CUHK electronic theses & dissertations collection

Preliminary analyses of three kinds of promoter activity showed that a-actin gene promoter was chosen to initiate the hormone transcription for the first consideration. We have fused the cDNAs encoding the intact somatolactins in frame to a zebrafish a-actin gene promoter to generate transgenic zebrafish lines co-injected with a GFP protein driven by the same promoter. The transgenic zebrafish were selected from GFP expression and confirmed by genomic PCR and Southern blot analysis, then maintained as transgenic founders. Measurement of the transgenes' expressions and the expressions of marker genes in different pathways by using real-time PCR provided a general understanding of SLs' actions. The data obtained indicated that the over-expressing of SLalpha and SLbeta in vivo significantly enhance the transcriptions of the insulin-like growth factors, IGF1 (5.46-fold and 6.77-fold), IGF2a (4.38-fold and 4.35-fold) and IGF2b (2.83-fold and 3.94-fold), but down-regulated IGF3 (a novel member found specifically in gonad) in larvae. However, the stimulation by administration of recombinant proteins (SLalpha and SLbeta) only showed a slight induction of the mRNA levels of IGFs (IGF1, IGF2a and IGF2b) on ZFL cells in vitro.Somatolactin (SL) is a novel member of pituitary polypeptide hormone found only in fish; it shares significant structural homology with prolactin and growth hormone. Since somatolactin receptor (SLR) was first defined as GHR1 and orthologous to the growth hormone receptor GHR2, SL and GH may share similar actions in growth and development. Recently, two SLs have been identified as SLalpha and SLbeta with similar structures, freshwater fish have these two isoforms found in the same species and only one isoform (SLalpha) is found in marine species. The two isoforms of SL may have different functions and physiological actions. To investigate the roles of SLs on vertebrate development and embryogenesis, we generated transgenic fish models with "all zebrafish" elements in origin to study the physiological functions of SLs in zebrafish.The ectopic expression of somatolactins also results in up-regulating gene expression of insulin, leptin, sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FAS), as well as the expression of vitellogenin and melanocyte-stimulating hormone (MSH) levels while causing reduction of catalase (CAT) and glutathione S-transferase (GST) levels in larvae. The results here represent the similar function between SLalpha and SLbeta and reveal more details in fish of the endocrinology system involvement in growth development, glucose synthesis, lipid metabolism, reproduction, pigmentation and antioxidant defense system through the actions of SLs.Three different gene promoters of zebrafish have been isolated to initiate the ectopic expression of somatolactins in vivo, which including a constitutional beta-actin gene promoter, a liver specific transferrin gene promoter and a zinc ion inducible metallothionein (MT) gene promoter. The promoter activities were tested in fish cell-line by using luciferase reporter assay. In MT gene promoter, two alleles of a zebrafish metallothionein II gene (zMT-II) promoter (zMT-IIA and zMT-IIB) containing 10 MREs in the 5'-flanking region (1,514 bp) were identified in zebrafish. These putative MREs were confirmed via electrophoretic mobility shift assay (EMSA) to have binding activities from the cellular and nuclear extracts of a zebrafish cell line, ZFL. Transient gene expression studies using zebrafish liver (ZFL) cell lines also confirmed that the most distal cluster of MREs contributed to the maximal induction of zMT-IIA activity by Zn2+ and the Zn 2+ induction was dose-dependent. EMSA also identified transcription factor(s) of two different sizes from the cytoplasmic and nuclear extracts of the ZFL cells that were able to bind with the MREs, but no increase in MRE binding was detected in the extracts of these cells after Zn2+ or Cd2+ treatment, compared with untreated control cells. The mechanisms of MT gene transcription induction via metal ions are discussed herein.Wan, Guohui.Adviser: Chan King Ming.Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: .Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.Includes bibliographical references (leaves 139-163).Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.Abstract also in Chinese