29ºC. Além disso, as moscas mth são mais resistentes ao stress oxidativo e à restrição calórica. O gene mth codifica um recetor acoplado a proteína G (GPCR), apresentando um domínio Mth que possui dez resíduos de cisteína conservados no seu N-terminal e um domínio 7-transmembranar no C-terminal. Estas caraterísticas são denominadas como as características típicas da proteína Mth. A função desta proteína ainda não é conhecida, mas além de estar implicada em estudos de longevidade em D. melanogaster, mostrou evidências de que poderá estar envolvida no controlo da eficácia das sinapses na junção neuromuscular glutamatérgica, no aumento a resistência a stress, a uma maior frequência do batimento das asas assim como na coordenação visuomotora durante o voo, bem como na manutenção da taxa de divisão das células estaminais com a idade. Este gene só se encontra presente no subgrupo de melanogaster e tem uma idade estimada de cerca de dez milhões de anos. Em D. melanogaster, o gene mth pertence a uma família composta por 16 membros: o gene mth e os seus parálogos, os genes mth-like. No entanto, esta família de genes só possui dois membros em Bombyx mori e sete em Anopheles gambiae. Por isso, foi determinada a história da família mth-like através do género de Drosophila e concluído que o número de membros desta família não é mantido entre as espécies de Drosophila. Consequentemente, coloca-se a questão se outros genes mth-like também influenciam a longevidade numa espécie que está distantemente relacionada com D.melanogaster, como por exemplo, D. americana. Esta questão coloca-se porque associação entre polimorfismos aminoacídicos, que ocorrem naturalmente na proteína Mth, e a longevidade foram descritos em D. melanogaster. D. americana encontra-se a divergir de D. melanogaster há cerca de 40 milhões de anos e não possui nenhum gene ortólogo do gene mth. Contudo, aqui mostrase que marcadores para genes mth-like que possuem as características típicas da Are all mth-like genes involved in lifespan determination? xii Figure 14: Box plot representing the association results obtained for mth-like 11 in F2 individuals ................................................................................................ 39 Figure 15: Box plot representing the association results obtained for mth-like 14 in F2 individuals ................................................................................................ 41 Figure 16: Box plot representing the association results obtained for mth-like 8 in F2 individuals .................................................................................................... 43 Figure 17: Box plot representing the association results obtained for mth-like 9 in F2 individuals .................................................................................................... 44 Figure 18: Box plot representing the association results obtained for mth-like 10 in F2 individuals ................................................................................................ 46 Figure 19: Box plot representing the association results obtained for GJ12490 ortholog in F2 individuals .................................................................................. 47 Figure 20: Compilation of the association study results .................................... 49 Figure 21: Schematic diagram of the structure of the nine mth-like genes present in D. americana ................................................................................................. 50 Figure 22: Lifespan variation observed in the 453 individuals of the second F2 association experiment ..................................................................................... 51 Figure 23: Fold change in mth-like genes expression in two strains of D. americana – H5 and W11 – using the2-ΔΔCT method ............................................ 55 Are all mth-like genes involved in lifespan determination? xiii LIST OF TABLES Table 1: Summary of mammalian and Drosophila GPCRs ..................................... 6 Table 2: Molecular markers designed for all nine mth-like genes present in D. americana genome ........................................................................................... 18 Table 3: Specific primers designed for seven mth-like genes and RpL32 tested for qRT-PCR ............................................................................................................ 20 Table 4: Comparative study concerning the annotation of mth-like genes present in D. virilis genome and their orthologous in D. melanogaster .......................... 29 Table 5: Comparison of the annotation of mth-like 14 in the twelve species of Drosophila, whose genome is publically available .............................................. 30 Table 6: Conserved domains encoded by mth-like genes of D. americana ......... 31 Table 7: Localization of mth-like genes in D. americana genome and respective type of marker .................................................................................................. 32 Are all mth-like genes involved in lifespan determination? xiv CHAPTER I – INTRODUCTION Are all mth-like genes involved in lifespan determination? 1 1. INTRODUCTION 1.1. Prelude Lifespan is a life-history component that varies significantly among organisms in natural populations (Promislow et al., 1996). The increased or decreased Drosophila lifespan has been studied when some genes are manipulated (reviewed by (Paaby and Schmidt, 2009), but the major factors that contribute for a genetic variance for longevity in natural populations are still uncharacterized (Paaby and Schmidt, 2008). For instance, many induced mutations in single genes belonging to the insulin/insulin-like growth factor-like signaling (IIS) pathway result in lifespan changes in divergent species such as nematodes, Drosophila and mouse (Giannakou and Partridge, 2007). Nevertheless, these genes may be under strong purifying selection and thus may contribute little to the observed variation in lifespan in natural populations (Paaby and Schmidt, 2008). In Drosophila melanogaster, some complementary tests using mutant strains have shown that naturally occurring variation at many genes affects lifespan (reviewed by(Paaby and Schmidt, 2009), including methuselah (mth) gene. The mth gene was described for the first time in 1998 as being associated to increased lifespan (Lin et al., 1998), and for this, mth gene was proposed as a candidate gene that may be implicated in lifespan determination. A mutant line of mth lived 35% longer than the wild type flies at both 25ºC and 29ºC. Moreover, mth flies were resistant to oxidative stress, heat and starvation (Lin et al., 1998). Additionally, studies in D. melanogaster showed that the protein encoded by this gene may be implicated in many roles as regulation of neurotransmitters exocytosis in larval glutamatergic neuromuscular junctions (Song et al., 2002), higher wing beat frequency and coordinated visuomotor entrainment during simulated flight (Petrosyan et al., 2007), as well as, maintaining the rate of stem cell division with age (Wallenfang et al., 2006). The mutant line has a P-element inserted into the third intron of the mth gene (Lin et al., 1998). Both the precise and imprecise excision of the P-element were generated in mth flies and caused a reversion back to the wild type phenotype. Two additional lines were isolated with a deletion of the DNA adjacent to the P- CHAPTER I – INTRODUCTION Are all mth-like genes involved in lifespan determination? 2 element insertion site and revealed to be null alleles of mth gene. In embryos, they are homozygous lethal, which suggests that mth plays a major role in early development. mth gene encodes a protein whose predicted sequence shows a leader peptide and seven hydrophobic regions, suggestive of a transmembranar domain. Then a gapped Blast search revealed that Mth protein is similar to G protein-coupled receptors (GPCR) (Lin et al., 1998), a large and important group of receptors proteins involved in signal transduction (Schmidt et al., 2000). In Drosophila, GPCRs are classified into four groups: Rhodopsin-like receptor family, Secretinlike receptor family, Metabotropic glutamate receptor-like family and Atypical family (Brody and Cravchik, 2000). The gene mth is the founder member of methuselah-like (mth-like) family, a subfamily inside the secretin-like receptor family, which is found in Drosophila but not in Caenorhabditis elegans or vertebrates. Accordingly to Nordström et al. (2009), the mth-like family in D. melanogaster comprises fifteen members including the mth original and fourteen paralogous called mth-like genes. Little is known about the other members of the mth-like family and all the studies about mth gene were performed in D. melanogaster. 1.2. GPCRs and signaling through G proteins GPCRs are the largest family of membrane proteins that mediate most of the cellular response to hormones and neurotransmitters and are responsible for many senses like vision, olfaction and taste. Their classical tridimensional structure comprises a seven spanning αhelical segments separated by alternating intra and extracellular loops (figure 1). Each GPCR has unique signaltransduction activities that involve various kinds of G-proteins in order to establish communication between the internal and external environments of the cell. After the binding of an agonist, the GPCR is active and can activate a specific heterodimeric G protein leading to the transmission of the signal through the modulation of downstream effectors. Every ligand triggers a specific signaling pathway via stabilization of an exclusive collection of molecules, allowing the correct interaction with them and which then will defuse a biological response (reviewed by(Rosenbaum et al., 2009). CHAPTER I – INTRODUCTION Are all mth-like genes involved in lifespan determination? 3 Like all GPCRs, the members of Mth-like family are coupled to intracellular GTPbinding proteins (G proteins) in order to transmit the signal through the cell. The G proteins are composed by three subunits: Gα, Gβ and Gγ. Once bound to ligand, the GPCR suffers a conformational change and acts as a guanine exchange factor (GEF) inducing the swap of GDP for GTP. Once the GTP binds Gα, the G proteins dissociate into Gα and Gβγ components, modulating an intracellular signal by activation of downstream effectors and second messengers in the cell (reviewed by(McCudden et al., 2005, Strader et al., 1994). Gα and Gβγ subunits can interact with different downstream effectors, activating different signaling pathways (figure 2). There are three ways to inactivate the signal transducing by G proteins: GTP hydrolization, through regulators of G protein signaling (RGS) proteins or by G protein coupled receptor kinases (GRKS). In the first form, the intrinsic guanosine triphosphatase (GTPase) activity of Gα subunit causes the hydrolysis of GTP to GDP, leading to its inactive state. The reassociation of subunit Gα and dimmer Gβγ terminates all effectors interactions. The second form is through RGS proteins, which are able to act like a GTPase accelerating protein (GAP) in order to facilitate the hydrolyzing of GTP and therefore deactivate Gα subunit (Neubig, Figure 1: Representation of crystal structure of the human GPCR A2A receptor. The protein crystal was obtained by Jaakola et al. in 2008 and this image was created in PyMol (Schrodinger, 2010). In orange are marked the seven transmembrane domains with respective numeration, in green the three intracellular loops (ICL) and in blue the three extracellular loops (ECL). In magenta is represented the eighth helix of human A2A receptor, which is in a parallel position relatively to the cellular membrane and is the intracellular region of the protein. Protein is represented in cartoon. (Jaakola et al., 2008) CHAPTER I – INTRODUCTION Are all mth-like genes involved in lifespan determination? 4 2002). In the third form, GRKs phosphorylates the active GPCR, converting it into a target for high affinity binding Arrestin followed by Clatherin-induced endocytosis of the receptor, blocking the activation of G-proteins (Wilden, 1995, Krupnick et al., 1997). GPCRs are membrane proteins and are highly expressed once they are responsible for mediating the interaction between cells and their environment. Signals such as light, odors and taste or intracellular stimuli as hormones, peptides and neurotransmissors are detected by GPCRs. Since so many types of ligands activate them, they play a role in many biological processes like neurotransmission, growth, development, cellular differentiation, inflammation and the immune response (Horn et al., 1998). For example, in Anopheles gambiae, GPCRs mediate the many sensory pathways that are related with malaria transmission, because the olfactory sense plays an important role in the choice of Figure 2: Diversity of G protein-coupled receptor signaling (Dorsam and Gutkind, 2007). CHAPTER I – INTRODUCTION Are all mth-like genes involved in lifespan determination? 5 human host (Hill et al., 2002); and in humans the over expression of GPCRs and their activation by agonists released by tumor or stromal cells is the tactic most used by tumor cells to stimulate GPCRs and the signaling pathways activated by them (Dorsam and Gutkind, 2007). Due to the fact that these membrane receptors control key physiological functions, their dysfunction contributes to some of the most prevalent human diseases and GPCRs are the target, both directly or indirectly, of about 50 to 60% of all therapeutic agents nowadays (Pierce et al., 2002). However, when comparing invertebrates with vertebrates, the latter ones have a larger number of GPCRs than invertebrates, and thus vertebrates may be more sensible to sensory information from the environment that allows for a more complex homeostasis regulation. A large amount of diversified GPCRs members might enable a better adaptation of the individual to new environments and new situations (Strotmann et al., 2011). 1.3. The Secretin-like Receptor Family of GPCRs The GPCRs are one of the largest protein families in mammalian genomes with about 800 members in human genome (reviewed by(Lagerstrom and Schioth, 2008) divided into five groups in GRAFS classification: Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2 and Secretin (table 1) (Fredriksson et al., 2003). These five families are present in most of bilateral species including Caenorhabditis elegans, Drosophila melanogaster, Anopheles gambiae, Strongylocentrotus purpuratus, Ciona intestinalis and the vertebrate species (Fredriksson and Schiöth, 2005, Whittaker et al., 2006, Schiöth et al., 2007). The Drosophila genome contains approximately 200 genes coding for GPCRs (Adams et al., 2000) classified into four families based on primary and secondary structure predictions: Rhodopsin-like, Secretin-like, Metabotropic glutamate-like and Atypical seven-transmembrane proteins (table 1) (Brody and Cravchik, 2000). The Secretin-like family includes many hormone receptors such as receptors for secretin, calcitonin, vasoactive intestinal peptide, parathyroid hormone and related peptides. These receptors are characterized by long NH2-terminal domains CHAPTER I – INTRODUCTION Are all mth-like genes involved in lifespan determination? 6 with five cysteine residues highly conserved that form disulfide bonds and by a short cytoplasmatic domain (Brody and Cravchik, 2000). Table 1: Summary of mammalian and Drosophila GPCRs reviewed by Fredriksson et al. 2003 and Broady and Cravchik 2000. BAIs, brain-specific angiogenesis-inhibitory receptors; CALCR, calcitonin receptor; CASR, calcium-sensing receptor; CELSRs; cadherin EGF laminin-A sevenpass G-type receptors; CRHRs, corticotropin-releasing hormone receptors; EMRs, EGF-like module containing, GCGR, glucagon receptor; GHRHR, growth hormone-releasing hormone receptor; GIPR, gastric inhibitory polypeptide receptor; GLPRs, glucagon-like peptide receptors; GRM, metabotropic glutamate receptors; LECs, lectomedin receptors; PACAP, pituitary adenylyl cyclase-activating protein; PTHR, parathyroid hormone receptors; SCTR, secretin receptor; TAS1 and TAS2, taste receptors; VIRP, vasoactive intestinal peptide receptor. In D. melanogaster the latrophilin-like receptor gene is thought to have a role in the control of synaptic exocytosis and was identified as a member of Secretin-like receptor family (Brody and Cravchik, 2000). Its endogenous ligands are unknown but the neurotoxin α-latrotoxin binds to latrophilin-like receptor and stimulates Glutamate GRM, GABA receptors, CASR, TAS1 Glutamate-like receptors Glutamate receptors, GABA receptors, Calcium receptors Rhodopsin α, β, γ and δ Rhodopsin-like receptors Opsins, GPCRs for biogenic amines, related compounds and purines, peptide GPCRs Adhesion CELSRs, BAIs, LECs and EMRs Adhesion (Atypical) Stan and Boss Frizzled/Taste2 Frizzled receptors, TAS2 receptors Frizzled (Atypical) Frizzled-like proteins Secretin CALCR, CRHRs, GCGR, GIPR, GLPRs, GHRHR, PTHR, PACAP, SCTR, VIPR Secretin-like receptors Hormone receptors, mth-like receptor family latrophilin-like receptor Mammalian GPCRs Subgroups Drosophila GPCRs Subgroups CHAPTER I – INTRODUCTION Are all mth-like genes involved in lifespan determination? 7 the massive neurotransmitter release which leads to nerve terminal degeneration (Krasnoperov et al., 1997, Holz and Habener, 1998). This evidence supports the hypothesis of a putative role of Secretin-like receptor family in neutransmission. In 2003, Johnson et al. showed that another member of Secretin-like receptor family is indeed a functional diuretic hormone 44 receptor in Drosophila, once the expression of this receptor in HEK cells elevate the intracellular calcium concentrations in response to diuretic hormone 44. The Mth-like family is a subfamily within Secretin-like family and phylogenetic studies showed similarities between this subfamily and the Adhesion GPCRs (Nordström et al., 2009). The evolutionary link between Adhesion GPCRs and Secretin GPCRs suggests that the latter family could also play an important role in the development of nervous system and hence mth-like genes (Nordström et al., 2009). 1.4. Methuselah and Methuselah-like family In 1998, when Lin et al. described the relationship between lifespan and mth gene, it was also predicted that the sequence of this gene encoded a leader peptide (Mth ectodomain) and seven hydrophobic regions which suggested that it had a transmembrane domain. Using Blast tool, this sequence showed high homology with GPCR (Lin et al., 1998). Brody and Cravchik first annotated the mth-like family in 2000 in D. melanogaster. At that time, ten mth-like genes were identified and a phylogenetic tree, showing their evolutionary relashionships, was created using the neighbor joining method (figure 3) (Brody and Cravchik, 2000). In 2009, Nordström et al. described the other members of mth-like family, which from that moment till now comprises fifteen members, including mth gene and Figure 3: Phylogeny of the mth-like family performed by Brody and Cravchik, 2000. CHAPTER I – INTRODUCTION Are all mth-like genes involved in lifespan determination? 14 CHAPTER II – METHODS Are all mth-like genes involved in lifespan determination? 15 2. METHODS 2.1. Identification of mth-like genes in Drosophila genus and phylogenetic analyses In order to identify how many mth-like genes are present in the annotated genome of twelve species of Drosophila genus publically available, a tBLASTn search as implemented in FlyBase (http://flybase.org) using the amino acid sequence of mth gene from D. melanogaster as query was performed. From this search, a total of 141 sequences were obtained. Nucleotide sequence alignments were performed with the help of Clustal tool as implemented in DAMBE (Xia and Xie, 2001). The amino acid sequences were first aligned and then used as a guide to align the corresponding nucleotide sequences. Phylogenetic Bayesian analyses were run for nucleotide sequences using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The GTR model of sequence evolution was used, thus allowing for among-site rate variation and a proportion of invariable sites. Moreover, third codon positions were allowed to have a gamma distribution shape parameter that is different from that of first and second codon positions. Two simultaneous and completely independent analyses were run for 500.000 generations (each with one cold and three heated chains), starting from random trees. Samples were taken every 100th generation. The first 1250 samples were discarded (burn-in). Then, we rooted the resulting gene tree in order to make inferences on the age of Drosophila mth-like genes. This was obtained by using, in addition to the compilation of 141 retrieved protein sequences of Drosophila, a set of fifty proteins from non-insect species that, when using BLASTp show the highest similarity to the D. melanogaster Mth protein, and the Minimum Evolution algorithm, as implemented in MEGA (Kumar et al., 2008). The resulting tree showed that the D. melanogaster mth-like 1, mth-like 5 and mth-like 14 genes are the oldest members of mth gene family, and therefore the Bayesian tree was rooted accordingly. Two mth-like sequences (one from D. pseudoobscura – GA26708 – and another from D. yakuba – GE26296) were not used, once they would introduce a large number of alignment gaps and thus compromise the use of many alignment positions. It should be noted that Mth ectodomain is only CHAPTER II – METHODS Are all mth-like genes involved in lifespan determination? 16 found in insects (Nordström et al., 2009), and for that reason, the inferred relationships are based on the non Mth-ectodomain region of the proteins. 2.2. Detection of protein domains Protein domains were detected using the Conserved Domains tool at NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Marchler-Bauer et al., 2011). 2.3. F2 association experiments Two different experiments were performed during this work. The first D. americana F2 association study was performed using five strains – W11, H5, O57, W29 and W46 – forming 5 crosses H5♂ x W11♀, W11♂ x W46♀, W29♂ x O57♀, O57♂ x H5♀, W46♂ x W29♀ named crosses AA, AT, TN, NA and TT, respectively. This F2 association study comprises 433 male individuals: 88 from cross AA, 75 from AT cross, 87 from TN cross, 94 from NA cross and 89 from TT cross. After the eclosion, all these individuals were collected and maintained at 25ºC under 12h light and dark cycle conditions up to their death with the aim to measure the lifespan of each individual. These 433 individuals were genotyped for all the nine mth-like genes present in D. americana genome (http://evolution.ibmc.up.pt) (see results) – mth-like 1, GJ16328 ortholog, mth-like 11, mth-like 5, mth-like 14, mth-like 8, mth-like 9, mth-like 10 and GJ12490 ortholog. The second D. americana F2 association study was performed using strains H5 and W11, described in detail in Reis et al. 2011, and was used to test for associations between mth-like genes and longevity too. This F2 association study is composed by 453 F2 D. americana males showing extreme phenotypes for developmental time, chill-coma recovery time, abdominal size and lifespan, that are descendents of three F0 H5 x W11 crosses and were selected out of 975 individuals. These 453 individuals were genotyped for 3 mth-like genes (GJ12490 ortholog, mth-like 5 and mth-like 11). Not all individuals could be genotyped due to unknown reasons. CHAPTER II – METHODS Are all mth-like genes involved in lifespan determination? 17 In the F2 association study there is only one generation of recombination and therefore associations are expected between phenotype and polymorphic sites in the region(s) where the causative variant(s) are located. 2.4. Genomic DNA extraction Genomic DNA from single males was extracted using the QIAamp DNA Mini Kit from QIAGEN (Izasa Portugal, Lda.) 2.5. Molecular markers for mth-like genes Specific primers (table 2) based on both the annotated D. virilis (http://flybase.org) and non-annotated D. americana (http://evolution.ibmc.up.pt) genomes were developed for all mth-like genes present in the genome of both species. For all genes, standard amplification conditions were 35 cycles of denaturation at 94ºC for 30 seconds, primer annealing (table 2) for 45 seconds and primer extension at 75ºC for 2 minutes. The DNA fragments were run in a 1,5% agarose gel stained with ethidium bromide, using SGTB buffer (Grisp, Portugal). PCR products obtained with primers developed for mth-like 1, GJ16328 ortholog, mth-like 5, mth-like 14, mth-like 8, mth-like 9 and GJ12490 ortholog were then digested with restriction enzymes (table 2) and run in a 2% agarose gel stained with ethidium bromide, using SGTB buffer. The mth-like 11 and mth-like 10 show alleles with different sizes, and those were used as a marker. CHAPTER II – METHODS Are all mth-like genes involved in lifespan determination? 18 Table 2: Molecular markers designed for all nine mth-like genes present in D. americana genome. Ta, annealing temperature; RE, restriction enzyme. Gene Primer Sequence (5’ 3’) Ta (ºC) RE mth-like 1 mthl1_GJ14760_F CTACGACAGCGGCACCAA 61 MspI mthl1_GJ14760_R CCAGCAGCAGGCAGAATA GJ16328 ortholog GJ16328_ex1_F GTCCAGAGCACAGCAGAT 53 HpyCH4I V GJ16328_int1_R TAAACAAAGCGAAAAATG mth-like 5 129ex4F AGCCATCGTCTATTTTAT 50 BclI Mth_129w11poly_R ACATCACCAGACACATCA mth-like 11 mth_60_amer_F TGTTTGAGGTTTGAGTTT 52 − mth_60_amer_R CACGCCGAAGATGTAATA mth-like 14 mthl4_g16090_70F CAAAAACGGAAATGAGTC 51 Sau3AI mthl4_g16090_641R TGGTGGTATGAAGTAACG mth-like 8 GJ12496_mthl8_F CTGATTGCGGCTGGCTAT 59 Sau3AI mthl8_alter_R TGGTGAACAGATGCTCCC mth-like 9 GJ12495_mthl9_ F CAACAACAACTGCGACAA 57 Sau3AI GJ12495_mthl9_ R ATTGAAGAGCCTCCGAAT mth-like 10 GJ12492_mthl10ex1_F TACGAACAGTTTGTGGTG 52 − GJ12492_mthl10ex2_R AAGGGTATGGAGAAGAGC GJ12490 ortholog Mth_29_F GTTCTTTCCGAGCAGCAA 53 Sau3AI Mth_29_R CAGAGCACACAGCAGAGC 2.6. Cloning and DNA sequencing The GJ16328 gene was not localized in D. virilis genome, but there is a hit of this gene in D. americana non-annotated genome. For that reason, some experiments were performed in order to certify that it is the same gene. Firstly, flanking primers were designed based on the D. virilis GJ16328 gene in order to amplify this gene. Standard amplification conditions were 35 cycles of denaturation at 94ºC for 30 seconds, primer annealing at 50ºC for 45 seconds, and primer extension at 75ºC for 2 minutes. An amplification product was obtained for two individuals, one from strain H5 and one from strain W11. Secondly, the PCR products were cloned using the TOPO-TA Cloning Kit for sequencing (Invitrogen, CHAPTER II – METHODS Are all mth-like genes involved in lifespan determination? 19 Spain). Then, positive colonies were picked randomly, grown in 5 mL of LB with Ampicillin, and plasmids were extracted using the QIAprep Spin Miniprep Kit from QIAGEN (Izasa, Portugal). The plasmidic DNA of three colonies was sequenced in order to correct for possible nucleotide miss-incorporations that may have occurred during the PCR reaction. Sequencing reactions were performed using ABI PRISM Big Dye cycle-sequencing kit version 1.1 (Perkin Elmer, CA, USA) and the primers for the M13 forward and reverse priming sites of the pCR2.1 vector. Sequencing runs were performed by STABVIDA (Portugal). The sequence of mth-like 1 was not complete in the non-annotated genome of D. americana because there is a gap in the sequence of this gene. So, a similar approach was used in order to have access to the full sequence. We started by designing flanking primers for mth-like 1 based on the sequence of this gene in D. virilis genome. Standard amplification conditions were those described above as well as all the process till the sequencing. 2.7. Gene expression analyses The expression levels of seven mth-like genes – mth-like 1, GJ16328 ortholog, mth-like 11, mth-like 8, mth-like 9, mth-like 10 and GJ12490 ortholog – were determined for eight sets of three males each from the H5 and W11 strains at four different times of longevity – 0 days, 10 days, 30 days and 60 days. After eclosure 0, 10, 30 and 60 days old, individuals were frozen in liquid nitrogen. Total RNA was isolated from each set of individuals using TRIzol Reagent (Invitrogen, Spain) according to the manufacturer’s instructions and treated with DNase I (RNase-Free) (Ambion, Portugal). cDNA was then synthesized by reverse transcription with SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen, Spain), using random primers. No-template controls and reactions with RNA that was not reverse transcribed were performed in order to confirm the absence of DNA contamination. Specific primers with efficiency between 90% and 100% (table 3) for all the seven genes were used when performing qRT-PCR experiments using the isolated cDNA. CHAPTER II – METHODS Are all mth-like genes involved in lifespan determination? 20 Table 3: Specific primers for the seven mth-like genes and RpL32 tested for qRT-PCR. bp, base pair. Gene Primer Sequence (5’ 3’) Fragment size mth-like 1 mth1_RT3_F TTGAATGGAACGACGCTG 129 bp mth1_RT3_R TGGCTGGAACTTGATGAA GJ16328 ortholog 16328_RT3_F CACATTCCTTTGGCTCTT 128 bp 16328_RT3_R CAAGTAACCCCGCAGATA mth-like 11 mth60_RT_F CTCTGGTTCGTCATTTCG 139 bp mth60_RT_R TATGCGTTGGTTTTCTCA mth-like 8 mthl8_RT2_F CTCACGCTGATACGCATC 130 bp mthl8_RT2_R CGCAAACGAAATAGGTAA mth-like 9 mthl9_RT_F TCTCCGTGTATTTGGTGG 151 bp mthl9_RT_R AGCAGCAGAATGAACCCG mth-like 10 mthl10_RT2_F CGGAGAGCGTCACATTGG 115 bp mthl10_RT2_R CTGGCAGTGTTGGCAAAC GJ12490 ortholog mth29_RT_F GGAGAGAAGGAGTCGGTG 110 bp mth29_RT_R GTGCGTTCATTGCTGTCG RpL32 RpL32_RT_F ACAACAGAGTGCGTCGTC 208 bp RpL32_RT_R ATCTCCTTGCGTTTCTTC All the experiments were performed in duplicate and with the iQ SYBR Green Supermix (Bio-Rad, Portugal) according to the manufacturer’s instruction on a BioRad iCycler with the following program: 3 minutes at 95ºC; 40 cycles of 30 seconds at 94ºC, 30 seconds at 58ºC and 30 seconds at 72ºC. Specific primers were also designed for the endogenous ribosomal protein L32 (RpL32) (table 3) that was used as the reference gene. Fold change in expression was calculated using the 2-ΔΔCT (Livak) method (Livak and Schmittgen, 2001). 2.8. Statistical analyses All statistical testes and genotype-phenotype associations were tested using nonparametric tests and the software SPSS Statistics 17.0 (SPSS Inc., Chicago, Illinois). CHAPTER II – METHODS Are all mth-like genes involved in lifespan determination? 21 Linear regression analyses were performed in order to estimate the percentage of variation in lifespan that can be explained by the molecular variation present at mth-like genes. CHAPTER II – METHODS Are all mth-like genes involved in lifespan determination? 22 CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 23 3. RESULTS 3.1. The rise of mth-like genes 3.1.1. The evolutionary origin of Mth ectodomain After performing BLASTp searches at NCBI (http://www.ncbi.nlm.nih.gov) in nonDrosophila insects using Mth ectodomain from D. melanogaster as a query, we were able to find that Mth-like proteins with the typical Mth features (a Figure 6: Sequence alignment of the Mth protein from D. melanogaster and a protein from Daphnia pulex (gi321478728). Arrows and boxes mark the five out of the ten cysteine residues of Mth ectodomain that are conserved in Daphnia pulex. CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 30 Table 5: Comparison of the annotation of mth-like 14 in the twelve species of Drosophila, whose genome is publicaly available. bp, base pair. Drosophila species CDS size (bp) Number of Exons Size of exons (bp, 5’3’) D. simulans 1605 7 114, 422, 189, 423, 76, 118, 255 D. sechellia 1605 7 115, 422, 189, 423, 76, 118, 255 D. melanogaster 1602 7 115, 419, 189, 423, 76, 118, 255 D. yakuba 1677 7 115, 494, 189, 423, 76, 118, 255 D. erecta 1590 7 115, 413, 189, 423, 76, 112, 255 D. ananassae 1590 6 100, 401, 189, 500, 133, 261 D. pseudoobscura 1200 5 124, 186, 500, 124, 261 D. persimilis 1200 5 124, 189, 500, 117, 261 D. willistoni 756 5 124, 183, 50, 127, 267 D. mojavensis 1509 6 19, 401, 687, 133, 85, 178 D. virilis 1380 5 292, 687, 133, 85, 178 D. grimshawi 1425 5 337, 687, 133, 89, 178 3.2.2. Annotation of mth-like genes in D. americana and searching for conserved domains In order to design specific molecular markers for each mth-like gene present in D. americana genome, firstly it was necessary to annotate all these genes in the genome of both strains. After annotating, all the mth-like genes present in D. americana, with the help of Conserved Domains tool at NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml), it was possible to identify which conserved domains are codified in these genes. From this study, we were able to predict that D. americana has three mth-like genes without Mth ectodomain, and six which have the Mth ectodomain. However, as we can see on table 6, mth-like 8 and mth-like 9 have an Mth ectodomain but do not show a 7tm domain. Instead of a 7tm domain, mth-like 8 has a wcaJ_sugtrans domain (undecaprenyl-phosphate glucose phosphotransferase). Besides that, mth-like 1 seems to have only a 7tm domain. The latter belongs to the group of the oldest CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 31 mth-like genes, which also comprises two more members that do not show any conserved domains – mth-like 5 and mth-like 14. Finally, we can say that D. americana encodes four mth-like genes that have the Mth typical features, in other words Mth ectodomain and a 7tm domain – orthologous of GJ16328 and GJ12490, mth-like 11 and mth-like 10. Table 6: Conserved domains encoded by mth-like genes of D. americana. mth-like genes Conserved Domains mth-like 1 7tm2 GJ16328 ortholog Mth and 7tm2 mth-like 5 − mth-like 11 Mth and 7tm2 mth-like 14 − mth-like 8 Mth and wcaJ_sugtrans mth-like 9 Mth mth-like 10 Mth and 7tm2 GJ12490 ortholog Mth, 7tm2 and 7tm6 3.3. Lifespan variation in D. americana 3.3.1. Molecular markers design Then it was found out all amino acid and nucleotide polymorphisms as well as differences in introns size existing between both D. americana strains (H5 and W11) in the same gene. After this identification, a molecular marker that distinguishes the polymorphism selected for each mth-like gene was designed. It was given preference for molecular markers that distinguish amino acid polymorphisms, since a change of an amino acid can interfere with the normal function of a protein. It was possible to design molecular markers for all mth-like genes present in D. americana (table 7). However it was not ruled out the possibility that the influence of these genes on lifespan is due to differences in the gene expression and not just due to a polymorphism. CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 32 Table 7: Localization of mth-like genes in D. americana genome and respective type of marker. mth-like genes Chromosome Type of Marker mth-like 1 X amino acid GJ16328 ortholog 2 amino acid mth-like 5 2 nucleotide mth-like 11 2 intron size difference mth-like 14 3 amino acid mth-like 8 3 amino acid mth-like 9 3 nucleotide mth-like 10 3 intron size difference GJ12490 ortholog 3 nucleotide 3.3.2. Lifespan of individuals from an F2 association study All the markers listed above were then tested in all individuals from the first F2 association study previously described. This F2 association study comprises five crosses among five different strains from the species in study – D. americana – with the aim to test if there is association between the different mth-like genes and lifespan variation. As previously stated, D. americana is a native species of United States and has a large effective population size. This species can be found in the central and eastern regions of United States from the south to the north, and this influenced the choice of strains used. The strains W11 and H5 were chosen since their genomes were recently sequenced. The other three species were selected in order to reproduce in this experiment what happens in the natural environment. So a strain typical from the north of distribution (O57) as well as two strains characteristic from the south of distribution (W29 and W46) were chosen, in order to include a high number of individuals and most of the relevant variation that exists at the molecular level. CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 33 H5♂ x W11♀ AAs W11♂ x W46♀ ATs W29♂ x O57♀ TNs O57♂ x H5♀ NAs W46♂ x W29♀ TTs A B C D E Figure 9: Lifespan variation observed in F2 individuals of each cross of the F2 association study. (A) cross between H5♂ x W11♀ (AAs), Mean=55,06 days, Standard Deviation=18,27, N=88; (B) cross between W11♂ x W46♀ (ATs), Mean=64,88 days, Standard Deviation =20,61, N=75; (C) cross between W49♂ x O57♀ (TNs), Mean=54,59 days, Standard Deviation =17,86, N=87; (D) cross between O57♂ x H5♀ (NAs), Mean=49,01 days, Standard Deviation =16,49, N=93; (E) cross between W46♂ x W29♀ (TTs), Mean=46,47 days, Standard Deviation =17,24, N=89. CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 34 As we can see on figure 9, the number of individuals varies according to the crosses, as well as the average lifespan of individuals from each cross. And since there is variation in phenotype, there should also be some variation in the genotype that justifies the difference in longevity. In the D. americana F2 association study there is a single generation of recombination, and thus closely linked markers will give similar information. The localization of all mth-like genes present in D. americana genome is shown in figure 10 and from this it is possible to see that there is a cluster of four mth-like genes in chromosome 3 (Muller’s element E). Moreover, there is a mth-like gene localized in chromosome X (Muller’s element A) – mth-like 1 -, and once only males were tested, there is only one allele present in each individual and there are not heterozygous individuals. The markers were firstly typed on the ten F0 individuals and in the crosses where the marker was segregating then the F2 individuals were genotyped. For the mth-like 1 gene, that is localized in chromosome X, the marker was segregating in two crosses: AA and AT. After digesting the amplification product with MspI, two different genotypes were found in these crosses, namely homozygous 0 (no digestion) and homozygous 1 (fully digested). In AA cross, the F0 male was homozygous 1 and the female was homozygous 0 and in AT cross, as expected F0 male was homozygous 0 and female was homozygous 1. In the F2 association experiment, AA individuals homozygous 1 (49.3±2.75 days; N=44) and homozygous 0 (61.4±2.51 days; N=43) show a significant association with lifespan (Non-parametric Kruskal-Wallis Test; P=0.000). In the individuals originated from AT cross, homozygous 1 (64.0±4.60; N=22) and homozygous 0 (66.1±3.11 days; N=43) were found, but no association was observed with lifespan (Non-parametric Kruskal-Wallis Test; P=0.708). Therefore, genotype 1 Figure 10: Chromosomal localization of mth-like genes identified in D. americana. CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 35 from H5 seems to be associated with short lifespan and genotype 0 from W11 seems to be associated with normal lifespan (figure 11). Moreover, allele 1 from W46 could have differences in comparison with allele 1 from H5, once in AT cross, individuals homozygous for allele 1 lived about more 15 days than individuals homozygous for allele 1 in AA cross. The amplification product of orthologous gene of GJ16328 after the digestion with HypCH4IV in F0 individuals showed two different genotypes: homozygous 1 (fully digested) and heterozygous for allele 1 and 0 (half-digested). However, only two out of the five crosses were segregating the marker: AA and AT. The AA male was homozygous for allele 1 and the AA female was heterozygous for allele 1 and 0 (1/0). On the other hand, AT male was heterozygous 1/0 and the female was homozygous for allele 1. The other six F0 individuals were homozygous for allele 1. The F2 descendent individuals from AA and AT crosses were then genotyped for this marker. Three genotypes were found in F2 individuals from AA cross: homozygous for allele 0 (60.8±4.03 days; N=16), homozygous for allele 1 (44.6±3.42 days; N=16) and heterozygous 1/0 (57.3±2.64 days; N=53) and an association between genotype and lifespan was found in this cross (NonA B Figure 11: Box plot representing the association results obtained for gene mth-like 1 in F2 individuals from cross AA (A) and cross AT (B). The top of the box represents the 75th percentile, the bottom of the box represents the 25th percentile, and the line in the middle represents the 50th percentile that corresponds to the median. The whiskers represent the highest and lowest values that are not outliers or extreme values. Circles beyond the whiskers represent outliers CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 36 parametric Kruskal-Wallis Test; P=0.028). The individuals originated from AT cross also showed three different genotypes: homozygous for allele 0 (70.9±4.66 days; N=14), homozygous for allele 1 (60.7±2.30 days; N=39) and heterozygous 1/0 (68.6±6.72 days; N=17), but no association was found between genotype and lifespan in this cross (Non-parametric Kruskal-Wallis Test; P=0.148). With these crosses, it is not possible to define which allele is dominant, once there is a common allele in the individuals from F0. However, it is clear that the genotype homozygous 1 is associated with short lifespan in both crosses (figure 12). Individuals homozygous 1 from AA lived about 16 days less than homozygous 0 and 13 days less than heterozygous 1/0 from the same cross. In the F2 individuals originated from AT cross we can see the same behaviour: homozygous 1 lived approximatly 11 days less than homozygous 0 and 8 days less than heterozygous 1/0. From these results, besides concluding that allele 1 is associated with short lifespan, we are able to say that allele 0 balances the effect of allele 1 once the heterezygous 1/0 show an average lifespan similar to the homozygous 0 in both crosses. A B Figure 12: Box plot representing the association results obtained for GJ16328 ortholog in F2 individuals from cross AA (A) and cross AT (B). The top of the box represents the 75th percentile, the bottom of the box represents the 25th percentile, and the line in the middle represents the 50th percentile that corresponds to the median. The whiskers represent the highest and lowest values that are not outliers or extreme values. Circles beyond the whiskers represent outliers. CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 37 For mth-like 5 the marker is segregating in three crosses – AA, TN and NA. After digesting the amplification product with BclI three genotypes were observed: homozygous 0 (no digestion), homozygous 1 (fully digested) and heterozygous for allele 0 and 1 (1/0). However, the F0 individuals only showed two of the three genotypes predicted: in AA cross, the male was heterozygous 1/0 and the female was homozygous 0; in TN cross, the male was homozygous 0 and the female was heterozygous 1/0; and in NA cross, the male was homozygous 0 and the female was heterozygous 1/0. F2 individuals from AA cross were homozygous 0 (57.8±2.76 days; N=47), only one homozygous 1 (43 days), and heterozygous 1/0 (52.43±2.95 days; N=37) but no association with lifespan was found (Nonparametric Kruskal-Wallis Test; P=0.489). In F2 individuals derived from TN cross Figure 13: Box plot representing the association results obtained for mth-like 5 in F2 individuals from AA cross (A), TN cross (B) and NA cross (C). The top of the box represents the 75th percentile, the bottom of the box represents the 25th percentile, and the line in the middle represents the 50th percentile that corresponds to the median. The whiskers represent the highest and lowest values that are not outliers or extreme values. Circles beyond the whiskers represent outliers. A B C CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 38 no homozygous 1 was found. Although, it was possible to find homozygous 0 (53.4±2.58 days; N=60) and heterozygous 1/0 (56.4±2.35 days; N=25) individuals and no association between genotype and lifespan was found (Nonparametric Kruskal-Wallis Test; P=0.458). In the individuals originated from the last cross – NA – an association between this genotype and lifespan was not also found (Non-parametric Kruskal-Wallis Test; P=0.742). In the descendents of this cross it was possible to find homozygous 0 (48.3±4.09 days; N=11), homozygous 1 (46.1±4.01 days; N=15) and heterozygous 1/0 (50.4±2.30 days; N=64). It seems that no matter what allele an individual has, the mean longevity for each genotype in each cross is very similar (figure 13). However, it is noted that individuals homozygous 1 are very rare. The marker for mth-like 11 is the only one that is segregating in all five crosses. There is, however, only one cross where an association was found between genotype and lifespan - AA cross (Non-parametric Kruskal-Wallis Test; AA cross P=0.003; AT cross P=0.452; TN cross P=0.173; NA cross P=0.444; and TT cross P=0.353). This marker showed fourteen types of genotypes in F2 individuals: homozygous 1, homozygous 2, homozygous 3, homozygous 4, homozygous 5 and heterozygous 1/2, heterozygous 1/3, heterozygous 1/4, heterozygous 2/3, heterozygous 2/4, heterozygous 2/5, heterozygous 3/4, heterozygous 3/5 and heterozygous 4/5. In the AA cross, the F0 male was heterozygous 2/4 and the female heterozygous 3/4, so allele size 4 was shared by both strains. Thus, in the F2 individuals from this cross we found five out of the six possible genotypes, once no homozygous 2 was detected: homozygous 3 (61.6±4.99 days; N=13), homozygous 4 (56.6±4.79 days; N=9), heterozygous 2/3 (52.4±2.96 days; N=19), heterezygous 2/4 (37.9±2.60 days; N=12) and heterozygous 3/4 (61.0±3.92 days; N=30). In the founder members of AT cross, the male was heterozygous 3/4 and the female heterozygous 1/2, so in the F2 individuals we found ten different genotypes: homozygous 1 (60 days; N=1), homozygous 2 (52.0±10 days; N=2), homozygous 3 (82.3±18.48 days; N=3), homozygous 4 (68.7±5.74 days; N=14), heterozygous 1/2 (56.9±2.40 days; N=10), heterozygous 1/3 (61.6±11.00 days; N=8), heterozygous 1/4 (63.7±5.91 days; N=9), heterozygous 2/3 (72.2±5.86 days; N=12), heterozygous 2/4 (60.4±7.04 days; N=7) and heterozygous 3/4 (75.5±12.9; N=4). In the F0 individuals of TN cross, the male was homozygous 2 and the female was heterozygous 3/5, thus in the F2 CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 39 Figure 14: Box plot representing the association results obtained for mth-like 11 in F2 individuals from AA cross (A), TN cross (B), TT cross (C), AT cross (D) and NA cross (E). The top of the box represents the 75th percentile, the bottom of the box represents the 25th percentile, and the line in the middle represents the 50th percentile that corresponds to the median. The whiskers represent the highest and lowest values that are not outliers or extreme values. Circles beyond the whiskers represent outliers. A C B D E CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 46 originated from H5 strain, it seems that allele 3 from H5 is associated with short lifespan. The amplification product of ortholog gene of GJ12490 after the digestion with Sau3AI showed two different genotypes in F0 individuals: homozygous 0 (no digested) and homozygous 1 (fully digested). From the five crosses, only two A B C Figure 18: Box plot representing the association results obtained for mth-like 10 in F2 individuals from AA cross (A), TN cross (B) and NA cross (C). The top of the box represents the 75th percentile, the bottom of the box represents the 25th percentile, and the line in the middle represents the 50th percentile that corresponds to the median. The whiskers represent the highest and lowest values that are not outliers or extreme values. Circles beyond the whiskers represent outliers and star represents an extreme outlier. CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 47 segregate this marker: AA and AT crosses. F0 male of AA cross was homozygous 0 and the female was homozygous 1, so in the F2 individuals three different genotypes were found: homozygous 0 (42.0±2.54 days; N=12), homozygous 1 (57.7±3.24 days; N=31) and heterozygous 1/0 (57.1±2.90 days; N=44). In the founder members of AT cross, the male was homozygous 1 and the female homozygous 0, thus in F2 individuals there were homozygous 0 (62.2±4.38 days; N=17), homozygous 1 (76.7±5.90 days; N=16) and heterozygous 1/0 (62.5±3.00 days; N=39). An association between genotype and lifespan was found in the individuals from both crosses (Non-parametric Kruskal-Wallis Test; P=0.028 and P=0.049, respectively). The average lifespan is very similar between the homozygous 1 and heterozygous 1/0 in AA cross and they lived much longer than homozygous 0 (figure 19 A). Therefore, genotype 0 seems to be associated with short lifespan. However, when we look to the results of AT cross, the average lifespan is similar between homozygous 0 and heterozygous 1/0, who lived lesser than homozygous 1 (figure 19 B). From this cross we are able to conclude that genotype 1 seems to be related with longer lifespan. Nevertheless, when taken together no inference could be made in terms of allele dominance. A B Figure 19: Box plot representing the association results obtained for GJ12490 ortholog in F2 individuals from AA cross (A) and AT cross (B). The top of the box represents the 75th percentile, the bottom of the box represents the 25th percentile, and the line in the middle represents the 50th percentile that corresponds to the median. The whiskers represent the highest and lowest values that are not outliers or extreme values. Circles beyond the whiskers represent outliers and star represents an extreme outlier. CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 48 Grouping all the results, some more conclusions can be taken. As we can see on figure 20, in the genes that previously showed association with lifespan determination, the homozygous that carry the allele from H5 lived approximately 20 days less than the homozygous for the allele originated in the other founder strain. Moreover, the H5 allele seems to be recessive. This is especially notorious when we look for AA cross, where the allele from W11 seems to compensate the short lifespan determinated by allele from H5 strain (figure 20 B, F, G and I). On the other hand, in relation to genes that did not showed association with lifespan, these differences are not so notorious, once homozygous for the allele from H5, homozygous for the allele from the other founder strain and heterozygous lived approximately the same. Taking all this into account, we are able to say that the allele that came from H5 strain is in some way related to short lifespan. This means, that individuals that belong to H5 strain should live, in general, less than individuals from the other strains. In order to try to understand why some genes show strong association than others it is essential to comprehend what differences are present in these proteins that might put in question their normal function. In figure 21, a scheme representative of the protein structure of each mth-like studied, as well as the amino acid differences between the two strains of D. americana – H5 and W11 – and the foreseen N-glycosylation sites are shown. The major difference is in the length of the proteins codified by these nine mth-like genes. mth-like 1 and the ortholog of GJ12490 are much longer than the other genes. Moreover it is possible to see that the major part of amino acid polymorphisms occur inside Mth ectodomain, which is likely important for the protein to be functional, once the active site of this receptor is likely be here. Another difference is the fact that ortholog of GJ12490 has an additional 7tm domain (figure 21). Many efforts were done in order to understand if this 7tm domain is part of this gene or not, but no conclusion was reached. However, when a BLASTp is performed in NCBI (http://blast.ncbi.nlm.nih.gov/) using the sequence of the beginning of the gene till the end of the first 7tm domain as a query, there is a hit with odorant receptor of D. pseudoobscura pseudoobscura. So probably, this 7tm domain belongs to another gene that is next to GJ12490 ortholog. CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 49 Figure 20: Compilation of the association study results, where it is possible to compare the genotypes and the lifespan that is associated to them in all mthlike genes present in D. americana. mth-like 1 (A), GJ16328 ortholog (B), mth-like 5 (C), mth-like 11 (D), mth-like 14 (E), mth-like 8 (F), mth-like 9 (G), mth-like 10 (H) and GJ12490 ortholog (I). * represents an allele that is shared by the two individuals of F0. A B C D E F G H I CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 50 Figure 21: Schematic diagram of the structure of the nine mth-like genes present in D. americana. Green boxes represent the amino acid differences between W11 (top of the box) and H5 (bottom of the box) strains. N* represent the N-glycozylation sites. A, alanine; C, cysteine; D, aspartic acid; E. glutamic acid; F, Phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine. MTH, Mth ectodomain; 7tm, seven-transmembrane domain. CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 51 3.4. The second F2 association study In order to test if the association between mth-like genes and lifespan determination remained untouched when the sample is bigger, another F2 association study was performed. The individuals of this F2 association experiment show a high diversity of phenotypes (figure 22), and for that reason some variation in genotypes might explain the variety in individuals’ lifespan. In this F2 association study only three markers of mth-like genes of D. americana were tested. These genes were chosen due to its association or not with lifespan in the previous F2 association study. Firstly, the mth-like 5 was chosen in order to guarantee that when the sample is enlarged, mth-like genes without Mth ectodomain keep on showing no association with lifespan determination. Secondly, two mth-like genes, which show a strong association with lifespan, were chosen – ortholog of GJ12490 and mth-like 11 – in order to confirm its association with lifespan. mth-like 5 and mth-like 11 are located in chromosome Figure 22: Lifespan variation observed in the 453 individuals of the second F2 association experiment (Mean=58.64 days, Standard Deviation=20.51, N=453). CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 52 2 (Muller’s element E) and GJ12490 ortholog is located in chromosome 3 (Muller’s element D). For mth-like 11, in this F2 association study, the size genotypes found were homozygous 3 (62.7 days±12.1 days; N=18) and homozygous 4 (64.2±19.8 days; N=173), and heterozygous for alleles size 2 and 3 (57.2±19.4 days; N=56), 2 and 4 (47.1±17.6 days; N=125), and 3 and 4 (67.2±21.6 days; N=65). Three alleles are present in this F2 association study because isofemale rather than isogenic strains were used. A significant association between this mth-like size genotype and lifespan was found (Non-parametric Kruskal-Wallis Test; P<0.001). When performing all possible pair wise allele size genotype comparisons, using Nonparametric Mann-Whitney tests, significant differences regarding lifespan are found for the following comparisons: 3 and 2/4 (P<0.001), 4 and 2/3 (P<0.05), 4 and 2/4 (P<0.001), 2/3 and 2/4 (P<0.001), 2/3 and 3/4 (P<0.005), and 2/4 and 3/4 (P<0.001). After applying the sequential Bonferroni correction for multiple testing, all significant comparisons with the exception of the one involving genotypes 4 and 2/3 are significant (P<0.05). These findings are not easily accounted for under a simple scenario, but we note that four out of the six most significant comparisons (P<0.001) involve genotype 2/4. Moreover, two out of the three comparisons involving genotype 2/3 are significant, although one of them is not significant after Bonferroni correction for multiple testing. These observations suggest that allele size 2 is associated with short lifespan and that is dominant over the alleles 3 and 4, though the latter two alleles are clearly not equivalent, because when comparing genotypes 2/3 and 2/4 it is possible to detect significant differences. On average, individuals that do not have allele size 2 live 28.8 % longer than individuals carrying allele size 2 (the average for individuals that do not have allele size 2 and for those that have allele size 2 is 64.9 and 50.4 days, respectively). In D. melanogaster, more than 40 genes have been implicated in lifespan determination (Paaby and Schmidt, 2009). Therefore, the possibility that another gene in the vicinity of mth-like 11 is the one showing variation that influences lifespan must be taken into account. One of these candidate genes is four wheel drive (fwd) and this gene is in the middle of the genomic region defined by mthlike 11 and mth-like 5. For that reason, and under the hypothesis that variation at fwd rather than variation at mth-like 11 is the responsible for lifespan variability observed in the F2 experiment, an association should be found when looking CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 53 both at mth-like 11 and mth-like 5. For the latter gene, after the digestion of amplification product with BclI, two genotypes were found in this F2 association experiment: homozygous 0 (no digestion) (57.0±19.2 days; N=227), and heterozygous 1/0 (half digested) (60.2±22.1 days; N=200). It is not clear why no males homozygous for allele 1 were found, and this is in agreement with the results obtained in the previous association study, since in the AA cross was only observed one individual homozygous 1. However, and as previously reported in the first F2 association study, no association was found between this genotype and lifespan (Non-parametric Mann-Whitney Test; P>0.05). The GJ12490 ortholog, when digesting the amplification product with Sau3AI, we found three genotypes in this F2 experiment study: homozygous 0 (no digestion) (50.2±20.1 days; N=75), 1 (fully digested) (60.2±19.6 days; N=109) and heterozygous for alleles 0 and 1 (half-digested) (61.2±20.8 days; N=237). A significant association between this genotype and lifespan was found (Nonparametric Kruskal-Wallis Test; P<0.001). There are no significant differences regarding lifespan when genotypes 1 and 1/0 are compared (Non-parametric Mann-Whitney Test; P>0.05) but significant changes are observed when comparing genotypes 0 and 1/0 (Non-parametric Mann-Whitney Test, P<0.001), and when comparing genotypes 0 and 1 (Non-parametric Mann-Whitney Test; P<0.005). These results are significant after applying the sequential Bonferroni correction for multiple testing (P<0.05). Therefore, genotype 0 seems to be associated with short lifespan. Individuals with genotypes 1/1 and 1/0 show a 21.3% average increase in lifespan in comparison with individuals with genotype 0/0. Nevertheless, as said before, more than 40 genes are described as being implicated in lifespan determination in D. melanogaster, so it is conceivable that variation at another gene in the neighborhood of GJ12490 ortholog is the one showing variation that influences lifespan, and that this mth-like gene plays no role in the setting of lifespan. CHAPTER III – RESULTS Are all mth-like genes involved in lifespan determination? 54 3.5. The mth-like genes expression in D. americana Once we have found out which mth-like genes might be involved in lifespan determination, the interest on their expression levels arose. In order to test if there are differences in the expression levels of each one of the seven genes analyzed, a qRT-PCR study was performed. The use of these seven genes tested by qRT-PCR was considered, once they have showed strong association with lifespan in at least one F2 association cross. The seven genes tested were mthlike 1, GJ16328 ortholog, mth-like 11, mth-like 8, mth-like 9, mth-like 10 and GJ12490 ortholog. Since the aim of this experiment was to test the relationship between mth-like genes expression and lifespan, we used eight different sets of samples in order to measure the expression levels of these genes along time. To perform this study, only two strains were used – H5 and W11 – once they were the founder members of the cross where we saw the largest number of association between genotype and lifespan (AA cross). The expression levels were measured in males only, since the association studies were performed with males only. From the eight sets, four are for strain H5 and the other four are for W11 strain. The four kinds of sets for each strain correspond to four lifespan time points, i.e., 0 days, 10 days, 30 days and 60 days of life. With this sample it was possible to get a notion on how the expression of these genes varies over the life of the individuals from D. americana species. RpL32 was used as an internal control. Nevertheless, the expression of this gene may vary with time and thus it is difficult to infer how the expression of mth-like genes varies along time. It should be noted that the concentration of RNA was normalized to 1μg upon the cDNA synthesis and the same cDNA was used to test the expression of all mth-like genes. The results of qRT-PCR were analyzed with the use of 2-ΔΔCT (Livak) method (Livak and Schmittgen, 2001). We only believe that the expression of these genes is different between the two strains (H5 and W11) when there are great differences (more than two-fold) that are consistent along the four lifespan time points. CHAPTER III– RESULTS Are all mth-like genes involved in lifespan determination? 55 A1 A2 B1 B2 C1 C2 D1 D2 F2 F1 E2 E1 G1 G2 Figure 23: Fold change in mth-like genes expression in two strains of D. americana - H5 and W11 – using the 2-ΔΔCT method. There are two different representations of the results: (1) the housekeeping gene was used to normalize the expression levels; and (2) the value of threshold cycle (CT) of H5 at 0 days was used to normalize the expression levels. (A) mth-like 1, (B) GJ16328 ortholog, (C) mth-like 11, (D) mth-like 8, (E) mth-like 9, (F) mth-like 10 and (G) GJ12490 ortholog. CHAPTER IV – DISCUSSION AND CONCLUSIONS Are all mth-like genes involved in lifespan determination? 62 CHAPTER V – REFERENCES Are all mth-like genes involved in lifespan determination? 63 5. 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