Dissertations / Theses on the topic 'Gene cloning'
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Sakuntabhai, Anavaj. "Positional cloning of Darier's disease gene." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302392.
Full textGlod, Frank. "PCR generated gene probes for cloning fungal polykeptide synthase genes associated with squalestatin biosynthesis." Thesis, University of Bristol, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268525.
Full textSeto, Nina Oi Ling. "The copper-zinc superoxide dismutase gene from Drosophila melanogaster : attempts to clone the gene using two mixed sequence oligonucleotide probes." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26534.
Full textMedicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
Monk, Sarah. "Positional cloning of the Darier disease gene." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325681.
Full textLam, W. F. E. "Gene cloning of human placental alkaline phosphatase." Thesis, University of Liverpool, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.384394.
Full textBallance, David James. "Transformation and gene cloning in Aspergillus nidulans." Thesis, University of Bristol, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.248172.
Full textHill, Russell. "Gene cloning studies in two nocardioform bacteria." Doctoral thesis, University of Cape Town, 1988. http://hdl.handle.net/11427/21896.
Full textNocardioforms are Gram-positive, aerobic actinomycetes and are a metabolically diverse group which produce antibiotics, useful enzymes, are important in the biotransformation of organic compounds and the decomposition of organic wastes and are important medically. A gene cloning vector designated pLR591 was constructed from the broad host range, multicopy Streptomyces plasmid pIJ702 and the positive selection Escherichia coli plasmid pEcoR251. This plasmid has useful features for the construction of actinomycete genomic libraries. Cloning of DNA into the unique Bg1II endonuclease site of pLR591 inactivated the lethal EcoRI gene derived from pEcoR251, thereby selecting for recombinant plasmids containing inserted DNA. The thiostrepton resistance gene derived from pIJ702 was shown to be functional in Streptomyces lividans enabling selection of recombinant pLR591 plasmids containing foreign DNA in S. lividans. The vector pLR591 therefore functions as a positive selection Streptomyces-E. coli shuttle vector facilitating construction of actinomycete genomic libraries in E. coli and subsequent transfer of recombinant plasmids into S. lividans.
Moser, Bernhard. "Molecular cloning, characterization and expression of the endoglucanase C gene of Cellulomonas fimi and properties of the native and recombinant gene products." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/29036.
Full textScience, Faculty of
Microbiology and Immunology, Department of
Graduate
Bates, Nancy Carol. "Characterization of cbg : a cloned gene encoding an extracellular [beta]-glucosidase from Cellulomonas fimi." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26163.
Full textScience, Faculty of
Microbiology and Immunology, Department of
Graduate
Zhang, Ling 1962. "Molecular cloning and characterization of the chicken ornithine decarboxylase gene." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=22831.
Full textLi, Xiang. "Cloning and Regulation of Bovine and Porcine Comparative Gene Identification-58 Gene." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1345044694.
Full textWoodruff, Wendy Anne. "Cloning and characterization of the oprF gene for protein F from Pseudomonas aeruginosa." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/29218.
Full textScience, Faculty of
Microbiology and Immunology, Department of
Graduate
Howard, J. J. "Cloning of a Schwanniomyces castellii debranching glucoamylase gene." Thesis, Cranfield University, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.352929.
Full textMarshall, Daina Elisabeth. "Cloning and expression of a novel neural gene." Thesis, University of Edinburgh, 1997. http://hdl.handle.net/1842/29868.
Full textMammadov, Jafar. "Towards Cloning the Leaf Rust Resistance Gene Rph5." Diss., Virginia Tech, 2004. http://hdl.handle.net/10919/28704.
Full textPh. D.
Avela, Kristiina. "Positional cloning of the mulibrey nanism gene (MUL)." Helsinki : University of Helsinki, 2000. http://ethesis.helsinki.fi/julkaisut/laa/haart/vk/avela/.
Full textChapman, J. W. "A study of Bacillus subtilis sporulation genes cloned on plasmids." Thesis, Open University, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484403.
Full textMcKie, Norman. "Methylmalonyl CoA mutase from Saccharopolyspora erythraea." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259763.
Full textBarton, Jenny Loretta. "IL-1L1 : a novel gene in the human interleukin-1 cluster." Thesis, University of Sheffield, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366223.
Full textWakarchuk, Warren William. "The molecular cloning and characterization of a Beta-glucosidase gene from an Agrobacterium." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/27559.
Full textScience, Faculty of
Microbiology and Immunology, Department of
Graduate
Baker, N. E. "Wingless : A gene required for segmentation in Drosophila." Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377244.
Full textSkeggs, Patricia Ann. "Cloning of aminoglycoside-resistance determinants in Streptomyces." Thesis, University of Leicester, 1986. http://hdl.handle.net/2381/35152.
Full textXie, Weiqiao Hope Lila W. "Isolation and characterization of a gene required for peroxisome biogenesis." Oregon Health & Science University, 1993. http://content.ohsu.edu/u?/etd,234.
Full textMolecular Biology
This thesis describes the cloning and analysis of PER6, a gene required for peroxisome biogenesis in Pichia pastoris. The gene was cloned by functional complementation of a per6 P. pastoris mutant strain that was one of a number of peroxisome-deficient mutants isolated in this laboratory. The complementing activity was localized to a small DNA fragment by subcloning and Northern filter hybridization analysis and the DNA sequence of the fragment was determined. The sequence revealed a 1296-bp open reading frame which potentially encodes a 432-amino acid protein of 49 kD. The gene was transcribed into a message of 1.4 kilobases that was constitutively expressed but induced several-fold in cells growing on methanol. A mutant strain with a deletion of a large portion of the open reading frame was constructed and used to genetically demonstrate that the cloned gene was identical to the defective gene in the originally isolated per6 mutant. The predicted amino acid sequence of the PER6 product revealed several interesting features, including a significant regional similarity to PAF-1, a gene known to be defective in some patients with Zellweger syndrome, a lethal human genetic disease caused by peroxisome deficiency. Finally, the PER6 product was produced in E. coli and purified to serve as antigen for antibody production.
Xie, Weiqiao. "Isolation and characterization of a gene required for peroxisome biogenesis /." Full text open access at:, 1993. http://content.ohsu.edu/u?/etd,234.
Full textOza, Kalpesh. "Cloning of a DNA repair gene (uvsF) from Aspergillus." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59577.
Full textMoonen, Nele. "The cloning of the clrA gene of Ideonella dechloratans." Thesis, Karlstads universitet, Fakulteten för hälsa, natur- och teknikvetenskap (from 2013), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kau:diva-62977.
Full textWarner, Simon A. J. "Cloning and characterization of an asparagus wound-induced gene." Thesis, University of Leicester, 1992. http://hdl.handle.net/2381/35363.
Full textSimons, Kristin Jean. "Cloning and characterization of the wheat domestication gene, Q." Diss., Manhattan, Kan. : Kansas State University, 2005. http://hdl.handle.net/2097/135.
Full textDiez, Eduardo. "Positional cloning of the Legionella pneumophila-resistance gene Lgn1." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85151.
Full textThus, in our last publication we have proposed that Naip5 (recently named Birc1e) is the gene within the Lgn1 locus responsible for differential permissiveness to intracellular L. pneumophila replication in mice.
Varela, Mosquera Anabel. "The positional cloning of the mouse deafness gene whirler." Thesis, Imperial College London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270783.
Full textHo, Mengfatt. "Isolating the gene for McLeod syndrome by positional cloning." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259962.
Full textClaesen, Jan. "Cloning and analysis of the cypemycin biosynthetic gene cluster." Thesis, University of East Anglia, 2010. https://ueaeprints.uea.ac.uk/25828/.
Full textDesai, Suchita Umesh. "TOWARDS CLONING THE CLK-3 GENE IN CAENORHABDITIS ELEGANS." UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_theses/549.
Full textIwanejko, Lesley Ann. "Cloning the enterotoxin gene from Clostridium perfringens type A." Thesis, University of Nottingham, 1991. http://eprints.nottingham.ac.uk/12195/.
Full textFisher, Simon E. "Positional cloning of the gene responsible for Dent's disease." Thesis, University of Oxford, 1995. http://ora.ox.ac.uk/objects/uuid:22f6e7a5-4f00-41c9-a1d3-1b05899f22c0.
Full textStarkey, Michael Peter. "Cloning of a delta-endotoxin gene from Bacillus thuringiensis." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335286.
Full textTurner, Gaynor Phillippa Linley. "The cloning, expression and mutagenesis of M13 gene 5." Thesis, University of Portsmouth, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293115.
Full textSmith, E. P. "Analysis of herpesvirus saimiri gene expression by cDNA cloning." Thesis, University College London (University of London), 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371058.
Full textWinter, Andrew Gavin. "Cloning, characterisation, and regulation of the NRD convertase gene." Thesis, Glasgow Caledonian University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267548.
Full textKourdi, Zerikly Malek. "Cloning, sequencing and analysis of spirotetronate biosynthetic gene clusters." Thesis, University of Warwick, 2011. http://wrap.warwick.ac.uk/38545/.
Full textLin, Bor-do, and 林博淂. "Cloning of Thrombomodulin Gene." Thesis, 1997. http://ndltd.ncl.edu.tw/handle/18670767785317658157.
Full text國立成功大學
生物化學研究所
85
Thrombomodulin (TM) is a vascular endothelial cell receptor that is a critical cofactor in a major physiologically natural anticoagulant system. The pathway is initiated when thrombin binds to the endothelial cell thrombin binding protein﹐ thrombomodulin. TM-bound thrombin has lost its procoagulant properties﹐but is a potent activator of protein C. Activated protein C strongly inhibits activation of the blood coagulation pathway via the degradation of Va and VIIIa。 The human genome has only one contains a single TM gene,which is located on chromosome 20 position p12-cen. The TM gene is unique in that it contains no intron. The mature human TM is a single- chain membrane glycoprotein which consisted of 557 amino acids and is structurally divided into five domains. It contains an N- termainal lectin-like domain(residues 2-154)﹐a hydrophobic region (residues 155- 222)﹐six epidermal growth factor-like domain (residues 223~462)﹐a serine/ threonine- rich region(residues 463-497)﹐a 23 amino acid resicues-long transmembrane domain(residues 498-521)﹐and a 35 amino acid residues-long cytoplasmic tail(residues 522-557)。 Studies of the biochemical, enzymatic, and structural poperties of a protein would be facilitated by access to large quantities of purified, native protein. We used baculovirus expression system to expression human TM peptide fragments, include (1)TM0.5 the lectin-like domain (residues -17~157) , (2)TM0.8 the EGF-domain (residues 223~497) to serine/ threonine-rich domain, and (3)TM1.5 the lectin-like domain to serine/ threonine-rich domain(residues -17~497).
Cai, Lian-Ding, and 蔡連錠. "Desaturase gene cloning from zabrafishzeng." Thesis, 1995. http://ndltd.ncl.edu.tw/handle/68587551602485885443.
Full textLin, Ta-fang, and 林大方. "Cloning of rice RNase gene." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/97581984348214403984.
Full text國立臺灣師範大學
生命科學研究所
92
In the previous study, we have known that rice TN5-9 suspension cultured cells will secrete a 17kDa RNase to the medium upon phosphate-starvation. It indicates that RNase production can be induced to reuse the organic phosphate during phosphate-starvation. However, it is unclear, how many RNase genes in the rice genome and what is the gene expression pattern of RNases upon phosphate-starvation. In this study, we try to clone RNase gene from rice by subtractive cDNA PCR and RT-PCR. Sixteen clones induced by phosphate-starcation were isolated. After sequences alignment, none of 16 clones belonged to RNase of T2/S family, but one of them was a putative RNase gene shich was very similar to pathogenesis-related protein 10a gene (PR-10a). Northern and RT-PCR results indicate that rice PR-10a gene is highly expressed during phosphate-starvation, especially from 6 hour to 24 hour after treatment of phosphate-starvation. In order to check whether PR-10a protein has RNase activity. We use E. coli BL21 (DE3) to expressed recombinant PR-10a protein. After protein purification and enterokinase digestion for removing of Nus tag fragnment, a 17kDa recombinant rice PR-10a protein was obtained. However, this mature recombinant PR-10a protein was inactive for RNA digestion. After β-ME, DTT, EDTA, or CIAP treatment, only recombinant PR-10a protein that treated with CIAP exhibited RNase activity in digestion of rRNA and tRNA.
Wu, chieng-hung, and 巫建宏. "Gene cloning of fish interferon." Thesis, 1996. http://ndltd.ncl.edu.tw/handle/88236632054435075280.
Full text國立海洋大學
水產養殖學系
84
In order to study the antivrial machanism induced by poly I:C, we firsttested and found that the poly I:C dose not cause cytotoxicity to fishcell lines. We treated 5 different fish cell lines with poly I:C thentreated with T42G strain IPNV, and found that RTG-2 cell line preasenthighest antiviral ability in response to the poly I:C treatment. Whendifferent concerntractions of poly I:C were applied to the RTG-2 cells,the antiviral ability of these cells increased as the dosage of poly I:Cincreased, up to 6 ug/ml of poly I:C. SDS-PAGE electrophoresis to analysisthe poly I:C treated RTG-2 cell culture medium showed the present of an extra-protein band of about 17.4KD by commassie blue staining. In order to confirm that this protein is induced by poly I:C, we labled the poly I: Ctreated RTG-2 cells with 35S-methionine,and showed that a labled protein of about 17.4KD also present in the culture medium, and the radioactivity ofthis protein band is stronger than the one in the medium of control not treated with poly I:C. So we proved that this protein is induced by poly I:Ctreatment. According to the above data, we think that the interferon gene is exist inthe RTG-2 cells, so we synthesized squence-specific primers according to thecDNA squence of flatfish interferon published by Tamai, to amplify and squenced, which showen 48% homology when compared with flatfish interferon cDNA. Since there are stop codens with in the fragment. We need further studyto evaluate whether this fragment really respresent a part of fish interferongene.
Su, Hui-Chun, and 蘇惠君. "Gene cloning, characterization and gene recombination of maltooligosyltrehalose trehalohydrolase." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/26699917361522816934.
Full text國立臺灣海洋大學
食品科學系
95
Maltooligosyltrehalose trehalohydrolase (MTHase) mainly hydrolyzes the α-1,4 linkage adjacent to the α-1,1 bond of maltooligosyltrehalose to release trehalose, and it also hydrolyzes the α-1, 4 linkage at the reducing end of maltooligosaccharides to release glucose leading to the decrease of the trehalose yield. BvtreZ gene from Brevibacterium helvolum ATCC 11822 was cloned and inserted into an expression vector pET21b. The stop codon of BvtreZ gene was changed to glycine codon by site-directed mutagenesis, therefore, the expressed BvMTHase possessed a His-Tag on its C-terminal region. The recombinant BvMTHase was expressed in the host E. coli BL21 (DE3) and was purified by Ni2+-affinity chromatography. The purified recombinant BvMTHase exhibited optimum activity at 40℃ and pH 6.0. The enzyme was quite stable at 30~40℃ and pH 5.5~6.5. The activity of recombinant BvMTHase would be completely inhibited by 4 mM Cu2+ and Zn2+ ions. The SStreZ and SAtreZ gene from pET-15b-△H-TreZ recombinant plasmid and Sulfolobus acidocaldarius ATCC 33909, respectively, were amplified and inserted into an expression vector pET21b. The BvtreZ, SStreZ and SAtreZ genes were used as template DNA in genetic recombination. These three amino acid sequences were 40~60% identical. DNA family shuffling was first carried out according to Stemmer’s method. The DNA fragments of 50-100 bp were prepared by DNase I cleavage. Since the sequence homology is not high, cannot be amplified by this method. In addition, DNA family shuffling was also carried out according to Kikuchi’s method using DNA fragments of 100-300 bp prepared by different restriction enzyme digestion. The fragments are reassembled into full-length genes of 1.5 kb, and inserted into an expression vector pET21b. The sequencing result shows that the sequences of RMTHase only contain partial original template, and RMTHase even lost the conserved regions of α-amylase family regions.
Wen, Chi-jen, and 溫啟仁. "Patent Protection against Microbial Gene Cloning." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/44164790445188671373.
Full text東吳大學
法律學系
93
The primitive motive of this thesis was triggered by how the invention, that is, a biotechnological product involving microbial gene cloning, which was made by the author studying in the graduate institute of Agricultural Chemistry of National Taiwan University a decade ago, can obtain protection in view of patent law. Assuming the invention could be protected, what would be scope of the protection is? In addition, in the aspect of practical patent application, what kind of especially care-taking tips should be looked out in order to be granted a patent smoothly? To stretch out the motive mentioned above, there were four objectives that the thesis was going to reach: A) To review, conclude and introduce the patent protection system. B) As the Unites States is a progressive nation in the field of biotechnology, the author tried to understand how her legal system protects against biomaterial patent, including gene cloning. C) To find out some status quo and to conclude out some of the characteristics regarding (microbial) gene cloning in Taiwan by way of real-case analysis. D) Retuning to the primitive motive of the author, this thesis sought how the invention could be legally protected. The first chapter of the thesis is the motive, objective, means and scope of the study. The second chapter is to make a simple introduction to the background technology of gene cloning. The third chapter is to introduce the patent system, and, taking-example-of US legal system, how the biomaterial, including microbes, can be protected. The fourth chapter is to introduce some distinctive parts of biomaterial patent against that of others. The fifth chapter is to conduct domestic case study of granted patents of gene cloning. The sixth chapter, that is, the last one, is to make some conclusion and advice. There were two major points about the conclusion and advice raised from the thesis: 1) The first item of article 24 of up-to-date valid Taiwan Patent Law, which conferred to TRIPs and EPC, did not cover the wording of “or the products thereof”, whereas the two treaties mentioned above did use the wording. Though there aren’t any disagreements while explaining the application of the article, however, in the search of the precision of legal wording, the author recommends to add the wording “or the products thereof” onto the article to make consistency between legal wording and scholar explanation. 2) The process of technology regarding gene cloning could be patented as long as it meets the requirements of patent application. The transformant, that is, the host containing foreign DNA could be patented as well. As Taiwan has released the patent towards new microbial species, it could be seemed as a new species and turned out to be a patent-grantable object. However, the review of Non-obviousness should be made carefully. The recombinant plasmid principle-wise could be the object of patent application. However, in case both the vector and cutting-in gene(s) were well known, and the recombinant plasmid thereafter was obvious in view of persons skilled in the field, the latter will be considered in lack of non-obviousness. As the invention of gene was considered a kind of discovery and Taiwan has recognized that discovery could be the object of patent (e.g., the second indication of medicine), gene could be the object of patent granting, on condition that it could be determined through the base pair sequence and/or the amino acid sequence of the protein that was encoded by the gene. Moreover, the requirement of industrial application should also be stated while applying gene-discovering patent.
HU, LUN, and 胡倫. "Cloning □-sarcin gene and its substrate." Thesis, 1991. http://ndltd.ncl.edu.tw/handle/74469941623425586473.
Full textLien, Te-Sheng, and 連德昇. "Production and Gene Cloning of Chitinases." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/02800838386864837209.
Full text大葉大學
生物產業科技學系
95
In this study, Aeromonas sp. DYU-Too7 was stimulated to produce extra- cellular chitinases using various substrates, and the chitinases were then purified with chromatographic methods. Results indicated that the chitinases could be produced, when DYU-Too7 was cultivated in a medium containing either chitin or glucosamine (GlcN) as the sole carbon source. In the chitin medium, maximal chitinase production occurred when using a medium containing 2% chitin. Maximal glucosamine production occurred in the medium containing 0.1% GlcN. The content of this experimentally obtained 36 kDa chitinase (denoted as Chi36) was highest among the chitinases. The Chi36 chitinases produced using both media were chromatographically purified separately, and their properties were compared. There were no significant differences in the enzymatic properties of Chi36 produced with the chitin medium versus the glucosamine medium. For instance, both of the Chi36s had an optimal reacting pH of 5.0 and showed high thermal stability between 10 and 60℃ and high pH stability in the range of pH 5.0 - 8.0. Using either Chi36 in the hydrolysis of colloidal chitin produces chitobiose, making the chitinase of Aeromonas sp. DYU-Too7 an exo-type enzyme. However, the Chi36 produced using a chitin versus glucosamine medium showed differing levels of activity in certain environments. For instance, after reacting the two Chi36s with 10 mM Hg2+ for 1 h, the Chi36 produced in a chitin medium retained 55% of its original activity, while the Chi36 produced in a GlcN medium retained 67% of its original activity. The feasibility study of protein collection and purification was performed through adsorption using modified nano-diamond as the absorber. The Chi36 purified in the previous study was used as the target protein to explore the optimal operating conditions. Results indicated the following optimal conditions: the ratio between the protein and modified nano-diamond was 75, the pH environment was 4.1, and the efficiency of protein adsorption was 86%. The enzymatic properties had no significant change after the chitinase was adsorbed. The efficiency of protein desorption from modified nano-diamond was almost 100% in a Tri-HCl buffer of pH 8.0 or 9.0, however, the desorption efficiency decreased to 76% in a buffer of pH 7.0. Next, modified nano-diamond was used to collect protein from a culture broth, replacing the often used ammonium sulfate precipitation procedure. Results showed that 87% of the crude protein was recovered after the adsorption step. The efficiency of protein desorption from modified nano-diamond was 88% in a Tri-HCl buffer of pH 8.0, and the properties of crude protein were not signify- cantly altered through adsorption of modified nano-diamond and desorption from nano-diamond. The gene encoding Chi36 produced by Aeromonas sp. DYU-Too7 was amplified using a polymerase chain reaction (PCR). Results indicated that the open reading frame of the gene, chi36, is 1,080 bp long encoding the protein of Chi36 with 360 amino acids, of which 27 amino acids compose the N-terminal signal peptide. In addition, the sequence 137FDGIDIDLE145 of Chi36 is homologous with the consensus catalytic sequence of family 18 of glycosyl hydrolases. Furthermore, the chi36 genes with and without the signal peptide were inserted into E. coli to produce recombinant proteins. Results showed that the recombinant protein produced from the gene, including a signal sequence to produce a signal peptide, showed chitinolytic activity, and produced chitobiose as the main hydrolysate in the hydrolysis of colloidal chitin. However, when the recombinant gene did not include the signal sequence, the recombinant protein produced thereafter did not show chitinase activity.
"Goldfish (Carassius auratus) somatolactin: gene cloning and gene expression studies." 1999. http://library.cuhk.edu.hk/record=b5889873.
Full textThesis (M.Phil.)--Chinese University of Hong Kong, 1999.
Includes bibliographical references (leaves 123-133).
Abstracts in English and Chinese.
ACKNOWLEDGMENTS --- p.i
ABSTRACT --- p.ii
槪論 --- p.iii
ABBREVIATIONS --- p.iv
AMINO ACIDS SHORTHAND --- p.vi
TABLE OF CONTENTS --- p.vii-x
Chapter CHAPTER 1 --- LITERATURE REVIEW
Chapter 1.1 --- Introduction --- p.1
Chapter 1.2 --- Structural Analysis of SL --- p.1
Chapter 1.3 --- Location of SL-producing cells and Expression of SL --- p.5
Chapter 1.4 --- Possible Functions of SL --- p.9
Chapter 1.4.1 --- Adaptation to various backgrounds and Intensities of Illuminations --- p.9
Chapter 1.4.2 --- Control of Reproduction and Maturation --- p.10
Chapter 1.4.3 --- Responses to Stress --- p.12
Chapter 1.4.4 --- Regulation of P034- and Ca2+ Metabolism --- p.12
Chapter 1.4.5 --- Acid - Base Balance --- p.14
Chapter 1.4.6 --- Regulation of Energy Metabolism --- p.15
Chapter 1.4.7 --- Regulation of Fat Metabolism --- p.15
Chapter 1.5 --- Regulation of SL Gene Expression --- p.19
Chapter 1.5.1 --- Pit-1 Related Gene Regulation --- p.19
Chapter 1.5.2 --- Regulation of Hormone Secretion --- p.21
Chapter 1.5.2.1 --- Hypothalamic Factors --- p.21
Chapter 1.5.2.2 --- Steroids --- p.23
Chapter 1.6 --- Aims of Thesis --- p.23
Chapter 1.6.1 --- Identification of SLII from Goldfish (Carassius auratus) --- p.23
Chapter 1.6.2 --- Aims --- p.27
Chapter CHAPTER 2 --- PCR ANALYSIS OF GFSLII GENE AND ITS EXPRESSION IN GOLDFISH TISSUE
Chapter 2.1 --- Introduction --- p.28
Chapter 2.2 --- Materials and Methods --- p.31
Chapter 2.2.1 --- Materials --- p.31
Chapter 2.2.2 --- Methods --- p.33
Chapter 2.2.2.1 --- Subcloning and DNA Sequencing of the Goldfish SLII Amplified by PCR --- p.33
Chapter 2.2.2.1.1 --- PCR Cloning of Goldfish SLII Gene --- p.33
Chapter 2.2.2.1.2 --- Restriction Enzyme Digestion of the PCR Clones --- p.33
Chapter 2.2.2.1.3 --- Subcloning of the Digested Fragments --- p.33
Chapter 2.2.2.1.4 --- DNA Sequencing of the Subcloned Fragments --- p.34
Chapter 2.2.2.2 --- Tissue Distribution Studies Using RNA Assay --- p.35
Chapter 2.2.2.2.1 --- Tissue Preparation --- p.35
Chapter 2.2.2.2.2 --- Total RNA Extraction --- p.35
Chapter 2.2.2.2.3 --- Electrophoresis of RNA in Formadehyde Agarose Gel --- p.36
Chapter 2.2.2.2.4 --- First Strand cDNA Synthesis --- p.37
Chapter 2.2.2.2.5 --- Goldfish SLII Specific PCR --- p.37
Chapter 2.2.2.2.6 --- PCR to Test DNA Contamination --- p.38
Chapter 2.3 --- Results --- p.39
Chapter 2.3.1 --- Subcloning and DNA Sequencing of the Goldfish SLII Amplified by PCR --- p.39
Chapter 2.3.2 --- Tissue Distribution Studies Using RNA Assay --- p.40
Chapter 2.4 --- Discussion --- p.45
Chapter 2.4.1 --- Subcloning and DNA Sequencing of the Goldfish SLII Amplified by PCR --- p.45
Chapter 2.4.2 --- Tissue Distribution Studies Using RNA Assay --- p.46
Chapter CHAPTER 3 --- ANALYSIS OF GOLDFISH SLII GENE
Chapter 3.1 --- Introduction --- p.47
Chapter 3.2 --- Materials and Methods --- p.49
Chapter 3.2.1 --- Materials --- p.49
Chapter 3.2.2 --- Methods --- p.54
Chapter 3.2.2.1 --- Screening of Goldfish Genomic Library --- p.54
Chapter 3.2.2.1.1 --- Preparation of the Plating Host --- p.54
Chapter 3.2.2.1.2 --- Preparation of the Probe --- p.54
Chapter 3.2.2.1.3 --- Primary Screening of Goldfish Genomic Library --- p.55
Chapter 3.2.2.1.4 --- Isolation of the Positive Clones --- p.56
Chapter 3.2.2.1.5 --- Phage Titering --- p.56
Chapter 3.2.2.1.6 --- Purification of the Positive Clones --- p.57
Chapter 3.2.2.1.7 --- Phage DNA Preparation --- p.57
Chapter 3.2.2.1.8 --- Find out the Target Gene Size of the Positive Clones --- p.58
Chapter 3.2.2.1.9 --- Cloning of the PCR Fragments into pUC18 Vector --- p.59
Chapter 3.2.2.1.10 --- Checking the Cloned Insert Size --- p.60
Chapter 3.2.2.1.11 --- Restriction Enzyme Digestion to Release the Inserts --- p.61
Chapter 3.2.2.1.12 --- Mini prep of the Positive Clones for Further Investigations --- p.61
Chapter 3.2.2.1.13 --- DNA Sequencing of the Positive Clones --- p.61
Chapter 3.2.2.1.14 --- Restriction Enzyme Mapping of the Positive Clones --- p.62
Chapter 3.2.2.1.15 --- Subcloning of Clone 2A and5A
Chapter 3.2.2.1.16 --- Determination of the Promoter Region of Clone 2A Using Universal Genome Walker Kit --- p.63
Chapter 3.2.2.2 --- Southern Blot Analysis of Goldfish and Catfish Genomic DNA --- p.66
Chapter 3.2.2.2.1 --- Genomic DNA Preparation from Goldfish and Catfish Tissues --- p.66
Chapter 3.2.2.2.2 --- Restriction Enzyme Digestion of the Genomic DNA --- p.67
Chapter 3.2.2.2.3 --- Alkaline Transfer of the Digested Genomic DNA --- p.67
Chapter 3.2.2.2.4 --- Hybridization of the Digested Genomic DNA --- p.67
Chapter 3.3 --- Results --- p.69
Chapter 3.3.1 --- Screening of the Goldfish Genomic Library --- p.69
Chapter 3.3.2 --- Mapping the Target Genes --- p.69
Chapter 3.3.3 --- DNA Sequencing of the 2 Positive Clones --- p.69
Chapter 3.3.4 --- Southern Blot Analysis of Goldfish and Catfish Genomic DNA --- p.81
Chapter 3.4 --- Discussion --- p.83
Chapter CHAPTER 4 --- EXPRESSION OF RECOMBINANT GOLDFISH SOMATOLACTIN IN ESCHERICHIA COLI (E. COLI)
Chapter 4.1 --- Introduction --- p.87
Chapter 4.2 --- Materials and Methods --- p.89
Chapter 4.2.1 --- Materials --- p.89
Chapter 4.2.2 --- Methods --- p.96
Chapter 4.2.2.1 --- Transformation of the Recombinant Protein Carrying Plasmid into E. coli. (BL21) --- p.96
Chapter 4.2.2.2 --- Small Scale Expression of Recombinant Goldfish SLII Protein --- p.96
Chapter 4.2.2.3 --- Large Scale Expression of Recombinant Goldfish SLII Protein --- p.97
Chapter 4.2.2.4 --- Preparation of the Recombinant Protein for Purification --- p.99
Chapter 4.2.2.5 --- Protein Purification Using Novagen His-Bind Resin Kit --- p.99
Chapter 4.2.2.6 --- Production of Polyclonal Antibody in Rabbits --- p.100
Chapter 4.2.2.7 --- Enzyme Linked Immunosorbant Assay (ELISA) --- p.101
Chapter 4.2.2.8 --- Western Blot Analysis of the Recombinant Hormones --- p.103
Chapter 4.3 --- Results --- p.105
Chapter 4.3.1 --- Expression of the Recombinant Goldfish SLII --- p.105
Chapter 4.3.2 --- Purification of the Recombinant Goldfish SLII --- p.105
Chapter 4.3.3 --- ELISA Analysis --- p.105
Chapter 4.3.4 --- Western Blot Analysis --- p.110
Chapter 4.4 --- Discussion --- p.113
Chapter 4.4.1 --- Expression of the Recombinant Goldfish SLII --- p.113
Chapter 4.4.2 --- Purification of the Recombinant Goldfish SLII --- p.114
Chapter 4.4.3 --- Analysis of the Recombinant Goldfish SLII --- p.114
Chapter CHAPTER 5 --- GENERAL DISCUSSION AND CONCLUSIONS --- p.116
REFERENCES --- p.123
Chang, Chih-Hsiang, and 張智翔. "Cloning and characterizing crumbs gene in mouse." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/33892538080205334709.
Full text東海大學
生物學系
89
Crumbs is a cell polarity gene that encodes a protein containing 30 EGF-like domains, 4 laminin-AG domains and a short cytoplasmic domain in Drosophila. Loss-of-function mutations in crumbs lead to loss of cell polarity in most ectodermally derived epithelia in Drosophila. Immunocytochemical studies showed that Crumbs localized at the apical domain of the epithelial cells. All together, crumbs is thoμght to play an important role in establishing cell polarity in epithelial cells. In addition to Drosophila crumbs, several crumbs homologues have been cloned and sequenced in Caenorhabditis elegans, Xenopus laevis, and Homo sapiens. Histological studies showed gene products of crumbs homologues are expressed in most epitheliμm and central nervous system. Moreover, in hμman deficient of crb-1 causes photoreceptor degeneration and resμlts in retinitis pigmentosa. However, the regμlatory mechanism of crumbs in setting cell polarity and neuronal degeneration remains unclear. To elucidate the function of crumbs in mammals, we cloned the crumbs gene in mouse and characterize the expression pattern of the gene during development. Using RACE, we obtained full-length of mouse crumbs. This gene contains 6166 nucleotides which encode 1315 amino acids. Peptides conserve motif analysis suggests that mouse Crumbs is a secretory protein containing 17 EGF-like domains repeats and 3 laminin-AG domains. RT-PCR shows mouse crumbs is expressed in the brain, eye and kidney. Temporal studies of expression show mouse crumbs expression throμghout the developmental stages in the eye, from embryonic stage to adμlt, but the expression decreased gradually in the brain and kidney. The persistent expression of mouse crumbs in the eye suggests that this gene may participate in the eye development and in the maintenance of photoreceptors.