Rozprawy doktorskie na temat „Gene cloning”
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1
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.
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Glod, 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.
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3
Seto, 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.
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Superoxide dismutase is an enzyme which scavenges superoxide radicals and is thought to be a longevity determinant, as there exists a positive correlation between superoxide dismutase concentration and maximum life span potential. The cytosolic CuZn superoxide dismutase in D. melanogaster has been purified and sequenced, but the gene has not been cloned. However, when it is available the CuZn SOD gene may be reintroduced into the Drosophila genome via the P-element transformation system so its effects on the life span potential of Drosophila may be studied. This study describes attempts to clone the CuZn SOD gene from D. melanogaster using two mixed sequence oligonucleotide probes. The SI probe corresponds to amino acids 43-48 of the protein sequence and contains 128 different oligonucleotide sequences representing all possible codon combinations predicted from the amino acid sequence. The GT3 probe is targeted to amino acids 90-95 of the protein. In this probe, deoxyguanosine was placed in positions where all four nucleotides may occur to decrease probe heterogeneity. The probes were used to screen D. melanogaster Canton-S and Oregon-R genomic lambda libraries. Three positive clones isolated from the Canton-S library had identical nucleotide sequence in the GT3 probe binding region, and sequencing of the probe binding site revealed that one member of the GT3 probe had formed a 15 bp duplex with the phage DNA. Screening of the Oregon-R library produced four clones which hybridized with both GT3 and S1 probes. When these phage DNA were hybridized to polytene chromosomes by in situ hybridization, none mapped to 68AB on the third chromosome, the location of the CuZn SOD gene. These results suggest that modification of the classical strategy used in this study is necessary, and implications on probe design are discussed.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
4
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.
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Lam, 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.
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6
Ballance, 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.
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Hill, Russell. "Gene cloning studies in two nocardioform bacteria". Doctoral thesis, University of Cape Town, 1988. http://hdl.handle.net/11427/21896.
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Bibliography: pages 147-177.Nocardioforms 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.
8
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.
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In addition to substrate-associated cellulases, Cellulomonas fimi secretes endoglucanases ( endo-1, 4-β-D-glucan glucanohydrolases, EC 3.2.1.4. ) which are recovered from the cellulose-free culture supernatant of cells grown on microcrystalline cellulose. Two such enzymes, C3.1 and C3.2 with Mrs of 130'000 and 120'000, respectively, were purified to homogeneity. The two endoglucanases were shown to share the same N-terminal amino acid sequence and to hydrolyze carboxymethylcellulose ( CMC ) with similar efficiencies ( 236u/mg protein for C3.1 and 367u/mg protein for C3.2 ).
The recombinant lambda vector L47.1-169 was identified from a C.fimi DNA-lambda library on the basis of hybridization with C3.1/2-specific oligonucleotide probes. The subclone pTZ18R-8 only moderately expressed CMCase activity. The 5'-terminus of cenC ( the gene coding for C3.1/2 ) was localized in the insert by Southern transfer experiments and nucleotide sequence analysis. Results from total C.fimi RNA-DNA hybrid protection analyses defined the boundaries of cenC in pTZ18R-8 and led to the tentative identification of -10 and -35 promoter sequences.
To improve the expression of cenC, its entire coding sequence, except for the start codon GTG, was fused in frame to the ATG codon of a synthetic ribosomal binding site ( PTIS ) and placed under the transcriptional control of the lac p/o system. Induction of the resulting clone ( JM101[pTZP-cenC] ) led to impaired growth in liquid cultures because overproduction of CenC inhibited cell division'" and eventually led to cell death. Analysis of cell fractions by SDS-PAGE revealed a dominant ( >10% of total cell extract proteins ), clone-specific protein with a Mr of approximately 140'000 which was found exclusively in the cytoplasmic fraction. Conversely, 60% of the total CMC-hydrolyzing activity was localized in the periplasmic fraction indicating that the export of CenC is required for maximal expression of endoglucanase activity.
Isolation of the cellulolytic activities from an osmotic shockate led to the purification to homogeneity of two recombinant cellulases, CenC1 and CenC2, with Mr of 130'000 and 120'000, respectively. Both enzymes hydrolyzed CMC with similar efficiencies ( 278u/mg protein for CenC1 and 390u/mg protein for CenC2 ). In addition, amino acid sequence analyses showed the two enzymes to have the same N-termini as the native enzymes and proved furthermore that the CenC leader peptide was functional in Escherichia coli.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
9
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.
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A group of Escherichia coli clones harbouring recombinant pBR322 plasmid, containing Cellulomonas fimi DNA inserts, that reacted with antiserum to C.fimi culture supernatant, was screened for their ability to hydrolyze carboxymethyl cellulose (CMC) and 4-methylumbeliferyll-β-D-cellobioside (MUC). A clone, pEC62, hydrolyzed MUC but did not hydrolyze CMC. The recombinant enzyme encoded by pEC62 was shown to be a β-glucosidase (cellobiase). C.fimii itself was shown to encode an extracellular β-glucosidase in C.fimi. This is the first report of an extracellular β-glucosidase from a bacterium.
Deletion analysis localized the cloned gene (cbg)to the tet promoter proximal region of the 7.0 kilobase insert of pEC62. Further analysis and sequence data showed a highly active derivative of pEC62 contained a translational gene fusion between lacZ of pUC13 and cbg. From this data, a location for the cbg start site was proposed.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
10
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.
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Ornithine decarboxylase (ODC) is the rate determining enzyme in the biosynthesis of polyamines which are essential for cell growth. In chickens, significantly higher bioactivity is reported in broiler than in egg layer strains of chickens (Bulfield et al., 1988). To characterize the genetic differences in growth rates and ODC levels in chickens, an ODC cDNA and genomic gene were cloned and sequenced. Sequencing of ODC cDNA revealed that this clone (pODZ3: 2,052 bp) was not a full length of ODC cDNA and contained 2 putative introns. The open reading frame (introns deleted) coded for a protein of 404 amino acids which had about 85% amino acid identity with human ODC. Sequencing of genomic ODC clone (pODG2-8: 5098 bp) represented the 3$ prime$ end of ODC gene from downstream of intron 7. Northern blotting of chicken RNA probed with the insert of pODZ3 revealed 2 hybridizable messages of 1.6 and 2.1 kb, respectively. In addition, analysis of MspI restriction fragment length polymorphism (RFLP) using the 3$ prime$ end of ODC gene as a probe suggested that two MspI RFLPs present in the lean line of broiler chickens was related to selection of high lean body mass.
11
Li, 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.
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Woodruff, 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.
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The oprF gene encoding porin protein F from Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed in large amounts in E. coli and retained its heat- and reduction-modifiable and immunological characteristics. The cloned oprF gene product was purified from E. coli and characterized with respect to pore-forming ability in black lipid bilayers. Small channels, with an average single channel conductance of approximately 0.4 nS, were observed. A similar small channel size was observed for native protein F. The oprF sequences were used as a DNA-DNA hybridization probe with chromosomal DNA from the 17 IATS (International Antigen Typing Scheme) strains of P. aeruginosa, 52 clinical isolates and the non-aeruginosa Pseudomonads. Conservation of oprF sequences was observed among all the P. aeruginosa strains and to a lesser extent among the non-aeruginosa strains of the P. fluorescens rRNA homology group.
Insertion mutations in the oprF gene were created in vivo by Tn1mutagenesis of the cloned gene in E. coli and in vitro by insertion of the streptomycin-encoding Ω fragment into the cloned gene, followed by transfer of the mutated protein F gene back into P. aeruginosa and homologous recombination with the chromosome. The oprF mutants were characterized by gel electrophoresis and immunoblotting, and it was shown that the mutants had lost protein F. The P. aeruginosa oprF mutants were characterized with respect to growth rates, antibiotic permeability and cell surface hydrophobicity. The results of these studies indicated that major alterations in the cell surface had occurred and that the cells were unable to grow in a non-defined liquid medium without added electrolytes. Marginal differences were observed in MICs (minimum inhibitory concentrations) of hydrophilic antibiotics for the oprF mutants compared with their protein F-sufficient parents.
The putative roles of protein F in antibiotic permeability and general outer membrane permeability are discussed. Evidence for extensive homologies between protein F, the OmpA protein of E. coli and PHIII of Neisseria gonorrhoeae are presented. A role for protein F in prophylactic anti-Pseudomonas therapy, as a target for vaccine development, is proposed.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
13
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.
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Marshall, Daina Elisabeth. "Cloning and expression of a novel neural gene". Thesis, University of Edinburgh, 1997. http://hdl.handle.net/1842/29868.
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Five novel cDNA clones were isolated and identified from a differentiated glial subtractive library. They were shown to be specific to the nervous system by Northern blotting analysis. In situ hybridisation (ISH) studies were carried out on cultured glial cells to determine if any of the clones were expressed by specific glial cell types. Oligodendrocytes were identified by the monoclonal antibody O4 and astrocytes were identified with an anti-GFAP monoclonal antibody. Clone OL0755, one of the novel brain specific clones identified, was chosen for further investigation. Two cDNA clones, believed to be full length, were obtained by hybridisation screening of a rat cDNA library. Clone OL0755-A is 2.7 kb and encodes a protein of approximately 60 kD. Clone OL0755-B is 2 kb in size and encodes a protein of approximately 50 kD. Sequence analysis showed these clones to be two alternatively spliced forms of a common gene. An antibody (Ab755) raised against a 23 amino acid N-terminus peptide common to both clones (EASFVQTTMALGLPSKKASSRNV) identifies two proteins with the predicted sizes, 50 kD and 60 kD. Both the mRNAs and the proteins are developmentally regulated. Both mRNAs increase in abundance in the postnatal rat brain peaking at postnatal day 21 (P21). Expression of the smaller, more abundant protein also increases during development peaking at P21 while the larger protein is less abundant and downregulates slightly with age. Neither protein is phosphorylated or N-glycosylated. ISH studies on p21 rat brain sagittal sections with clone OL0755 (common to both OL0755-A and OL0755-B) showed a very strong neuronal pattern of expression but also a weaker expression in glial cells which was confirmed by ISH on rat optic nerves and which was consistent with the ISH studies on cultured glial cells. The appearance of these proteins in both glial cells and neurons during the active phase of myelination suggests a possible role in this process either as a structural component or as part of a signalling mechanism between the cell types.
15
Mammadov, Jafar. "Towards Cloning the Leaf Rust Resistance Gene Rph5". Diss., Virginia Tech, 2004. http://hdl.handle.net/10919/28704.
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Leaf rust caused by Puccinia hordei is an important disease of barley (Hordeum vulgare) in many regions of the world. Yield losses up to 62% have been reported in susceptible cultivars. The Rph5 gene confers resistance to the most prevalent races (8 and 30) of barley leaf rust in the United States. Therefore, the molecular mapping of Rph5 is of great interest. Genetic studies were performed by analysis of 93 and 91 F2 plants derived from the crosses 'Bowman' (rph5) x 'Magnif 102' (Rph5) and 'Moore' (rph5) x Virginia 92-42-46 (Rph5), respectively. Linkage analysis positioned the Rph5 locus to the extreme telomeric region of the short arm of barley chromosome 3H at 0.2 cM proximal to RFLP marker VT1 and 0.5 cM distal from RFLP marker C970 in the Bowman x Magnif 102 population. Synteny between rice chromosome 1 and barley chromosome 3 was employed to saturate the region within the sub-centimorgan region around Rph5 using sequence-tagged site (STS) markers that were developed based on barley expressed sequence tags (ESTs) syntenic to the phage (P1)-derived artificial chromosome (PAC) clones comprising distal region of the rice chromosome 1S. Five rice PAC clones were used as queries to blastn 370,258 barley ESTs. Ninety four non-redundant EST sequences were identified from the EST database and used as templates to design 174 pairs of primer combinations. As a result, 10 EST-based STS markers were incorporated into the 'Bowman' x 'Magnif 102' high-resolution map of the Rph5 region. More importantly, six markers, including five EST-derived STS sequences, co-segregate with Rph5. Genes, represented by these markers, are putative candidates for Rph5. Results of this study demonstrate the usefulness of rice genomic resources for efficient deployment of barley EST resources for marker saturation of targeted barley genomic region.Ph. D.
16
Avela, Kristiina. "Positional cloning of the mulibrey nanism gene (MUL)". Helsinki : University of Helsinki, 2000. http://ethesis.helsinki.fi/julkaisut/laa/haart/vk/avela/.
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Chapman, 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.
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McKie, Norman. "Methylmalonyl CoA mutase from Saccharopolyspora erythraea". Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259763.
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Barton, 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.
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Wakarchuk, 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.
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The β-glucosidase (Abg) from ATCC 21400, an Agrobacterium species, was purified to homogeneity. The protein was cleaved with cyanogen bromide and the peptides were purified by reversed phase high pressure liquid chromatography. The partial amino-acid sequences for three CNBr peptides, CNBr1, CNBr2 and CNBr3, were determined by automated Edman degradation. A sequence from CNBr2 was used to synthesize a mixture of oligonucleotides which was used as a hybridization probe to identify a recombinant DNA clone carrying the gene for β-glucosidase. A single clone was isolated which expressed an enzymatic activity that hydrolyzed several β-glucosides. The enzymatic activity produced by this clone could be adsorbed by rabbit antiserum raised against the Agrobacterium enzyme. The direction of transcription of the β-glucosidase gene was determined by verifying the DNA sequence 3' to the oligonucleotide probe binding site. After subcloning the gene a high level of expression was obtained in the plasmid vector pUC18 using the lacZ gene promoter.
The nucleotide sequence of the 1599 bp insert in pABG5 was determined using the chain terminator method. The start of the protein coding region was determined by aligning the amino terminal sequence of the protein with the predicted amino acid sequence of the cloned gene. The open reading frame was 1387 nucleotides and contained 458 codons. The molecular weight calculated from the deduced amino acid sequence agreed with that observed from both the native and recombinant enzymes. The predicted amino acid composition from the open reading frame matched with that determined for the native β-glucosidase. The stop codon of this coding region was followed by a potential stem loop structure which may be the transcriptional terminator. There was a region of the deduced Abg sequence which had homology to a region from two other β-glucosidase sequences. This region of homology contained a putative active site by analogy with the active site of hen egg white lysozyme.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
21
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.
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22
Skeggs, Patricia Ann. "Cloning of aminoglycoside-resistance determinants in Streptomyces". Thesis, University of Leicester, 1986. http://hdl.handle.net/2381/35152.
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Xie, 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.
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M.S.Molecular 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.
24
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.
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Oza, 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.
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In order to clone the DNA repair gene of Aspergillus nidulans, uvsF$ sp-$ pyrG$ sp-$ strains were transformed with a genomic library in a plasmid vector that carried the pyr-4 gene of Neurospora which complements pyrG mutants of Aspergillus. Out of the several transformants obtained, four were like wild type. For rescuing plasmids, transformant DNA was digested with Bg/II and self ligated, and used for transformation of E. coli. Two types of plasmids were obtained; these two had a region in common ($<$1.0 kb) that was not a simple overlap and gave evidence for rearrangements. Surprisingly, only the plasmids with the larger insert of Aspergillus DNA were able to complement uvsF$ sp-$ in the secondary transformation. Northerns of polyA$ sp+$-enriched mRNA, probed with this plasmid, showed three bands. However, its subclone which spans the shared region hybridized to only one of them (1.0 kb). Two cDNA and five genomic clones were identified. The two cDNA clones though not identical, cross-hybridized. Three out of five genomic clones were identical. The cDNA hybridized to a short segment (2.2 kb) of one of the three types of genomic clones, locating the putative uvsF gene sequence.
26
Moonen, 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.
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Warner, Simon A. J. "Cloning and characterization of an asparagus wound-induced gene". Thesis, University of Leicester, 1992. http://hdl.handle.net/2381/35363.
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Following previous studies, Asparagus officinalis single cell suspensions were hypothesized to be a rich source of wound-inducible mRNAs. A previously isolated clone, DDl-34, was shown to hybridize to wound-inducible transcript. This sequence was used to isolate the AoPR1 (Asparagus officinalis Pathogenesis Related cDNA clone 1). Data from the isolation and analysis of genomic clones hybridizing to DDl-34 probe suggested that these clones were unlikely to contain the upstream regulatory sequences of the AoPR1 gene and that the genomic arrangement of these sequences is complex. Inverse polymerase chain reactions (IPCR) were used to amplify AoPR1 genic sequences directly from the asparagus genome. Two products were cloned and sequenced, demonstrating that the correct sequences, upstream and downstream of the primers, had been amplified. The downstream IPCR product's sequence overlaps with AoPR1 coding sequence and contains an intron sequence. The upstream IPCR product partially overlaps with the start of AoPR1 coding sequence and was successfully used in transcript mapping experiments. Translational fusions were constructed between this fragment and the -glucuronidase (gus) reporter gene. GUS analysis demonstrated that this fragment, containing the AoPR1 promoter, was sufficient to drive wound-inducible transcription in transgenic tobacco. A smaller upstream fragment was insufficient to drive wound-inducible transcription. GUS expression was also detectable in tissues such as the xylem parenchyma, mature pollen and coloured regions of the petal. AoPR1-gus transgene expression correlates with the spatial expression patterns of phenylpropanoid biosynthesis pathway genes. The nature of the fusion suggested that the AoPR1 protein is intracellular. This is the first example of the cloning and analysis of a monocotyledon gene belonging to the 'intracellular pathogenesis related protein' class. The analysis and application of AoPR1 sequences are discussed.
28
Simons, Kristin Jean. "Cloning and characterization of the wheat domestication gene, Q". Diss., Manhattan, Kan. : Kansas State University, 2005. http://hdl.handle.net/2097/135.
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Diez, 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.
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Legionella pneumophila is an intracellular bacterium that causes an acute form of pneumonia called Legionnaires' disease. Segregation analyses using macrophages from susceptible and resistant inbred mice previously indicated that a single genetic locus, named Lgn1 , could determine permissiveness to intracellular replication of L. pneumophila. A positional cloning strategy was undertaken, which makes use of genetic and molecular biology techniques to identify the gene responsible for a particular phenotype, based mostly on its location within a chromosome. The work described in this thesis covers three aspects of Lgn1: (1) Building upon the work of others, the Lgn1 genetic interval was narrowed to 0.32 cM within distal mouse chromosome 13. The corresponding 140 Kb Lgn1 physical interval contains only two known transcripts: the Neuronal Apoptosis Inhibitor Protein ( Naip) genes Naip2 and Naip5. (2) The expression profile of the Lgn1 candidates was investigated both at the mRNA and protein levels. Expression of both Naip2 and Naip5 in mouse macrophages strengthened their candidacy for the Lgn1 locus. (3) Transfer of BAC clones from the critical interval into transgenic mice was successfully used to functionally complement the Lgn1 susceptibility phenotype of A/J mice with cloned DNA from non-permissive 129X1 or C57BL/6J origins. Two independent rescuing BAC clones were identified, with a 56-Kb overlap where the entire Lgn1 transcript must lie. The only known full-length transcript coded in this reduced genomic region is Naip5.Thus, 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.
30
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.
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Ho, 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.
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Claesen, Jan. "Cloning and analysis of the cypemycin biosynthetic gene cluster". Thesis, University of East Anglia, 2010. https://ueaeprints.uea.ac.uk/25828/.
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Desai, Suchita Umesh. "TOWARDS CLONING THE CLK-3 GENE IN CAENORHABDITIS ELEGANS". UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_theses/549.
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Mutations in the clk-1, clk-2, clk-3 and gro-1 genes in Caenorhabditis elegans show alterations in developmental and behavioral timing and lifespan, collectively termed the Clk phenotype. While the clk-1, clk-2, and gro-1 genes have been cloned, clk-3 gene has not been identified. Gene expression changes in clk-3 mutant worms were determined using microarray expression data. I examined genes in the region to which clk-3 gene maps, for strongly reduced expression in the clk-3 mutants and identified thirteen clk-3 candidate genes. RNAi feeding vectors for all these candidate genes were picked and cultured from the RNAi library. Knock-down worm strains were generated by feeding RNAi and analyzed for Clk phenotypes. Of all the candidate genes tested, the Y48E1B.5 gene showed the most similar phenotypic profile to the clk-3 mutants. The Y48E1B.5 gene shows weak homology to a mammalian mitochondrial ribosomal protein. Primers were designed to amplify all 9 exons of the Y48E1B.5 gene. Sequence analysis was carried out on the resulting PCR products from clk-3 mutants. An amino acid change was found in exon 4.
34
Iwanejko, Lesley Ann. "Cloning the enterotoxin gene from Clostridium perfringens type A". Thesis, University of Nottingham, 1991. http://eprints.nottingham.ac.uk/12195/.
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A C. perfringens type A genomic library was constructed in E. coli by banking overlapping 6-10 kbp Hind III fragments of chromosomal DNA from the enterotoxin (CPE) positive strain NCTC 8239 into the pUC derived vector pHG165. The library was screened by colony hybridization with a degenerate 26 bp oligonucleotide probe, derived from the amino acid sequence CPE9_17A. complex mixture of plasmid DNA was isolated from the only hybridization positive clone. A second round of screening picked out a single plasmid, with an apparently altered copy number, pLWl, that carried the CPE gene, cpe, on a 6.8 kbp insert. A sequence deduced primer strategy for direct plasmid sequencing was initiated using a primer deduced in a similar manner to the 26 bp probe, obviating the need for prior mapping and subcloning of the insert. The amino acid sequence for the conceptual gene product of the single open reading frame differed only slightly from the known CPE sequence but lacked the C terminal residues. The biased cpe codon usage reflected the low %G+C content of the DNA. The %G+C content was even lower in the upstream region and possessed properties characteristic of bent DNA. The region 5' to the ATG translational start codon contained a Shine-Dalgarno sequence and several sequences with significant homology to the putative transcriptional control regions for the tetanus toxin gene. The N-terminal coding region contained a direct repeat of an upstream sequence that shared considerable homologies with the crossover point in site 1 of the Tn3 res region. Southern blot analyses of chromosomal and plasmid DNAs from several isolates indicated that the majority of strains were cpe-. The chromosomal location and architecture of cpe appeared identical in all cpe+ strains. A second copy, pLW2, of the 5' end of cpe, on a 4.5 kbp Pst I/Eco RI restriction fragment, was cloned during one of many unsuccessful attempts to clone the 3' end. A separate re-cloning experiment isolated several different clones that contained the 0.6 kbp Hind III located = 2.5 kbp 5' to the ATG codon of both cloned copies of cpe but none of them carried the CPE gene. The fragment was used as a DNA probe to show that it was present in high copy number in some strains of C. perfringens but completely absent from others. An hypothesis describing the possible involvement of a mobile genetic element in C. perfringens enterotoxin production offers explanations for the cloning of a complex mixture of plasmids, the apparent alteration in plasmid copy number, the identification of putative DNA crossover points, the failure to clone the 3' end of cpe and the isolation of a novel DNA fragment.
35
Fisher, 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.
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The hypervariable locus DXS255 in human Xp11.22 has a heterozygosity exceeding 90% and has therefore facilitated the localization of several disease genes which map to the proximal short arm of the X chromosome, including the immune deficiency Wiskott-Aldrich syndrome and the eye disorders retinitis pigmentosa, congenital stationary night blindness and Aland Island eye disease. In addition, a microdeletion involving DXS255 has been identified in patients suffering from Dent's disease, a familial X-linked renal tubular disorder which is characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis (kidney stones) and eventual renal failure. Two YAC contigs were constructed in Xp11.23-p11.22 in order to aid transcript mapping; the first centred on the DXS255 locus, the second mapping distal to the first and linking the genes GATA, TFE3 and SYP to the OATL1 cluster. Eleven novel markers were generated, one of which contains an exon from a novel calcium channel gene. Four putative CpG islands were detected in the region. Analysis of the microdeletion associated with Dent's disease using markers from the DXS255 contig demonstrated that it is confined to a 370kb interval. A YAC overlapping this deletion was hybridized to a kidney-specific cDNA library to isolate coding sequences that might be implicated in the disease aetiology. The clones thus identified detect a 9.5kb transcript which is expressed predominantly in kidney, and originate from a novel gene (CLCN5) falling within the deleted region. Sequence analysis indicates that the 746 residue protein encoded by this gene is a new member of the C1C family of voltage-gated chloride channels. The coding region of CLCN5 is organized into twelve exons, spanning 25-30kb of genomic DNA. Using the information presented in this thesis, other studies have identified deletions and point mutations which disrupt CLCN5 activity in further patients affected with X-linked hypercalciuric nephrolithiasis, confirming the role of this locus in renal tubular dysfunction.
36
Starkey, 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.
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Turner, 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.
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38
Smith, 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.
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39
Winter, 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.
Pełny tekst źródła
40
Kourdi, Zerikly Malek. "Cloning, sequencing and analysis of spirotetronate biosynthetic gene clusters". Thesis, University of Warwick, 2011. http://wrap.warwick.ac.uk/38545/.
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Spirotetronate antibiotics are natural products that have been shown to have a broad spectrum of biological activities, including antiviral, antibacterial, antitumor and antimalarial activities. Two representatives of this group are quartromicins and chrolactomycin, which are produced by Amycolatopsis orientalis and Streptomyces sp. 569N-3 respectively. Based on other related natural products with known biosynthetic pathways, the biosynthetic routes for quartromicins and chrolactomycin were proposed and several strategies for locating the putative gene clusters directing the biosynthesis of these natural products were used. An FkbH-like protein was proposed to be specifically involved in the biosynthetic incorporation of a glycerol-derived three-carbon unit into the tetronate moieties of quartromicins and chrolactomycin. It was selected as a target to locate the biosynthetic gene clusters of tetronate natural products. After aligning sequences of known FkbH-like proteins, a set of degenerate oligonucleotide primers for amplification of fkbH-like genes was designed and tested. Fragments of fkbH-like sequences were amplified from several available strains and sequenced. Several genomic libraries were created and screened by PCR using the degenerate fkbH primers. After validating the library, clones containing the putative biosynthetic gene cluster were identified and sequenced. The obtained sequence data was then assembled and analysed. Coding sequences were identified, protein functions assigned and a biosynthetic route was proposed for the biosynthesis of quartromicins and chrolactomycin.
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Lin, Bor-do, i 林博淂. "Cloning of Thrombomodulin Gene". Thesis, 1997. http://ndltd.ncl.edu.tw/handle/18670767785317658157.
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碩士國立成功大學
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).
42
Cai, Lian-Ding, i 蔡連錠. "Desaturase gene cloning from zabrafishzeng". Thesis, 1995. http://ndltd.ncl.edu.tw/handle/68587551602485885443.
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43
Lin, Ta-fang, i 林大方. "Cloning of rice RNase gene". Thesis, 2004. http://ndltd.ncl.edu.tw/handle/97581984348214403984.
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碩士國立臺灣師範大學
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.
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Wu, chieng-hung, i 巫建宏. "Gene cloning of fish interferon". Thesis, 1996. http://ndltd.ncl.edu.tw/handle/88236632054435075280.
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碩士國立海洋大學
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.
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Su, Hui-Chun, i 蘇惠君. "Gene cloning, characterization and gene recombination of maltooligosyltrehalose trehalohydrolase". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/26699917361522816934.
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碩士國立臺灣海洋大學
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.
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Wen, Chi-jen, i 溫啟仁. "Patent Protection against Microbial Gene Cloning". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/44164790445188671373.
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碩士東吳大學
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.
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HU, LUN, i 胡倫. "Cloning □-sarcin gene and its substrate". Thesis, 1991. http://ndltd.ncl.edu.tw/handle/74469941623425586473.
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48
Lien, Te-Sheng, i 連德昇. "Production and Gene Cloning of Chitinases". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/02800838386864837209.
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博士大葉大學
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.
49
"Goldfish (Carassius auratus) somatolactin: gene cloning and gene expression studies". 1999. http://library.cuhk.edu.hk/record=b5889873.
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by Yeung Sze Mang.Thesis (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
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Chang, Chih-Hsiang, i 張智翔. "Cloning and characterizing crumbs gene in mouse". Thesis, 2001. http://ndltd.ncl.edu.tw/handle/33892538080205334709.
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碩士東海大學
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.