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Characterization of the human gonadotropin-releasing hormone receptor gene at the molecular level Fan, Fan, Nancy C. Nancy C. 1995

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CHARACTERIZATION OF THE HUMAN GONADOTROPINRELEASING HORMONE RECEPTOR GENE AT THE MOLECULARLEVELbyNancy C. FanB.Sc., University of British Columbia, 1991A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THEREQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY)(REPRODUCTIVE AND DEVELOPMENTAL SCIENCES PROGRAM)We accept this thesis as conforming to the required standardTHE UNIVERSITY OFJuLy 1995COLUMBIA© Nancy C. Fan jCj9In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis br scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature____________________________Department of S*e/7/cS 7AJ 6 4QThe University of British ColumbiaVancouver, CanadaDate__________DE-6 (2/88)ABSTRACTThe gonadotropin - releasing hormone (GnRH) receptor is a plasma membraneassociated receptor and a member of the GTP - binding protein coupled receptorfamily. The interaction of the ligand, GnRH, and the GnRH receptor is a criticalevent in the endocrine control of reproduction. This coupling stimulates thesynthesis and release of both luteinizing hormone and follicle stimulating hormonefrom the anterior pituitary. In addition, GnRH - GnRH receptor binding acts locallyto regulate human chorionic gonadotropin secretion in the placenta andsteroidogenesis in the ovary. The objective of this thesis was to isolate andcharacterize the gene for the GnRH receptor in human. The human GnRH receptor(GriRH-R) gene was isolated from a human genomic library derived from placentaltissue. The genomic clones obtained encompassed the entire gene including itscoding region (987 bp) as well as substantial 5’ and 3’ sequences. Sequence analysisrevealed a structural organization consisting of three exons and two intronsdistributed over 18.9 Kb. Exon II contains only 219 bp and the remainder of theapproximately 4.7 Kb transcript is distributed between exon I (1915 bp) and III(3321 bp). Sequence analysis and restriction endonuclease mapping revealed thesizes of intron A and B to be approximately 4.2 and 5.0 Kb, respectively.Sequencing of the 5’ end of the gene revealed the presence of five consensus TATAiisequences clustered within a 700 nucleotide region. Primer extension analysisdetected multiple transcription initiation sites associated with this group of TATAsequences. Transcription of this region up to the most 5’ initiation site wasdemonstrated by the reverse transcription - polymerase chain reaction (RT-PCR)method. The 5’ nontranslated region has a length between 703 and 1393 bp,depending on which initiation site is used. Several consensus cis - acting regulatorysequences were identified within the 5’ end. These include sites for GATA-i,WAP, PEA-3, AP-1, and Pit-i. In addition, cAMP response element (CRE) - likeand glucocorticoid / progesterone response element (GRE / PRE) - like sequenceswere found. The ability of these response elements to bind to their respectiveregulatory proteins (CRE binding protein for CRE; progesterone receptor for GRE!PRE) was investigated by mobility shift assays. No DNA - protein complexes wereobserved for these response - like elements suggesting that the mismatches incurredcould not be recognized by the respective regulatory proteins. The 3’ end of thegene was also sequenced and five classical polyadenylation signals were foundscattered over a region of 800 nucleotides. RT-PCR conducted on the 3’nontranslated region confirmed transcription up to the most 3’ locatedpolyadenylation signal. Factoring in the location of the most 5’ initiation site andthe most 3’ polyadenylation signal, the total transcript covers a region of 5455 bp.The finding of multiple transcription intiation sites and polyadenylation signals raisesiiithe possibility of tissue - specific regulation and the existence of variable transcriptsfor the human GnRH receptor. Genomic Southern blot analysis indicated thepresence of a single copy of the gene encoding for the GnRH-R gene within thehuman genome. Using somatic hybrid analysis, the GnRH-R gene was also assignedto human chromosome 4. This study represents the first report on the isolation andcharacterization of the GnRH-R gene in any species. The characterization of thehuman GnRH-R gene should facilitate future investigative efforts on the delineationof possible genetic disorders for this gene, the mechanisms involved in its regulation,and on generation of improved GnRH analogues currently in use for severalreproductive disorders and diseases.ivTABLE OF CONTENTSPageABSTRACT iiT&L ô,t Ct)rJjr VLIST OF TABLES ixLIST OF FIGURES xLIST OF ABBREVIATIONS xiiACKNOWLEDGEMENTS xiiiI. INTRODUCTION 11.1 Physiological role 31.2 Distribution of the GnRH receptor 71.2.a. Pituitary 71.2.b. Brain 81.2.c. Gonads 81.2.d. Placenta 91 .2.e. Neoplastic tissues and cell lines 101.3 Regulation of the GnRH receptor 111.4 Activation of the GnRH receptor and signal transduction 151.5 Molecular biology of the GnRH receptor 241.5.a. General 241.5.b. Structure 251 .5.c. Interspecies relationship at the mRNA level 281.6 Binding characteristics 301.7 Clinical applications 31V1.8 Objective 32II. MATERIALS AND METHODS 342.1 Genomic DNA library screening 342. l.a. Preparation of filters 342.1 .b. Preparation of radioactive cDNA probes 352. i.e. Hybridization with radioactive probes/Washing 362.1. d. Screening 372. i.e. Preparation of liquid lysate 382.1 .f. Preparation of lambda DNA from phage lysates 382.2 Cloning of DNA 392.2.a. Competent cells 392.2.b. Ligation 402.2.c. Transformation 412.3 DNA sequencing 422.3.a. General 422.3 .b. Preparation of template 422.3. c. Annealing of primer 432.3.d. Labelling reaction 432.3.e. Termination reaction 442.3.f. Denaturing gel electrophoresis 442.4 Characterization of the 5’ terminus of the humanGnRH-R mRNA 452.4.a. Isolation of total RNA 452.4.b. Primer extension analysis 462.5 Reverse-transcription polymerase chain reaction (RT-PCR) 502.5.a. Preparation of eDNA 502.5.b. Polymerase chain reaction (PCR) 512.6 Gel Mobility shift assay 522.6.a. Cell culture 522.6.b. Preparation of nuclear extract 532.6.c. Preparation of radiolabelled double-strandedoligonucleotide probes 54vi2.6.d. DNA-protein binding assay 572.6.e. Nondenaturing PAGE 582.7 Southern analysis 592.7.a. Genomic Southern analysis 602.8 Northern analysis 622.9 Chromosome assignment 63III. RESULTS 643.1 Isolation of the human GnRH-R gene 643.2 The human GnRH-R gene sequence 703.3 Comparative analysis of the human GnRH-R gene 823.4 Structural organization of the human GnRH-R gene 943.5 Promoter region of the human GnRH-R gene 993.6 Characterization of the 5’ terminus of thehuman GnRH-R mRNA 1043.7 RT-PCR analysis of the 5’ end of the human GnRH-R gene 1073.8 Transcription factor binding sites(in addition to TATA and CAAT)and regulatory elements 1133.9 Analysis of GRE/PRE-like and CRE-like sequences inthe human GrIRH-R gene 1183.10 Analysis of the 3’ end of the human GnRH-R gene 1243.11 Genomic Southern blot analysis 1303.12 Chromosomal assignment 135vii.3.13 Northern blot analysis of the human GnRH-R mRNA 140IV. DISCUSSION 1434.1 Analysis of the 5’ end of the human GnRH-R gene 1524.2 Analysis of the 3’ end of the human GriRH-R gene 170V. SUMMARY AND CONCLUSIONS 175VI. REFERENCES 182viiiLIST OF TABLESPageTable 1. Oligonucleotides used in this investigation 47Table 2. Oligonucleotides used in mobility shift assays 55Table 3. Interspecies sequence comparison of the GnRH-R gene and cDNA 84Table 4. The exon-intron organization of the human GnRH-R gene 97Table 5. Chromosome contents of human-hamster hybrid cell lines andthe assignment of the human GnRH-R gene 138ixLIST OF FIGURESPageFig. 1. Diagram of the hypothalamic-pituitary-gonadal axis 4Fig. 2. Signal transduction mechanism of the GnRH receptor 16Fig. 3. Structure of the GnRH receptor 26Fig. 4. Southern blot analysis of genomic fragments of the humanGnRH-R gene 65Fig. 5. Southern blot analysis of 5’ and 3’ oriented genomic fragments ofthe human GriRH-R gene 67Fig. 6. The nucleotide sequence of the human GnRH-R gene 71Fig. 7. Nucleotide sequence of the human GnRH-R coding region 78Fig. 8. DNA sequence alignment and comparison of GnRH-R codingregions among various species 86Fig. 9. Alignment of the amino acid sequences of various species 91Fig. 10. The human GnRH-R gene structure 95Fig. 11. The 2.7 Kb Hindill subclone 100Fig. 12. Organization of the human GnRH receptor promoter region 102Fig. 13. Analysis of the human GnRH-R gene by primer extension analysis 105Fig. 14. Analysis of the 5’ end of the human GnRH-R gene 108Fig. 15. f3-Actin controls 111Fig. 16. Cis-acting DNA regulatory sequences of the human GnRH-R gene 115Fig. 17. Mobility shift assay of the CRE-like element 120Fig. 18. Mobility shift assay of the GRE/PRE-like element 122Fig. 19. Analysis of the 3’ end of the human GriRH-R gene 125Fig. 20. Genomic Southern blot analysis of the human GnRH-R gene 131Fig. 21. Chromosomal assignment of the human GnRH-R gene tochromosome 4 136Fig. 22. Northern blot analysis of human pituitary and intestinal RNA 141xLIST OF ABBREVIATIONSATP adenosine 5’-triphosphatebp base pairdegrees CelsiuscAMP adenosine 3’, 5’ -cyclic-monophosphatecDNA complementary deoxyribonucleic acidcGMP guanosine 3’, 5’-cyclic-monophosphateCi Curiecpm counts per minuteDEP diethylpyrocarbonatedNTP deoxyribonucleoside triphosphatesdATP deoxyadenosine 5 ‘-triphosphatedCTP deoxycytidine 5’-triphosphatedGTP deoxyguanosine 5’-triphosphatedI.dC polydeoxyinosiriic acid and polydeoxycytidylic acid(dl. dC)-(dI dC) homopolymer of dl. dCdTTP deoxythymidine 5’-triphosphateddATP dideoxyadenosine 5’ -triphosphateddCTP dideoxycytidine 5’ -triphosphateddGTP dideoxyguanosine 5’ -triphosphateddTTP dideoxythymidine 5’ -triphosphateDNA deoxyribonucleic acidDNase deoxyribonucleaseDTT dithiothreitolE. coli Escherichia coliEDTA ethylene diaminetetraacetic acidg acceleration of gravityh hour(s)IPTG isopropyl- 1 -thio-13-D-galactosideKb kilobaseLB Luria-BertanimCi millicuriemm minute(s)ml milliliterMOPS 3-(N-morpholino)propane sulfonic acidMW molecular weightmRNA messenger RNAxing nanogramnM nanomolarnt nucleotideNZY NZ amine/Yeast/NaC1/MgSO4mediaOD optical densityPAGE polyacrylamide gel electrophoresispcv packed cellular volumePEG polyethylene glycolPIPES piperazine-N, N’-bis(2-ethanesulfonic acid)PMSF phenylmethylsulfonyl fluoridepnv packed nuclear volumeRNA ribonucleic acidrpm revolutions per minuteRT-PCR reverse transcription-polymerase chain reactionSDS sodium dodecyl sulphates second(s)Taq Therinus aquaticus, source of a DNA polymeraseTEMED N, N, N’, N’-tetramethylethylenediamineTris tris(hydroxy methyl) aminomethanetRNA transfer RNAUV ultravioletU unit(s)vol volumeX-Gal 5-bromo-4-chloro-3-indoyl-fl-D-galactopyranosidemicrograma alphabetagammaA lambdaxiiACKNOWLEDGEMENTSThroughout the duration of this thesis many individuals have generously giventheir time, shared their experiences and expertise as well as provided support andguidance. To everyone who gave time unselfishly and shared in the objective andcritical evaluation of this thesis, I extend my sincerest thanks.Grateful acknowledgement must be extended in particular to a special groupof individuals who made the submission of this thesis possible:My personal and deep appreciation goes especially to my supervisors Drs.Peter C.K. Leung and Johann Krisinger (Department of Obstetrics and Gynaecology,University of British Columbia), under whose counsel, guidance, and encouragementthe experimental program was carried out. To each I extend my heartfeltappreciation.I also wish to express my gratitude to the British Columbia Research Institutefor Child and Family Health who provided financial support during my studies.xiiiAdditionally, appreciation is also extended to my supervisory committeemembers, Drs. Shirley Gillam (Department of Pathology, University of BritishColumbia) and Gregory Lee (Department of Obstetrics and Gynaecology, Universityof British Columbia) for their much valued involvement in this thesis.I would also like to thank Dr. Chun Peng and Pearly Lee. Thanks for all thememorable times we shared, the two of you made it interesting.Finally, I would like to acknowledge my family: Dale, Peter, Noah, P.P.,and my parents, William and Shou-Chin Fan. Truly without their support,understanding, and love, the submission of this thesis would not be possible. Theycreated an environment in which I could devote the many hours required for thecompletion of this thesis. To my family, my deepest and sincerest thanks.This thesis is dedicated to my mother and father, two people who have alwaysencouraged me to follow my dreams and who were my source of strength andinspiration throughout the compilation of this work. They, above all, understood thetrials and tribulations associated with research and they prepared me well for thechallenge. Mom and Dad, this is for you.xlvI. INTRODUCTIONThe gonadotropin - releasing hormone (GnRH) receptor is a member of theGTP - binding (G) protein coupled receptor superfamily. The binding of thisreceptor to its ligand, GnRH, plays a pivotal role in reproduction. Among itsphysiological functions, GnRH - receptor coupling stimulates the synthesis andrelease of gonadotropins such as luteinizing hormone (LH) and follicle stimulatinghormone (FSH) from the anterior pituitary. In addition, GnRH and GnRH receptorsare of importance locally for gonadal steroidogenesis and for the maintenance ofpregnancy. Signal transduction pathways for the GnRH receptor are generally of thephosphoinositol - phospholipase C nature (Leung and Steele, 1992).In terms of the receptor ligand, GrRH, the existence of this neuropeptide waspredicted in the 1950s (Harris, 1950), but its structure was not reported until the1970s (Matsuo et al 1971; Burgus et al., 1972). Since then, concerted efforts havefocused on its structural analysis (Sherwood et al., 1993). Comparatively fewerstudies were focused on the equally important other half, the receptor. It istherefore not surprising that for the past 20 years, relatively limited information wasknown about the GnRH receptor. And until the early part of this decade, no:1.information was available on its molecular biology. However, despite the initiallyslow start, information on the GnRH receptor, particularly its molecular biology, isgrowing steadily.Binding sites for the GiiRH receptor have been identified in pituitary for anumber of vertebrate species (Naor et al., 1980; Limonta et al., 1986; Peter et al.,1992; Weil et al., 1992; Pal et al., 1992; Schulz et al., 1993) including human(Wormald et al., 1985). Several extrapituitary GnRH receptor binding sitesincluding the brain (Millan et al., 1985), gonads (Clayton et al., 1979; Clayton etal., 1980a; Clayton et al., 1980b; Bourne et al., 1980; Hazum et al., 1982), placenta(Currie et at, 1981; Iwashita et at, 1986), and neoplastic tissues (Lamberts et al.,1982; Eidne et al., 1985; Miller et al., 1985; Pahwa et al., 1989; Fekete et al.,1989; Qayum et al., 1990; Emons et al., 1992) have also been identified.21.1 Physiological roleThe GnRH receptor is an integral plasma membrane protein involved inintercellular and intracellular communication. In essence, the GnRH receptor actsas a link connecting the extracellular world to the internal control centers. Thereceptor itself accepts specific external cues called the first messenger (ligand;GnRH; agonists; antagonists) and transfers them into uniformly comprehensiblesignals, known as second messengers. These messages can then be passed onto thenucleus eliciting protein phosphorylation or dephosphorylation and eventuallytranscriptional activation or inactivation.The primary physiological response of pituitary GnRH receptor binding toGnRH, a hypothalamic decapeptide, is the release of gonadotropins such asluteinizing hormone and follicle stimulating hormone into the peripheral circulation(Fig. 1). These gonadotropins in turn affect gonadal function. As well, othercellular responses such as biosynthesis of gonadotropins and GnRH receptors, upand down - regulation of GnRH receptors, and desensitization of gonadotropes arealso prominent effects of GnRH receptor activation.In addition to the pituitary, GnRH via its receptor has been suggested to act3Fig. 1 The hypothalamic-pituitary-gonadal axis.Schematic representation of the physiological role of GnRH. GnRH =gonadotropinreleasing hormone; LH =luteiriizing hormone; FSH = follicle stimulating hormone.40 F-I.CD 5:Cl)4‘1 Cl)/04_directly on the ovary in an autocrine and I or paracrine fashion (Peng et al., 1994;Dong et al., 1993). In the ovary, GnRH has both stimulatory and inhibitory actionsdepending mostly upon experimental conditions and on the maturational stage of theovarian follicles. Inhibitory effects of GnRH on ovarian function include reductionsin gonadotropin receptor biosynthesis, gonadotropin - mediated steroidogenesis, andfollicular development. Stimulatory effects of GnRH in the ovary includeaugmentation of steroidogenic and ovulatory processes.Further to extrapituitary GnRH receptors, the importance of the GnRHreceptor in the placenta cannot be overlooked. GnRH, present in both cyto - andsyncytiotrophoblast, is the major candidate for stimulation of human chorionicgonadotropin (hCG) release (Belisle et al., 1984) from the placenta via possibleautocrine and / or paracrine mechanisms. Levels of hCG peak at 11 to 14 weeksof gestation and thereafter decline steadily. Placental GnRH mRNA levels do notvary significantly during pregnancy (Lin et al., 1995), suggesting that other factors,including GnRH receptor down - regulation and desensitization, may be involved inthe decline of hCG after the first trimester. Functions of hCG include themaintenance of the early corpus luteum to ensure continued progesterone until thisfunction is taken over by the trophoblast. As well, hCG plays a role in relaxinsecretion by the ovary, early development of the fetal adrenal gland, and the6regulation of male sexual differentiation (Seron-Ferre et al., 1978; Siler-Khodr,1983).1.2 Distribution of the GnRH receptor1.2.a. PituitarySpecific high affinity binding sites for GnRH, GnRH agonists, and antagonistshave been characterized in the pituitaries of several species (Naor et a!., 1980;Limonta et a!., 1986; Peter et al., 1992; Weil et a!., 1992; Pa! et al., 1992; Schulzet a!., 1993) including human (Wormald et al., 1985). Among the anterior pituitarycell types, sublocalization of the GnRH receptor to the gonadotrope is prevalent forall species studied. For some species, the gonadotrope is the exclusive pituitary celllocale for the receptor (Hyde et al., 1982). However this is not necessarily the casefor all species. In species, such as the goldfish, GnRH has also been shown throughelectron microscopy studies to bind directly to somatotropes (Cook et al., 1991) andelicit GH release (Chang et al., 1990). However in general, pituitary GnRHreceptors for varying species were found to share widely similar properties. Asdemonstrated by photoaffinity labelling, all possessed a common theme of two7subunit components for the GnRH receptor.1.2.b. BrainGnRH receptor binding sites have been shown to exist in several areas of thebrain. Localization of GnRFI receptors to the lateral septal nucleus, anteriorcingulate cortex, subiculum, entorhinal cortex, and hippocampus are among theidentified regions (Millan et al., 1985).1.2.c. GonadsOutside of the central nervous system, several reproductive tissues havedemonstrated expression of the GnRH receptor. In the ovary, the GnRH receptorhas been identified in granulosa and luteal cells using mainly photoaffinity labellingstudies (Clayton et al., 1979; Hazum et al., 1982). Recently, using RT-PCRanalysis the GnRH receptor has also been identified in the rat ovary (Moumni et al.,1994) and in human preovulatory granulosa cells (Peng et al., 1994). In testiculartissue, receptors have been localized to the Leydig cells, but not to the Sertoli cells8(Clayton et al., 1980a; Bourne et al., 1980). Photolabelled GnRH receptors in therat gonads were found to be similar to those of the rat pituitary gland, involving twodistinct subunits of 43 and 53 kilodaltons (kDa) (Iwashita and Catt, 1985).Additionally, sequencing of the rat (Olofsson et al., 1994) and human (Peng et al.,1994) ovarian GnRH-R cDNAs indicated that they were identical to the pituitaryGnRFI-R cDNA sequences. With identical primary structures revealed, these datafurther support that the rat and human ovarian GnRH receptors are identical to theirpituitary counterparts.1.2.d. PlacentaThe placenta contains low - affinity GnRH binding sites distinct from thepituitary GnRH receptors (Currie et al., 1981; Iwashita et al., 1986). There remainsome similarities however, in the binding subunit itself which suggests that theplacenta receptor may likely be a variant of the pituitary GnRH receptor.91.2.e. Neoplastic tissues and cell linesGnRH receptors have been identified in pituitary adenomas (Snyder, 1985),human breast cancer tissue (Eidne et al., 1985; Miller et al., 1985), human epithelialovarian carcinomas (Lamberts et a!., 1982; Pahwa et al., 1989; Emons et al., 1992),and prostate tumours (Fekete et al., 1989; Qayum et al., 1990).Additionally, GnRH binding sites have been identified in immortalized a-T3gonadotropes (Horn et al., 1991), human breast cancer cell lines (Eidne et al., 1987;Kakar et al., 1994), human endomethal cancer cell lines (Emons et al., 1993), andin human ovarian cancer cell lines (Kakar et a!., 1994). Ovarian cancer GnRHreceptors are very similar to other extrapituitary GnRH binding sites of the lowaffinity and high capacity type (Emons et al., 1992). Antiproliferative effects ofGnRH analogues on ovarian tumour cells (Emons et al. ,1993), prostate cancers, andon non - neoplastic prostatic tissue (Fekete et al., 1989; Qayum et al., 1990; Janakyet a!., 1992) have been observed.101.3 Regulation of the GnRH receptorRegulation of GnRH receptor levels in the pituitary gland has been wellcharacterized during various physiological conditions. Ontogenic experiments havedemonstrated changes in GnRH receptor level with developmental stage (Chan et al.,1981). Estrous cycle experiments demonstrated maximum numbers of GriRHreceptors during the proestrous period prior to the onset of the preovulatory surgeof luteinizing hormone (LH) (Clayton et al., 1980b; Crowder et al., 1984; Nett etal., 1987). These levels were sustained for several hours after the surge, followedby a marked decrease in receptor levels at metestrus, and a subsequent slightincrease in receptor numbers during the day of estrus (Bauer-Dantoin et al., 1993).In contrast, during pregnancy and lactation, lower levels of GnRH receptors havebeen observed than during the estrous cycle. Further physiological studies involvingcastration and hypothalamic lesions have demonstrated GnRH receptor number up-regulation (Marian et al., 1981). Specifically, pituitary GnRH-R mRNA levelsincreased several fold in ovariectomized female and orchidectomized male rats(Kaiser et al., 1993) as well as in castrated sheep (Illing et al., 1993). These studiesclearly indicate the regulation of GnRH receptors in vivo and point towards potentialendocrine factors such as ovarian steroid hormones and protein hormones, includingGnRH, as potential regulators of the GnRH receptor.11The ability of GnRH to regulate the expression of its own receptor in thepituitary has been well documented. In in vitro studies, treatment of pituitary cellcultures with physiological concentrations of GnRH have resulted in a biphasicchange in GnRH receptor numbers (Conn et al., 1984). Initially, receptor activationleads to a down - regulation of receptors for GnRH (< 4 h post - treatment) whichis subsequently followed by an increase in the number of GnRH receptors (9 h post -treatment). The initial down - regulation of receptors is reflective of desensitizationof gonadotropes to GnRH resulting from uncoupling of receptors to secondmessenger systems. Mechanisms underlying homologous up - and down - regulationappear to be independently regulated.Down - regulation of GnRH receptors occurs within a time frame of 3 to 4h after GnRH treatment and appears to be independent of extracellular calcium.Supporting studies which raised intracellular calcium or chelated extracellularcalcium (Conu et al., 1984) have and have not resulted, respectively, in receptordown - regulation. Up - regulation of GnRH receptors, on the other hand, occursseveral hours following down - regulation. Increasing the numbers of GnRHreceptor is dependent upon extracellular calcium and can be stimulated bycompounds that raise intracellular calcium (Young et a!., 1985). As well, receptorup - regulation requires that intact protein synthesis and microtubular function takes12place (Conn et al., 1984; Young et al., 1984). The mechanisms of up - and down-regulation of GnRH receptors are complex and data indicate that these processesoccur through different pathways. However, for both cases, occupancy of theGnRH receptor alone is not responsible for induction of receptor up - or down-regulation.GnRH mediated GnRH receptor up-regulation can be mimicked by treatmentwith adenosine 3’, 5’-monophosphate analogues as well as by depolarization ofpituitary cells with KC1 (Young et al., 1984) or calcium ionophore, A23 187 (Younget a!., 1985). More recent studies on the homologous regulation of the GnRHreceptor mRNA in rat pituitary cells parallel previous findings on the ability ofGnRH to regulate its own receptor (Kaiser et al., 1993). Continuous stimulation byGnRH resulted in no observable change in receptor mRNA, while pulsatileadministration of GnRH increased levels of receptor mRNA.In addition to the regulation of the GnRH receptor by its own ligand, GnRH,several other factors may be involved in receptor regulation as well. In particular,gonadal steroids assume a likely role. Studies involving estradiol and testosteronereplacement in ovariectomized female and orchidectomized male rats, respectively,have resulted in decreased receptor mRNA levels for both cases (Kaiser et al.,131993). Similarily, in the absence of intact hypothalamic - pituitary connections,treatment with 17f3-estradiol has been shown to increase GnRH receptor levels(Gregg and Nett, 1989). Further to gonadal steroids, treatment of sheep pituitarycell cultures with estradiol (Laws et aL, 1990b; Sealfon et al., 1990) andprogesterone (Laws et al., 1990a; Sealfon et al., 1990) could increase and decrease,respectively, the numbers of GnRH receptors. Treatment with inhibin has alsoresulted in a significant increase in GnRFT receptor levels (Laws et al., 1990a;Sealfon et al., 1990). Estradiol and inhibin treatments performed in combinationadditively increased GnRH-R mRNAs to a higher extent than with each treatmentalone. Similar results were obtained when RNA levels were measured by Northernblots (Miller et al. ,1993).However, in direct contrast to the above studies, Wang et al. (1988) havedemonstrated a decreased GnRH receptor number in rat pituitary cell cultures treatedwith inhibin. This decrease in receptor number was in part due to the ability ofinhibin to antagonize GnRH - stimulated synthesis of GnRH receptors (Braden et al.,1990). It is clear that further detailed studies will be necessary in order to clarifythe role of inhibin in the regulation of GnRH receptor numbers.141.4 Activation of the GnRB receptor and signal transductionThe GnRH receptor is a GTP - binding (G) protein coupled receptor andfunctions in calcium mobilization. Activation depends largely upon reversiblebinding of the ligand, GnRH. Signal transduction (Fig. 2) across the plasmamembrane is dependent upon three proteins: the receptor, G protein, and theeffector(s). In the case of GnRH, the effectors are enzymes involved in secondmessenger production and calcium ion channels. The first step in the mechanism ofaction of GnRH receptor involves ligand binding. Following binding of the agonist,GnRH receptors aggregate and become internalized. After internalization, thereceptors become associated with lysosomes to undergo a degradation pathway and!or become associated with the Golgi apparatus and LH granules to undergo receptorrecycling (Hazum and Conn, 1988). As well, binding of GnRH to a pocket formedby the transmembrane regions of the GnRH receptor leads to conformation changesin the receptor. These changes allow the receptor to associate with heterotrimeric(cx, (3, and -y subunits) G protein(s) and mediate the stimulatory actions of GnRH(Andrews et al., 1986; Perrin et al., 1989; Hawes et al., 1992; Stojilkovic et al.,1993).All of the three subunits of heterotrimeric G proteins share structural and15Fig. 2 Signal transduction mechanism of the GnRB receptor.DG = Diacyiglycerol; 1P3 =Inositol 1, 4, 5-triphosphate; PIP2 Phosphoinositol 4, 5-bisphosphate; PLC=Phospholipase C; PKC=Protein Kinase C;PLA2=Phospholipase A2; GTP =guanosine 5’ triphosphate; GDP =guanosine 5’diphosphate.16HG.Pro(ein1P3(5-upoxygenase)LeukotrknesF.functional diversity. Studies have revealed that the a, /3, and ‘y subunits are encodedby at least 15, 4, and 8 genes, respectively. Although the major phospholipase C(PLC) activating component is the a subunit in most tissues, fry subunits have alsobeen shown to be involved in PLC activation (Spiegel et al., 1992). In generalthough, the 0 proteins have been divided into two distinct classes: pertussis toxinsensitive and pertussis toxin insensitive. Those that are categorized under pertussistoxin sensitive include (t=transducin), 0t2 (i=inhibitory), °j2’ G13, and Gand have cGMP - phosphodiesterase, adenylate cyclase, K and Ca2 channels, andCa2 mobilizing receptors as their possible effectors. Pertussis toxin insensitive Gproteins include G (s = stimulatory), G01f (olfactory), Gq (q = members of aubiquitously distributed family of 0 proteins), G11, G12, G13, 014, and 015,16 (0protein subtype) and have /31- and /32-PLC as well as adenylate cyclase as theireffectors (Bourne et al., 1991). Studies have indicated that members of the pertussistoxin insensitive class of G proteins mediate PLC activity via GnRH in pituitarygonadotropes (Hsieh et al., 1992).Once the activated receptor has contacted the 0 protein, this results in therelease of a subunit bound GDP in exchange for GTP. This exchange is permitteddue to the fact that GTP is present in the cell at higher concentrations than is GDP.Dissociation of the 0 protein into a and fry subunits leads to the activation of the /3 -18forms of PLC (Spiegel et al., 1992). Activation of PLC leads to the cleavage ofmembrane phospholipids: phosphatidylinositol (P1), phosphatidylinositol 4-phosphate(PIP), and phosphatidylinositol 4, 5-phosphate (PIP2) into inositol 1, 4, 5-triphosphate (1P3) and diacyiglycerol (DAG). PIP2 has been identified as the majorsubstrate during GnRH stimulated activation of PLC in gonadotropes (Morgan et al.,1987).1P3 is the major second messenger molecule involved in calcium mobilizationand represents both an early and sustained response to GnRH stimulation(Guillemette et a!., 1987). GnRH stimulates a quick rise in intracellular [Ca2],followed by a smaller but sustained level that is in agreement with IP3 release (Izumiet aL, 1989). The initial peak of Ca2 is independent of extracellular Ca2, whilethe later sustained peak is dependent on entry of Ca2 through voltage sensitive andinsensitive Ca2 channels (Virmam et al., 1990). Thus the early peak of Ca2 isreflective of Ca2 released from intracellular stores, as a result of 1P3 mediated Ca2release from the endoplasmic reticulum. The sustained phase supports direct 1P3mediated Ca2 entry into gonadotropes.IP3 is a comparatively more hydrophilic molecule than DAG and diffuses intothe cytoplasm where it elicits the release of calcium from intracellular stores distinct19from the mitochondria. Further support for 1P3 as a calcium mobilizer was obtainedin patch clamp studies involving single gonadotropes being injected with 1P3 (Tseand Hille, 1992). Catabolic routes for the termination of this second messenger arevia the 5-phosphate pathway where 1P3 is dephosphorylated to inositol 1, 4-bisphosphate, inositol 4-phosphate, and inositol. Also catabolism via the 3-kinaseroute to inositol 1, 3, 4, 5-tetraphosphate and subsequent dephosphorylation toinositol 1, 3, 4-triphosphate has been demonstrated (Zheng, 1993).Several lines of evidence support the idea that calcium functions as a secondmessenger in the acute release of LH in response to GnRH. First, whenextracellular calcium is blocked, GnRH - induced LH release is inhibited (Stern andConn, 1981; Conu et al., 1979). However when Ca2 replacement occurred, LHrelease was re - established (Bates and Corn, 1984). Second, agents that increaseintracellular Ca2, specifically, also stimulate the release of LH. Third, GnRH -induced LH release, caused a measurable increase in intracellular Ca2 levels.Finally, treatment of pituitary cells with Ca2 channel blockers such as verapamil ormethoxyverapamil blocked stimulated LH release (Corn et al., 1983), whereasutilization of a calcium channel agonist, maitotoxin, resulted in LH release (Cornetal., 1987).20Once intracellular Ca2 concentrations are elevated through mobilization ofintracellular stores and extracellular sources, several responses take place. Calciumbinds to Ca2 binding proteins such as calmodulin and protein kinase C (PKC)resulting in protein phosphorylation or dephosphorylation. In addition, severalreports have eluded to phospholipase D (PLD) as a Ca2 sensitive enzyme,suggesting that Ca2 may have a direct role in activating PLD as well.In addition to 1P3 and Ca2,DAG is the third potential second messenger andthe other product of the PLC reaction. This second messenger functions to activateprotein kinase C directly. Characteristically, DAG is a hydrophobic molecule thattends to remain within the phospholipid bilayer of the plasma membrane. It is fromthis locale that DAG elicits many of its GnRH stimulated responses. Initially, DAGproduction temporally parallels the formation of 1P3. The early rise in DAG isfollowed by a slow sustained phase of DAG production. However, the sustainedphase is several magnitudes higher than 1P3 levels. These data suggest that othersources of DAG may be involved, including phosphatidylcholine (PC). PC ishydrolyzed readily by PLC and PLD (Billah and Anthes, 1990) into choline andphosphatidic acid (PA; interconverts with DAG). Also, receptor - coupled activationof PLD gives rise to DAG formation and may play a role in sustaining the activationof PKC.21In addition to being metabolized by PLC and PLD, PC is also hydrolyzed byphospholipase A2 (PLA2). A PA - specific PLA2 that utilizes PA in the arachidonicacid (AA) cascade has also been associated with receptor - mediated cell activation(Axeirod et al., 1988). Turnover of DAG is rapidly achieved by conversion to PAand subsequently to P1 (Nishizuka, 1992).All three of these second messengers converge to activate PKC. The PKCfamily of proteins are single polypeptides consisting of a regulatory and a kinasedomain. There exists at least 10 closely related isozymes of PKC (Huang andHuang, 1993). Compounds such as Ca2, DAG, AA, phosphatidyinositolphosphates, and phosphatidylserine for example can unmask the catalytic domain ofthese enzymes and cause activation. Activation of PKC results in the production ofcAMP from ATP which can then proceed to influence several well documentedcellular responses, including phosphorylation. In addition, a role of PKC in thecontrol of exocytosis has also been documented (Stojilkovic and Catt, 1992).Generally speaking, PKCs are proteins that are involved in the control of cellulargrowth and differentiation. De - regulation of these proteins can lead to detrimentaleffects and eventual cell death.In terms of cyclic nucleotide signalling in GnRH - mediated action, early22studies (Naor et a!., 1975; Sundberg et al., 1976) have shown that under certainconditions, GnRH can induce a slight increase in both cAMP and cGMP. Morerecent studies have also suggested a role for cGMP on the regulation of GnRHstimulated phosphoinositide turnover (Naor, 1990). However, in direct contrast,studies by Corin et al. (Conn et a!., 1979) have shown that cyclic nucleotides are notinvolved in GnRH - stimulated LH release and it is generally accepted that cAMPis not a second messenger for GIiRH. More detailed studies involving cyclicnucleotides may be necessary to end this debate.231.5 Molecular biology of the GnRH receptor1.5.a. GeneralUntil the early part of this decade, there was very limited informationconcerning the molecular biology of the GnRH receptor. Isolation of the cDNA orgene had not been accomplished as yet in any species. Chromosomal assignment ofthe gene for the GnRH receptor as well as basic structural data on the receptor atany level was lacking. However, data on the isolation and sequencing of the GnRHR cDNA in several species were to follow. The cloning of the GnRH-R cDNA wasto be preceded by expressional studies using Xenopus laevis oocytes injected withpituitary RNA (Yoshida et al., 1989; Sealfon et al., 1990). These studies servedtwo purposes. The first was the necessary confirmation of the presence of RNAencoding for the GnRH receptor and the second was the identification of a readilyavailable source of mRNA for construction of cDNA libraries. The isolation of thefirst GnRH-R cDNA clone was derived from screening of an a-T3 (mouse) derivedcDNA library. RNA produced from the mouse cDNA clone was demonstrated toencode for a functional receptor when expressed in Xenopus oocytes (Tsutsumi etal., 1992). Isolation of the mouse GnRH receptor cDNA (Reinhart et al., 1992;Tsutsumi et al., 1992; Perrin et al., 1993) has lead to the cloning of the cDNA for24the rat (Eidne et al., 1992; Kaiser et al., 1993; Kakar et a!., 1994) GnRH receptor.More recently, cDNAs for sheep (Brooks et al., 1993; hung et al., 1993), bovine(Kakar et al., 1993), pig, and human (Kakar et a!., 1992; Chi et a!., 1993) GnRHreceptors have been isolated.1.5.b. StructureData indicate that GnRH receptors are members of the G protein coupledreceptor superfamily. These receptors consist of a single polypeptide chaincontaining seven hydrophobic transmembrane domains (TM) as indicated throughhydropathy plots (Fig. 3). These hydrophobic regions are joined to hydrophilicextracellular and intracellular loops (El, E2, E3; Ii, 12, 13). Analysis of threedimensional conformations for G protein coupled receptors indicate that the TMdomains are predominantly c - helical in nature and arranged so as to form a centralhydrophulic ligand binding pocket (Baldwin, 1993). Potential N - linkedglycosylation sites are present in the extracellular domain. Potential sites forphosphorylation by cAMP - dependent protein kinases and PKC are located withinthe first and third cytoplasmic domains.25Fig. 3 Structure of the GnRII receptor.Amino acid sequence of the GnRH receptor (mouse; Perrin et al., 1993; Reinhartet al., 1992; Tsutsumi et a!., 1992) is represented in the single letter amino acidcode. Amino acids are numbered as shown and the transmembrane domains areboxed.26M —1200,300ExflcefluMembcnbflcdkAaA highly conserved tripeptide sequence located at the junction between the endof the third transmembrane domain and the beginning of the second cytoplasmicdomain is implicated in G protein coupling. This conserved sequence consists of anAsp-Arg-Tyr triplet which is found is many G protein coupled receptors to date.Cytoplasmic carboxyterminal tails have been described for all G protein receptorscharacterized to date. This region has been linked to the coupling of G proteins,desensitization, and internalization of G protein coupled receptors (Dohiman et al.,1991). Preliminary data on mouse and rat GnRH receptors indicate completeabsence of this carboxyterminal region.1.5.c. Interspecies relationship at the mRNA levelUtilization of molecular biology techniques has lead to the examination ofmRNA levels encoding for the GnRH receptor across various species. Northernanalyses of a-T3- 1 and mouse pituitary mRNA have identified GnRH-R transcriptsof sizes 3.5 and 1.6 Kb (Tsutsumi et al., 1992; Reinhart et al., 1992; Kaiser et al.,1992). Similarly for the rat, studies using Northern analyses and reversetranscription - polymerase chain reaction (RT-PCR) (Perrin et a!., 1993) haveidentified a major transcript ranging in size from 4.5 (Reinhart et al., 1992) to 5 -285.5 Kb (Kaiser et a!., 1992) as well as a smaller 1.8 Kb transcript. Multipletranscripts for the sheep have also been identified. Sizes of 5.4 - 6, 3.6 - 4, 2.3,and 1.3 Kb were reported (Brooks et al., 1993; Illing et al., 1993). Northernanalysis of bovine pituitary RNA revealed the presence of four different transcripts(5.0, 3.5, 2.5, and 1.5 Kb), of which the 5.0 Kb was the most abundant (Kakar eta!., 1993). Northern analyses conducted on human pituitary RNA have detected asingle mRNA of 4.7-5.0 Kb (Chi et al., 1993; Zhou et al., 1994) encoding for theGnRH receptor.Northern analyses performed on rat extrapituitary tissues such as ovary,Leydig cell, and testis have identified the GnRH receptor transcripts as well(Reinhart et al., 1992; Kaiser et al., 1992). No transcripts were observed usingNortherns for rat placenta however (Reinhart et al., 1992; Kaiser et a!., 1992). Insitu hybridization studies have also identified receptor mRNA in the rat hippocampusand hypothalamus (Jennes and Wright, 1993). Northern analyses performed onhuman testis, prostate, placenta, breast, and ovary did not detect any GnRH-RmRNA (Reinhart et al., 1992; Kakar et al., 1992; Clii et a!., 1993). More sensitiveanalysis through RT-PCR was necessary in order to detect mRNAs from humanplacenta, testis, prostate, and breast tissue (Kakar et al., 1994).291.6 Bmdmg characteristicsThe cloned mouse GnRH receptor (receptor expressed from cloned cDNA)has dissociation constants which are similar to those of its native receptor (Kd)values of 0.5 nM and 0.5 to 2.9 nM in pituitary and x-T3 cells, respectively. Rat,sheep, and human cloned GnRH receptors possess Kd values in the order of 0.6 nM,0.13 nM, and 0.9 nM when measured with agonist, des-GlylO-[D-A1a6jN-GnRH-N-ethylamide. Additionally, for sheep and human cloned GnRH receptors, Kd valuesof 4.9 nM and 2.8 nM were measured with GnRH. These Kd values are similar tothose of the native receptor for rat (0.2 nM), sheep (0.3 nM, agonist; 4.1 nM,GnRH), and human (0.32 nM, agonist; 4.8 nM, GnRH) as measured in pituitarycells.301.7 Clinical applicationsSeveral important clinical applications involving the GnRH receptor have beendescribed mainly through pharmacological studies. This involvement of the GnRHreceptor in treatment is based on the knowledge that the administration of bothGnRH agonists and antagonists results in the effective suppression of gonadotropinoutput. This desired effect is of importance in various therapeutic applications suchas in the treatment of precocious puberty, endometriosis, hormone - dependentneoplasia (breast and prostate), hirsutism, and uterine leiomyomata. As well,potential use of GnRH analogues as contraceptive agents are being currentlyresearched. On the other hand, stimulation of GnRH mediated actions has also ledto the successful treatment of hypogonadotropic hypogonadism and infertility (Millaret al., 1987). These studies indicate that detailed molecular information regardingGnRH - GnRH receptor coupling is of particular value in the improvement of GnRHanalogue based treatment.311.8 ObjectiveUnderstanding the molecular biology of the GnRH receptor is currently ofmajor interest to many reproductive endocrinologists, molecular biologists, and toall those who remain interested in understanding G protein mediated signaltransduction. The importance of GnRH - GnR}{ receptor coupling has been wellcharacterized as the neuroendocrine control of reproduction and is required fornormal reproductive function. Reproductive roles in addition to LH and FSHsynthesis and secretion include proper maintenance of pregnancy and gonadalfunction as well as use in clinical treatment of certain reproductive diseases andneoplasia.The recent increase in GnRH receptor related studies is evidenced by the bodyof work that has surrounded the GnRH receptor of late. Molecular cloning of therat and mouse GnRH-R cDNAs were reported by various groups and coincided withthe initiation of this project. However, no reports had ever been published on thegene encoding for these receptors in any species. By the time this thesis wascompleted, additional GnRH-R cDNAs had been isolated in sheep, cow, pig, andhuman. As well the gene encoding for the mouse GnRH receptor was subsequentlyreported in addition to various regulatory studies on the GnRH receptor at the32mRNA level.This investigation was primarily concerned with the isolation andcharacterization of the gene encoding for the human GnRH receptor. As mentionedthis is as yet unchartered territory for the GnRH receptor. The results of thispresent investigation delineate the organization of the human GnRH-R gene in termsof structure, copy number, and location in the genome. Additionally, the findingsof this thesis conthbute to the better understanding of the mechanistic action and theregulatory control of the GnRH receptor.33II. MATERIALS AND METHODS2.1 Genomic DNA library screening2.1.a. Preparation of filtersA human genomic placental library (Stratagene, La Jolla, CA), constructedin XFIX II vector - Xho I site, was plated at a density of 20,000 to 30,000 plaqueforming units (pfu) per 150 mm petri dish plate on host strain E. coli SRB(P2).Approximately 2 x 106 plaques were screened in the isolation of the hGnRH-R gene.The procedure for screening was conducted according to standard protocols(Ausubel et al., 1992). Briefly, NZY broth supplemented with 0.02 % maltose wasiimoculated with a single colony of SRB(P2) and grown to saturation at 37 °C underconditions of good aeration (shaker, 275 rpm). A mixture of 600 1d of host cellsand approximately 25,000 to 30,000 phages (100 d of a 10 dilution of a 2.2 x 1010pfu I ml library) was incubated for 20 mill at 37°C. Ten ml of 0.7 % NZY topagar were added and the mixture was poured onto NZY agar plates. Incubationperiods of 7 to 8 h at 37 °C followed. Plates were collected and placed at 4 °C for34at least 1 h before applying filters.Plaque lifting was conducted with nylon filters which were labelled and placedface down on plates for 1 mm. Orientations of the filters were marked using an 18-gauge needle and subsequently lifted from the plates and placed face up indenaturing solution (0.2 M NaOH / 1.5 M NaC1) for 2 mm. Filters were thenneutralized in 1.5 M NaCl, 0.4 M Tris-Ci, pH 7.6 for 5 mm, washed in 2X SSC(20X SSC stock: 3M NaC1 and 0.3 M Na3citrate 2H0, pH 7.0) for 30 s, andplaced on filter paper to dry. After drying, filters were wrapped in clear plastic andplaced under UV light for 2 mm on each side for DNA cross - linking. At thispoint filters were subjected to hybridization with a partial human GnRH-R cDNAprobe (760 bp; +904 to +1663; Peng et al., 1994) and autoradiography withintensifying screens (overnight exposure).2db. Preparation of radioactive cDNA probesRecombinant plasmids were constructed and digested with appropriaterestriction endonucleases and the inserts were subsequently isolated by 5 %polyacrylamide gel electrophoresis (PAGE). Bands containing target DNAs were35excised and recovered by electroelution and ethanol / sodium acetate precipitation.Preparation of DNA (25 to 50 ng) probes was performed using a RandomPrimers DNA Labelling Kit (Gibco - BRL, Burlington, Ontario, Canada). Labellingof target DNAs was done in accordance to instructions supplied by the manufacturer.DNA was denatured at 100 °C for 5 mm and placed on ice. Two l each of thefollowing deoxyribonucleoside triphosphates : dATP, dGTP, and dTTP were added.Also 15 d of random priming buffer, 50 Ci of cx[32P]dCTP (3000 Ci / mmol;Amersham, Oakville, ON, Canada), and 3 U of Kienow fragment were added.After an incubation period of 2 h at 25°C, the reaction mixture was denatured at 100°C for 5 mm prior to hybridization. The specific activity of probes obtained was 108cpm / g.2.1.c. Hybridization with radioactive probes I WashingFilters were incubated in prehybridization solutionDenhardts [1X Denhardts solution: 0.02 % ficoll, 0.02 %0.02 % polyvinylpyrrolidone], and 5X SSPE [1X SSPE:Na3PO4, 20 mM EDTA, pH 7.4, 0.1 % SDS, and 0.1 mg(50 % formamide, 5Xbovine serum albumin,3 M NaC1, 200 mM/ ml denatured salmon36sperm DNA]) for 1 to 2 h at 42 °C. Hybridization with radioactive probes wasperformed overnight at 42 °C in a hybridization oven.Filters were washed once with lx SSC (20X SSC stock: 3M NaC1 and 0.3M Na3citrate2H20,pH 7.0) / 0.1 % SDS for 20 mm at 42 °C and at least twicewith 0. 1X SSC /0.1 % SDS at 52° to 65°C for 15 mm. After washing, filters weremounted wet in plastic wrap for autoradiography with Kodak XAR5 film andintensifying screens at -70 °C for overnight exposure.2.1.d. ScreeningPositive plaques were identified by aligning primary library filters and plateswith their respective autoradiograms using needle punctures as reference points. Acircular agar plug containing the positive plaque was removed and placed in phagedilution buffer (20 mM Tris-HC1, pH 7.4, 100 mM NaC1, and 10 mM MgSO4)forat least 5 h. Positive plaques were replated at a density of 500 pfu per 90 mm plateand subsequent 2° and 3° screening followed. Third round positive plaques werepurified and single isolates for each positive were replated and kept as stock forfuture analysis.372.1.e. Preparation of liquid lysateA concentrated phage lysate was made for each positive plaque and processedusing the following protocol. NZY broth supplemented with 0.02 % maltose wasinnoculated with E. coli SRB(P2) and grown overnight at 37 °C. A single plaquewas added to 10 ml of NZY broth containing 10 mM MgSO4 and 0.1 ml ofovernight SRB(P2) cells. This mixture was shaken vigorously at 37°C until lysisoccurred (6 to 8 h). After lysis, 0.1 ml of chloroform was added to lyse anyremaining cells and incubated for 1 mm. Centhfugation at 3000 x g at roomtemperature for 10 mm followed. The supernatant was retained and 0.1 ml of 1 MMgSO4was added to 10 ml of lysate. Lysates were processed immediately to isolatelambda DNA. Ample quantities (several micrograms) of lambda DNA wereprepared from an aliquot of the lysate for future restriction analysis.2.1.f. Preparation of lambda DNA from phage lysatesLysate was transferred to a corex tube and the following were added: 10 mlof TM buffer (50 mM Tris-HC1, pH 7.4 / 10 mM MgSO4), 10 l of 5 mg / mlDNase, and 20 l of 10 mg I ml RNase. Lysate was incubated for 1 h at 37 °C.38Thereafter 2 ml of 5 M NaC1 and 2.2 g of solid PEG-6000 were added and themixture was further incubated for 1 h on ice. Recovery of the phage DNA pelletwas obtained after centrifugation at 10,000 x g for 10 mm at 4 °C. The pellet wasresuspended in 1 ml of TM buffer, subjected to two phenol / chloroform extractions,one chloroform / isoamyl alcohol (24:1) extraction, and an isopropanolprecipitation.2.2 Cloning of DNAFour prominent genomic DNA segments, encompassing the entire span of thehuman GnRH-R gene, were cloned into pUC19 vector. Three EcoRT genomicfragments of sizes 7.7, 7.4, and 3.8 Kb were cloned in addition to a 2.7 Kb Hindlilfragment.2.2.a. Competent cellsCells were made competent using the calcium chloride / rubidium procedure(Ausubel et al., 1991). An overnight culture of E. coli DH5a was prepared and a39portion (1 ml) added to 250 ml of LB broth. The culture was incubated at 37°C,while shaking at 275 rpm. Cell density was monitored using a spectrophotometerand allowed to reach an O.D.6 of 0.5 (2 to 3 h). Cells were harvested bycentrifugation at 4,000 x g for 10 mm at 4 °C and resuspended in 125 ml of solutionA (10 mM 3-(N-morpholino) propanesulfonic acid (MOPS), pH 7.0 / 10 mM RbCl).Cells were centrifuged again, resuspended in 125 ml of solution B (100 mM MOPS,pH 6.5, 50 mM CaC12, and 10 mM RbC1), and incubated on ice for 15 mm. Cellswere resuspended in solution B to a final volume of 25 ml with 15 % (v / v)glycerol. Competent cells were frozen in 0.1 ml aliquots using an ethanol / dry icebath and stored at -70 °C.2.2.b. LigationTarget DNAs were cleaved to completion with the appropriate restrictionendonuclease(s). Isolation of the target DNA by agarose gel electrophoresis orPAGE and subsequent purification of the DNA followed. Vectors were preparedand digested with the same restriction endonuclease(s) as the target DNA. Ligationreactions consisted of a 1: 10 ratio of vector to insert and 20 U of T4 DNA ligasein a final volume of 40 JLl. The ligation mixture was incubated overnight at 15°C40prior to E. coli DH5o transformation.2.2.c. TransformationA mixture of 0.1 ml of competent cells with 1 - 200 ng of the ligationreaction were combined and incubated on ice for 30 mm. Several controls were alsoimplemented as well. Positive and negative controls consisted of competent cellsplus recombinant plasmid and competent cells without DNA or with non-recombinantplasmid, respectively. Following the ice bath incubation, the reaction was heatshocked for 1 mm at 42 °C, 1 ml of LB broth was added, and the mixture wasincubated for an additional 1 h at 37 °C. The ligation mixes were then plated on LB- ampicillin plates supplemented with X - Gal (40 il of 20 mg / ml stock solution)and IPTG (40 d of a 100 mM stock solution). Plates were incubated overnight at37 °C and positive colonies were identified by the blue (negative) - white (positive)selection pattern. Confirmation of positive colonies was achieved byminipreparations of plasmid DNA using the alkaline lysis procedure, restrictiondigests, and Southern analysis. Subsequent large scale preparations of recombinantplasmid DNA were conducted for each positive clone of interest.412.3 DNA sequencing2.3.a. GeneralDNA fragments were sequenced by the dideoxy chain termination method(Sanger et a!., 1977) using the T7 Sequencing Kit (Pharmacia, Vancouver, B.C.,Canada) and conducted as per manufacturer’s instructions with slight modifications.All sequencing was performed using both M13 universal forward and reverseprimers as well as sequence specific oligonucleotides. Analysis of the nucleotidesequences was conducted with the assistance of a program from the GeneticsComputer Group Inc. (Madison, WI).2.3.b. Preparation of templateDouble - stranded DNA templates were denatured by alkali treatment priorto sequencing. Approximately 2 pg of each template DNA were combined with 2d of 2 M NaOH I 2 mM EDTA in a reaction volume of 10 pJ. Following anincubation period of 10 mm at room temperature, a sodium acetate I ethanolprecipitation (7 d of H,O, 3 l of 3 M sodium acetate, pH 4.8, and 60 l of 95 %42ethanol) was performed. Precipitated DNA was collected by centrifugation at14,000 rpm for 10 mm, washed briefly with 70 % ethanol, and dried in a speedvacevaporator.2.3.c. Annealing of primerEach denatured DNA template was resuspended in 10 l of 1120, combinedwith 2 d of primer (50 ng / p1), and 2 p1 of annealing buffer (1 M Tris-HC1 pH7.6, 100 mM MgC12, and 160 mM DTT). The reaction was incubated for 20 millat 37 °C and allowed to cool to room temperature (10 mm) in order to promoteannealing.2.3d. Labelling reactionAfter the annealing reaction, 3 pi of Labelling Mix-dATP, 10 Ci of [-35S]dATP, and 2 p1 of diluted T7 DNA Polymerase (3 U) were added and incubatedfor a further 5 mm at room temperature.432.3.e. Termination reactionFour sequencing mixes, one for each nucleotide, were prepared. Into each“A” tube, 2.5 l of A-Mix (840 1M each dCTP, dGTP, and dTTP; 93.5 M dATP;14 iM ddATP; 40 mM Tris-HC1 pH 7.6, and 50 mM NaC1) were dispensed.Consequently for the other nucleotides, 2.5 l of C-Mix, G-Mix, and T-Mix(identical components as A-mix, except 14 pM of ddATP is replaced by 14 1M ofthe respective nucleotide as identifiably labelled) were dispensed into their respectivetubes. The four sequencing mixes were pre - warmed for at least 1 mm beforeaddition of 4.5 d of the labelling reaction to each mix. An incubation period for5 mm at 37 °C followed. Reactions were stopped with 5 1 of Stop Solution(0.3 % each Bromophenol Blue and Xylene Cyanol FF; 10 mM EDTA pH 7.5, and97.5 % deionized formamide). Each stopped reaction was heated for 5 mm at100 °C prior to loading (3 jl per well) onto a sequencing gel.2.3.f. Denaturing gel electrophoresisSequencing gel apparatuses were obtained from Gibco - BRL, Burlington,Ont., Canada. Polyacrylamide 6 % / 7 M urea sequencing gels of dimensions 3844x 50 cm and 0.4 mm thickness were prepared and prerun at 45 W constant powerfor 30 mm prior to loading. Multiple staggered loadings (3) for each reaction wereconducted and gels were electrophoresed at 70 W constant power for 6 to 7 h.Subsequently, gels were processed, dried at 80°C for 1 h 20 mm using a gel dryer,and subjected to autoradiography at room temperature.2.4 Characterization of the 5’ terminus of the human GnRH-R mRNA2.4.a. Isolation of total RNATotal RNA was prepared using the guanidinium isothiocyanate - cesiumchloride procedure (Ausubel et al., 1992). Approximately 2 g of each tissue samplewere homogenized using a polytron homogenizer (two or three 10 s bursts) in 15 mlof 4 M guanidinium isothiocyanate buffer containing 5 mM sodium citrate pH 7.0,0.5 % sarcosyl, and 1 % 13-mercaptoethanol. The mixture was then centrifuged for10 mm at 15,000 rpm, 4°C and the supernatant was recovered. Cesium chloridewas added to the supernatant at a concentration of 1 g I 2.5 ml of supernatant andthen layered onto 3 ml of 5.7 M CsC1 containing 0.1 M EDTA, pH 7.5. Sampleswere centrifuged at 28,000 rpm in an ultracentrifuge using an SW4O rotor for 18 h45at 18°C. RNA pellets were washed with 1 ml of diethylpyrocarbonate water andthen resuspended in 10 mM Tris-HC1 pH 7.4, 5 mM EDTA, and 1 % SDS. Theresuspended RNA was subjected to a chloroform : n-butanol (4:1) extraction,followed by a 1 / 10 vol 3M sodium acetate pH 5.2 and 2.2 vol 95 % ethanolprecipitation at -20 °C overnight. RNA pellets were resuspended indiethylpyrocarbonate water and stored at -70 °C. RNA concentration and puritywas assessed by optical density readings at 260 nm and 280 mn. RNA integrity wasassessed by the identification of the 28S and 1 8S ribosomal bands through denaturingagaraose gel electrophoresis in the presence of ethidium bromide. The use of humantissue (intestine; placenta) was approved by the Clinical Screening Committee forResearch and Other Studies Involving Human Subjects of the University of BritishColumbia. Human brain total RNA and human pituitary poly-A RNA were obtainedfrom Clonetech, Palo Alto, CA.2.4.b. Primer extension analysisPrimer extension analysis was conducted using three oligonucleotides, PE-1,PE-2, and PE-3 (Table 1). These primers were designed such that they wouldencompass the span of TATA boxes found in the DNA sequence of the genomic46Table 1. Oligonucleotides used in this investigation.The location and orientation of primers used in this study are listed accordingly.The corresponding sequence is also shown. Numbering is sequential with the most3’ transcription initiation site designated as +1.47Name Sequence PositionPE-1 5’AGCTTCTCTGTGTACTGGCTG3’-583 to -603PE-2 5’CTGTTATTACACATTAATGCA3’-36 to -56PE-3 5’TTTATTAATCAATCTrACTGAT3’ +74 to +53A 5’GCTTGAAGCTCTGTCCTGGGA3’ +679 to +699B 5’TTTATGGTCACAAATCTCA3’-782 to -764C 5’AGAGAAGCTGGTAATI’CTG3’-690 to -672D 5’GATGCTGTTGTTGATGGC3’ +766 to +749E 5’ATGAATCTCTCCATCTGGGAA3’ +1733 to + 1753F 5’CACACTTGTACAGATACAA3’ +3591 to +3609G 5’TGTATCTGTACAAGTGT3’ + 3608 to + 3592H 5’CATATGAGCACAATGTAT3’ +4766 to +4749I 5’AACTATAGGAGGGAAAGTTG3’ +2189 to +2170J 5’TCTTAAGGTTCAATATGT3’ + 4062 to + 4079K 5’AAGTAGGATTTACACTTAAGT3’ + 1793 to + 181348clones obtained in this study. Each oligonucleotide was end - labelled in a reactioncontaining 100 Ci [-y-32P]ATP (3000 Ci / mmol; Amersham, Oakville, ON,Canada), 100 ng of oligonucleotide, 2 l lox polynucleotide kinase buffer, and 4U of T4 polynucleotide kinase. The mixture was incubated for 1 h at 37 °C and theenzyme was subsequently inactivated at 65 °C for 5 mm. Approximately 2 x l0cpm of each oligonucleotide primer as determined by Cerenkov counting washybridized with 40 g of total RNA from human brain and intestine in a reactionmixture containing 0.5 M NaC1, 40 mM PIPES pH 6.8, and 1 mM EDTA for 1 hat 42°C. Annealed primer - RNA reaction mixtures were diluted 1: 10 into reversetranscription mix containing 1 mM of each dNTP, 50 mM Tris-HC1 pH 7.6, 60 mMKC1, 10 mM MgC12, 1 U RNAsin (Promega, Madison, Wis., USA), 1 mM DTT,and 10 U of AMV reverse transcriptase per 30 l reaction. The reaction mixturewas incubated for 2 h at 37°C and the primer extended products were subsequentlyanalyzed on a 6 % polyacrylamide / 7.0 M urea gel. DNA sequencing reactions ofM13 were used as a size standard.492.5 Reverse-transcription polymerase chain reaction (RT-PCR)2.5.a. Preparation of cDNATen ug of total RNA per tissue sample used were reverse transcribed intocDNA using the First Strand eDNA Synthesis Kit (Pharmacia, Vancouver, B.C.,Canada). RNA was resuspended in a reaction volume of 8 d, heated at 70 °C for10 mm and then chilled on ice. Thereafter, the following components were addedto the heat - denatured RNA mix: 5 l of Bulk First-Strand Reaction Mix(containing Murine Reverse Transcriptase, RNAsin, BSA, dATP, dCTP, dGTP, anddTTP in aqueous buffer), 200 mM DTT solution, and 1 ,ig of oligo d(T)12..8 Thereaction mixture was incubated at 37 °C for 1 h and followed by a 10 mm heatingat 95 °C. Reverse transcribed cDNAs were stored at -20 °C. One pJ aliquots wereused as template for susbsequent polymerase chain reaction (PCR) amplification.In addition to total RNA being reversed transcribed, 2 g of human pituitarypoly-A RNA were also subjected to reverse transcription for 1 h at 37 °C using theFirst Strand cDNA Synthesis Kit under the same conditions mentioned above.502.5.b. Polymerase chain reaction (PCR)Primers used in PCR amplification are indicated in Table 1 and were designedbased on the human GnRH-R gene sequence determined in the preceedingexperiments. PCR reactions were conducted in a final reaction volume of 25 l inthe presence of 10 mM Tris-HC1 pH 8.3, 50 mM KC1, 2.0 mM MgC12, 50 mMdNTPs, 0.01 % gelatin, 50 pmol / l of sense and antisense oligonucleotides, 1 Uof Taq DNA polymerase (Gibco - BRL, Burlington, ON, Canada), and overlayedwith 50 d of mineral oil. The PCR cycle profile consisted of a denaturation stepat 96 °C for 30 s, an annealing step at 50 °C for 30 s, and an extension step for 90s at 72 °C. PCR was conducted for 35 cycles in a DNA thermal cycler (PerkinElmer) with a final extension for 15 mm at 72 °C after the last cycle ofamplification. Ten pl of the PCR reaction mixture were fractionated on a 1 %agarose gel and stained with ethidium bromide. Several controls were implementedduring the PCR reactions. Negative controls consisted of PCR reaction mixturewithout cDNA in order to examine the possibility of cross - contamination betweensamples. Also, the possibility of genomic DNA contamination was excluded withthe incorporation of nonreverse transcribed RNA as a template. PCR conductedwith human f3 - Actin derived primers (antisense 5’-ggacctcactgactacctcatgaa-3; sense5’-ggtggaaggtggtcaacacctag-3’; Peng et al., 1993) was used as a positive internal51control in order to eliminate the possibility of RNA degradation and any technicalproblems associated with reverse transcription.2.6 Gel Mobility shift assay2.6.a. Cell cultureMCF-7, a human breast cancer cell line (obtained from Dr. J. Emerman,Department of Anatomy, University of British Columbia), was used as a source ofprogesterone receptors for DNA-protein binding studies. Cells were grown inDulbecco’s modified Eagle’s medium supplemented with F12 (Gibco - BRL,Burlington, ON, Canada) and 10 % fetal bovine serum and maintained at 37 °C ina humidified atmosphere with 5 % CO2. Treatment of cells with 10 nM estradiol(Sigma, St. Louis, MO, USA) for 24 h prior to harvesting was conducted in orderto increase progesterone receptor levels. Confluent monolayers of MCF-7 wereharvested by cell scraping for the preparation of nuclear extract. In addition toMCF-7 extract, HeLa cell nuclear extract (Promega) was obtained for preliminarystudies involving CRE binding.522.6.b. Preparation of nuclear extractApproximately 100 x 106 cells were harvested and incubated with 10 ml ofmedia containing 10 nM R5020 for 1 h at 37 °C. Cells were then centrifuged at1850 x g for 10 mm at 4 °C and allowed to swell on ice for 10 mm in 5 packed cellvolumes (pcv) of hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgC12, 10 mMKC1, 0.2 mM PMSF, and 0.5 mM DTT). Cells were pelleted again andresuspended in 2 pcv of hypotonic buffer prior to homogenization (50 strokes) witha Dounce homogenizer using pestle type B. The homogenate was centrifuged for20 mm at 3,300 x g, resuspended in ½ packed nuclear volume (pnv) ice cold lowsalt buffer (20 mM HEPES pH 7.9, 25 % glycerol, 1.5 mM MgC12,0.02 M KC1,0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT). Addition of ‘/2 pnv of highsalt buffer (20 mM HEPES pH 7.9, 25 % glycerol, 1.5 mM MgCl2, 1.2 M KC1, 0.2mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT) to a final concentration ofapproximately 300 mM KC1 was followed by a 30 mm incubation at 4 °C with gentlestirring. The nuclear precipitate was harvested by centrifugation at 25,000 x gfor 30 mm and was dialyzed for 4 to 5 h against dialysis buffer (20 mM HEPES pH7.9, 20 % glycerol, 100 mM KC1, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mMDTT). Nuclear extracts were aliquoted into tubes and stored at -70 °C. Proteinconcentrations were assessed using the Bradford assay (Ausubel et al., 1992).532.6.c. Preparation of radiolabelled double-stranded oligonucleotide probesOligonucleotides used in mobility shift assays are indicated in Table 2. CyclicAMP response element (CRE) and progesterone response element (PRE) concensussequences were obtained from Promega Gel Shift Assay Systems. Targetoligonucleotides containing the CRE - like and PRE - like sequences were designedaccording to the human GnRH-R gene sequence revealed in this study.Oligonucleotides were labelled using 1 il of T4 polynucleotide kinase (10 U / id),1 1 of lOX kinase buffer, 100 ng of oligonucleotide, 50 Ci of -y-[32P]ATP, andH20 to a final volume of 10 d. After an incubation period of 1 h at 37 °C, theenzyme was heat inactivated at 70°C for 10 mm. A 1.5 M excess of unlabelledcomplementary oligonucleotide and 1 d of 20X oligonucleotide annealing buffer(200 mM Tris-HC1 pH 7.9, 40 mM MgC12, 1 M NaCl, and 20 mM EDTA) wereadded. The reaction mixture was heated to 65 °C for 15 mm and allowed to coolto room temperature over a period of 2 h in order to promote annealing. TE buffer(pH 7.6) was added to a final volume of 100 Ll and the mixture was passed througha NAP 5 column (Pharmacia, Vancouver, B.C., Canada) to separate the labelledoligonucleotides from the unincorporated nucleotides. Eluates of labelledoligonucleotides were collected and stored at -70 °C. The level of radioactivityobtained for labelled oligonucleotides ranged from 0.5 to 1 x 106 cpm / l.54Table 2. Oligonucleotides used in mobility shift assays.Response elements are underlined within the oligonucleotide sequence. Alloligonucleotides were annealed to its complement before experiments. CRE=cAMPresponse element; GRE/PRE = glucocorticoid/progesterone response element;m mutant.55ClUE 5’-AGAGATTGCCTGACGTCAGAGAGCTAG-3CRE-like 5’-CTCCAGATCTGAAGTCTGCCTAATA-3GRE/PRE 5’-CGACTGGTACACAGTGTTCTGCTAC-36 1 1 — 6GRE/PRE-like 5’-CAGCAGTTACACAGTATTCTCTTCA-3mGRE/PRE 5’-CAGCAGGTATACAGTATTCTCTTCA-3562.6.d. DNA-protein binding assayBinding reactions were carried out in a reaction volume of 15 JLl at roomtemperature. Binding buffer containing 10 mM HEPES pH 7.5, 10 % glycerol, 50mM KC1, 2.5 mM MgC12,200 ng of poly (dI.dC)- (dI.dC) (Pharmacia, Vancouver,B.C., Canada), 0.1 mM EDTA, and 1 mM DTT was added to 10 p.g of totalprotein. This reaction mixture was allowed to incubate for 10 mm at roomtemperature. Following this incubation, 30,000 cpm of labelled oligonucleotide wasadded and a further incubation period occurred. The reactions were stopped withthe addition of 1 1d of gel loading lOX buffer and loaded onto 4 % nondenaturingPAGE.572.6.e. Nondenaturing PAGEMobility shift assays were conducted using low - ionic strength PAGE. Gelswere prepared with 6.8 mM Tris-HC1 pH 7.9, 1 mM EDTA pH 7.9, 4 %acrylamide, 0.05 % bisacrylamide, and 2.5 % glycerol and allowed to preelectrophorese with low - ionic strength buffer (6.7 mM Tris-HC1 pH 7.9, 3.3 mMsodium acetate pH 7.9, and 1 mM EDTA pH 8.0) for at least 30 mm at a constantvoltage of 150. Gels were run at a constant 200 V with recirculation of runningbuffer every 30 mm for approximately 2 h, dried under vacuum in a gel drier, andautoradiographed overnight.582.7 Southern analysisOne of plasmid DNA per sample was digested to completion with variousresthction endonucleases, electrophoresed on either 1 % agarose gels or 5 %polyacrylamide gels, and stained with 0.5 g I ml of ethidium bromide solution.Photographs of the gels were taken with a ruler under long - wavelength UV lightin order to document the positions of the bands. Gels were treated with 0.2 MNaOH I 0.6 M NaCl for 30 mm at room temperature with gentle rocking. Afterdenaturation, the gels were neutralized in 1 M Tris-HC1 pH 7.4 / 0.6 M NaCl for30 mm at room temperature. Gels were transferred to a Whatman 3MM paper wickresting on a support. The wick was placed in a large tray containing severalhundred milliliters of 20X SSC (3 M NaCl and 0.3 M Na3citrate 21120, pH 7.0) andwetted with 20X SSC. The gel was then transferred on top of the Whatman wickand any remaining air bubbles were carefully removed. Nylon membrane cut to thesame or slightly smaller dimensions of each gel was wetted with 20X SSC andplaced onto the gel. A dry piece of Whatman 3MM paper followed by a 10 cmthick stack of paper towels was placed on top of the nylon membrane. A glass platewas placed on top of the paper towels and the entire apparatus was compressed bythe addition of a 0.4 kg weight. This transfer setup was left overnight at roomtemperature and dismantled the next day in order to allow for capillary transfer of59the DNA from the gel onto the membrane. Nylon membranes were dryed and thenirradiated by UV light for 2 mm each side in order to cross-link the DNA to themembrane. Hybridization was conducted overnight with various human GnRH-RcDNA probes consisting of 396 (+904 to + 1300), 364 (+1300 to + 1663), or 760(+904 to + 1663) bp as well as various sequence specific oligonucleotides. Blotswere washed and subjected to overnight autoradiography with intensifying screensat -70 °C.2.7.a. Genomic Southern analysisHuman placental genomic DNA was prepared for genomic Southern analysis.Human placental tissue, approximately 1 to 2 g, was obtained and quickly mincedand frozen in liquid nitrogen. The tissue was ground to a fine powder with aprechilled mortar and pestle. Ground tissue was resuspended in 1.2 ml of digestionbuffer (100 mM NaCl, 10 mM Tris-Ci pH 8.0, 25 mM EDTA pH 8.0, 0.5 % SDS,and 0.1 mg I ml of freshly prepared proteinase K) per 100 mg of tissue. Sampleswere incubated overnight at 50 °C with gentle rocking. Afterwards, samples wereextracted with an equal volume of phenol chloroform : isoamyl alcohol andcentrifuged for 10 mm at 3,000 rpm. Aqueous layer was transferred to a new tube60and ½ vol of 7.5 M ammonium acetate and 2.2 vol of 95 % ethanol were added.The mixture was then centrifuged for 5 mm at 3,000 rpm. The DNA pellet waswashed with 70 % ethanol and resuspended in TE buffer at a concentration of 1 mg/ ml. Ten g of human placental genomic DNA were used for each restrictionendonuclease digestion (EcoRI, Hindlil, BamHI, and PstI). Digested genomic DNAwas size fractionated on 0.8 % agarose gels overnight at a constant 50 V. The gelswere then denatured, neutralized, and prepare for DNA transfer and blottingaccording to the same conditions mentioned previously for Southern analysis. Filterswere then hybridized with a [o-32P]dCTP - labelled 760 bp human GnRH-R cDNAand PstI - digested fragments, 396 bp and 364 bp in length, respectively.612.8 Northern analysisHuman pituitary poly-A RNA was subjected to Northern analysis. Five ,igof poly-A RNA was denatured in 50 % formamide / 2.2 M formaldehyde andincubated at 60 °C for 15 mm. Samples were then electrophoresed on 1 %denaturing agarose gels (20 mM MOPS, 2.2 M formaldehyde, 8 mM sodiumacetate, and 1 mM EDTA pH 8.0) at a constant voltage of 140 for 2.5 h. Capillarytransfer of RNA to nylon membranes was conducted overnight in 20X SSC. Nylonmembranes were subsequently irradiated by UV light for 2 mm each side in orderto cross-link the RNA to the membrane. Hybridization was conducted with a364 bp eDNA probe overnight, washed, and exposed to Kodak XAR-5 filmovernight at -70 °C with intensifying screens.622.9 Chromosome assignmentThe chromosomal localization of the human GnRH-R gene was determinedby using PCR with genomic DNA from 25 human - hamster somatic hybrid celllines (BIOS laboratories, New Haven, CT, USA). The primers K (sense; Table 1)and I (antisense; Table 1) were used. The reaction mixtures, containing 200 ng ofhybrid cell line genomic DNA, were subjected to 30 cycles of PCR using similarconditions as previously described. The reaction products were then electrophoresedon a 1 % agarose gel and visualized by ethidium bromide staining.63III. RESULTS3.1 Isolation of the human GnRH-R geneUpon screening of the human genomic library with a 760 bp (position; +904to + 1663; Peng et a!., 1994) cDNA probe under high stringency hybridizationconditions, 12 genomic clones were isolated from an initial screening ofapproximately 1.5 x 106 plaques. After three consecutive rounds of screening,positive clones were purified to homogeneity and ADNA was prepared for eachpositive clone and digested with various restriction enzymes. Genomic DNAfragments were analyzed by Southern blot analysis using the human GnRH-R cDNAas well as oligonucleotides corresponding to the 5’ and 3’ nontranslated regions ofthe gene. One X-clone (X-1) contained a 7.7 Kb EcoRI fragment corresponding tothe 5’ end of the gene (Fig. 4 and 5) and another X-clone (X-2) contained 3’ orientedEcoRI fragments of 7.4 Kb and 3.8 Kb in size (Fig. 4 and 5). All of thesehybridized with either the cDNA or oligonucleotide probes. These fragmentsspanned the entire gene for the human GnRH-R and were subsequently subclonedinto pUC19 vector and sequenced by the dideoxy method.64Fig. 4. Southern blot analysis of genomic fragments of the human GnRH-Rgene.Restriction digests performed on A-i and X-2. Positive signals identified byhybridization to a 760 bp human GnRH-R eDNA probe (+ 904 to +1663). X =XbaT; E = EcoRL650) 0)>m >< m m >< mFig. 5. Southern blot analysis of 5’ and 3’ oriented genomic fragments of thehuman GnRH-R gene.(A) Several 5’ oriented fragments as identified by hybridization with oligonucleotideA. The 7.7 Kb EcoRI fragment was subcloned into pUC19 and sequenced. (B)Multiple 3’ oriented restriction fragments as identified through hybridization to a 364bp (+ 1300 to +1663) human GnRH-R cDNA probe. EcoRI fragments, 3’oriented, of sizes 7.4 and 3.8 Kb were subcloned into pUC19 and sequenced. X =XbaI; E = EcoRI.67m0)>x mAmo) CDwx mAA.3.2 The human GnRH-R gene sequenceApproximately 6.4 Kb of the human GnRH-R gene was sequenced and isshown in Fig. 6 along with its exon - intron splice junctions and deduced proteintranslation. This sequence contains 905 bp of 5’ flanking region (from the most 5’transcription initiation site), the entire 5’ nontranslated sequence (1393 bp), theentire protein coding region (987 bp; Fig. 7), the exon - intron splice sites, the entire3’ nontranslated region (3076 bp), and additional 3’ sequence. Once the sizes of theintrons have been subtracted, the size of the structural gene corroborated well withthe size of the major transcript of approximately 4.7 Kb and indicated full coverageof the mRNA.Additional analyses of the human GnRH-R gene sequence are detailed furtherin forthcoming sections.70Fig. 6. The nucleotide sequence of the human GnRH-R gene (excluding intronicsequence).The coding region of exons are shown in upper case letters whereas the flanking andnontranslated regions are depicted in lower case letters. TATA boxes are denotedby patched underlining and CAAT boxes are underlined. Enlarged andbold nucleotides indicate the start of transcription. The translational start codon andstop codon are represented by bold lettering. Exon - intron splice junctions areindicated by the following symbol V. Classical polyadenylation signals in the 3’ endof the gene sequence are double - underlined whereas ATTAAA signals are denotedwith an asterisk (*). ATTTA motifs in the gene sequence are underlined.Nucleotides of the transcript are numbered sequentially and the most 3’ transcriptioninitiation site was arbitrarily assigned the +1 position.71—1595 aagctttctg aagcataaat ctggccatac ctaccgtata ttactccatt-1545 ctttatatag gtaaagccta aactcctttt cttggaatat aggctctcca-1495 gatctgaagt ctgcctaatt atttacactt ttgctttcac ataccctttg-1445 aactttctca cattgtcttc gtgtttgcat gtgctgctcc agcttctaag—1395 catgccctcc ctgtcctcat accccattcc ccagccactt attacgctat-1345 atgctgtagt cccattcagc tcggttgcaa cctcttccct aatgaatcag-1295 tccatcatta acaaagaaag ggagggaggg agggaggaag agaggaatga-1245 aaggaggaaa agggaaggaa ggggaaggga aaggggaagg gaaggggaag-1195 ggaagggaag gggaagggaa ggaatgggag gaaaagggac aaataatgaa-1145 tgatatgctc taatcttttt cccctagata tagaagacaa agagcaaaat-1095 atacttcact aaattgattt ttacataaat tttctttcct ttgttttttg-1045 gttgctggtc cacttacaaa cacttttcat atttgtatgt ctttccaatg-995 gttatcctgt tttgttcatt tcaggcatat ggccctgatc agattaactg-945 acatgatgta tatgcaaagc cttttgagtt cttcagaaaa ataaattatc-895 ttattcaaga ctgattgctt ataaggaact tattatagct aatatagtag-845 gcacaatttt tttttgtaat tctcctagat gagtcagaac ttagttttga-795 tgtaggtaaa aattttatgg tcacaaatct caggtgtgag aaaatctctt-745 tccttgatac tctatataaa tagaggatat aaatatttca agtctggaagAAAAAA AAAAAAA-695 tagtgagaga agctggtaat tctggaCata tagtgacagt caaaaaggag-645 ctcaggtaca ggactggtct aagctgctca agattcagga gacagccagt—595 acacagagaa gctgaggaaa taatacagat atatctaaaa cacttatcta-545 accttctgtg gtaacaagct ccttaaaggg gctggatgat gttgtgttca-495 ctttttatca ccagcaaagg ctaagataat gtatatagta aatatttagt—445 aaccatttat taaataaata aatatttaag acagaataaa caagtataat72—395 aaatgaacca ataagaatgc accatctaag tcaaaatagc cacttttatc-345 cttaacattg tacctgcttt ggctgctgca gaagcaaact tgttggcatt—295 agacaaatca agctggtgat ttaataaatt ccaatgtaag tcttaccagt—245 attgatgaat aactatccag cactcaccat gaaagttaaa gaagcaacac—195 agaaaaagtt cctaagtggt cccaatttga aatgatcaga taacctataa—145 aagaacatat tcatattata ctaacataaa cacatataaa tgcaCttacaA AAAAAA—95 gcagttacac agtattctct tcaataacta gtttccttat gcattaatgt—45 gtaataacag caactacaat atttagataa ttataaaaac caaggCaataA A A A A A6 atttaaaaac tgattaaccg ttttactcta acttaagcat ggattggatc56 agtaagattg attaataaat ttgaatgcag tcagttggat tgattctaat106 ttaaagtttt aatttgttgt agaataattt taagtgaata tatttgtcca156 gtgttcgagt gctcaacagt gtgtttgaaa aggaaaacaa agaaatgttt206 ttgagaaatg tgttaattcc ttaagacaat ggattttaat tggatctagt256 tgttttcatt tttcttcatt atcattatac atctgtatgt tggacagaac306 actaacacta aatagttttt agaaaaattt tttaaagtta tttaaatcat356 aatatcatga ctgacatttt aaattcaaaa ttaggctgtg actatccttc406 ttcacttagg aagagtgttg tgaaagccag accatctgct gaggtgctac456 agttacatgt ggccctcaga atgcgtttgg cctgctctgt tttagcactc506 tgttggatta ccaatcacaa aacaagttaa ccttgatctt tcacattaag556 tatctcaggg acaaaatttg acatacgtct aaacctgtga acgtttccat606 ctaaagaagg cagaaataaa acaggacttt agattcggtt acaataaaat656 atcagatgca ccagagacac aaggcttgaa gctctgtcct gggaaaatAT706 GGCAAACAGT GCCTCTCCTG AACAGAATCA AAATCACTGT TCAGCCATCA756 ACAACAGCAT CCCACTGATG CAGGGCAACC TCCCCACTCT GACCTTGTCT73806 GGA.AAGATCC GAGTGACGGT TACTTTCTTC CTTTTTCTGC TCTCTGCGAC856 CTTTAATGCT TCTTTCTTGT TGAAACTTCA GAAGTGGACA CAGAAGAAAG906 AGAAAGGGAA AAAGCTCTCA AGAATGAAGC TGCTCTTAAA ACATCTGACC956 TTAGCCAACC TGTTGGAGAC TCTGATTGTC ATGCCACTGG ATGGGATGTG1006 GAACATTACA GTCCAATGGT ATGCTGGAGA GTTACTCTGC AAAGTTCTCA1056 GTTATCTAAA GCTTTTCTCC ATGTATGCCC CAGCCTTCAT GATGGTGGTG1106 ATCAGCCTGG ACCGCTCCCT GGCTATCACG AGGCCCCTAG CTTTGAAAAG1156 CAACAGCAAA GTCGGACAGT CCATGGTTGG CCTGGCCTGG ATCCTCAGTAV1206 GTGTCTTTGC AGGACCACAG TTATACATCT TCAGGATGAT TCATCTAGCA1256 GACAGCTCTG GACAGACAAA AGTTTTCTCT CAATGTGTAA CACACTGCAG1306 TTTTTCACAA TGGTGGCATC AAGCATTTTA TAACTTTTTC ACCTTCAGCT1356 GCCTCTTCAT CATCCCTCTT TTCATCATGC TGATCTGCAA TGCAAAAATCV1406 ATCTTCACCC TGACACGGGT CCTTCATCAG GACCCCCACG AACTACAACT1456 GAATCAGTCC AAGAACAATA TACCAAGAGC ACGGCTGAAG ACTCTAAAAA1506 TGACGGTTGC ATTTGCCACT TCATTTACTG TCTGCTGGAC TCCCTACTAT1556 GTCCTAGGAA TTTGGTATTG GTTTGATCCT GAAATGTTAA ACAGGTTGTC1606 AGACCCAGTA AATCACTTCT TCTTTCTCTT TGCCTTTTTA AACCCATGCT1656 TTGATCCACT TATCTATGGA TATTTTTCTC TGTGAttgat agactacaca1706 agaagtcata tgaagaaggg taaggtaatg aatctctcca tctgggaatg1756 attaacacaa atgttggagc atgtttacat acaaacaaag taggatttac1806 acttaagtta tcattctttt agaaactcag tcttcagagc ctcaattatt1856 aaggaaaagt cttcaggaaa aatactaaaa tattttctct tcctcataag1906 cttctaaatt aatctctgcc ttttctgacc tcatataaca cattatgtag1956 gtttcttatc actttctctt tgcataataa tgtactaata tttaaaatac2006 cttcagccta aggcacaagg atgccaaaaa aacaaaggtg agaaaccaca742056 acacaggtct aaactcagca tgctttggtg agtttttctc caaaaggggc2106 atattagcaa ttagagttgt atgctatata atacatagag cacagagccc2156 tttgcccata atatcaactt tccctcctat agttaaaaag aaaaaaaatg2206 aatctatttt tctctttggc ttcaaaagca ttctgacatt tggaggagtc2256 agtaaccaat cccaccaacc actccagcaa cctgacaaga ctatgagtag2306 ttctccttca tcctatttat gtggtacagg ttgtgaagta tctctatata2356 aagggaaatt ttagaggggt taggatttgg acaggggttt agaacattcc2406 tctaagctat ctagtctgtg gagtttgtgg caattaattg ccataaaata2456 acaatgtttc caaatgcaac taagaaaata ctcatagtga gtacgctcta2506 tgcatagtat gacttctatt ttaatgtgaa gaattttttg tctctctcct2556 gatcttacta aatccatatt tcataaataa ctgagaataa ttaaaacaaa2606 attaagcaaa tgcacaagca aaaagatgct tgatacacaa aaggaactct2656 ggagagaaaa ctacagcttc agtctgtaca gatcaaagaa gacagaacat2706 gtcaggggaa ggagggaaag atcttgatgc agggtttctt aacctgcagt2756 ctatgcacaa cactatattt ccatgtaatg tttttatttc agccctattt2806 gtattatttt gtgcatttaa aaaacacaat cttaagggga tagactagac2856 tgccacagca gcccatggca caactaacac ctactgatat tcacattaaa2906 tagtatggtt tccaaaatat gtctgcacaa caagacctct ttatgtaatt2956 caggcttgtg tctacctctt ccatgaaaaa tggaaaggga tgaaaataat3006 gggagtataa tacccattta atgtgaaaaa cataagagtc ttaaaagaaa3056 ttaagccatt taacattttt taaataggta agataccatt atatttatat3106 gagctatgta ctgccacaaa aaaagatgaa atgtaatttc taaatactcc3156 aggtgtgtgg tattatggaa agcaaattgc caactaatgg cacgtccttt3206 ctttctttga ttttctcctc tcatacttca gttttatagt gttgtgttgt3256 tgtttttttc atatcctacc ttactttcca attctgtctc aattgaactc753306 cctctgtcta ctcactcttt cattcatagc ttcttttcca ttaaactcat3356 acctttaatt aaccaattca3406 gctaaaattc tgtagtgtgc3456 tccaagattt tatagttctt3506 aacaaaacca gtgtcctcta3556 ataattctta cttctggatt3606 acaatctaag ggctttatca3656 gttcttcttg tgtgctcatt3706 ggcatttttt ttttttacac3756 tcagggttct taacctacat3806 attttttatt ttagtctatt3856 cctgagaggg atggaccaga3906 gttaagaagt cctagttgac3956 taaaaatata acataatcta4006 taaatattga aacaagattg4056 aaactctctt aaggttcaat4106 ttctatatga atattatggt4156 ataatgtaat atataaacta*ttgaaattaa agatattccaacaatcattt_attttatgtcaaaaaaggaa atgttccacattctatcttg gtttctcttcttggaatttc ttttagcaat*tgacaataaa ttaaacaaagtggcccagtt ctacagttgataaaatgctc aagttggcacgtagataaca cagggatgtaagtctctatc atatacttattaaaatcaaa aataacacaccacacgtgtt aacgaatgtaatggatctct ctgtcttaggattaactaaa gggctattcgttcatgcaaa aaatatatattgtattattt tatgcatttactgccacagc agctcatagctttgtatata tataaagaaattcatctatt_tatatgcaaacttcaatatg cttattgtttatgtaataaa aaacataacatcataaatta taatgtataaaaatttatgg cacaaaagatctcaacagac aatatttcatctattataat_aaaaggtgaggagtcaatct aatatatcagctttacttag cctataaaactcagtttagt acaggagtgacctacattag ttcaatttaaattggacaagataaacccatgataagttgatcctaaactgttgtacagattctcagcttgaattgcctcaaaatcttgacatttcaatgtaaaacacagtacaaaaaaagtctattacaacataaaaatgtcaaaccaaccaaataattatctatacattaaatatggctatttgatattgactccttgtatattggagatagttaaaaacattaactaagcctattcaa4206425643064356440644564506 cagaaatata gaaatatagt agctaaaaaa atactctggg gaaggtacca764556 caaacattat ctaccaggga acatagcata aattagtctg aaatttcctg4606 agagtgactt tgtcttagaa cttaggtggt agtcatgaag agataatgtt4656 tttaggcagt taaaatactt ctagaactcc atctatttta cctgtggtcc4706 actttcctac attgaaccaa tgccttgggc ttctctaatt actatacatt4756 gtgctcatat gaataaaaga aattttaaaa gaaaaaaaa77Fig. 7. Nucleotide sequence of the human GnRR-R coding region.The nucleotide sequence of the complete protein coding region for the human GnRHR is shown. Numbering of the nucleotides begins with +1 at the translational start(ATG) codon. The amino acid translation is represented below its respectivenucleotide sequence in its three and one letter code.781 .ATGGCAAACAGTGCCTCTCCTGAACAGAATCAAAATCACTGTTCAGCCATCAACAACAGC+ + + + + +TACCGTTTGTCACGGAGAGGACTTGTCTTAGTTTTAGTGACAAGTCGGTAGTTGTTGTCGMetAlaAsnSerAlaSerProGluGlnAsnGlnAsnHisCysSerAlal leAsnAsnSerMANS ASP EQ N Q N H C S A INNSATCCCACTGATGCAGGGCAACCTCCCCACTCTGACCTTGTCTGGAAAGATCCGAGTGACG+ + + + + +TAGGGTGACTACGTCCCGTTGGAGGGGTGAGACTGGAACAGACCTTTCTAGGCTCACTGCI leProLeuMetGlnGlyAsnLeuProThrLeuThrLeuSerGlyLys I leArgValThrI P L M Q G N L PT L T L S G K I R VTGTTACTTTCTTCCTTTTTCTGCTCTCTGCGACCTTTAATGCTTCTTTCTTGTTGAAACTT+ + + + + +CAATGAAAGAAGGAAAAAGACGAGAGACGCTGGAAATTACGAAGAAAGAACAACTTTGAAValThrPhePheLeuPheLeuLeuSerAlaThrPheAsnAlaSerPheLeuLeuLysLeuVT F FL FL L SAT F N A SF L L K LCAGAAGTGGACACAGAAGAAAGAGAAAGGGAAAAAGCTCTCAAGAATGAAGCTGCTCTTA+ + + + + +GTCTTCACCTGTGTCTTCTTTCTCTTTCCCTTTTTCGAGAGTTCTTACTTCGACGAGAATGlnLysTrpThrGlnLysLysGluLysGlyLysLysLeuSerArgMetLysLeuLeuLeuQ K W T Q K K E KG K K L SR M K L L LAAACATCTGACCTTAGCCAACCTGTTGGAGACTCTGATTGTCATGCCACTGGATGGGATG+ + + + + +TTTGTAGACTGGAATCGGTTGGACAACCTCTGAGACTAACAGTACGGTGACCTACCCTACLysHisLeuThrLeuAlaAsnLeuLeuGluThrLeul leValMetProLeuAspGlyMetK H L T LAN L LET LI VM P L D GMTGGAACATTACAGTCCAATGGTATGCTGGAGAGTTACTCTGCAAAGTTCTCAGTTATCTA+ + + + + +ACCTTGTAATGTCAGGTTACCATACGACCTCTCAATGAGACGTTTCAAGAGTCAATAGATTrpAsnl leThrValGlnTrpTyrAlaGlyGluLeuLeuCysLysValLeuSerTyrLeuW NIT V Q WY A GEL L C K V L S Y LAAGCTTTTCTCCATGTATGCCCCAGCCTTCATGATGGTGGTGATCAGCCTGGACCGCTCC+ + + + + +TTCGAAAAGAGGTACATACGGGGTCGGAAGTACTACCACCACTAGTCGGACCTGGCGAGGLysLeuPheSerMetTyrAlaProAlaPheMetMetValVall leSerLeuAspArgSerK L F SM YAP A F MM V VI S L DR S79CTGGCTATCACGAGGCCCCTAGCTTTGAAAAGCAACAGCAAAGTCGGACAGTCCATGGTT+ + + + + +GACCGATAGTGCTCCGGGGATCGAAACTTTTCGTTGTCGTTTCAGCCTGTCAGGTACCAALeuAlalleThrArgProLeuAlaLeuLysSerAsnSerLysValGlyGlnSerMetValLA IT R P LA L K S N S K V G Q S MVGGCCTGGCCTGGATCCTCAGTAGTGTCTTTGCAGGACCACAGTTATACATCTTCAGGATG+ + + + + +CCGGACCGGACCTAGGAGTCATCACAGAAACGTCCTGGTGTCAATATGTAGAAGTCCTACGlyLeuAlaTrplleLeuSerSerValPheAlaGlyProGlnLeuTyrl lePheArgMetG LAW IL S S V FAG P Q L Y IF R MATTCATCTAGCAGACAGCTCTGGACAGACAAAAGTTTTCTCTCAATGTGTAACACACTGC+ + + + + +TAAGTAGATCGTCTGTCGAGACCTGTCTGTTTTCAAAAGAGAGTTACACATTGTGTGACGI leHisLeuAlaAspSerSerGlyGlnThrLysValPheSerGlnCysValThrHisCysI H LADS S G Q T KV F SQ CV T H CAGTTTTTCACAATGGTGGCATCAAGCATTTTATAACTTTTTCACCTTCAGCTGCCTCTTC+ + + + + +TCAAAAAGTGTTACCACCGTAGTTCGTAAAATATTGAAAAAGTGGAAGTCGACGGAGAAGSerPheSerGlnTrpTrpHisGlnAlaPheTyrAsnPhePheThrPheSerCysLeuPheSFS Q W W H Q A F Y N F FT F SC L FATCATCCCTCTTTTCATCATGCTGATCTGCAATGCAAAAATCATCTTCACCCTGACACGG+ + + + + +TAGTAGGGAGAAAAGTAGTACGACTAGACGTTACGTTTTTAGTAGAAGTGGGACTGTGCCI lel leProLeuPhel leMetLeulleCysAsnAlaLysilel lePheThrLeuThrArglIP L F I ML IC N A K lIFT L T RGTCCTTCATCAGGACCCCCACGAACTACAACTGAATCAGTCCAAGAACAATATACCAAGA+ + + + + +CAGGAAGTAGTCCTGGGGGTGCTTGATGTTGACTTAGTCAGGTTCTTGTTATATGGTTCTValLeuHisGlnAspProHisGluLeuGlnLeuAsnGlnSerLysAsnAsnl leProArgV L H Q D PH EL Q L N Q S K N NI PRGCACGGCTGAAGACTCTAAAAATGACGGTTGCATTTGCCACTTCATTTACTGTCTGCTGG+ + + + + +CGTGCCGACTTCTGAGATTTTTACTGCCAACGTAAACGGTGAAGTAAATGACAGACGACCAlaArgLeuLysThrLeuLysMetThrValAlaPheAlaThrSerPheThrValCysTrpAR L K T L K MT VA FAT SF TV C W80ACTCCCTACTATGTCCTAGGAATTTGGTATTGGTTTGATCCTGAAATGTTAAACAGGTTG+ + + + + +TGAGGGATGATACAGGATCCTTAAACCATAACCAAACTAGGACTTTACAATTTGTCCAACThrProTyrTyrValLeuGlyl leTrpTyrTrpPheAspProGluMetLeuAsnArgLeuT P Y Y V L G 1W Y W F D P E ML N R LTCAGACCCAGTAAATCACTTCTTCTTTCTCTTTGCCTTTTTAAACCCATGCTTTGATCCA+ + + + + +AGTCTGGGTCATTTAGTGAAGAAGAAAGAGAAACGGAAAAATTTGGGTACGAAACTAGGTSerAspProValAsnHisPhePhePheLeuPheAlaPheLeuAsnProCysPheAspProSD P V N H F F FL F A FL N PC F D PCTTATCTATGGATATTTTTCTCTGTGA 987+ +GAATAGATACCTATAAAAAGAGACACTLeul leTyrGlyTyrPheSerLeuEndLI Y G Y F S L *813.3 Comparative analysis of the human GnRB-R geneSoftware from the Genetics Computer Group, Inc. was employed in theanalyses of the human GnRH-R gene. “FASTA” searches were performedcomparing the human GnRH-R gene to the entire data base of GenBank and EMBL(European Molecular Biology Laboratory). “BESTFIT”, “GAP”, and “PILEUP”alignments were implemented in the 5’ flanking and 5’ nontranslated as well as inthe translated, 3’ flanking, and 3’ nontranslated species comparisons.FASTA searches revealed profiles of sequence similarity between the humanGnRH-R gene and other genes. These genes included those for human wglel,human STS, human j3-globin, and orangutan f3- and ô-globins. In particular, oneprominent area of sequence similarity (-1300 bp to -1150 bp) was shared by all.This stretch of repetitive sequence, classified as an Alu repeat, was 74.6 % (wglel),70.8 % (STS), 68.5 % (human 13-globin), and 64.7 % (orangutan f3- and ô-globin)identical to the human GnRH-R Alu repeat.Comparison of the human GnRH-R gene to that of the mouse gene as well asto the cDNAs of other species revealed notable sequence identity. Sequencecomparisons for some species in the 5’ and 3’ ends were made on very limited82lengths of nontranslated regions. The results of the sequence comparisons areillustrated in Table 3 and Fig. 8.As expected, the coding region for all species examined exhibited the highestdegree of sequence identity. The sheep, cow, pig, mouse, and rat shared 89.5 %,89.1 %, 88.4 %, 85.4 %, and 85.2 % sequence identity, respectively, with thehuman GnRH-R coding region. Notable sequence identity existed between thehuman and the mouse in the 5’ end (5’ of translational start) over a distance of 1.5Kb. Lower sequence identity of 36.6 % in the 5’ end ( 700 bp) was evidencedbetween the rat and human. Short stretches (all that was available) of notablesequence identity also existed for the other species, in particular, for the cow (70.5%) and sheep (58.8 %). Sequence comparisons of the 3’ end (3’ of the stop codon),with lengthier sequences of up to 1.3 Kb, demonstrated relatively high sequenceidentity between the sheep (70.2 %), cow (68.1 %), pig (64.0 %), and humanspecies. Lower scores were obtained for both the mouse (48.5 %) and rat (47.4 %).Amino acid comparisons of the protein coding region of the human GriRH-Rgene with the above mentioned species were also conducted (Fig. 9). High aminoacid identity was observed between species. Obvious species polarity existed interms of the number of amino acids which code for the receptor. Human, pig, cow,83Table 3. Interspecies sequence comparison of the GnRR-R gene and cDNA.Comparisons between species of the 5’ end, 3’ end, and coding region for theGnRH-R gene (mouse) and cDNA (other species) are shown. Comparisons arerepresented numerically as percentage of sequence identity to the human gene. Inaddition, the length of sequence comparison is numerically indicated in parentheses.84Species 5’ End Coding Region 3’ EndHuman ************** ************** **************(—1 Kb)Cow 70.5% (—5Obp) 89.1% 68.1% (—1 Kb)Sheep 58.8% (—40 bp) 89.5% 70.2% (—1 Kb)Pig 88.4% 64.0% (—1 Kb)Mouse 57.3% (‘-1.5 Kb) 85.4% 48.5% (—1.3 KbRat 36.6% (—700 bp) 85.2% 47.4% (—1 Kb)85Fig. 8. DNA sequence alignment and comparison of GnRII-R coding regionsamong various species.The nucleotide sequences of the mouse (m), rat (r), cow (b), sheep (s), human (h),and pig (p) GnRH-R coding regions as compared using the “PILEUP” alignmentfrom the GCG (The Genetics Computer Group, Inc., Madison WI) software system.Identical nucleotides among species are indicated with an asterisk (*).861 50m CACTCTTGAA GCCTGTCCTT GGAGAAA.TA TGGCTAACAA TGCATCTCTTr CACTCTTGAA GCCCGTCCTT GGAGAAA. TA TGGCTAACAA TGCGTCTCTTb GAGTCTTGAA GCTGCATCAG CCATAAAGGA TGGCAAACAG TGACTCTCCTs GAGTCTTGAA GCTGTATCAG CCATAAAGGA TGGCAAACGG TGACTCTCCTh AAGGCTTGAA GCTCTGTCC. TGGGAAAATA TGGCAAACAG TGCCTCTCCTp TGGATCAGT TGGGGAAGGA TGGCAAACAG TGCCTCTCCT* ****** * ** * **** *** ** **** *51 100m GAGCAGGACC CAAATCACTG CTCGGCCATC AACAACAGCA TCCCCTTGATr GAGCAGGACC AAAATCACTG CTCAGCCATC AACAACAGCA TCCCCCTGACb GAACAGAATG AAAACCACTG TTCAGCGATC AACAGCAGCA TCCCTCTAACs GATCAGAATG AAAACCACTG TTCAGCGATC AACAGCAGCA TCCTACTAACh GAACAGAATC AAAATCACTG TTCAGCCATC AACAACAGCA TCCCACTGATp GAGCAGAATC AAAATCACTG CTCAGCCATC AACAGCAGCA TCCTGCTGAC** *** * *** ***** ** ** *** **** **** *** *101 150m ACAGGGCAAG CTCCCGACTC TAACCGTATC TGGAAAGATC CGAGTGACCGr ACAGGGCAAG CTCCCGACTC TAACCTTATC TGGAAAGATC CGAGTGACGGb ACCAGGCAGC CTCCCCACCC TGACCCTATC TGGAAAGATC CGAGTGACAGs ACCGGGCAGC CTCCCCACCC TGACCCTATC TGGAAAGATC CGAGTGACTGh GCAGGGCAAC CTCCCCACTC TGACCTTGTC TGGAAAGATC CGAGTGACGGp GCAGGGCAAC CTTCCCACCC TGACCTTATC TCCAAACATC CGCGTGACAG* **** ** ** ** * * *** * ** * *** *** ** ***** *151 200m TGACTTTCTT CCTTTTCCTA CTCTCTACTG CCTTCAATGC TTCCTTCTTGr TGACTTTCTT CCTTTTCCTA CTCTCTACTG CCTTCAATGC CTCTTTCTTGb TTACTTTCTT CCTTTTTCTA CTCTCCACAA TTTTCAACAC TTCTTTCTTGS TTACTTTCTT CCTTTTTCTA CTCTCCACAA TTTTCAACAC TTCTTTCTTGh TTACTTTCTT CCTTTTTCTG CTCTCTGCGA CCTTTAATGC TTCTTTCTTGp TCACTTTCTT CCTTTTCCTA CTCTCCACAG CTTTCAATGC TTCTTTCTTG* ******** ****** ** ***** * ** ** * ** ******201 250m TTGAAGCTGC AGAAGTGGAC TCAGAAGAGG AAGAAAGGAA AAAAGCTCTCr GTAAAGCTGC AGAGGTGGAC CCAGAAGAGG AAGAAAGGAA AAAAGCTCTCb TTGAAACTTC AGAATTGGAC TCAAAGGAAA GAGAAGAGGA AAAAACTCTCS TTGAAACTTC AGAATTGGAC TCAAAGGAAA GAGAAGAGGA AAAAACTCTCh TTGAAACTTC AGAAGTGGAC ACAGAAGAAA GAGAAAGGGA AAAAGCTCTCp TTGAAACTTC AGAAATGGAC TCAAAGGAAA GAGAAAGGGA AAAAACTCTC* ** ** * *** ***** * * ** **** * * **** *****251 300m AAGGATGAAG GTGCTTTTAA AGCATTTGAC CTTAGCCAAC CTGCTGGAGAr AAGGATGAAG GTGCTTTTAA AGCATTTGAC CTTAGCCAAC CTCCTTGAGAb GAGAATGAAG TTGCTTTTAA AACATTTGAC TTTAGCCAAC CTGCTGGAGAs AAAAATGAAG GTGCTTTTAA AACACTTGAC TTTAGCCAAC CTGCTGGAGAh AAGAATGAAG CTGCTCTTAA AACATCTGAC CTTAGCCAAC CTGTTGGAGAp AAGAATGAAG GTGCTTTTAA AACACTTGAC TCTAGCCAAC CTGTTGGAGA* ****** ********* * ** **** ******** ** * ****301 350m CTCTGATCGT CATGCCACTG GATGGGATGT GGAATATTAC TGTTCAGTGGr CTCTAATCGT CATGCCGCTG GATGGGATGT GGAACATCAC TGTTCAGTGGb CTCTGATTGT TATGCCACTG GATGGAATGT GGAACATAAC TGTTCAATGGs CTCTGATTGT TATGCCACTG GATGGAATGT GGAACATAAC TGTTCAATGGh CTCTGATTGT CATGCCACTG GATGGGATGT GGAACATTAC AGTCCAATGGp CTCTGATTGT CATGCCGCTG GATGGCATGT GGAACATCAC CGTGCAATGG**** ** ** ***** *** ***** **** **** ** ** ** ** ***87351 400m TATGCTGGGG AGTTCCTCTG CAAAGTTCTC AGCTATCTGA AGCTCTTCTCr TATGCTGGAG AGTTCCTTTG CAAAGTTCTC AGCTATCTGA AGCTCTTCTCb TATGCTGGAG AGCTCCTTTG CAAAGTCCTC AGCTATCTGA AGCTTTTCTCs TATGCTGGAG AGCTCCTTTG CAAAGTCCTC AGCTATCTGA AGCTTTTCTCh TATGCTGGAG AGTTACTCTG CAAAGTTCTC AGTTATCTAA AGCTTTTCTCp TATGCCGGAG AGTTCCTCTG CAAAGTCCTC AGCTACCTGA AGCTTTTCTC***** ** * ** * ** ** ****** *** ** ** ** * **** *****401 450m TATGTATGCC CCAGCTTTCA TGATGGTGGT GATTAGCCTG GACCGCTCCCr TATGTATGCC CCAGCCTTCA TGATGGTGGT GATTAGCCTG GATCGCTCCCb CATGTACGCC CCCGCCTTCA TGATGGTGGT GATCAGCCTC GACCGCTCGCs CATGTACGCC CCCGCCTTCA TGATGGTGGT GATCAGCCTC GACCGCTCCCh CATGTATGCC CCAGCCTTCA TGATGGTGGT GATCAGCCTG GACCGCTCCCp CATGTATGCC CCCGCCTTCA TGATGGTGGT GATTAGCCTG GACCGCTCGC***** *** ** ** **** ********** *** ***** ** ***** *451 500m TGGCCATCAC TCAGCCCCTT GCTGTACAAA GCAACAGCAA GCTTGAACAGr TGGCCGTCAC TCAGCCCTTA GCTGTCCAAA GCAAGAGCAA GCTTGAACGGb TGGCGATCAC CAAGCCTCTA GCAGTGAAAA GCAACAGCAA GCTTGGACAGS TGGCCATCAC CAGGCCTCTA GCAGTGAAAA GCAACAGCAA GCTCGGACAGh TGGCTATCAC GAGGCCCCTA GCTTTGAAAA GCAACAGCAA AGTCGGACAGp TGGCCATCAC AAGGCCCCTC GCTGTGAAAA GCAACAGCAG GCTCGGACGG**** **** *** * * * *** **** **** * * ** *501 550m TCTATGATCA GCCTGGCCTG GATTCTCAGC ATTGTCTTTG CAGGACCACAr TCTATGACCA GCCTGGCCTG GATTCTCAGC ATTGTCTTTG CGGGACCACAb TTCATGATTG GCTTGGCCTG GCTCCTCAGT AGCATCTTTG CTGGACCACAs TTCATGATTG GCTTGGCCTG GCTCCTCAGT AGCATCTTTG CTGGACCACAh TCCATGGTTG GCCTGGCCTG GATCCTCAGT AGTGTCTTTG CAGGACCACAp TTCATGATTG GCTTGGCCTG GCTCCTCAGT AGCATCTTTG CTGGACCACA* *** ** ******* * * ***** * ****** * ********551 600m GTTATATATC TTCAGGATGA TCTACCTAGC AGACGGCTCT GG. GCCCAr GTTATATATC TTCAGGATGA TCTACCTAGC CGACGGCTCT GG. . GCCAGb GCTATACATC TTTGGGATGA TCCATTTAGC AGATGACTCT GGACAGACTGs GTTATACATC TTTGGGATGA TCCATTTAGC AGATGACTCT GGACAGACTGh GTTATACATC TTCAGGATGA TTCATCTAGC AGACAGCTCT GGACAGACAAp GTTATACATC TTCAGGATGA TCCATTTAGC AGACAGCTCT GGACAGACAG* **** *** ** ****** * * **** * **** ****** *601 650m CAGTCTTCTC GCAATGTGTG ACCCACTGCA GCTTTCCACA GTGGTGGCATr CAGTTTTCTC GCAATGTGTG ACCCACTGCA GCTTTCCGCA ATGGTGGCATb AAGGTTTCTC TCAGTGTGTA ACACACTGCA GTTTTCCACA GTGGTGGCATS AAGGTTTCTC TCAATGTGTA ACACACTGCA GTTTTCCACA GTGGTGGCATh AAGTTTTCTC TCAATGTGTA ACACACTGCA GTTTTTCACA ATGGTGGCATp AAGGTTTCTC TCAATGTGTA ACACATGGCA GTTTTCCACA ATGGTGGCAT** ***** ** ***** ** ** *** * *** * ** *********651 700m CAGGCCTTCT ACAACTTTTT CACCTTCGGC TGCCTCTTCA TCATCCCCCTr GAAGCCTTCT ACAACTTTTT CACCTTCAGC TGCCTGTTCA TCATCCCTCTb CAAGCCTTTT ATAACTTTTT CACCTTCAGC TGCCTCTTCA TCATCCCTCTS CAAGCCTTTT ATAACTTTTT CACCTTCAGC TGCCTCTTCA TCATCCCTCTh CAAGCATTTT ATAACTTTTT CACCTTCAGC TGCCTCTTCA TCATCCCTCTp CAAGCCTTTT ATGACTTTTT CACCTTTAGC TGCCTCTTCA TTATCCCTCT* ** ** * * ******* ******* ** ***** **** ******* **88751m TCCTTCATCAr TCCTTCATCAb TCCTTCATCAS TCCTTCATCAh TCCTTCATCAp TCCTTCAGCA******* **AGACCCACGCGGACCCACGCGGATCCCCACGGATCCCCACGGACCCCCACGGATCCCCAC** ** * *AAACTACAGCAAACTACAGCAAACTACAACAAACTACAACGAACTACAACAACCTGCAGC* ** * *TGAATCAGTCTGAATCAATCTGAATCAGTCTGAATCAGTCTGAATCAGTCTGAATCAATC******* **800CAAGAATAATCAAGAATAATCAAGAACAATCAAGAACAATCAAGAACAATCAAGAACAAC****** **801m ATCCCAAGAGr ATCCCAAGAGb ATACCACGAGs ATACCACAAGh ATACCAAGAGp ATACCACGAG** *** **CTCGGCTGAGCACGGCTGAGCTCGGCTGAGCTCGGCTGAGCACGGCTGAACTCGGTTGAG* *** ***AACGCTAAAGAACTCTAAAGGACCCTAAAGGACCCTAAAGGACTCTAAAAGACTCTGAAG** ** **ATGACAGTCGATGACAGTGGATGACGGTTGATGACGGTTGATGACGGTTGATGACAGTTG***** ** *850CATTCGCTACCATTTGCCACCATTTGCCACCATTTGCGACCATTTGCCACCATTTGCTGC**** ** *851m CTCCTTTGTCr CTCCTTTGTCb TTCATTTACTs TTCATTTACTh TTCATTTACTp TTCATTTATT** ***GTCTGCTGGAATCTGCTGGAGTCTGCTGGAGTCTGCTGGAGTCTGCTGGAGTCTGCTGGA*********CTCCCTACTACTCCCTACTACGCCCTACTACGCCCTACTACTCCCTACTACTCCCTACTT* *******TGTCCTAGGCCGTCCTAGGATGTCCTTGGATGTCCTTGGATGTCCTAGGAAGTCCTAGGA***** **900ATTTGGTACTATCTGGTACTATTTGGTATTATTTGGTATTATTTGGTATTATTTGGTACT** ***** *901m GGTTTGATCCr GGTTTGATCCb GGTTTGATCCS GGTTTGATCCh GGTTTGATCCp GGTTTGATCC** ** * * * ** *AGAAATGTTGGGAAATGTTATGACATGGTATGACATGGTATGAAATGTTACGAAATGGTA** *** *AACAGGGTGTAACAGGGTGTAACAGGGTGTAACAGGGTGTAACAGGTTGTAACAGGGTGT****** ***CAGAGCCAGTCAGAGCCAGTCAGATCCAGTCAGATCCAGTCAGACCCAGTCAGATCCAGT**** *****950GAATCACTTTCAATCACTTCAAATCACTTCAAATCACTTCAAATCACTTCCAATCACTTC*** ** * * *951rn TTCTTTCTCTr TTCTTTCTCTb TTCTTTCTCTs TTCTTTCTCTh TTCTTTCTCTp TTCTTTCTCT*** ** ** * * *TTGCTTTCCTTTGCTTTTCTTTGCTTTTTTTTGCTTTTTTTTGCCTTTTTTTGCTTTTTT**** ** *AAACCCGTGCAAACCCGTGCAAATCCATGCAAATCCATGCAAACCCATGCAAATCCATGC*** ** ***TTCGACCCACTTCGACCCACTTTGATCCACTTTGATCCACTTTGATCCACTTTGATCCAC** ** ****1000TCATATATGGTTATATATGGTTATATATGGTTATATATGGTTATCTATGGTTATATACGG* ** ** **1001m GTATTTCTCTr GTATTTCTCTb ATATTTCTCTs ATATTTCTCTh ATATTTTTCTp ATATTTCTCT***** ***TTGTAGTTGGTTGTAATTGGCTATAATTGTCTGTAATTGTCTGTGATTGACTGTAATTTT* * **GAGACTACACGAGACTACCCTAGACTGCATTAGATTGCATTAGACTACACGTTAGACTAT*AAGAA.. . CAACCA.AGAAAG.AGAAAG....AAGAAGTCATATACAGAATT*1051TCAGATAGAATTGTGCTGAA• TCAAAGAA• TCAAAGAAATGAAGAAGGATATAAAGAA701 750m CCTCATCATG CTAATCTGCA ATGCCAAAAT CATCTTTGCT CTCACGCGAGr TCTCATCATG CTAATCTGCA ATGCCAAAAT CATCTTCGCC CTCACACGAGb TCTCATCATG GTGATCTGCA ATGCAAAAAT CATCTTTACC CTAACAAGGGs TCTCATCATG CTGATCTGCA ATGCAAAAAT CATCTTCACC CTAACAAGGGh TTTCATCATG CTGATCTGCA ATGCAAAAAT CATCTTCACC CTGACACGGGp CCTCATCATG TTGATCTGCA ACGCAAAAAT CATGTTCACT CTGACAAGAG******** * ******* * ** ***** *** ** * ** ** * *89and sheep all possessed 328 amino acids, whereas, the rat and mouse receptorspossessed only 327 amino acids. Identical amino acids shared between species arealso indicated in Fig. 9.90Fig. 9. Alignment of the ammo acid sequences of various species.The derived amino acid sequences for the human, pig, cow, sheep, rat, and mouseGnRH receptors are aligned. Commonly conserved residues are depicted with thenumber symbol (if). Regions of the proposed transmembrane domains are doubleunderlined (= = =).91HUMAN MANSASPEQNQNHCSAINNSI PLMQGNLPTLTLSGKIRVTVTFFLTLLSA 50PIG MANSASPEQNQNHCSAINSS ILLTQGNLPTLTLSPNIRVTVTFFLFLLST 50COW MANSDSPEQNENHCSAINSSIPLTPGSLPTLTLSGKIRVTVTFFLTLLST 50SHEEP MANGDSPDQNENHCSAINSSILLTPGSLPTLTLSGKIRVTVTFFLFLLST 50RAT MANNASLEQDQNHCSAINNS IPLTQGKLPTLTLSGKIRVTVTFFLFLLST SOMOUSE MANNASLEQDPNHCSAINNS I PLIQGKLPTLTVSGKIRVTVTFFLFLLST SO### # # ####### ## # # ##### # ######### ###TFNASFLLKLQKWTQKKEKGKKLSRMKLLLKHLTLANLLETLIVMPLDGM 100AFNASFLLKLQKWTQRKEKGKKLSRMKVLLKHLTLANLLETLIVMPLDGM 100IFNTSFLLKLQNWTQRKEKRKKLSRNKLLLKHLTLANLLETLIVMPLDGM 100IFNTSFLLKLQNWTQRKEKRKKLSKMKVLLKHLTLANLLETLIVMPLDGM 100AFNASFLVKLQRWTQKRKKGKKLSRNKVLLKHLTLANLLETLIVMPLDGM 100AFNASFLLKLQKWTQKRKKGKKLSRMKVLLKHLTLANLLETLIVMPLDGM 100## ### ### ### # ####### ######################WNITVQWYAGELLCKVLSYLKLFSMYAPAFMMVVISLDRSLAITRPLALK 150WNITVQWYAGEFLCKVLSYLKLFSMYAPAFMMVVI SLDRSLAITRPLAVK 150WNITVQWYAGELLCKVLSYLKLFSMYAPAFMMVVISLDRSLAITKpLAVK 150WNITVQWYAGELLCKVLSYLKLFSMYAPAFMMVVI SLDRSLAITRPLAVK 150WNITVQWYAGEFLCKVLSYLKLFSMYAPAFMMVVI SLDRSLAVTQPLAVQ 150WNITVQWYAGEFLCKVLSYLKLFSMYAPAFMMVVI SLDRSLAITQPLAVQ 150########### ############################## # ###SNSKVGQSMVGLAWILSSVFAGPQLYIFRMIHLADSSGQTKVFSQCVTHC 200SNSRLGRFMIGLAWLLSSIFAGPQLYIFRMIHLADSSGQTEGFSQCVTHG 200SNSKLGQFMIGLAWLLSSIFAGPQLYIFGMIHLADDSGQTEGFSQCVTHC 200SNSKLGQFMIGLAWLLSSIFAGPQLYIFGMIHLADDSGQTEGFSQCVTHC 200SKSKLERSMTSLAWILSIVFAGPQLYIFRMIYLADGSGPA—VFSQCVTHC 199SNSKLEQSMISLAWILS IVFAGPQLYIFRMIYLADGSGPT—VFSQCVTHC 199# 7# # ### ## ,######### ## ##)# ## ########SFSQWWHQAFYNFFTFSCLFIIPLFIMLICNAKIIFTLTRVLHQDPHELQ 250SFPQWWHQAFYDFFTFSCLFI IPLLIMLICNAKIMFTLTRVLQQDPHNLQ 250SFPQWWHQAFYNFFTFSCLFIIPLLIMVICNAKIIFTLTRVLHQDPHKLQ 250SFPQWWHQAFYNFFTFSCLFIIPLLIMLICNAKIIFTLTRVLHQDPHKLQ 250SFPQWWHEAFYNFFTFSCLFIIPLLIMLICNAKIIFALTRVLHQDPRKLQ 249SFPQWWHQAFYNFFTFGCLFIIPLLIMLICNAKIIFALTRVLHQDPRKLQ 249## #### ### #### ####### ## ###### ##### ### ##92LNQSKNNIPRARLKTLKMTVAFATSFTVCWTPYYVLGIWYWFDPEMLNRL 300LNQSKNNIPRARLRTLKMTVAFAASFIVCWTPYLVLGIWYWFDPEMVNRV 300LNQSKNNIPRARLRTLKMTVAFATSFTVCWTPYYVLGIWYWFDPDMVNRV 300LNQSKNNIPQARLRTLKMTVAFATSFTVCWTPYYVLGIWYWFDPDMVNRV 300LNQSKNNIPRARLRTLKMTVAFATSFVICWTPYYVLGIWYWFDPEMLNRV 299LNQSKNNIPRARLRTLKMTVAFATSFVVCWTPYYVLGIWYWFDPEMLNRV 299######### ### ######### ## ##### ########## # #SDPVNHFFFLFAFLNPCFDPLIYGYFSL* 328SDPVNHFFFLFAFLNPCFDPLIYGYFSL* 328SDPVNHFFFLFAFLNPCFDPLIYGYFSL* 328SDPVNHFFFLFAFLNPCFDPLIYGYFSL* 328SEPVNHFFFLFAFLNPCFDPLIYGYFSL* 327SEPVNHFFFLFAFLNPCFDPLIYGYFSL* 327# ##########################AminoSpecies acid %IdentityHumanPig 91.8 %Cow 91.2%Sheep 90.5 %Rat 87.8 %Mouse 89.3 %933.4 Structural organization of the human GnRH-R geneDNA sequencing and sequence comparison to the published human GnRH-RcDNA sequences (Kakar et al., 1992; Chi et a!., 1993) revealed that structurally theGnRH-R gene consists of three exons and two introns (Fig. 10). Exon II consistsof 219 bp while exon I and exon III consist of up to 1915 and 3321 bp, respectively.GT - AG consensus rules were followed for all splice junctions found (Table 4).Additionally, RT-PCR was conducted with human pituitary eDNA as a template inorder to confirm transcription up to the most 5’ and 3’ ends of the major 4.7 Kbtranscript (Fig. 13 and 18). Amplification of a 1456 bp product of the expected sizeresulted when PCR was conducted with oligonucleotides C and D (Table 1).However, when oligonucleotide B, a primer located upstream of the most 3’transcription initiation site, was used in combination with D no amplification productwas observed (Fig. 13). Additionally, oligonucleotide combinations of E with G andF with H resulted in the amplification of products of the expected sizes of 1876 and1176 bp, respectively (Fig. 18). For all primer combinations employed, no bandswere amplified from negative controls consisting of no cDNA, intestinal eDNA, ornonreverse transcribed human pituitary poly-A RNA as a template. As well,amplification using f3 - Actin primers on cDNA templates resulted in the successfulproduction of fragments of the expected size (Fig. 14).94Fig. 10. The human GnRH-R gene structure.A schematic representation of the human GnRH-R gene. (A) Organization of thethree genomic EcoRI (B) subclones. (B) Sequencing strategy employed for thehuman GriRH-R gene. Arrows indicate the orientation of oligonucleotides and theextent of sequence obtained. (C) Exon-intron localization. (D) Structure of thehuman GnRH-R cDNA. Open boxes indicate the protein coding regions and hatchedboxes are the putative transmembrane domains. (E) The relative positions of thehuman cDNA probes used in this study. Representative scale is illustrated.95A clonel clone2 clone3BDE5’PstI2kbI I0.1kbE1 IE ECE5’INIllsgenomic subclonessequencing strategy3’ gene structure3’ eDNAeDNA probesTMI II III IV V VI VII5’ AB3,C96Table 4. The exon- mtron organization of the human GnRII-R gene.The splice-junctions share the consensus sequences for the donor and acceptor sitesdescribed by Breathnach and Chambon, 1981. Numbers in parentheses are relativeto the translational initiation codon.97—_______Sequence of exon - intron junctionsE Exon Intronx size 5’Boundary 3’Boundaryo (kb)n (bp)ACCACAG gtgaa...tacag TTATACA1 1915 (+ 1125)4.2CCCCACG gtatg...aacag AACTACA2 219 (+1445) ii. A5.03 3319 GAAAAAAAA(+ 4766)983.5 Promoter region of the human GnRH-R geneA 7.7 Kb EcoRI genomic fragment was subjected to restriction and Southernanalyses resulting in the subcloning of a 2.7 Kb Hindlil genomic fragmentcontaining the 5’ end of the human GnRH-R gene (Fig. 11). The 3’ end of thissubclone contained the segment of exon I carrying the translational start site for thehuman GnRH receptor. Nucleotide sequencing of the 2.7 Kb fragment wasperformed and analysis of the 5’ end of the human GnRH-R gene revealed thepresence of five consensus TATA boxes (TATAAA; -14, -111, -150, -718, and -731; Fig. 5 and 12) residing in close proximity to one another in a cluster - likearrangement. Several CAAT boxes were also found well interspersed among theTATA boxes.In addition, inspection of the 5’ nontranslated region for the human GnRH-Rgene revealed the presence of several ATG (AUG; mRNA) codons upstream of themajor reading frame (Fig. 6).99Fig. 11 The 2.7 Kb HmdIII subclone.Configurational alignment of the 2.7 Kb Hindlil subclone (containing the promoterregion of the human GnRH-R gene) within the 7.7 Kb EcoRI genomic clone.Corresponding gene structure is depicted below. Stippled and solid black boxesindicate nontranslated region and coding region of the human GnRH-R gene,respectively. Intronic regions are shown as solid lines. E=EcoRI; H =HindIII.100CGeneStructureI2.7kbIIIIIIIH-ClonesEHHEIIFig. 12. Organization of the human GnRII receptor promoter region.The positions of the TATA and CAAT sequences are emphasized. The mRNA capsites (v) for the human GnR}I receptor transcripts are indicated by the respectivenucleotides in lower case letters. Nucleotides of the transcript are numberedsequentially and the most 3’ transcription initiation site was arbitrarily assigned the+1 position.1025’erid—1004 ———CCAAT -—884 -CAAT --731 -718-765 TATAAA TATAAAV Va c—644—524—404 CCAAT-284 CCAATCCAATT---150 V -111-164 TATAAA t TATAAAV---C CAAT-14 V-44 CAAT TATAAA cJ.Lexon I1033.6 Characterization of the 5’ terminus of the human GnRH-R mRNAContinued analyses on the 5’ end of the gene proceeded. Experiments todetermine the transcription initiation site(s) for the human GnRH-R gene wereconducted using primer extension analysis. Moreover, since several potentialpromoters for the human GnRH-R gene existed, mapping of the 5’ terminus (ortermini) of the human GnRH-R mRNA was undertaken to examine which one(s)may be functional. Five transcription initiation sites were identified through primerextension analysis. Primer extension was performed on human brain total RNAusing three synthetic oligonucleotides, PE-1, PE-2, and PE-3 (Table 1). It shouldbe noted that in preliminary studies, human pituitary poly-A RNA was used inaddition to brain total RNA. Because early results demonstrated similar findings forboth the pituitary and the brain, utilization of the more available source of humanbrain total RNA for primer extension experiments was undertaken. PE- 1 resultedin two bands at positions -669 bp and -690 bp, respectively (Fig. 13A). PE-2 andPE-3 revealed three signals (-101 and -124 bp, Fig. 13B; +1, Fig. 13C). Humanintestinal total RNA, as a negative control, showed no signals when extended withPE- 1, PE-2, or PE-3. All five transcription initiation sites were found to bepositioned in close proximity to the TATA boxes (Fig. 12).104Fig. 13. Analysis of the human GnRII-R gene by primer extension analysis.Oligonucleotides PE- 1 (panel A), PE-2 (panel B), and PE-3 (panel C) were end -labelled and hybridized to 40 g each of human brain (B) and intestinal (I) totalRNA. Extended products were analyzed on a 6 % polyacrylamide/7.0 M Urea gel.Standard lanes A, C, G, and T indicate M13 DNA sequencing reactions which wereused as a size standard. Positions of the positive bands are indicated numericallyand by arrows.105ABCStandardRNAIIIIACGTBIStandardRNAIIIIACGTBIStandardRNAIIIIACGTBI0 0)•-*-690bp-669bp-.4--124bp•-+1bp-101bp3.7 RT-PCR analysis of the 5’end of the human GnRB-R geneTo confirm the results of the primer extension analysis, PCR was performedusing oligonucleotide B, a primer positioned upstream of transcription initiation site-690, and oligonucleotide D, which is located downstream of transcription initiationsite -690 (Table 1). No cDNA amplification product was observed (Fig. 14).However, when PCR was performed using C, an oligonucleotide locatedimmediately 3’ to transcription initiation site -690, and oligonucleotide D (Table 1),amplification of a 1456 bp product of the expected size resulted (Fig. 14). No bandswere amplified from nonreverse transcribed human pituitary poly-A RNA as atemplate (Fig. 14).107Fig. 14. Analysis of the 5’ end of the human GnRH-R gene.A) PCR amplification of the 5’ nontranslated region of the human GnRH-R gene.Two ug of human pituitary poly-A RNA, reverse-transcribed (Pit) or nonreversetranscribed (C), were amplified by PCR using primers B + D (1) and C + D (2)for 35 cycles. Molecular size standard is indicated as MW. A product consistingof 1456 bp is indicated. B) Southern blot analysis using a PE-3 oligonucleotideprobe of the 1456 bp DNA fragment obtained through PCR analysis. Abbreviationsand markings are the same as for Fig. 14A.1082036bp—1 635bp—lOl8bp—MWC1C2PhiPit2—1456bpl456bpMW Cl C2 Pitl Pt2110Fig. 15. fi - Actin controls.Ethidium bromide stained gels showing DNA fragments of expected size (524 bp)generated through RT-PCR with f3-Actin derived primers. Molecular size standardis depicted by MW, P indicates pituitary cDNA as a template, and I representsintestinal cDNA as a template.111-‘ 1%)/3-ActinMWP1516bp4524bp3.8 Transcription factor binding sites (in addition to TATA and CAAT) andregulatory elementsThe Genetics Computer Group, Inc. “TF sites” file, with a database of 2,036recognition sites for transcription factors, was used to detect the presence ofpotential binding sites in the 5’ end of the human GnRH-R gene. The TF sitescomputer file targeted only those binding sites which were perfect matches to thehuman GnRH-R gene. Additionally, a BESTFIT comparison was conducted toidentify potential cis - acting regulatory elements.Using BESTFIT analysis, two putative regulatory elements were located. Aputative cAMP responsive element (CRE) (Roesler et al., 1988) was identified atposition -1490 to -1483 (Fig. 16). Analysis of the sequence for the CRE (5’-TGAAGTCT-3’) revealed that it was identical to the published consensus sequence(5’-TGACGTCA-3’) at six out of eight base pairs. A putative glucocorticoid /progesterone response element (GRE / PRE; 5’ -GTTACACAGTATTCT-3; Fig.16), with one base pair deviation from a functional GRE / PRE (Beato et al., 1989),was located at position -78 to -92.Consensus binding sites for some typical transcription factors (Faisst et al.,1131992) were also identified. Notably, those for GATA-1, WAP, engrailed protein(Ohkuma et al., 1990), PEA-3 (Xin et al., 1992), AP-1, and Pit-i were found (Fig.16).114Fig. 16. Cis - acting DNA regulatory sequences of the human GnRII-R gene.Sequences of gene regulatory protein bindng sites for the human GnRH receptor.The +1 position was assigned to the most 3’ located transcription initiation site.115—1595—CRE-like-1495 -[TGAAGTCT)—1395PEA-3-1295 [AGGAAG)PEA-3[AGGAAG]—1195—1095—995—895AP-1[TGAGTCAG]—795—695—595116—495 ——395 ——295 —PIT-i(ATGAATAA)engrailed-195 [AGATAACCTATAAGATA-1GRE/PRE-like-95 ---[GTTACACAGTATTCT)WAP6 —— [TTTAAA]exon I1173.9 Analysis of GRE I PRE - like and CRE-like sequences in the human GnRIIR geneComputer analysis of the human GnRH-R gene revealed the presence of twopotential regulatory elements which possess sequence similarity to a GRE I PRE anda CRE, respectively. The nucleotide sequence and position of these motifs areshown in Fig. 6. As well, all oligonucleotides used in this study are shown in Table2. The CRE - like sequence differed in two positions from the consensus CREwhereas the GRE/PRE - like sequence differed in one position from a functionalGRE I PRE (two positions from concensus sequence). Experiments involvingmobility shift assays were conducted in order to examine the binding of therespective proteins to these sequences.Nuclear extract from hormone - treated (E2; R5020) MCF-7 cells were usedas a source of progesterone receptors for analysis of GRE / PRE sequence - proteinbinding. CRE studies were performed with nuclear extract from HeLa cells(preliminary experiments; data not shown) and MCF-7 cells. Similar results wereobtained for either extract. Thereafter for ease and consistency, all gel shift assayswere performed with MCF-7 extract. Negative controls consisted of bindingreactions without extract. In addition, for the GRE I PRE - like motif, a double118point mutation of the consensus sequence also functioned as a negative control.Positive controls consisted of consensus sequences for both CRE and GRE / PRE.The results of gel shift assay experiments are shown in Fig. 17 and 18. RetardedDNA - protein complexes were observed for the positive controls as expected.CRE- and GRE I PRE- like sequences yielded no retardation complexes andtherefore failed to bind to their respective protein factors. As well, no binding ofproteins were observed for negative controls.119Fig. 17. Mobility shift assay of the CRE - like element.All gel shift assays were performed with 30,000 cpm of labelled oligonucleotide [1,2 (consensus CRE), and 3 (CRE - like)], 200 ng of poly (dl. dC) - (dl. dC) [1, 2, and3), and with (2 and 3] or without (1) nuclear extract. Bound protein complexes areindicated.120DNA-proteincomplexfree DNA probe123121Fig. 18. Mobility shift assay of the GRE I PRE - like element.All gel shift assays were performed with 30,000 cpm of labelled oligonucleotide (1,2 (GRE/PRE consensus), 3 (GRE/PRE - like), and 4 (GRE/PRE mutant), 200 ngof poly (dI.dC) - (dI.dC) (1, 2, 3, and 4), and with (2, 3, and 4) or without (1)nuclear extract.1221 2 34i DNA-proteincomplexfree DNAprobe1233.10 Analysis of the 3’ end of the human GnRH-R geneThe 3’ end of the human GnRH-R gene was contained within a 3.8 Kb EcoRIgene fragment. Configurational alignment of the 3.8 Kb clone within the humanGnRH-R gene structure is depicted in Fig. 10. A 3095 bp segment of the 3’ end ofthe gene was sequenced (Fig. 6). Five classical AATAAA polyadenylation signals(+3954, +4080, +4283, +4460, and +4767) compatible with the reported size ofthe human GnRH-R mRNA were identified. They are located entirely within an 800bp nucleotide region in a cluster - like format similar to the TATA boxes in the 5’end. Several ATTAAA sequences (+4212 and +4465), a functional variant of theclassical AATAAA signal were also identified. Additionally, several ATTTAmRNA instability motifs (+3841, +3983, +4178, +4263, and +4490) (Shaw andKamen, 1986) were found.PCR analysis was conducted with pituitary cDNA as a template in an attemptto localize the major polyadenylation site. Various primer combinations wereemployed in order to amplify the 3’ nontranslated region (Table 2). Oligonucleotidecombinations of E with G and F with H resulted in the amplification of products ofthe expected sizes of 1876 and 1176 bp, respectively (Fig. 19). Using nonreversetranscribed RNA as a template for all primer combinations employed, no DNA124Fig. 19. Analysis of the 3’ end of the human GnRII-R gene.A) PCR amplification of the 3’ nontranslated region of the human GnRH receptor.Two pg of human pituitary poly-A RNA, reverse-transcribed (Pit) or nonreversetranscribed (C), were amplified by PCR using pimers E + G and F + H (2) for 35cycles. Molecular size standard is indicated as MW. The correspondingamplification products of the expected sizes (1876 and 1176 bp) are indicated. B)and C) Southern blot analyses of the 1876 bp (B) and 1176 bp (C) productsgenerated through PCR amplification. (B) was probed with oligonucleotide I and(C) was probed with oligonucleotide J. All abbreviations are as in Fig. 19A.125MWClC2PitlPit2- r%) 0)2036bp—1635bp—lOl8bp——1876bp1176bp1876bpMW Cl 02 Pitl Pit2127MW Cl C2 Pitl P1t21176bp128fragments were amplified (Fig. 19A). Results obtained confirm transcription of theentire 3’ nontranslated region up to the polyadenylation signal at position + 4767.1293.11 Genomic Southern blot analysisGenomic Southern blot analysis was performed to detect the GnRH-R genedirectly from human genomic DNA. Genomic DNA from human placenta wasdigested with each of four restriction endonucleases: EcoRI, BamHI, Hindill, andPstT. Each digest generated multiple fragments that hybridized to the 760 bp humanGnRH-R cDNA probe (Fig. 20A). However, when this probe was segmented withPstI digestion to generate a 5’ oriented 396 bp probe (Fig. 20B) and a 3’ oriented364 bp probe (Fig. 20C), the number of DNA fragments decreased. As well, therewas a marked polarization of results consistent with the type of probe used. Forexample, three fragments (7.7, 7.4, and 3.8 Kb) hybridized to the 760 bp probe(Fig. 20A), only the 7.7 Kb fragment hybridized to the 396 bp probe (Fig. 20B),and the 7.4 and 3.8 Kb moieties hybridized to the 364 bp probe (Fig. 20C). Thehybridization pattern of the genomic Southern blot is in agreement with therestriction map obtained from the isolated clones and indicates that the gene for thehuman GnRH-R spans approximately 19 Kb and is present as a single copy in thehuman genome.130Fig. 20. Genomic Southern blot analysis of the human GnRII-R gene.Ten ,ttg of human genomic DNA was digested with each EcoRI, BamHI, Hindlil,and PstI and hybridized with either 760 bp (+904 to 1663; A), 396 bp (+904 to+ 1300; B) or 364 bp (+ 1300 to 1633; C) human GnRH-R cDNA probes.1313)ro0)01CD0IIIIVCa)Ca)0)IIICA)JOD0VI0)roII[0I3.12 Chromosomal assignmentThe human GnRH-R gene was assigned to a specific human chromosome byconducting somatic cell hybrid analysis. DNA isolated from 23 human - hamstersomatic hybrid cell lines representing coverage of all human chromosomes and theirparental cell lines (BIOS laboratories) were used to examine the presence of theGnRH-R gene by PCR using primers (E and I) specific for the human GnRHreceptor. Segregation of the human GnRH-R gene to a unique human chromosomewas based on the observable pattern of the human band in the various hybrids. Asshown in Fig. 21, PCR conducted on the cell line DNAs, electrophoresed on 1 %agarose, and stained with ethidium bromide, detected a major product of expectedsize (397 bp) from a human cell line and two hybrid cell lines, #852 (9) and #1079(22). However, this 397 bp product was not observed in the hamster cell line or therest of the hybrid cell lines. Analysis of the PCR results with the provided humanchromosome localization tabulation sheet identified chromosome 4 as the carrier ofthe GnRH-R gene (Table 4).135Fig. 21. Chromosomal assignment of the human GnRH-R gene to chromosome4.DNA from 25 human - hamster somatic hybrid cell lines and their parental cell lineswere amplified by 30 cycles of PCR using primers derived from the human GnRHreceptor sequence. A 397 bp product was obtained from two hybrid cell lines (nos.852 and 1079) and the human parental cell line, but not from the hamster parentalcell line or the other hybrid cell lines (representative cell line no. 983).136397 bp—*506/516394344(bp)137Table 5. Chromosome contents of human-hamster hybrid cell lines and theassignment of the human GnRB-R gene.The gene content of each hybrid was determined through PCR analysis. Thepresence [+J or absence[-J of the gene is indicated. [+] = chromosome present in atleast 70 %; [(+)j = chromosome present in 40-60 %; [-] = chromosome absent;[(-)] = chromosome present in 5-30 %; d=multiple deletions in 5q; D = deleted at5q15. l-5q15.2.138/f/iII)(..** • *—. . .. 4. .*a , , .+ . * . * . .., . . * 4. + 0 0 . * 4. * + 0 0 . . * * * . CH+ +1393.13 Northern blot analysis of the human GnRII-R mRNAThe human GnRH receptor possesses a mRNA which is expressed atrelatively low levels. Therefore, the use of poly-A RNA was incorporated in thestudy of human GnRH-R mRNA expression in the pituitary. Human pituitary poiy -A RNA was subjected to Northern analysis using a 364 bp human GnRH-R cDNAas a probe. An approximately 4.7 Kb transcript specific for the GnRH receptor wasdetected from 5 pg of poly-A RNA (Fig. 22). No signal was obtained from humanintestinal poly-A RNA which served as a negative control (Fig. 22).140Fig. 22. Northern blot analysis of human pituitary and intestinal RNA.Five pg of poly-A RNA from the pituitary (1) and intestine (2) were analyzed.Hybridization was performed with a 364 bp human GnRH-R eDNA probe (+1300to + 1663).141. 4.7kb12142IV. DISCUSSIONAt the outset, very little information regarding the gene for the GnRHreceptor was available. The isolation of the GnRH-R gene itself had not beenaccomplished in any species. I had at my disposal, however, a human GnRH-ReDNA clone which was isolated through cDNA library (pituitary) screening with arat GnRH-R cDNA probe (Peng et al., 1994). From this point, I had a basis fromwhich to begin my work on the isolation and characterization of the human GnRH-Rgene. The work presented here represents the first report on the isolation andcharacterization of the GnRH receptor gene and was published in 1994 (Fan et al.,1994). Additional information on the gene was reported subsequently in 1995 (Fanetal., 1995).Prior to the isolation of the human GnRH-R gene, various analytical studieswere first conducted. Among these studies, in particular, the copy number of theGnRH-R gene in the human genome was assessed. Genomic Southern blot analysiswas performed on restriction digested human genomic DNA. Evidence indicated abanding pattern that is consistent with the notion of a single copy gene encoding forthe GnRH receptor. Elaborating further on the digestion pattern obtained, a multiple143and complex banding of 3, 3, 4, and 4 was observed for each endonuclease, EcoRI,BaniHI, Hindill, and PstI, respectively, with human GnRH-R cDNA probing (760bp). All bands could be accounted for with additional probing with the 364 bp and396 bp cDNA probes and are in agreement with restriction mapping of the humanGnRH-R genomic clones which were later obtained. As well, the EcoRI genomicfragments as identified by genomic Southern analysis are identical to the 7.7, 7.4,and 3.8 Kb genomic clones obtained subsequently through genomic libraryscreening. The implications of these findings on the extrapituitary GIIRH receptorsthat have been identified are far reaching. These findings indicate that despite thetissue-specific differences of the GnRH receptor, the receptors for GriRH areencoded for by one gene. Therefore other control mechanisms at the level of thegene must be important for these tissue-related differences in the GnRH receptor.Chromosomal assignments studies were also conducted on the GnRFI receptor.Use of somatic cell hybrid analysis was successful in segregating the gene for theGnRH receptor to chromosome 4. The assignment of the human GnRH-R gene tochromosome 4 is identical with the localization of several other genes belonging tothe superfamily of G protein coupled receptors. These include the genes encodingfor a2-C4 adrenergic receptor (Kobilka et al., 1987), cholecystokinin receptor A (deWeerth et al., 1993), D5 dopamine receptor (Polymeropoulos et al., 1991; Eubanks144et al., 1992), endothelin-A receptor (Hosoda et al., 1992), and neuropeptide Y Y 1receptor (Herzog et al., 1993). Recent analysis on the subchromosomal localizationof the human GnRH-R gene was performed using DNA isolated from these studies(Leung et al., 1995). Using fluorescent in situ hybridization (FISH), the humanGnRH-R gene was mapped to 4q21 .2. Incorporating the use of a larger 15 Kb X-2genomic clone as a probe served to increase the precision of chromosomelocalization. Additionally, Kaiser et al., (Kaiser et a!., 1994) using somatic cellhybrid analysis have localized the GnRH-R gene to an adjacent band on humanchromosome 4q13. l-q21. 1. This work was confirmed by Morrison et al., (Morrisonet al., 1994) using a 1.9 Kb cDNA probe localizing the human GnRH-R gene to4q13.2-13.3. The mapping of the GnRH-R gene to its precise chromosomal siteshould facilitate further studies on the identification of closely linked genes. Aswell, investigations of possible chromosomal rearrangements involving the GnRH-Rgene and its consequent implications on fertility may be further studied.Studies focusing on the mRNA for the GnRH receptor were also conducted.Northern analysis performed on human pituitary poly-A RNA using a 364 bp humanGnRH-R cDNA probe identified a 4.7 Kb transcript. This size of mRNA for thehuman GnRH receptor was consistent with values of 4.7 and 5.0 Kb reported fromother laboratories (Kakar et al., 1992; Chi et al., 1992). Comparison of this data145to the sequence for the human GnRH-R cDNA (Kakar et aL, 1992; Chi et a!., 1992)revealed that the entire coding region (987 bp) for the receptor encompassed lessthan half of its mRNA, leaving ample sequence for 5’ and 3’ nontranslated regions.More importantly from these data, it became evident that a considerable proportionof the transcript was unaccounted for in the isolated cDNA clones of the GnRHreceptor. Moreover, from these data it was expected that the gene for the GnRHreceptor would likely be greater than 15 Kb.Isolation of the human GnRH-R gene through high stringency genomic libraryscreening proved my initial hypothesis regarding gene size correct. Based onSouthern analysis, restriction map analysis, and sequence data, the gene encodingfor the human GnRH receptor spans approximately 18.9 Kb.Sequencing of the entire coding region for the human GnRH receptor revealed100 % sequence identity with its corresponding cDNA sequence. Analyses revealedthat the gene structure for the human GnRH receptor consisted of a three exon- twointron motif. Two exon- intron splice sites were identified through sequencecomparison of genomic sequence to its cDNA counterpart as well as by using GT -AG consensus splice sequences. These findings classify the GnRH receptor asbelonging to the intron containing division of G protein coupled receptors. A146subsequent study on the mouse GnRH-R gene (Zhou and Sealfon, 1994)demonstrated identical findings of a three exon and two intron organization as wellas identical locales for splice junctions.Exon I of the human GnRH-R gene carries the 5’ nontranslated region andpart of the reading frame encompassing transmembrane domains I through III as wellas a portion of transmembrane domain IV. The second exon codes for theremainder of the IVth transmembrane domain together with the Vth transmembranedomain of the deduced amino acid sequence. The third exon for the human GnRHR gene encodes the carboxy - terminal part of the open reading frame and the 3’nontranslated region of the receptor. Of particular interest is the finding of twointrons present within the GnRH receptor gene, since many G protein coupledreceptor genes examined to date are intronless (Probst et al., 1992). The first intronwas determined to localize within the IVth transmembrane domain and the secondintron was assigned to the third intracellular loop of the deduced amino acidsequence. Among the genes encoding for G protein coupled receptors, introns (ifpresent) generally tend to be positioned between the transmembrane domains.Notwithstanding, there are several known exceptions to this as exemplified by thehuman rhodopsin and opsin genes (Nathans et al., 1984, 1986) and the humanserotonin (5-HT2) receptor gene (Chen et al., 1992). Interestingly, in terms of its147structure, the GnRH-R gene greatly resembles the human 5-HT2 receptor in severalrespects. Both possess a three exon- two intron gene structure and a gene size of20 Kb. Additionally, both receptors exhibit a major transcript of 5 Kb.High sequence identity among species for coding regions of the GnRHreceptors existed. The coding region of human shared greater than 85 % sequenceidentity with mouse, rat, sheep, cow, and pig. Notably, the human GnRH receptorwas encoded for by 328 amino acids which was identical to that of sheep, cow, andpig, but in contrast to the 327 amino acids described for both mouse and rat.Comparisons at the amino acid level, however, revealed similar results of greaterthan 85 % identity between human and other species. However, higher identity(>90 %) existed at the regions of the transmembrane domains. In particular,transmembrane domains II, III, V, VI, and VII are the most highly conserved.Regions of high conservation also existed in extracellular loop 1.Interestingly, the deduced amino acid sequence revealed that the human GnRHreceptor possessed several distinguishing features. Among these include the absenceof a carboxy - terminal tail which has been previously described for the mouse andrat receptors (similar case subsequently reported for cow, sheep, and pig). Thisregion has been linked to the coupling of G proteins, receptor desensitization, and148internalization (0’ Dowd et al., 1988; Dohlman et al., 1991) in several G proteinreceptors. Notable exceptions include the f3-adrenergic receptor which does notinternalize (Hertel et al., 1990) despite the presence of a carboxy tail. As for thecase of the GnRH receptor, it seems likely that throughout evolution the receptor hasdelegated those functions specific for the carboxy tail to other regions or that thereexists a level of functional redundancy within the receptor. In support of this, anumber of potential PKC phosphorylation sites, suggested to play a role in receptordesensitization, are present in transmembrane domains I and III for the human GnRHreceptor.The human GnRH receptor also did not retain the characteristic Asp-Arg-Tyrtripeptide motif of the G protein receptors. Instead, the receptor for GnRHsubstitutes a serine for the conserved tyrosine at position 140 thereby creatinganother potential phosphorylation site. In accord with these differences, the humanGnRH receptor contained a reciprocal interchange of an asparagine at position 87(other receptors; aspartate) and an aspartate (other receptors; asparagine) at position318. This situation has also been described for all other GnRH receptors isolatedto date. This amino acid switch is reportedly necessary for GnRH receptor - ligandbinding (maintenance of ligand- binding conformation) as well as for signaltransduction from the ligand - binding site to the regions involved in G protein149activation (Sealfon et al., 1993). It is interesting that these switches are necessaryfor the GnRH receptor when in other receptors the conservation of aspartate 87 andasparagine 318 are necessary for normal agonist binding and G protein coupling.In addition, two potential glycosylation sites at asparagines, position 18 and 102,have also been identified from the deduced amino acid sequence for the humanGnRH receptor. These glycosylation sites have been implicated to be important fornormal receptor expression and plasma membrane localization (Sealfon et al., 1993).The GnRH receptor distinguishes itself from other G protein coupled receptorsin other ways as well. In particular, the GnRH receptor possesses a long and highlybasic first intracellular loop uncharacteristic of the G protein receptor family. It alsopossesses a seventh transmembrane domain which has an unusually highphenylalanine content.Despite these unique differences, comparison of the human GnRH receptoramino acid sequence to other G protein coupled receptors also demonstratedevidence of similarity. The human GnRH receptor shares a number of amino acidswhich are highly conserved among the G protein receptors (Savarese and Fraser,1992; Baldwin et al., 1993). Most of these are located within the transmembranedomains and are likely to be constituents of the ligand binding pocket of the150receptor. Amino acids such as proline in transmembrane domains II, IV, V, VI, andVII are highly conserved. In addition, other conserved residues include asparagineat positions 53 and 315, tryptophan at positon 164, serine at position 167, andtyrosine at position 323. A highly conserved sequence of the G protein receptors,phenylalanine-X-X-cysteine-tryptophan-X-proline-tyrosine, is found intransmembrane domain VI. Additionally in the human GnRFI receptor, there aretwo cysteine residues located in extracellular loop 1 and 2 which have also beenfound to be highly conserved among G protein receptors. Many of these aminoacids mentioned above have suggested importance in either maintaining the structure(disulphide bridges, kinking of a-helices, etc.) or function (coupling to G protein,signal transduction) of G protein receptors suggesting why they are so highlyconserved (Zhang and Weinstein, 1993).The remarkable conservation of the transmembrane domains between theGnRH receptor and the other G protein coupled receptors suggests that these genesmay have evolved from a common precursor more than 1 billion years ago (Kleinet al., 1988). Mechanisms of convergent evolution that appear to have played a rolein generating the multiplicity of the G protein receptor family involve geneduplication (Ohno, 1970) and retroposition (Brosius, 1991).151In accord with this, the high conservation of amino acids seen among theGnRH receptors of several species also suggests that the receptor itself performs acrucial function in reproduction of the organism.Once the coding region for the human GnRH-R gene was sequenced andanalyzed, continued sequencing and analysis of both the 5’ and 3’ ends followed.Points of focus included determining the localization of the human GnRH-R gene’spromoter and control region. As well, analysis of the 3’ end of the gene wasundertaken to answer the question of polyadenylation and possible differential RNAprocessing.4.1 Analysis of the 5’ end of the human GnRH-R geneAs previously mentioned, a lengthy 5’ nontranslated region was anticipated.Therefore, sequencing was performed for greater than 1.5 Kb upstream from thetranslational methionine start site. Analysis of this sequence for a specific DNAsequence(s) which contained the start site for RNA synthesis and signals where RNAsynthesis should begin ensued. This sequence of interest, collectively called thepromoter, has been identified in many genes by an A - T rich consensus region or152a G - C rich island. Genes possessing TATA promoters are generally associatedwith more regulated genes. Those that have G - C rich promoters have traditionallybeen linked to housekeeping genes. As for the human GnRH-R gene, five A - Trich regions described as TATAAA consensus sequences were identified within the1.5 Kb stretch of 5’ sequence. TATAAA sequences are important in binding TF lIDwhich in turn directs the correct initiation of RNA polymerase II for RNA synthesis.Additionally, several CAAT motifs were also located within this 5’ sequence.CAAT sequences are located in most eukaryotic promoter regions at positions -50to -129 (from the transcriptional start) and are required for efficient transcription(Benoist et al., 1980). The CAAT motifs found within the human GnRH-R genepromoter region were located well within this range and found to cluster around therespective TATA sequences.Many genes in the literature have been characterized as having only onepromoter. However the finding of multiple TATA boxes within a single gene is notuncommon (Srilcantha et al., 1990; Kawamura et al., 1992). What is unusual is thepresence of consensus TATA boxes among the G protein coupled receptorssequenced to date. Instead, many of these genes have been identified as containingG - C rich promoter regions, including the genes for the mouse follicle stimulatinghormone receptor (Huhtaniemi et al., 1992), human thyrotropin releasing hormone153receptor (Gross et al., 1991), and the human endothelin-A receptor (Hosoda et al.,1992). Of special note is the interesting dichotomy that exists in the rat luteinizinghormone receptor (Tsai-Morris et al., 1991). This receptor possesses a “mixedpromoter”; several TATA boxes as well as G - C rich regions and, therefore,partially places itself along with the human GnRH receptor into the TATA -containing group of G protein coupled receptors.It should be noted however that relatively few G protein coupled receptorpromoter regions have been sequenced. Therefore whether or not the absence orpresence of TATA boxes in the promoter region constitutes an identifyingcharacteristic of the majority of this receptor family remains to be further clarified.Nevertheless, the finding of multiple putative promoters for the human GnRHR gene reveal several exciting possibilities. First, this finding suggests that thecontrol region for the human GnRH-R gene is complex and highly regulated whichis in accordance with the many detailed regulatory data that have been widelyreported. More importantly, the existence of multiple promoters suggests thealternative use of promoters, possibly in a tissue - specific manner. Tissue- specificuse of promoters found in the human GnRH-R gene may explain the differencesexhibited between the receptors in different tissues.154The finding of consensus TATA sequences in the 5’ end of the human GnRHR gene was one step forward in establishing that the entire gene, including the 5’regulatory region for the human GnRH receptor, had. been isolated. Furtherevidence came in the form of primer extension studies which defined the 5’ terminifor the human GnRH-R gene.All five transcription initiation sites found for the human GnRH-R gene werepositioned in close proximity to the TATA boxes. The compact and clusteringnature of both the TATA sequences and the transcription initiation sites madeassigning TATA boxes to their respective initiation sites a complex task. However,based on conforming spatial standards, TATA boxes at position -14, -111, and -150were found to be related to transcription initiation sites +1, -101, and -124,respectively. As well, TATA boxes positioned at -718 and / or -731 are likely tobe responsible for transcription initiation sites -669 and / or -690. Three out of fivetranscripts initiated from a cytosine base (+1, -101, and -669), while the remainderstarted from a thymine (-124) and an adenosine (-690). It has been observed thatmany but not all eukaryotic gene transcriptions initiate from an A residue (or anotherpurine) surrounded by a pyrimidine rich region. This is true for transcriptioninitiation site -690 which initiates from an adenosine. For the four other mRNA capsites, they are pyrimidines and do not seem to follow this generalization. However,155it is probable that the A I G nucleotides positioned immediately next to thesepyrimidines are the true sites of transcription imtiation. This error in measurement(± 1 nucleotide) is likely due to using a single stranded template as a ladder ratherthan a double stranded one.Major sites of transcription initiation for the human GIiRH-R gene were alsodocumented. Notable variability in the intensity of transcription initiation signalswere obtained through primer extension analysis. These differences were reflectiveof the message level for the human GnRH receptor. Transcription initiation sites+ 1, -101, and -669 were accessed as major start sites of transcription for the humanGnRH-R gene as evidenced by higher intensity levels. Transcription initiation -669was deemed the major start site based on band intensity from primer extensionresults and on supporting RT-PCR data and mRNA size. Notably weaker signalslocated at -690 and -124 were classified as minor sites of transcription initiation.The finding of multiple transcription initiation sites for the human GnRH-RmRNA brings forth the possibility that alternative transcription initiation sites areutilized in the pituitary, as well as, at extrapituitary sites and thus may in partaccount for some of the complexities observed regarding the regulation of expressionof this gene.156Further confirmation of the primer extension results came in the form of RT -PCR analysis. Secondary in nature, these findings also served to confirm theisolation of the human GnRH-R gene in its entirety as well as the complete structureof the human GnRH-R gene.PCR data confirmed transcription of the immediate 3’ sequence fromtranscription initiation site -690 through to the coding region of the human GnRH-Rgene. Identification of a 1393 bp 5’ nontranslated region corresponding totranscription initiation site -690 is consistent with the size of the human GnRH-RmRNA of approximately 4.7 Kb as well as with reported PCR findings of Chi et al.,(Chi et al., 1993) which predicted a size of at least 1.3 Kb of additional 5’nontranslated region (from the translational start) for the human GnRH receptor.Transcripts possessing extensive 5’ nontranslated regions such as is the case for thehuman GnRH-R gene are not uncommon among vertebrates in general. Examplesamong others include those for human GnRH (Seeburg and Adelman, 1984) andhuman preproenkephalin B (Horikawa et al., 1983). Further to the RT-PCR data,the finding of no amplifiable product upstream of transcription initiation site -690,excluded the possibility of additional 5’ initiation sites. Taken together, these datasupport the primer extension findings mentioned above.157It seems likely that the human GnRH-R gene is highly regulated at thetranscriptional level as revealed through the discovery of multiple initiation sites andputative promoters for this gene. More recent reports have suggested that anothercontrol switch, at the level of translation, may play a larger role in the regulation ofgenes than previously ascribed. This possibility of additional regulatory control inexistence for the human GnRH-R gene was supported by several lines of evidence.First, the presence of an extensive 5’ nontranslated region supports the notion ofadditional controls. In vitro studies have shown that a longer 5’ nontranslated regionincreases the efficiency of translation over that of a shorter one and may benecessary for the efficient initiation of translation in some genes (Kozak, 1991).Second, the presence of several ATG (AUG) codons upstream of the majorreading frame for the human GnRH-R gene also points toward additionaltranslational controls in effect. Of the vertebrate mRNAs analyzed to date, fewerthan 10 % have been found to contain upstream initiation codons (Geballe andMorris, 1994). Interestingly, many of these mRNAs were identified as those havinga fundamental role in cell proliferation and development. In particular, mRNAsspecific for proto - oncogenes, growth factors, and cell-surface receptors were ofspecial note.158It is therefore of interest when several ATG codons were located upstream ofthe reading frame for the human GnRH receptor since it is almost always from thefirst ATG codon that translation initiates. Examination of the flanking sequencesaround the upstream ATGs indicated poor sequence identity to conserved initiationmotifs. These data support the possibility of ribosomal ATG skipping, that isribosomes scan sequence for an optimal ATG match from which translation isinitiated. This somewhat rarely studied event has been observed in other genes(Geballe and Morris, 1994).Collectively, these data support the existence of potential translational controlsin operation. Detailed studies focusing on the translational control of GnRHreceptor expression and on the potential regulators themselves may be necessary inorder to fully understand the underlying mechanisms involved in GnRH receptorregulation.In addition to identifying several upstream ATGs, the human GnRH-R genewas evaluated for potential cis - acting regulatory sequences in an attempt tounderstand the receptor’s regulation at the genomic level.Physiological paradigms of GriRH receptor regulation abound in rat and sheep159models studied thus far. Similar detailed studies concerning the regulation of thehuman GnRII receptor are still lacking. Nevertheless in animal models, severalparallel observations can be drawn. Various regulators such as GnRH, 17f3-estradiol, progesterone, inhibin, and cAMP play a role in the regulation of thepituitary GnRH receptor (Miller et al., 1993; Sealfon et al., 1990; Laws et al.,1990b). The 5’ end of the human GnRH-R gene was therefore evaluated for bindingsites which have been demonstrated to confer responsiveness.No similar sequence to an estrogen responsive element (ERE) could be foundusing BESTFIT or TF SITES analysis. This was somewhat unexpected since severalregulatory studies in rat and sheep have demonstrated estradiol mediated up-regulation of GnRH receptors. However, these findings do not rule out that anERE may be found beyond the 5’ and 3’ sequence obtained or within the intronicregions of the gene. These data do suggest, more importantly, that estrogen mayhave a complex indirect role in the regulation of the GnRH receptor, such as, inactivating other genes whose products in turn trigger the delayed secondary responsein receptor expression.Further searches for potential responsive elements were successful however.Two responsive elements of particular interest were identified after computer160analysis. These two sequences were identified using BESTFIT comparisons andconsequently were not perfect matches to their respective consensus sequences. Inaddition, these two sequences were not identifiable using the “perfect match” TFSITES file. An element with sequence similarity to a CRE was located at -1490 to -1483. The consensus CRE sequence of 8 bp has also been identified in theregulatory region of other genes that are activated by cAMP. Further, this sequenceis recognized by a specific gene regulatory protein called the CRE - binding (CREB)protein. CREB is phosphorylated by protein kinase A in response to increasedcAMP levels which in turn stimulate transcription of these genes. The finding ofa CRE - like sequence supports regulatory studies which have shown cAMP (oranalog) involvement in mimicking GnRH mediated GnRH receptor up - regulation(Young et al., 1984) and suggests a direct stimulatory pathway for cAMP.Additionally, a 15 bp GRE I PRE - like sequence (-78 to -92) was alsoidentified within the 5’ end of the human GnRH-R gene using BESTFIT analysis.The consensus sequences for the GRE and PRE are identical. They are composedof two half site sequences of 6 bp separated by a variable region of 3 bp and bindto glucocorticoid and progesterone receptors (GR; PR). Regulatory studies in ratand sheep pituitary cell cultures have identified progesterone as a regulator of GnRHreceptor expression. Reports have indicated progesterone action as inhibitory on the161receptor’s expression (Laws et al., 1990b; Sealfon et al., 1990) while others haveestablished a stimulatory role for progesterone (Miller et al., 1993). A search fornegative response elements revealed limited information regarding these sequences.There is a total of 11 such elements published for the glucocorticoid receptor(Akerbiom and Mellon, 1991). These elements were searched for throughout thehuman GnRH-R gene and no observable sequence similarity existed.It should be noted as well that the human GnRH-R gene was also searched forpotential GRE I PREs using the “perfect match” TF SITES computer analysis. Thisanalysis identified 4 isolated and identically matching GRE/PRE half sites within thereceptor gene sequence. However the functional importance of isolated half sites isdoubtful. Genes which have been identified as having a single response elementhalf site have never demonstrated their involvement in gene regulatory control.Further, for genes with well isolated multiple half sites, data on their involvementin gene regulation have never been reported as well.It is evident that controversy still abounds regarding the regulatory effects ofpotential regulators. Whether these regulators have primary or secondary responsesin gene expression remains to be seen. However, it is hoped that the findings ofthese regulatory elements may facilitate the clarification of some of these162discrepancies. Initial steps towards this clarification were undertaken in subsequentstudies described below.Mobility shift assays were employed in order to examine potential CRE andGRE I PRE binding activity. This DNA - protein binding assay served to answerthe question of CRE and GRE I PRE function in vitro.Before these experiments could be performed, the first task was to obtainsufficient quantities of the CREB protein and the progesterone receptor. After adetailed review of the literature, HeLa cells and MCF-7 cells were chosen as sourcesfor these proteins. MCF-7 cells were pre-treated with estradiol in order to increasethe progesterone receptor levels by approximately five - fold and consequentlyincrease the efficiency of nuclear extract. Nuclear extract prepared from both celllines yielded similar results for CRE experiments indicating sufficient concentrationsof CREB in MCF-7 cells as compared to HeLa cells. This was not surprising sincereports have indicated cAMP involvement in the regulation of various genes in thesecell lines. Thus, all remaining experiments were conducted with MCF-7 as a sourcefor both CREB and progesterone receptor proteins. Once the initial steps wereaccomplished in these DNA - protein binding assays, experiments proceeded withthe actual testing of these regulatory elements.163As mentioned above, progesterone and cAMP have been implicated in theregulatory control of the GnRH receptor and the findings of these regulatoryelements at first seem to support these regulatory data. However, once mobilityshift assays were performed on both the CRE - like and GRE / PRE - likesequences, no binding of protein to these elements was found. The absence ofprotein- DNA complexes indicated that the CRE and GRE / PRE elements foundwithin the human GnRH-R gene were functionally void. These findings were notsurprising however based on the nucleotide alterations which these elementspossessed.Two mismatches out of a total of 8 bp, as demonstrated through gel shiftexperiments, represented a significant degree of dissimilarity for the CRE - likeelement. This amount of variability could not be compensated due to the inabilityof the CREB protein to recognize this sequence as a true CRE. In addition, thissuggests that the two mismatches of a “C” to an “A” and an “A” to a “T” arecrucial to CREB protein binding. Also, these data imply that cAMP either actsindirectly in the control of the human GnRH-R gene or a functional CRE may belocated elsewhere within the gene.Gel-shift assays also demonstrated no protein-DNA complex for the human164GnRH-R GRE / PRE related sequence. Detailed examination, of the base pairchanges for the GRE I PRE - like sequences found in the human GnRH-R gene,revealed insights as to why this sequence was shown to be nonfunctional in DNA -binding assays.The base pairs of crucial importance in GRE I PRE elements have beendefined (Beato et al. ,1993). At position 1 in the half site (Table. 2), the presenceof a thymine (T) base is necessary for glucocorticoid receptor (GR) and progesteronereceptor (PR) binding. The 5’ methyl group of the T base has been implicated asthe receptor contact site. Modification of this base has been shown to interfere withthe binding of GR (Cairns et al., 1991). Further, a T base residing in position 3,has been shown to be essential for discrimination of GRE / PRE sequences. Thebase at the fourth position is the only site not contacted by the receptor.Nonetheless, the preferred base in this position is still a pyrimidine. The fifthposition is occupied by a cytosine (C) and has been shown to be very important forbinding of the GR and PR. This site has been identified as the most highlyconserved position in GRE / PRE sequences isolated to date. The sixth and lastposition of the half site is occupied by a thymine in 60 % of all GRE/PREsequences, although mutation to a C base does not interfere with GR or PR binding.165All positions mentioned above are occupied by the requisite base in the humanGnRH-R GRE / PRE related sequence. However, the one position that is notconserved within the human GnRH-R GRE I PRE - like element is the guanine (G)base at position 2. Instead an adenosine is found in this position for the humanGnRH-R gene. Contact of the GR and PR (even the estrogen receptor) to theguanine at the second position of the half - site is achieved by interaction with theN-7 position as demonstrated by methylation experiments (Scheidereit and Beato,1984; Truss et a!., 1991). This highly conserved G base is of paramount importanceto a functional GRE / PRE since it is the essential component involved in the highaffinity binding of receptors to their hormone response elements. This base has beensuggested to be the contact site for an amino acid side chain, such as arginine 489,arginine 496 or arginine 466 in rat GR, which is conserved throughout the steroidhormone receptors. Therefore it is concluded that this G to A base switch in thehuman GnRH-R GRE / PRE, is the underlying reason why this steroid response -like element is nonfunctional in the human GnRH-R gene.Although, these data have supported mainly indirect regulatory pathways forGnRH receptor expression in humans, it should be remembered that the regulatorystudies at the physiological and mRNA level for the pituitary have been performedin mainly animal models only. Therefore the possibility of different regulatory166responses for cAMP, progesterone, and other hormone regulators in the human cannot be excluded. In accord with this, many genes have been reported to possessvery different responses in human tissues than in other animal models.In addition to investigating potential regulatory elements, consensus bindingsites for some typical transcription factors known to regulate eukaryotic promoterswere also examined using TF SITES analysis. As mentioned, this computer analysisidentifies only those sequences within the human GnRH-R gene which are perfectmatches to the transcription factor binding sites located within the database.Therefore all sites identified are most likely active within the human GnRH-R gene.A GATA- 1 binding site was found in close proximity to TATA -150 and maylikely conthbute to its regulation. The engrailed binding site was found to overlapwith TATA -150. The engrailed protein has been shown in in vitro studies tonegatively regulate genes by competing with transcription factor lID for bindingsites. This transcription factor may have a similar role in the regulatory control ofthe human GnRH-R gene. Additionally, several PEA-3 binding sites (Xin et al.,1992) were located upstream of TATA -731 and -718. Of special note, the bindingsite for PEA-3 has also been recognized to function as a phorbol ester binding sitein some genes. This finding is in accordance with several regulatory studies which167have shown a role for phorbol esters in the regulation of the human GnRH receptor.A binding site for WAP (whey acidic protein; Lubon and Hemiighausen, 1987), amammary gland specific protein, was also found within the promoter region.Interestingly, an AP-i binding site, which would confer protein kinase Cresponsiveness, was located in close proximity to TATA box -731 and -718. TheseTATA boxes are the suggested control region for the major 4.7 kb transcript of thehuman GiiRH receptor. These data suggest that AP-i may have a potential role inthe regulation of the GnRH receptor.Of special interest is the identification of a Pit-i binding site (Faisst et al.,1992) within the receptor sequence. Pit-i, an anterior pituitary specific transcriptionfactor, has been reported to be involved in the regulation of anterior pituitaryhormones, such as, in the activation of the growth hormone (Bodner et a!., 1988)and prolactin (Ingraham et al., 1990) genes. Similarly, Pit-i transcription factormay have possible implications on the regulation of the anterior pituitary GIIRHreceptors through activation of the human GnRH-R gene.Further analysis of the human GnRH-R gene sequence revealed the presenceof a truncated (150 bp) Alu repetitive element (Fig. 5; position -1300 to -1150;Kariya et al., 1987). Alu repeats are transposable elements, generally 300 bp in168size, and consist of two alternating monomer units. Alu sequences are able to moveand create target - site duplications when it inserts. Alu repeats are characteristicallypresent in primate species and are remarkably abundant within the genome.Approximately 500,000 repeats are present per haploid human genome constituting5 % of human DNA. In other words, Alu repeats are present on average about onceevery 5,000 nucleotide pairs.Functionally speaking, it is unclear as to what Alu sequences do. It has beensuggested that they are derived from retroposition of an internally deleted host - cellRNA gene, 7SL, which encodes for the RNA component of the signal-recognitionparticle (SRP) that functions in protein synthesis (Chen et al., 1985). As well, Alusequences have been implicated as start sites in DNA replication (Ariga, 1984),inhibitors of gene conversion (Hess et al., 1983), hot areas for recombination(Rogers, 1985), modulators of chromatin structure (Duncan et al., 1981), and asstabilizing sequences for cytoplasmic mRNA (Robertson and Dickson, 1984).Comparisons of the Alu sequence in the human GnRH-R gene to other Alu -like sequences of other genes indicate notable sequence similarity. In addition,based on species distribution and sequence similarity, evidence suggests that theserepetitive sequences have multiplied to high copy relatively recently (Deininger and169Daniels, 1986). It is tempting to speculate that these highly scattered and abundantsequences in the genome have had major effects on the expression of many nearbygenes, including that for the human GnRH receptor.4.2 Analysis of the 3’ end of the human GnRH-R geneOnce the analysis of the 5’ end of the GnRH-R gene had been accomplished,efforts were focused on examining the 3’ sequence. It was hoped that the 3’ endwould reveal some clues as to how the GnRH-R gene’s complex regulatorymechanisms might be achieved.The 3’ sequence revealed a complex pattern of multiple polyadenylationsignals much like the situation found for the TATA boxes and initiation sites at the5’ end of the human GnRH-R gene. Five polyadenylation signals were of theclassical AATAAA type found for eukaryotic genes. However, two additionalpolyadenylation signals possessed the sequence ATTAAA which has been shown tobe a functional variant of the classical termination signal. The impact of thesefindings suggested that there were potentially seven sites for 3’ termination. Furthersupport came in the finding of several ATTTA motifs (Shaw and Kamen, 1986),170which have been implicated in mRNA instability and are notably present in manyrapidly degraded mRNAs, suggesting that the site of transcriptional termination iscontained within this 3’ sequence obtained.However, without a full length human GnRH-R eDNA being available, themajor polyadenylation site remains unknown. In order to delineate the most 3’polyadenylation site used in the pituitary, PCR analysis of the 3’ end with pituitarycDNA was conducted. PCR amplification of the entire 3’ sequence up to thepolyadenylation signal at position + 4767 was successful. Several controlsimplemented excluded any confounding factors which may have compromised theseresults.Summing up the figures of approximately 1.4 Kb of 5’ nontranslated (most5’), 3.1 Kb of 3’ nontranslated, and 1 Kb of coding region brings the amount ofmRNA accounted for at 5.5 Kb. These values corroborate well with the size of thehuman GnRH-R mRNA of 4.7 Kb including 200 to 400 bp ailotted for a poly-A tail.Therefore these findings strongly indicate that the functional polyadenylation site forthis transcript is located well within the 3’ nontranslated region sequenced.Additionally, the finding of multiple polyadenylation signals is of significant171value, particularly for its potential impact on the expression pattern of the humanGnRH receptor. The likelihood of differential RNA processing, occurring throughthe use of alternative cleavage / polyadenylation signals and consequently theexistence of multiple human GnRH-R transcripts, must be considered in light ofthese present findings. Support for the potential use of alternative terminationsignals (and I or alternative promoters and initiation sites) in the regulation of thehuman GnRH receptor came in 1994 with the finding of minor transcripts of sizes2.5 and 1.5 Kb in the pituitary (Kakar et al., 1994). From the Northern datapresented here, polyadenylation signal + 4767 is likely to be responsible for themajor transcript of 4.7 Kb. As for the minor transcripts, examination of the genesequence reveals an additional termination signal located at + 2595, which in lightof the recent data, may likely be responsible for the smaller messages based onspatial location. It is highly doubtful that the 1.5 Kb transcript encodes for afunctional receptor and it is speculated that this message is a truncated transcript.Overall, the analysis of the human GnRH-R gene revealed that structurally,it possessed the characteristics of a highly complex and regulated gene. Discoveriesof multiple putative promoters, initiation sites, and polyadenylation signals elude tointricate regulatory mechanisms in operation. Any one of which could beattributable to its multi - factorial regulation.172One further analysis of the human GnRH-R gene was performed in an attemptto elucidate any closely related sequences to the GnRH-R gene. Overall analysis ofthe gene for the human GnRH receptor revealed several interesting discoveries.Comparison to the other 75 G protein coupled receptors isolated to date revealedlittle sequence similarity. This was of particular interest considering that a commonorigin for this receptor family has been predicted. Relatively little sequencesimilarity suggests that considerable divergence has occurred throughout evolutionor perhaps the single precursor theory should be expanded to include theinvolvement of several evolutionary precursors.Some sequence similarity existed, however, between the human GnRHreceptor and a few G protein receptors. These included the human oxytocin receptor(Kimura et al., 1992), the human V2 vasopressin receptor (Birnbaumer et al., 1992),the rat V2 vasopressin receptor (Lolait et al., 1992), and the rat ViA vasopressinreceptor (Morel et al., 1992) which all shared approximately 25 % identity to thehuman GriRH receptor.Interestingly, the GnRH receptors in general have no significant sequenceidentity to the yeast STE2 receptors (Burkholder and Hartwell, 1985; Marsh andHerkowitz, 1988), except to a minor degree in the transmembrane domains. The173ligand for the STE2 receptor is a-factor, a yeast pheromone, that has close sequencesimilarity to GnRH in its amino - terminus and is known to bind to GnRH receptors.In addition to its structural similarities, the a-factor and GnRH also share functionalcommonality. In rat pituitary gonadotropes, a-factor has been demonstrated tostimulate the release of gonadotropins, an action that was blocked by GnRHantagonists (Loumaye and Catt, 1982). However, despite these structural andfunctional similarities between a-factor and GriRH, no significant sequence identityis observed between their respective receptors.174V. SUMMARY AND CONCLUSIONSThis investigation has been concerned with the isolation of the human GnRHR gene and its consequent characterization at the genomic level.The major procedures employed during this thesis are summarized as follows:1) High stringency genomic library screening with a human GnRH-R cDNA probewas conducted. 2) Southern blot and restriction analyses of genomic clones wereperformed in order to confirm the authenticity of the clones as being specific for thehuman GnRH-R gene. 3) Subcloning of the 7.7 Kb, 7.4 Kb, 3.8 Kb, and 2.7 Kbgenomic fragments into pUC19 vector was performed. Dideoxy DNA sequencingof the isolated subclones was performed using universal forward and reverse primersand sequence specific oligonucleotides. All sequence analyses were conducted usingGCG Computer Group Inc. software. 4) Characterization of the 5’ terminus for thehuman GnRH-R gene was carried out using primer extension analysis, RT-PCRanalysis, and Southern analysis. 5) MCF-7 cell lines were cultured to confluencyas a source of PR and CREB proteins. 6) DNA - protein binding studies wereperformed using gel mobility shift assays. 7) Southern blot and RT-PCR analyseswere used to confirm both the structure and the 3’ terminus of the human GnR}{-R175gene. 8) Genomic Southern blot analysis was performed in order to detect thehuman GriRH-R gene directly from chromosomal DNA. 9) Somatic - cell hybridanalysis was employed in the segregation of the human GnRH-R gene to a uniquechromosome in the genome. 10) Northern blot analysis was employed in order toinvestigate the number and size of mRNA transcripts encoding for the human GnRHreceptor.The literature review dealt with the general aspects of GnRH receptors,including function, distribution, and regulation. The results of this thesis werepresented, illustrated, and discussed. It was found in this investigation that:1. Genomic library experiments resulted in the isolation of twelve positiveclones for the human GnRH-R gene after tertiary screening.2. After purification of X-clones (1 and 2) to homogeneity, a 5’ or 3’ orientedX-DNA was identified and confirmed by Southern analysis.3. Restriction endonuclease digestions revealed that X- 1 contained a 5’oriented 7.7 Kb EcoRI genomic fragment of interest. Also it was observedthat X-2 contained 3’ oriented EcoRI fragments of 7.4 Kb and 3.8 Kb insize.4. Nucleotide sequencing revealed that the entire gene for the human GnRH176R gene was contained within these isolated genomic clones. Also throughsequencing, the coding region was found to be identical to the subsequentlypublished human cDNA as well as exhibit >85 % sequence identity to otherspecies.5. Structurally, the human GnRH-R gene was demonstrated to possess threeexon and two introns distributed over a span of 18.9 Kb. Exon I, II, and IIIconsisted of 1915 bp, 219 bp, and 3321 bp, respectively. Intron A and Bconsisted of approximately 4.2 Kb and 5.0 Kb, respectively. The codingregion, 5’ nontranslated, and 3’ nontranslated were disthbuted over 987 bp,— 1.4 Kb, and —, 3.1 Kb, respectively.6. Five consensus TATA sequences were revealed in the control region ofthe human GnRH-R gene through sequencing.7. Five transcription initation sites spatially related to the TATA sequenceswere also identified by primer extension analysis.8. Consensus transcription factor binding sites were located within the 5’ endof the gene.9. A CRE - and GRE / PRE - like sequences were identified as potentialregulatory elements for the human GnRH-R gene.10. Mobility shift assays demonstrated the absence of protein binding to theseregulatory elements.17711. Five classical polyadenylation sites in addition to several ATTTA motifswere found within the 3’ end of the human GnRH-R gene.12. Genomic Southern analysis identified that a single gene encodes for thehuman GnRH receptor in the genome.13. Chromosomal localization studies assigned the GnRH-R gene to humanchromosome 4.14. Northern analysis identified a single major transcript of 4.7 Kb for thehuman GnRH-R gene.Prior to this work, molecular characterization of the GnRH receptor waslimited to the cDNA level. Similar studies on the gene had not been accomplished.In addition, the reported cDNA sequences represented only a small fragment of theGnRH receptor. Therefore this left several inherent and unique properties of theGnRH receptor unknown. Questions concerning its regulation, promoter, andcontrol region as well as its 3’ termination region, structural organization, andlocation in the genome remained unanswered. Experiments of this investigationserved to answer these questions in addition to others which could not beinvestigated by analysis of the cDNA alone but rather required the detailed study ofthe gene itself.178In summary, the experiments of this thesis demonstrated for the first time, theisolation and characterization of the GriRH-R gene in any species. The implicationsof the human GnRH-R gene being single copy in nature suggests that the tissue -specific differences observed for some GnRH receptors are likely attributable to fineregulatory controls at the genomic level. This hypothesis was supported by thefinding of an extensive 5’ leader sequence, multiple promoters, initiation sites, andpolyadenylation signals for the human GaRH-R gene. Structurally complex 5’ and3’ control regions as demonstrated in these present studies are consistent with themulti - factorial regulation of the GnRH receptor previously described and pointtowards their involvement in the complex expression pattern of the GnRH receptor.Equally significant, these data support the existence of variable transcripts encodingfor the receptor by the use of differential promoters and / or termination signals inthe expression of the GnRH receptor.Collectively, these data delineated the entire locus of the human GnRHreceptor and provided the foundation for subsequent DNA - binding studies todetermine regulatory sequences involved in the regulation of the human GnRH-Rgene. DNA - binding studies performed in this study support indirect regulatorypathways for cAMP and progesterone in the control of human GnRH receptorregulation via related sequences identified in the promoter region.179In addition, the assignment of chromosome 4 as the carrier for the humanGnRH-R gene should facilitate future studies on the identification of closely linkedgenes and on the examination of possible chromosomal rearrangements involving thehuman GnRH receptor. Of note, use of a genomic clone isolated from thisinvestigation has already resulted in the subchromosomal localization of the humanGnRH-R gene to 4q21 .2.The isolation and characterization of the human GnRH-R gene asdemonstrated in this present investigation establishes a basis for which analysesconcerning the evaluation and identification of possible genetic disorders of theGnRH receptor can be accomplished. Further, cloning and sequencing of the humanGnRH-R gene should allow for the production of improved analogues of GnRH foruse in clinical treatment of certain reproductive maladies. Moreover, cloning of thereceptor allows questions to be raised beyond the immediate world of peptidehormone biology. The receptor sequences revealed in this study should help in theunderstanding of what parts of the conserved structure are essential for properreceptor function (ligand binding; recognition of specific signal transduction Gproteins).Future characterizations of the GnRH-R gene in other species and tissues will180be necessary in order to fully understand both the regulation and mechanism ofaction of the GnRH receptor. Of primary importance will be the determination ofthe major promoter used in the pituitary and in other tissues. Technically, theseexperiments should be straightforward and would require the use of constructscontaining the 5’ promoter sequences and transfection assays. 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