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Дисертації з теми "Molecular cloning"

Автор: Grafiati

Опубліковано: 4 червня 2021

Оновлено: 29 липня 2024

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1

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

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2

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

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3

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

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4

Boyes, Barry Edward. "Molecular cloning of the human Substantia innominata : characterization of a brain large mRNA." Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/30960.

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Brain tissue samples were collected from individuals with histologically and biochemically confirmed Alzheimer's Disease (AD), as well as from a group of individuals without any signs of neurological disease (NNC). Ribonucleic acid (RNA) was extracted from these tissues, characterized by several chemical methods, and the yields were compared between AD and NNC groups. High molecular weight RNA could be effectively extracted from frozen postmortem human brain. In comparison to the NNC group, tissue RNA levels were reduced in the AD hippocampus, but not in the temporal cortex or substantia innominata (SI). No difference in the physical integrity of the RNA was apparent between AD and NNC groups. A high complexity complementary deoxyribonucleic acid (cDNA) library was prepared using RNA extracted from the NNC SI. Differential hybridization screening using a variety of cDNA probes was employed to identify mRNAs expressed differentially between AD and NNC tissue, and between SI and other human tissues. Many selected mRNAs were examined for specificity of expression in brain tissue and brain regions. The cDNA clone pSI3a-24 identified an mRNA, which, on Northern blot hybridization, was expressed in brain tissue but not in the other human tissues examined. The identified mRNA was unusually large, with a chain length estimated at 15,500 bases. Quantification of the brain tissue levels of this mRNA was carried out using a ribonuclease protection assay. Tissue levels were higher in the SI (40 pg/μg RNA) than in the temporal cortex (28.6 pg/μg), and were lowest in the cerebellum (11.2 pg/μ9). Levels of the mRNA in temporal cortex samples were increased 29% in the AD group, relative to NNC. No significant difference in the SI tissue levels was observed between AD and NNC groups. Hybridization analysis of human genomic DNA indicated that the mRNA was encoded by a single copy gene. Sequence analysis of the full 3 kilobases of cloned cDNA was completed. Computer database searches failed to identify any known nucleic acid sequence with homology to the cDNA. Examination of the cDNA sequence for potential polypeptide coding regions suggested that the corresponding mRNA has a 3' untranslated region of at least 3 kilobases.
Medicine, Faculty of
Graduate

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5

Lush, Michael John. "Molecular cloning of neuropathy target esterase." Thesis, University of Leicester, 1998. http://hdl.handle.net/2381/29627.

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A single ingestion of certain organophosphorus esters (OPs) can cause a syndrome known as Organophosphate Induced Delayed Polyneuropathy (OPIDP), a paralysing neuropathy with degeneration of long axons, developing after a latent period of approximately one to three weeks. The primary target of these OPs has been shown to be a 155kDa neural protein designated Neuropathy Target Esterase (NTE), and the toxic effects apparently due to the covalent inhibition and subsequent secondary modification of this protein. Recently NTE has been purified to apparent homogeneity using a novel biotinylated OP and sufficient pure protein was produced for limited problem sequencing. The aim of this project was to clone NTE cDNA using peptide sequence data. Initially, these sequences were used to design degenerate oligonucleotide primers for amplifying sections of brain cDNA by polymerase chain reaction (PCR). These approaches were unsuccessful. Subsequently, a database searching with the peptide sequences identified a number of Expressed Sequence Tags (EST)s; these could be aligned to form an initial contig of 2.2kbp which encoded the 3' end of NTE cDNA. The 5' end of NTE cDNA, comprising a further 2.2kbp, was obtained by PCR-based technique. The final 4.4kbp contig encoded a 1327 residue polypeptide predicted to have a molecular mass of 146kDa and at least one transmembrane domain. A novel serine esterase domain of approximately 200 residues was present near the C-terminus. NTE is unrelated to any known serine hydrolases but homologous proteins are predicted to be present in diverse prokaryotic and eukaryotic organisms. The homologue in Drosophila is associated with the swisscheese phenotype, an age-dependent neurodegeneration of the brain. NTE was also mapped to chromosome 19p 13.3 between markers D19216 and the D19S413 (using the UniGene database) and an OMIM search reveals that this is near the locus of cerebellar ataxia (Cayman type).

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6

Long, Graham Stanley. "Molecular cloning of bacteriophage K1E endosialidase." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.339539.

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7

McNair, Alan Thomas. "Molecular cloning of Fasciola hepatica antigens." Thesis, Queen's University Belfast, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335604.

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8

Choi, Wai To. "Molecular cloning of ribosome-inactivating proteins." HKBU Institutional Repository, 1996. http://repository.hkbu.edu.hk/etd_ra/63.

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9

Fisher, Adam B. "ex vivo DNA cloning." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3962.

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Genetic engineering of microbes has developed rapidly along with our ability to synthesize DNA de novo. Yet, even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. While technological advances have resulted in powerful techniques for in vitro and in vivo assembly of DNA, each suffers inherent disadvantages. Here, an ex vivo DNA cloning suite using crude cellular lysates derived from E. coli is demonstrated to amplify and assemble DNA containing small sequence homologies. Further, the advantages of an ex vivo approach are leveraged to rapidly optimize several parameters of the ex vivo DNA assembly methodology testing lysates from different engineered strains of E. coli, with various buffer components and using titrations of purified cloning enzymes. Finally, in order to complete the cloning suite, a vector expressing the Pyrococcus furiosis (Pfu) DNA polymerase was designed, constructed and expressed in E. coli to create a ‘functionalized lysate’ capable of ex vivo PCR. Not only do we demonstrate ex vivo cloning methodology as a complete cloning package capable of replacing the expensive cloning reagents currently required by synthetic biologists, but also establish ex vivo as an overarching approach for conducting molecular biology.

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10

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

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11

Mulcahy, Anthony Francis. "The molecular cloning and characterisation of autoantigens." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242453.

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12

Paine, Mark John Ingraham. "Molecular cloning of Echis carinatus venom genes." Thesis, University of Liverpool, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.291952.

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13

Caird, Mandy Ruth. "Molecular cloning studies on DNA polymerase alpha." Thesis, University of Aberdeen, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292388.

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The DNA polymerase α assay systems and partial purification techniques already in use in the laboratory were improved by isolating better-activated DNA and freezing cells prior to sonication. The suitability of monoclonal antibodies against Xenopus laevis and human KB cell (Tanaka et al., 1982) DNA polymerase α was established as a prelude to screening expression libraries. All were found to inhibit polymerase activity by approximately 50%. Two out of three anti-Xenopus laevis DNA polymerase α antibodies and three out of four anti-human DNA polymerase α antibodies bound antigen in HeLa cell cytoplasmic extracts. Human hepatoma, human foetal liver, human fibroblast and mouse bone marrow lambda gt11 expression libraries were screened with the above antibodies using the method of Young and Davis (1983) and various horseradish peroxidase and alkaline phosphatase conjugated second antibody systems in an attempt to isolate a human or mouse DNA polymerase α gene. A 1.9kb cDNA clone from a calf thymus expression library (Foster et al., 1986) was characterised. The molecular weight of 165-175kDa and polymerase activity of the β-galactosidase-calf fusion protein was confirmed but the fusion protein could not be detected with any of the antibodies mentioned above. The 1.9kb cDNA clone was used to show the regulation of DNA polymerase α activity in growth-regulated bovine kidney cell to be at the transcriptional level. The DNA sequence of the calf thymus cDNA fragment was determined. The DNA sequence of 2042 bases and all predicted open reading frames were compared with sequences contained in the GenBank, EMBL and NBRF (nucleic acid and protein) databanks of the University of Wisconsin Genetics Computer Group molecular biology package. No homology to any DNA polymerase α nucleic acid or protein sequence was found, suggesting that the sequence of Foster et al. (1986) might not code for a DNA polymerase α. In searches of international sequence databanks the 1.9kb DNA fragment shows the greatest similarity to human, bovine and rodent sequences. However, the similarity scores are very low suggesting that this sequence is unique and will require the determination of a longer sequence before location of an identity.

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14

Mackinnon, Charlotte M. "Molecular cloning of human complement component Cls." Thesis, University of Aberdeen, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327928.

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Cls cDNA clones, which together contained the entire coding region of the protein, were isolated from two human-liver cDNA libraries. The initial Cls clones were identified using a synthetic oligonucleotide probe which corresponded to a region of low degeneracy near the C-terminus of the Cls catalytic chain. Fragments of the Cls cDNA were used to screen a cosmid library in an attempt to isolate the Cls gene, but this proved unsuccessful and no positive clones were isolated. The complete primary sequence of Cls revealed that the homology between the Cls and Clr catalytic chains also extends throughout their non-catalytic chains. Like Clr, Cls can be divided into six structurally independent domains of which the sixth represents the catalytic B chain. Domains I and III in the A chain of Cls are internally homologous, as are domains IV and V. The latter domains are homologous to the internally repeating 60-residue sequences found in Factor B, C2 and other proteins. Domain II of Cls is similar to the 40-residue repeat sequences found in epidermal growth factor precursor and many of the vitamin K-dependent proteins. The assignment of these domains to the different regions of Cls tertiary structure has still to be achieved, but studies in this area should be facilitated now that the complete primary sequence for the Cls zymogen is available.

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15

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

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16

Doucette, Stephanie A. "Cloning of Bovine Placental Lactogen and Production in Vitro." Fogler Library, University of Maine, 2003. http://www.library.umaine.edu/theses/pdf/DoucetteSA2003.pdf.

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17

Lee, Rivera Irene. "Cloning and characterization of mTom40." Thesis, McGill University, 1998. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21588.

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The majority of mitochondrial proteins are synthesized on cytoplasmic polysomes and subsequently imported to a specific subcompartment within the organelle. In order to translocate cytosolic proteins into mitochondria a protein import system is located in the outer and inner mitochondrial membranes. The genetic composition of this complex has been well characterized in mitochondria of S. cerevisiae and N. crassa. However, little is known about the components of the import machinery in higher eukaryotes. Until today only homologues of Tom20 and Tom17 as well as two new proteins Metaxin and hTom34 have been identified. Nevertheless, none of the proteins forming the outer membrane translocation pore have been identified so far.
We have cloned a 35 kDa protein from a mouse cDNA library with a 25% overall aminoacid identity to yTom40 and 27% identity to nTom40. mTom40 contains two possible start codons 36 amino acids apart from each other. Interestingly, both the long and the short version of mTom40 can be imported in vitro into mouse mitochondria. This data suggests that the first 36 amino acids are not important for the import process of the protein.
The identified protein was shown to be deeply embedded into the outer membrane of mitochondria, although it is partially exposed to the intermembrane space. It possesses a sequence with very high homology to a similar region on both previously described homologues of N. crassa and S. cerevisiae. It is proposed that this region may be of physiological importance. To further characterize mTom40 we generated a yeast strain with a deletion of yTom40, and tried to rescue the lethal phenotype with the mammalian homologue. Finally we describe a bacterial overexpression system for mTom40 and a purification protocol for the fusion protein overproduced.

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18

Suen, Ki-Ling. "Cloning of protein kinase genes from a carrot cDNA library." Diss., Georgia Institute of Technology, 1991. http://hdl.handle.net/1853/25431.

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19

Law, Katherine Mary. "Molecular studies of Theiler's murine encephalomyletis virus." Thesis, University of Cambridge, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357801.

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20

Yang, Xiaolong. "Molecular cloning and characterization of human BAG-1." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0016/NQ47506.pdf.

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21

Tse, Chi-hang. "Molecular cloning of the goldfish dopamine D2 receptor." Click to view the E-thesis via HKUTO, 1998. http://sunzi.lib.hku.hk/hkuto/record/B42128511.

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22

Townsend, Paul Andrew. "The molecular basis of osteoblast adhesion." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263651.

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23

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.

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24

Freeman, John Douglas. "The cloning of polyhomeotic, a complex Drosophila locus required for segment determination and cuticular differentiation." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26256.

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The polyhomeotic (ph) locus of Drosophila melanogaster has been characterized genetically. Early studies showed that ph is a member of the Polycomb (Pc) group. These genes have similar phenotypes and are required for normal segment determination. Recent analyses of amorphic ph mutations show that the ph locus is complex, has a strong maternal effect and plays a role in cuticular development. To test the function of ph at the molecular level, the cloning of the ph locus was undertaken. One strain had been shown to contain a P element insertion near ph. A genomic library was prepared from this strain and a recombinant phage containing this P element insertion was isolated by transposon tagging. The DNA flanking the insertion was used as a starting point for a chromosomal walk. A series of overlapping phage spanning 170 kilobases was isolated. Southern blot analysis was used to determine the locations of important deficiency breakpoints within the region covered by the walk. A distance of approximately 35 kb was shown to separate the two deficiency breakpoints which include ph. This interval was found to contain rearrangements in four of the seven ph alleles which were examined by Southern blot analysis. The interval also contains a repeated sequence. The relationship between the genetic and molecular structure of ph is discussed.
Science, Faculty of
Zoology, Department of
Graduate

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25

Zhu, Tong. "Molecular cloning and characterization of a novel mammalian myosin I." Case Western Reserve University School of Graduate Studies / OhioLINK, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=case1062006565.

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26

Tu, Liwen. "Cloning and sequence analysis of multiple genes from Bifidobacterium infantis /." free to MU campus, to others for purchase, 2004. http://wwwlib.umi.com/cr/mo/fullcit?p3137758.

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27

Huo, Longfei, and 霍龍飛. "Molecular cloning and functional studies of cyprinid calmodulin." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B3016316X.

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28

Choi, Bo Yon Linda. "Molecular cloning and characterization of zebrafish connexin 43." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ36014.pdf.

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29

Greer, Peter Alan. "Molecular cloning of measles virus nucleic acid sequences." Thesis, McGill University, 1985. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=71952.

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A library of cDNA clones was prepared from measles virus infected cells. Six classes of viral specific cDNA sequences were identified by a combination of Northern and Southern blot hybridization analysis.
Clones corresponding to measles virus NP, P/C and M genes were initially identified by hybridization selection translation experiments. Restriction mapping analysis and comparison with the recently published nucleic acid sequences have confirmed the identities of these clones.
The cloned sequences corresponding to the NP and M genes were subcloned into a plasmid vector which contains the Salmonella phage SP6 promoter sequence. These constructions facilitated the in vitro synthesis of RNA. This cloned RNA was subsequently translated in vitro, giving rise to the measles virus NP and M proteins. In addition to the full length NP and M proteins, smaller peptides were observed in this in vitro expression system. The series of smaller peptides made from the cloned NP RNA appear to correlate with NP-related peptides which are seen in extracts prepared from infected cells.
The other three classes of viral specific clones could not be conclusively identified by hybridization selection translation experiments. However, northern blot hybridization analysis enabled a tentative assignment of these three cloned sequences to the measles virus HA, F and L genes. The clone corresponding to the F protein was subsequently expressed in vitro using the SP6 transcription vector system described above.

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30

謝志恒 and Chi-hang Tse. "Molecular cloning of the goldfish dopamine D2 receptor." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1998. http://hub.hku.hk/bib/B42128511.

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31

Kwok, Ho-yan Amy, and 郭可茵. "Molecular cloning and characterization of chicken prostaglandin receptors." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41508336.

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32

Martin, Samuel A. M. "Molecular cloning of Atlantic salmon pituitary hormone genes." Thesis, Queen's University Belfast, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359115.

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33

Cowin, John. "Molecular cloning and transformation studies in Aspergillus nidulans." Thesis, University of Essex, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292601.

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34

Schofield, Miles Alexander. "Molecular cloning of renal cysteine conjugate #beta#-lyase." Thesis, University of Surrey, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385104.

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35

Kwok, Ho-yan Amy. "Molecular cloning and characterization of chicken prostaglandin receptors." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41508336.

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36

胡可進 and Kejin Hu. "Molecular cloning and characterization of the cathepsin L gene from the marine shrimp metapenaeus ensis." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B3124421X.

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37

Churchman, Sarah M. "Cloning and characterization of a novel nitrilase from Rhodococcus ruber NCIMB 40757." Thesis, University of Huddersfield, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289409.

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38

Dean, Deyrick Osmond. "Isolation and phenotypic characterisation of deletion mutants of dnaK." Thesis, University of East Anglia, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292657.

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39

Zhang, Gaiping. "Bovine IgG Fc receptors." Thesis, University of Hertfordshire, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387187.

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40

Millar, N. S. "Molecular cloning and sequence analysis of Newcastle disease virus." Thesis, University of Newcastle Upon Tyne, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380750.

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41

Antic, Dragana. "Molecular genetics of hantaviruses: Cloning, sequencing, expression, and morphogenesis." Thesis, University of Ottawa (Canada), 1993. http://hdl.handle.net/10393/7653.

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Molecular characterization of the Seoul 80-39 virus, a prototype of the Seoul serotype, was carried out by cloning and sequencing. The large (L) genomic RNA segment is 6530 nucleotides long. The viral complementary-sense RNA contains a single open reading frame (ORF) from the AUG codon at nucleotide positions 37-39 to the UAA stop codon at nucleotide positions 6490-6492. This ORF could encode a polypeptide of 2151 amino acids (246,662 D) which likely corresponds to the L protein detected in purified viral particles (Elliot et al., 1984) and is assumed to be an RNA-dependent RNA polymerase molecule (Schmaljohn and Dalrymple, 1983). The M RNA segment is 3651 nucleotides long. A single ORF was detected in the viral complementary-sense RNA. This ORF could encode a polypeptide of 1133 amino acids which is cotranslationally processed into viral glycoproteins G1 and G2. The S genomic RNA segment was characterized from the 3$\sp\prime$ end to nucleotide 1332. One long ORF was detected from the AUG codon at nucleotide positions 43-45 to the UAA stop codon at nucleotide positions 1330-1332. This ORF has a capacity to encode a 48 kD protein which corresponds to the viral nucleocapsid (N) protein. A comparison of coding properties of the tripartite genomes of various hantaviruses allowed construction of the evolutionary tree for the Hantavirus genus. The second part of the study was focused on the maturation of Hantaan 76-118 virus glycoproteins G1 and G2 virus morphogenesis. Glycoproteins G1 and G2 form a complex. Experiments with the glycosylation inhibitor brefeldin A indicate that complex formation between the two glycoproteins occurs in the ER. Resistance of both G1 and G2 to endoglycosidase D suggests a structure with more than five mannose residues. Processing of a high mannose structures ((Man)$\sb8$(GlcNAc)$\sb2$ to (Man)$\sb7$(GlcNAc)$\sb2$ or (Man)$\sb6$(GlcNAc)$\sb2$) may take place in the cis-Golgi by the enzyme $\alpha$-mannosidase I. Virus budding was observed only in Hantaan virus-infected cells, in the trans-Golgi network. When the Golgi apparatus was destroyed by brefeldin A treatment, assembly of virus particles was not observed. The third project was to express nucleocapsid proteins of Seoul 80-39 virus and Hantaan 76-118 virus using baculovirus recombinants. Analysis of recombinant virus-infected Sf9 cell lysates by SDS-PAGE showed that N proteins were expressed in high levels and were identical to virion associated N proteins in size and antigenicity. (Abstract shortened by UMI.)

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42

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.

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43

Pasternak, Stephen H. "Subtractive cloning of cDNAs from motor neuron-like hybrid cells : a strategy for cloning cDNAs from rare cell types." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41742.

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Although motor neurons represent an important and well defined neuronal population, little is known about their unique gene expression. Because of the difficulties of purifying and culturing large numbers of primary motor neurons, we adopted a cloning strategy based on the NSC34 motor neuron-like hybrid cell line. This cell line was developed by fusing dissociated E14 mouse spinal cord cells with N18TG2 neuroblastoma cells and expresses a host of motor neuron markers and traits. We examined a cDNA library of the NSC34 cell line using a subtractive screening strategy in order to isolate cDNA copies of mRNAs preferentially expressed in the hybrid cells with respect to the N18 parents. Of 8 cDNAs isolated, two encode known neural genes, namely chromogranin B (a component of neurosecretory vesicles) and GAP43 (important for growth cone extension) and a third clone corresponds to a sequence which has been proposed to be the glutamate binding subunit of an NMDA receptor. One novel cDNA was isolated which appears to be preferentially expressed in brain and spinal cord (as determined by semi-quantitative PCR), and is clearly detectable in motor neurons by in situ hybridization. The remaining 4 cDNAs encode cytochrome oxidase subunit II, the mouse IAP element, and the mouse b1 element (2 copies). We conclude that this strategy is effective in isolating novel genes from rare cell types, but that large numbers of library phage must be examined and that hybrid cell-specific probes should be generated by subtraction with other hybrid cells (rather than the N18 parent) because of the large number of genes non-specifically activated by cell fusion.

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44

Douvile, Elizabeth. "Cloning and characterization of novel kinases from embryonic cells." Thesis, University of Ottawa (Canada), 1994. http://hdl.handle.net/10393/6903.

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Protein tyrosine kinases (PYKs) play key regulatory roles in the control of cell growth and differentiation. Attempts to identify novel PYKs through expression cloning strategies have led to the identification of a novel family of protein kinases, referred to as dual specificity kinases (DSKs). In addition to their immunoreactivity with antiphosphotyrosine antibodies, DSK family members have the ability to phosphorylate serine, threonine as well as tyrosine residues. A novel protein kinase, Esk (Embryonal carcinoma Ser/thr/tyr Kinase), has been isolated from an embryonal carcinoma (EC) cell line using an expression cloning strategy. Sequence analysis of two independent cDNA clones (2.97 and 2.85 Kb) suggested the presence of two Esk isoforms in EC cells. The Esk-1 cDNA sequence predicted an 857 amino acid protein kinase with a putative membrane spanning domain, while the Esk-2 cDNA predicted an 831 amino acid kinase which lacked this domain. Genomic analysis revealed that the Esk transcripts could arise through alternative splicing of the same primary transcript to generate the cytoplasmic and transmembrane isoforms of the kinase. In adult mouse, Esk messenger RNA levels were highest in tissues possessing a high proliferation rate or a sizeable stem cell compartment suggesting that Esk may play some role in the control of cell proliferation or differentiation. As anticipated from the screening procedure, bacterial expression of the Esk kinase reacted with antiphosphotyrosine antibodies on immunoblots. Furthermore, in in vitro kinase assays Esk was shown to phosphorylate both itself and the exogenous substrate myelin basic protein on serine, threonine and tyrosine residues confirming that Esk is a novel member of the dual specificity family of protein kinases. An antibody raised to the Esk kinase revealed that the protein was subject to developmental regulation; being highly expressed in rapidly proliferating cells, and absent in terminally differentiated cells and in adult mouse tissues. Finally, the Esk kinase was found to associate with the 85 kDa subunit of phosphatidylinositol 3-kinase (PI3K) in proliferating stem cells. In vitro binding studies suggested that the interaction of the Esk kinase with the 85 kDa subunit of PI3K could be mediated via both the SH2 and SH3 domains of this protein. The results presented in this thesis suggest that the Esk dual specificity kinase may play a role in the control of cell growth and differentiation and that the effects of the kinase could be mediated by the regulation of PI3K activity. The interaction of the Esk kinase with an SH2 domain containing protein, is the first indication for the physiological function of the tyrosine phosphorylating activity of this kinase in mammalian cells.

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45

Szatmari, George B. "Cloning and characterization of Mu-like transposable bacteriophage D108." Thesis, McGill University, 1987. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75453.

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Bacteriophage D108 is a temperate, transposable, mutator phage similar to phage Mu. These heteroimmune phages have 37 kb linear, double-stranded DNA genomes which are over 90% homologous.
We have isolated four independent insertions of an entire thermoinducible D108 c ts10 phage genome in the low-copy plasmid pSC101. We also characterized a previously isolated Mu c ts62 insertion in the same plasmid replicon. Fine-structure analysis of these insertions showed that lytically transposing Mu and D108 genomes caused 5 bp duplications of the target site. In addition, we cloned and sequenced the terminal 800 bp of the right end of D108, a region important for the transposition of the phage genome. This analysis has delineated the critical DNA sequences required for D108 transposition, and has also indicated that these sequences are not entirely homologous to those of phage Mu, despite the fact that the phages can transpose each other's DNA. Moreover, a 520 bp insertion of DNA found in the right end of D108, but not Mu, has also been characterized, and has begins 72 bp from the right end of D108, immediately adjacent to the cis -acting sequences required for D108 transposition. This insertion does not appear to be an insertion element, but rather an extra gene in the right end of D108 whose function is apparently non-essential for D108 growth. The sequence differences and rearrangements between Mu and D108 in this region indicate a complex evolutionary relationship between these phages at their right extremities.
In addition to characterizing the DNA differences between Mu and D108, we have also examined a difference occurring at the protein level. The L gene products of Mu and D108 have different molecular weights, but are encoded from a region of homology between the two phages. We cloned a region of D108 DNA that was able to rescue amber mutations in the L gene of Mu by homologous recombination, but not complement Mu L amber phages in vivo. This region produced a gene product which was truncated at the carboxy terminus, but was able to cross-react with anti-Mu phage serum. The production of this truncated protein was lethal to growing Escherichia coli cells, apparently interfering with cell wall biosynthesis.

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46

Lun, Hoi-man. "Characterization and molecular cloning of proteinases of Trichinella spiralis (Nematoda) /." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?B23621989.

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47

Fischer, Roland. "Molecular cloning of a human plasma membrane Ca²⁺-ATPase /." Zürich, 1989. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=8811.

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48

Truesdell, Peter. "Molecular cloning and expression analysis of Pseudaletia unipuncta allatotropin." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq20707.pdf.

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49

Boyd, Jason. "Molecular cloning and characterization of two Arabidopsis thaliana sulfotransferases." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ64039.pdf.

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50

Nilsson, Isabelle. "The cholecystokinin receptor family : molecular cloning and pharmacological characterization /." Linköping : Univ, 2003. http://www.bibl.liu.se/liupubl/disp/disp2003/med791s.pdf.

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