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Molecular cloning of growth hormone and growth hormone receptor in lower vertebrates.

by Lee Tsz On. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 148-155). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgments --- p.v / Contents --- p.vi / List of figures --- p.xii / List of table --- p.xiv / Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1. --- Growth hormone (GH) --- p.1 / Chapter 1.1.1. --- Introduction to GH --- p.1 / Chapter 1.1.2. --- Actions of GH --- p.2 / Chapter 1.1.3. --- Structure of GH --- p.3 / Chapter 1.1.4. --- The sequence of GH --- p.5 / Chapter 1.2. --- Growth hormone receptor (GHR) --- p.6 / Chapter 1.2.1 --- Introduction to GHR --- p.6 / Chapter 1.2.2. --- Structure of the extracellular domain of GHR --- p.9 / Chapter 1.2.3. --- The regulation of GHR --- p.12 / Chapter 1.2.4. --- GHR biosynthesis --- p.13 / Chapter 1.2.5. --- Tissue distribution of GHR --- p.14 / Chapter 1.3. --- Signal transduction mechanisms of GHR --- p.15 / Chapter 1.3.1. --- Dimerization of GH and GHR complex --- p.15 / Chapter 1.3.2. --- The Jak and Stat pathway --- p.18 / Chapter 1.3.3. --- The ras and other signaling pathways --- p.20 / Chapter 1.4. --- Project aim --- p.22 / Chapter Chapter 2 --- Material and Methods / Chapter 2.1. --- Preparation of ribonuclease free reagents and apparatus --- p.23 / Chapter 2.2. --- Isolation of total RNA --- p.23 / Chapter 2.3. --- Isolation of mRNA --- p.24 / Chapter 2.4. --- Spectrophotometric quantification and qualification of DNA and RNA --- p.24 / Chapter 2.5. --- First strand cDNA synthesis --- p.25 / Chapter 2.6. --- Agarose gel electrophoresis of DNA --- p.25 / Chapter 2.7. --- Formaldehyde agarose gel electrophoresis of RNA --- p.26 / Chapter 2.8. --- Vacuum transfer of DNA to a nylon membrane --- p.26 / Chapter 2.9. --- Nucleic acids purification by MicroSpin (S-200HR) columns --- p.27 / Chapter 2.10. --- DNA radioactive labeling by nick translation --- p.27 / Chapter 2.11. --- Southern blot analysis --- p.28 / Chapter 2.12. --- Autoradiography and molecular imager --- p.28 / Chapter 2.13 . --- Linearization and dephosphorylation of plasmid DNA --- p.29 / Chapter 2.14. --- Purification of DNA from agarose using QIAEX II kit --- p.29 / Chapter 2.15. --- 3'End modification of PCR amplified DNA --- p.30 / Chapter 2.16. --- Ligation of DNA fragments to linearized vector --- p.30 / Chapter 2.17. --- Preparation of Escherichia coli competent cells --- p.31 / Chapter 2.18. --- Transformation --- p.31 / Chapter 2.19. --- Mini preparation of plasmid DNA --- p.32 / Chapter 2.20. --- Maxi preparation of plasmid DNA --- p.34 / Chapter 2.21 . --- PCR sequencing --- p.35 / Chapter 2.22. --- cDNA library screening --- p.36 / Chapter 2.23. --- Preparation and sterilization of culture medium --- p.38 / Chapter 2.24. --- Preparation of frozen stock of culture cells --- p.39 / Chapter 2.25. --- Cell passage of CHO-Kl --- p.39 / Chapter 2.26. --- Counting of cells --- p.40 / Chapter 2.27. --- Proliferation assay performed on CHO-K1 cells (MTT method) --- p.40 / Chapter 2.28. --- Luciferase assay --- p.41 / Chapter 2.29. --- SDS-PAGE preparation --- p.42 / Chapter 2.30. --- SDS-PAGE analysis of proteins --- p.42 / Chapter 2.31 . --- Recombinant protein expression --- p.43 / Chapter 2.32. --- Small scale purification of recombinant proteins --- p.44 / Chapter 2.33. --- Restriction digestion of DNA --- p.45 / Chapter 2.34. --- Purification of PCR product using QIAquick PCR purification kit --- p.45 / Chapter 2.35. --- TA cloning of PCR fragment --- p.45 / Chapter 2.36. --- Transfection of plasmid into CHO-K1 cells --- p.46 / Chapter 2.37. --- Sources of hormones --- p.46 / Chapter 2.38. --- Buffer and reagents --- p.47 / Chapter Chapter 3 --- "Cloning, expression and tissue distribution of Xenopus laevis GHR" / Chapter 3.1. --- Introduction --- p.50 / Chapter 3.2. --- Materials and methods --- p.51 / Chapter 3.2.1. --- Molecular cloning of xGHR cDNA / Chapter 3.2.1.1. --- Animals and tissues --- p.51 / Chapter 3.2.1.2. --- Reverse transcribed´ؤpolymerase chain reaction (RT-PCR) --- p.51 / Chapter 3.2.1.3. --- Subcloning of PCR amplified DNA fragment --- p.53 / Chapter 3.2.1.4. --- Library screening of xGHR --- p.53 / Chapter 3.2.1.5. --- 5 'Rapid amplification of cDNA end (5' RACE) --- p.55 / Chapter 3.2.2. --- Tissue distribution of xGHR / Chapter 3.2.2.1. --- Animals and tissues --- p.56 / Chapter 3.2.2.2. --- RT-PCR and Southern blot --- p.56 / Chapter 3.2.3. --- Eukarytoic expression of xGHR and functional assay of xGHR / Chapter 3.2.3.1. --- Subcloning ofxGHR into pRc/CMV --- p.57 / Chapter 3.2.3.2. --- Expression of xGHR in CHO-K1 cell --- p.58 / Chapter 3.2.3.3. --- Proliferation assay --- p.58 / Chapter 3.3. --- Results --- p.60 / Chapter 3.3.1. --- RT-PCR of the partial fragment --- p.60 / Chapter 3.3.2. --- Library screening of xGHR cDNA library --- p.61 / Chapter 3.3.3. --- 5' RACE --- p.64 / Chapter 3.3.4. --- The full-length cDNA sequence of xGHR --- p.65 / Chapter 3.3.5. --- Tissue distribution of xGHR mRNA --- p.69 / Chapter 3.3.6. --- Functional assay of xGHR in CHO-K1 cells --- p.71 / Chapter 3.4. --- Discussion --- p.74 / Chapter Chapter 4 --- Cloning and expression of Xenopus laevis GH-A and GH-B / Chapter 4.1. --- Introduction --- p.78 / Chapter 4.2. --- Materials and Methods --- p.79 / Chapter 4.2.1. --- PCR amplification of xGH-A and xGH-B partial fragments --- p.79 / Chapter 4.2.2. --- cDNA library screening of xGH-A and xGH-B --- p.80 / Chapter 4.2.3. --- Rapid amplification of cDNA ends of xGH-B / Chapter 4.2.3.1. --- 3'RACE --- p.80 / Chapter 4.2.3.2. --- 5'RACE --- p.81 / Chapter 4.2.4. --- Expression of xGH-A and xGH-B / Chapter 4.2.4.1 --- Construction of the expression vector --- p.84 / Chapter 4.2.4.2. --- Protein expression of xGH-A and xGH-B --- p.85 / Chapter 4.2.5. --- Purification of recombinant xGH-A and xGH-B --- p.85 / Chapter 4.3. --- Results --- p.87 / Chapter 4.3.1. --- PCRof xGH-A and xGH-B partial fragment --- p.87 / Chapter 4.3.2. --- Library screening of xGH-A --- p.87 / Chapter 4.3.3. --- 5' RACE and 3' RACE of xGH-B --- p.91 / Chapter 4.3.4. --- Sequence analysis of xGH-A and xGH-B --- p.93 / Chapter 4.3.5. --- Protein expression and purification of recombinant xGH-A and xGH-B --- p.100 / Chapter 4.4. --- Discussion --- p.102 / Chapter Chapter 5 --- Molecular cloning and function expression of goldfish GHR / Chapter 5.1. --- Introduction --- p.105 / Chapter 5.2. --- Materials and methods --- p.106 / Chapter 5.2.1. --- Molecular cloning of the partial fragment of gfGHR / Chapter 5.2.1.1. --- Primer design --- p.106 / Chapter 5.2.1.2. --- Library PCR of gfGHR partial fragment --- p.108 / Chapter 5.2.2. --- Library PCR of gfGHR cDNA sequence --- p.110 / Chapter 5.2.3. --- Determination of 3' End and 5' End sequences of gfGHR cDNA --- p.112 / Chapter 5.2.4. --- Tissue distribution of gfGHR / Chapter 5.2.4.1. --- Animals and tissues --- p.115 / Chapter 5.2.4.2. --- Semi-quantitative R T-PCR --- p.115 / Chapter 5.2.5. --- Functional expression of gfGHR in CHO-K1 cell / Chapter 5.2.5.1. --- Construction of an expression vector containing gfGHR --- p.116 / Chapter 5.2.5.2. --- Functional assay of gfGHR expression on CHO-K1 cells --- p.117 / Chapter 5.2.5.3. --- Proliferation assay --- p.118 / Chapter 5.2.5.4. --- Spi luciferase assay --- p.118 / Chapter 5.3. --- Results --- p.120 / Chapter 5.3.1. --- PCR amplification of the partial sequence of gfGHR --- p.120 / Chapter 5.3.2. --- The library PCR of gfGHR cDNA sequence --- p.122 / Chapter 5.3.3. --- The sequence of gfGHR --- p.124 / Chapter 5.3.4. --- Tissue distribution of gfGHR --- p.131 / Chapter 5.3.5. --- Proliferation assay --- p.133 / Chapter 5.3.6. --- Spi luciferase assay --- p.135 / Chapter 5.4. --- Discussion --- p.137 / Chapter Chapter 6 --- General discussion and future works --- p.145 / References --- p.148 / Appendix --- p.156

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_323183
Date January 2000
ContributorsLee, Tsz On., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvi, 161 leaves : ill. (some col.) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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