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postgraduate thesis: Molecular characterizations of chicken and zebrafish prostanoid receptors and their implications on evolution of vertebrate prostanoidreceptor family

TitleMolecular characterizations of chicken and zebrafish prostanoid receptors and their implications on evolution of vertebrate prostanoidreceptor family
Authors
Issue Date2011
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Kwok, H. A. [郭可茵]. (2011). Molecular characterizations of chicken and zebrafish prostanoid receptors and their implications on evolution of vertebrate prostanoid receptor family. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4775260
AbstractProstanoid receptors (PG-Rs: prostaglandin D, E, F, prostacyclin and thromboxane receptors (DP, EP1-4, FP, IP and TP)) are known to mediate a diverse range of biological responses, such as cardiovascular homeostasis, nociception and reproduction, via binding to their respective ligands belonging to the five classes of prostanoids (PGs: class D, E, F, I and thromboxane). The majority of these findings were reported in mammals, and despite suggestive evidence provided by previous pharmacological and physiological studies in non-mammalian vertebrates, investigations on the mechanisms behind actions of PGs were impeded by the limited information on their receptors. In the present study, the full-length cDNAs of chicken (c-) and zebrafish (z-) prostanoid receptors – cEP3, cFPs, zEP1s and zFP – were identified from respective adult ovaries and their tissue distribution examined by RT-PCR. A novel middle-truncated splice variant, cFPb, which lacks 107 amino acids between transmembrane domains 4 and 6 but otherwise identical to cFPa was first identified. Three isoforms of zEP1 – zEP1a, zEP1b, zEP1c – were found, which might have subfunctionalized in their ligand binding and G protein coupling specificity, in addition to differential tissue distribution. Using various luciferase reporter systems (pGL3-CRE, pGL-NFAT-RE, pGL4-SRE), all the cloned receptors, except cFPb, were shown to potentially couple to intracellular cAMP, Ca2+, and/or MAPK signaling pathways. Owing to the proposed roles of PGs and its potential regulation by and/or on EGFR ligands and gonadotropins in mammals and chicken, genes involved in regulation of PG functions at various levels, including biosynthesis (COX1, COX2, mPGES1, mPGES2 and cPGES), availability (PGT) and signaling (cEPs and cFPs), were also characterized in granulosa cells during hen follicular development. Lastly, using our experimental data and systematic sequence retrieval from available databases, the PG receptor cascades from representative vertebrate species were pooled and analysed using phylogenetic analyses and synteny studies. Three putative clusters (IP-like, EP4-like and EP1-like cluster) were found in lamprey genome; meanwhile, only one PG-R-like cluster was identified from the Cephalochordate lancelet (amphioxus) genome. This concurs with the 1-2-4 rule proposed in first round/second round (1R/2R) whole genome duplication in which the missing lamprey cluster was presumably lost secondarily. With support from conserved orthologs-localization, the four PG-R paralogs (proto-EP4, proto-IP/EP2/DP, proto-TP/FP/EP1 & putative proto-EP3 genes) in the ancestral vertebrates might have further diversified via either localized- (e.g. EP2 and DP) or chromosomal segmental duplication (e.g. EP1, FP and TP) which resulted in the present array of vertebrate PGRs. Additional paralogs (e.g. EP1 and EP4) were identified from fishes, by which molecular dating coincide with and hint of their origins whence the ancient fishspecific whole genome duplication (3R) occurred ~350 million years ago. The present study offers the first glimpse and a better understanding of the roles of the PG-Rs and presents a higher resolution to the evolutionary history of each PG-R family member, consolidating that particular care has to be taken when studying non-mammalian PG-R functions in which some members are absent or present in multiples and propel the investigation of adaptational changes in the coding sequence during evolution of vertebrate PG-Rs.
DegreeDoctor of Philosophy
SubjectProstanoids.
Chicks - Genetics.
Zebra danio - Genetics.
Dept/ProgramBiological Sciences
Persistent Identifierhttp://hdl.handle.net/10722/181300

 

DC FieldValueLanguage
dc.contributor.authorKwok, Ho-yan, Amy.-
dc.contributor.author郭可茵.-
dc.date.accessioned2013-02-25T01:03:58Z-
dc.date.available2013-02-25T01:03:58Z-
dc.date.issued2011-
dc.identifier.citationKwok, H. A. [郭可茵]. (2011). Molecular characterizations of chicken and zebrafish prostanoid receptors and their implications on evolution of vertebrate prostanoid receptor family. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4775260-
dc.identifier.urihttp://hdl.handle.net/10722/181300-
dc.description.abstractProstanoid receptors (PG-Rs: prostaglandin D, E, F, prostacyclin and thromboxane receptors (DP, EP1-4, FP, IP and TP)) are known to mediate a diverse range of biological responses, such as cardiovascular homeostasis, nociception and reproduction, via binding to their respective ligands belonging to the five classes of prostanoids (PGs: class D, E, F, I and thromboxane). The majority of these findings were reported in mammals, and despite suggestive evidence provided by previous pharmacological and physiological studies in non-mammalian vertebrates, investigations on the mechanisms behind actions of PGs were impeded by the limited information on their receptors. In the present study, the full-length cDNAs of chicken (c-) and zebrafish (z-) prostanoid receptors – cEP3, cFPs, zEP1s and zFP – were identified from respective adult ovaries and their tissue distribution examined by RT-PCR. A novel middle-truncated splice variant, cFPb, which lacks 107 amino acids between transmembrane domains 4 and 6 but otherwise identical to cFPa was first identified. Three isoforms of zEP1 – zEP1a, zEP1b, zEP1c – were found, which might have subfunctionalized in their ligand binding and G protein coupling specificity, in addition to differential tissue distribution. Using various luciferase reporter systems (pGL3-CRE, pGL-NFAT-RE, pGL4-SRE), all the cloned receptors, except cFPb, were shown to potentially couple to intracellular cAMP, Ca2+, and/or MAPK signaling pathways. Owing to the proposed roles of PGs and its potential regulation by and/or on EGFR ligands and gonadotropins in mammals and chicken, genes involved in regulation of PG functions at various levels, including biosynthesis (COX1, COX2, mPGES1, mPGES2 and cPGES), availability (PGT) and signaling (cEPs and cFPs), were also characterized in granulosa cells during hen follicular development. Lastly, using our experimental data and systematic sequence retrieval from available databases, the PG receptor cascades from representative vertebrate species were pooled and analysed using phylogenetic analyses and synteny studies. Three putative clusters (IP-like, EP4-like and EP1-like cluster) were found in lamprey genome; meanwhile, only one PG-R-like cluster was identified from the Cephalochordate lancelet (amphioxus) genome. This concurs with the 1-2-4 rule proposed in first round/second round (1R/2R) whole genome duplication in which the missing lamprey cluster was presumably lost secondarily. With support from conserved orthologs-localization, the four PG-R paralogs (proto-EP4, proto-IP/EP2/DP, proto-TP/FP/EP1 & putative proto-EP3 genes) in the ancestral vertebrates might have further diversified via either localized- (e.g. EP2 and DP) or chromosomal segmental duplication (e.g. EP1, FP and TP) which resulted in the present array of vertebrate PGRs. Additional paralogs (e.g. EP1 and EP4) were identified from fishes, by which molecular dating coincide with and hint of their origins whence the ancient fishspecific whole genome duplication (3R) occurred ~350 million years ago. The present study offers the first glimpse and a better understanding of the roles of the PG-Rs and presents a higher resolution to the evolutionary history of each PG-R family member, consolidating that particular care has to be taken when studying non-mammalian PG-R functions in which some members are absent or present in multiples and propel the investigation of adaptational changes in the coding sequence during evolution of vertebrate PG-Rs.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.source.urihttp://hub.hku.hk/bib/B47752609-
dc.subject.lcshProstanoids.-
dc.subject.lcshChicks - Genetics.-
dc.subject.lcshZebra danio - Genetics.-
dc.titleMolecular characterizations of chicken and zebrafish prostanoid receptors and their implications on evolution of vertebrate prostanoidreceptor family-
dc.typePG_Thesis-
dc.identifier.hkulb4775260-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiological Sciences-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b4775260-
dc.date.hkucongregation2012-

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