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1
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
2
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
3
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
4
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
5
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
6
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.
7
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.
8
Pauliny, Angela. „Cloning and molecular characterisation of the zebrafish colourless gene“. Thesis, University of Bath, 2002. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393804.
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9
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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10
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.
11
Huo, Longfei, und 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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12
Yowe, David Langdon. „Molecular cloning and characterisation of the Barramundi growth hormone gene“. Thesis, Queensland University of Technology, 1994.
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13
Jiang, Qijiao. „Cloning and characterization of midgut-specific gene/gene products in the mosquito Aedes aegypti“. Diss., The University of Arizona, 1996. http://hdl.handle.net/10150/282179.
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The yellow fever and dengue fever carrying mosquito, Aedes aegypti, requires blood feeding for egg production. The blood proteins are digested in the midgut to yield amino acids which are the nutritional source for oogenesis. Serine proteases are important enzymes that participate in the process of blood protein digestion. The identification of the corresponding genes may have significant implications in the control of mosquito-borne diseases. A gut-specific chymotrypsin-like cDNA was isolated and sequenced. The 938 bp clone encodes a preproenzyme with a putative 18 amino acid signal peptide sequence, a 7 amino acid activation peptide sequence rich in serine and charged residues, and a mature enzyme of 268 amino acids. The deduced amino acid sequence has a typical catalytic triad region for serine proteases (His 57, Asp 102 and Ser 195 in bovine chymotrypsin numbering system), and the hydrophobic substrate binding pocket with most features of chymotrypsins. Six cysteine residues are present in the sequence which are characteristically involved in disulfide bond formation in invertebrate serine proteases. Characterization of the gene expression and the protein synthesis, as well as the enzymatic activity in the midgut, clearly demonstrated that (1) the chymotrypsin gene is newly transcribed after eclosion and the mRNA is present almost steadily during the digestion of a meal; (2) the chymotrypsin synthesis and its corresponding activity are induced and increased significantly by the ingestion of a meal. In vitro studies of the recombinant protease derived from the cDNA clone indicated several unique properties of the mosquito chymotrypsin compared with its bovine analog.
14
Liang, Rongti. „Molecular cloning and characterization of the ovine placental lactogen gene /“. free to MU campus, to others for purchase, 1996. http://wwwlib.umi.com/cr/mo/fullcit?p9717169.
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15
Driscoll, Barbara. „Cloning and expression of the bovine papillomavirus major capsid gene“. Diss., The University of Arizona, 1988. http://hdl.handle.net/10150/184475.
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In order to characterize the protein product of the major capsid protein gene of Bovine Papillomavirus Type 2, 93% of the L1 open reading frame was cloned into two different expression vectors. This coding sequence produced a hybrid product when cloned into the expression vector pKK233-2, which interacted weakly with anti-BPV antisera and proved unable to elicit neutralizing antibodies. When the sequence was cloned into the expression vector pRA10, a more native form of the major capsid protein was produced, which interacted well with anti-BPV sera. Antisera raised against this cloned product was able to neutralize BPV in a tissue culture transformation assay. The 3' end of the L1 open reading frame was cloned into pBA10 in order to characterize the immunogenic potential of the carboxy terminus of the major capsid gene. The carboxy terminus proved to have no greater ability to interact with anti-BPV antisera, showing that the immunogenic epitopes of the protein are probably evenly distributed along the linear sequence.
16
Bingle, Lewis Edward Hector. „The molecular genetics of polyketide biosynthesis in filamentous fungi“. Thesis, University of Bristol, 1997. http://hdl.handle.net/1983/15ebe2f2-ab67-47b5-ae4b-1f00140561a1.
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17
胡可進 und 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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18
Seto, Nina O. L. „Cloning and expression of the Drosophila melanogaster CuZn superoxide dismutase gene“. Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/30971.
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Aging and disease processes may be due to deleterious and irreversible changes produced by free radical reactions. The enzyme copper-zinc superoxide dismutase (CuZn SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1) performs a protective function by scavenging superoxide radicals. In order to determine whether additional SOD activity affects longevity and oxygen metabolism in Drosophila, our approach was to clone the Sod gene and introduce additional copies of the gene back into the genome via P element mediated transformation. The effects of increased SOD activity on Drosophila life span and oxygen free radical metabolism were investigated.
The CuZn SOD cDNA and gene were cloned from Drosophila melanogaster. The sequence of the Sod cDNA and gene revealed an additional C-terminal triplet coding for valine not found in the mature SOD protein. The nucleotide sequence of the coding region has 56% and 57% identity when compared to the corresponding human and rat Sod genes, respectively. A probe of the cloned gene hybridizes to position 68A4-9 on Drosophila polytene chromosomes. In wild-type Drosophila the Sod cDNA hybridizes to a 0.7-0.8 kb transcript which is greatly diminished in a SOD 'null' mutant that produces only 3.5% of the SOD protein.
A 1.8 kb EcoRI gene fragment containing the Sod gene was cloned into the P vector pUChsneo and microinjected into Drosophila embryos. Five transformed lines, each of which contain an additional copy of the Sod gene at different chromosomal sites were constructed. The chromosomal positions of the transposed Sod sequence were determined by in situ hybridization of the Sod gene to salivary gland polytene chromosomes. Analysis of RNA from the transformed flies revealed that the transposed Sod gene was expressed. The range of SOD activity for the five transformed lines was 131% to 170% of the value of wild-type. There was good correlation between the amount of Sod mRNA and the level of SOD activity in the transformed lines.
Increased SOD levels in the transformed lines did not confer greater resistance to paraquat-generated superoxide radicals, nor increase their lifespan. The SOD 'null' mutant with 3.5% of the wild-type SOD activity was hypersensitive to paraquat when compared to wild-type, whereas the heterozygous SOD deficiency Df(3L)1xd⁹/TM3SbSer with 50% of the wild-type SOD activity was not. Mutants lacking SOD are dramatically impaired in oxygen metabolism and a few percent of wild-type activity appears to provide significant protection against superoxide, while 50% of the wild-type levels confers essentially the same resistance as wild-type. Despite the observation that the SOD activities found in a wide range of animals correlates directly with their longevity, Drosophila melanogaster appears to be well protected against the toxic effects of oxygen by its native levels of SOD.Arts, Faculty of
Philosophy, Department of
Graduate
19
Hughes, John Michael Xavier. „Molecular analysis of small RNAs of Saccharomyces cerevisiae“. Thesis, University of Leicester, 1988. http://hdl.handle.net/2381/35256.
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RNA has many diverse functions in living organisms, from serving as genome for many viruses, to regulating DNA replication, transcription, translation and other metabolic processes. Some RNA, like protein, has been shown to have catalytic activity. The great proportion of the mass of RNA in living cells, in the form of ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA), constitutes the machinery of protein synthesis, the remainder (approximately 2%) consists of many heterogeneous RNA species of relatively small size, loosely termed "small RNAs", the functions of many of which are completely unknown. In an attempt to understand some of these functions, three hitherto undescribed small RNAs of the budding yeast Saccharomyces cerevisiae were identified and their genes were cloned. These three small RNAs, which lack polyadenylation at their 3' ends, appear to represent the three most abundant RNA species in this organism after rRNA and tRNA. The most abundant of the three was found to be mainly cytoplasmic and was therefore called "small cytoplasmic RNA 1" (scR1). The other two RNAs, named snR17 and snR30, were found to be enriched in nuclear fractions and to possess trimethyl guanosine cap structures at their 5 ends, identifying them as belonging to the ubiquitous class of "U" small nuclear RNAs (U snRNAs), of which several are required for the endonucleolytic cleavage and splicing reactions in the maturation pathways of nuclear precursor mRNAs (pre-mRNA). Whereas scR1 and snR30 are both encoded by single genes, snR17 is the only yeast small RNA found so far to be encoded by two genes. SnR17 was found to be essential: haploid yeast strains lacking intact copies of one or other of the genes appeared to grow normally, but strains lacking both genes were inviable. The nucleotide sequences of the snR17 genes were determined, and the primary and predicted secondary structures of the RNA, 328 nucleotides in length, were found to show significant similarities to those of U3 snRNA, an abundant U snRNA, the function of which is not known. SnR17 belongs to a family of S. cerevisiae snRNAs which, unlike those involved in pre-mRNA splicing, are located in the nucleolus hydrogen-bonded to pre-rRNA, and are associated with antigenic protein that is recognized by human antibodies specific for a 36 kD polypeptide of the U3 small nuclear ribonucleoprotein (U3 snRNP) in mammals. U3 snRNA is also nucleolar and associated with pre-rRNA. Given their structural similarities, snR17 and U3 snRNA are presumably homologous. Yeast snRNAs associated with the anti-(U3)RNP antigen share with U3 snRNAs a conserved nucleotide sequence element. This sequence element alone, however, when injected into Xenopus oocytes, was not sufficient to direct binding of the antigen. The association of snRNAs with pre-rRNA suggests that they function in ribosomal biogenesis.
20
Marcantonio, Daniela. „Cloning and characterization of a novel steroid hormone responsive gene“. Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37773.
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Estrogen modulates growth and function of female reproductive tissues, such as uterus and mammary gland, by eliciting an array of biochemical responses and is linked to mammary carcinogenesis. To further understand the involvement of estrogens in complex physiological processes, we sought to increase the spectrum of genes regulated by estrogens. We cloned a novel 3.719 Kb cDNA from rat uterine tissue, termed steroid sensitive gene-1 (SSG1), encoding a nuclear protein of 386 amino acids and demonstrated that it is regulated by 17beta-estradiol in the uterus and mammary gland. In the rat uterus, SSG1 mRNA is down-regulated by 17beta-estradiol in time- and dose-dependent manners. In the mammary gland, chronic 17beta-estradiol treatment resulted in a significant accumulation of the SSG1 protein, which was dependent on the presence of elevated levels of 17beta-estradiol. SSG1 was consistently over-expressed in estrogen-dependent 7,12-dimethylben(a)anthracene-induced rat mammary tumors which may be a consequence of an altered hormonal environment. In rat and human mammary tissue, SSG1 protein was localized to the myoepithelial cells of the mammary ducts and to the smooth muscle cells of the vasculature. In human mammary tumors SSG1 was predominant in the epithelial compartment. In males, we demonstrate that SSG1 protein expression, immunolocalized to the prostatic smooth muscle cells and to the smooth muscle cells of the vasculature, is dependent on the presence of androgens. We conclude that SSG1 expression is regulated by steroid hormones in both female and male reproductive systems. We propose that SSG1 is a target for both estrogen and androgen modulation and that it may be implicated in estrogen functions related to mammary carcinogenesis.
21
Finch, Jennie Louise. „Cloning and characterisation of the human uroplakin 1B gene /“. Title page, contents and summary only, 1998. http://web4.library.adelaide.edu.au/theses/09PH/09phf4918.pdf.
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22
Lai, Kun-Nan. „Cloning and expression of cambialistic Bacteroides fragilis superoxide dismutase gene“. Diss., This resource online, 1992. http://scholar.lib.vt.edu/theses/available/etd-05042006-164528/.
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23
Fan, Xiaohui. „Uroguanylin : molecular cloning and characterization of a potential natriuretic hormone /“. free to MU campus, to others for purchase, 1997. http://wwwlib.umi.com/cr/mo/fullcit?p9841285.
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24
Boddy, Lynn M. „Regulation and molecular cloning of an invertase gene from Aspergillus niger“. Thesis, University of Nottingham, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336021.
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25
Joseph, Rajiv. „Molecular cloning of neuronatin : a novel brain-specific mammalian developmental gene“. Thesis, University of London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243807.
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26
Raitt, Desmond C. „Molecular cloning and characterisation of the Aspergillus nidulans cytochrome c gene“. Thesis, Cranfield University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305383.
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27
Helps, Nicholas Royston. „Cloning and molecular analysis of an evolutionarily conserved Drosophila melanogaster gene“. Thesis, University of Leicester, 1993. http://hdl.handle.net/2381/35249.
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28
SAEDI, MOHAMMAD SAEED. „CLONING OF BACTERIOPHAGE-PHI29 GENE 15; ISOLATION, OVERPRODUCTION AND PURIFICATION OF PHI29 LYTIC ENZYME“. Diss., The University of Arizona, 1987. http://hdl.handle.net/10150/184008.
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A spontaneous deletion mutant of Bacillus phage φ29 (φ29Δ1) is characterized in the first part of this study. This mutant has a 1,112 base pair deletion, which covers the entire coding sequence of genes 14 and 15 including the early promoter, B2. While lysis is very delayed, the phage DNA synthesis and internal phage development appears to be normal in the cells infected with this deletion mutant. These results indicate that the early functions are intact in φ29Δ1. Results also suggest that genes 14 and 15 are dispensable for bacteriophage φ29 growth, and that the B2 promoter may also be despensable for the early functions of φ29. To further explore the function of gene 15, a DNA fragment of φ29 chromosome, encoding the entire sequence of this gene, has been cloned into the Escherichia coli expression vector pPLc245, under the control of the phage lambda major early leftward promoter, PL. Upon heat induction, a protein with an apparent size of 26 kdal was over-produced. This protein has been purified to near homogeneity and confirmed to be the product of gene 15 by amino acid sequence analysis of its N terminus. The purified product of gene 15 has a lysozyme activity similar to the other phage-type lysozymes: products of phage T4 gene e and of phage P22 gene 19. This is the first lysozyme to be cloned and purified from a gram positive system. Bacteriophage φ29 lysozyme has been characterized in the last part of this study. Results show that this enzyme seems to more active than hen egg-white lysozyme against B. subtilis, E. coli, and M. lysodeikticus cells. Most of the characteristics of φ29 lysozyme appears to be similar to the P22 and T4 lysozymes, however, φ29 lysozyme seems to be about 2 times more thermostable than the other two lysozymes.
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馬忠華 und Chung-wah Ma. „Genomic isolation and molecular analysis of a rat [alpha]-globin gene cluster“. Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1998. http://hub.hku.hk/bib/B31237514.
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Ma, Chung-wah. „Genomic isolation and molecular analysis of a rat [alpha]-globin gene cluster /“. Hong Kong : University of Hong Kong, 1998. http://sunzi.lib.hku.hk/hkuto/record.jsp?B19473047.
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Zvejnieks, Peter Andrew. „Cloning of an embryo-specific gene from Daucas Carota cDNA library“. Thesis, Georgia Institute of Technology, 1990. http://hdl.handle.net/1853/25408.
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32
Agarwal, Anika. „Molecular cloning and characterisation of the human oviduct-specific glycoprotein (HuOGP) promoter“. Thesis, Hong Kong : University of Hong Kong, 2002. http://sunzi.lib.hku.hk/hkuto/record.jsp?B25335273.
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Grove, Heather Lee. „Cloning and characterization of the Pichia Pastoris PMR1 gene“. Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/613.
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Pichia pastoris, a popular protein expression system, is limited in its ability to secrete heterologous proteins. The PMR1 gene, the disruption of which is known to improve the secretion of prochymosin, human prourokinase, and human tissue plasminogen activator in Saccharomyces cerevisiae, was cloned from P. pastoris. The pmr 1 mutant in S. cerevisiae also displayed a slow growth phenotype when grown on low Ca2+ medium. The putative P. pastoris PMR1 gene, encoding for a 924 amino acid P-type Ca2+ ATPase, was disrupted in P. pastoris and the secretion of horseradish peroxidase (HRP) and β-galactosidase (β-gal) analyzed. Secreted HRP activity was determined using 3,3',5,5' tetramethylbenzidine (TMB) colorimetric assay and western analysis. β-gal expression and secretion was determined by western analysis. Secretion in P. pastorius Δpmr1 for both heterologous proteins showed no appreciable difference compared to wild type, nor did P. pastoris Δpmr1 display the slow growth phenotype seen in S. cerevisiae Δpmr1 (Rudolph H. et al., 1989).
34
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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Lonie, Andrew. „Cloning and characterisation of the Polycomblike gene, a transacting repressor of homeotic gene expression in Drosophila“. Title page, contents and summary only, 1994. http://hdl.handle.net/2440/21504.
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Includes bibliographies.{59} leaves : ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
The Polycomblike gene of Drosophila melanogaster is required for the correct spatial expression of the homeotic genes of Antenapaedia and Bithorax Complexes. This thesis describes the isolation and molecular characterization of the Polycomblike gene.
Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1995
36
Hidalgo-Downing, Alicia. „Molecular cloning of patched and analysis of its role in intrasegmental patterning in D. melanogaster“. Thesis, University of Oxford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.258158.
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Smith, Simon A. „Molecular analysis of the yeast cell cycle : isolation and characterization of a new gene, TSM3721“. Thesis, University of Manchester, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.253128.
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Cluness, Martin James. „Cloning and molecular analysis of the cyanide hydrotase gene of Fusarium lateritium“. Thesis, University of Sunderland, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278926.
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39
Kosmidou, Effie. „Molecular cloning and characterisation of the pp2a#alpha# gene in Aspergillus nidulans“. Thesis, University of East Anglia, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361419.
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40
Moffat, K. G. „The molecular cloning of the mutT gene of Escherichia coli K-12“. Thesis, Cranfield University, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.373991.
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Benson, Fiona Elizabeth. „Molecular cloning and analysis of the ruv gene of Escherichia coli K12“. Thesis, University of Nottingham, 1988. http://eprints.nottingham.ac.uk/30593/.
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Mutations in the ruv gene of Escherichia coli K-12 result in an increased sensitivity to agents that damage DNA. Studies presented in this thesis demonstrate that the ruv gene product is required for conjugational recombination in certain genetic backgrounds. From this it was inferred that the role of the ruv gene product was in the recombination repair of daughter strand gaps and double strand breaks in damaged DNA. In addition, the ruv gene product is shown to be required for the efficient recovery of F' transconjugants in certain genetic backgrounds, suggesting that recombination between transferred F' and the recipient chromosome may be an obligatory event in these strains. Expression of ruv is regulated as part of the SOS response to DNA damage, by the lexA and recA gene products. The ruv gene product appears not to have any major role in its own regulation, however the basal level of expression of other SOS genes is increased in strains carrying ruv mutations. The ruv gene has been cloned on a 10.4kb HindIII fragment into the low copy number vector pHSG4l5, to give plasmid pPVA101, which has been demonstrated to complement the UV sensitivity of strains carrying any of the 10 different ruv mutations tested. Analysis of the proteins synthesised by pPVA101, its deletion derivatives, and derivatives with Tnl1000 insertions inactivating the ruv gene, allowed the identification of the ruv coding region, and suggested that the ruv gene encoded a 4lkd protein. In addition, regions of the cloned DNA coding for two further proteins of approximately 24kd and 33kd were identified. The sites of insertion of Mud(Ap)Rlac and Tn10 elements in the ruv gene were mapped, which allowed the direction of transcription to be determined, and suggested that the 4lkd protein may be cotranscribed with the 24kd protein from a promoter upstream of the smaller protein. This was substantiated by the demonstration that two of the ruv mutations studied were chromosomal inversions, one of which had its end point within the coding region for the 24kd protein, and by the isolation of an SOS inducible promoter derived from the region upstream of the 24kd protein. The nucleotide sequence of the ruv region revealed two open reading frames, designated ruvA and ruvB, with coding potential for proteins of 22087 daltons and 37177 daltons respectively, corresponding to the proteins with molecular weights estimated as 24kd and 41kd from SOS-polyacrylamide gels. A possible promoter, and two sequences with homology to the LexA binding site consensus sequence were identified upstream of the coding region of the 22kd protein. An amino acid sequence within the proposed RuvB protein was identified with homology to ATP binding sites of other proteins 950involved in DNA metabolism.
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„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
43
Li, Cheng-Fang, und 李承芳. „Molecular cloning and analysis of Alterococcus agarase gene“. Thesis, 2005. http://ndltd.ncl.edu.tw/handle/94976591728616652853.
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碩士東吳大學
微生物學系
93
Agar is an important gelifying agent for biochemical use and the food industry, mainly extracted from the red seaweeds belonging to the family of Rhodophyceae. Agarose is the gelling component comprising a linear chain of alternating 3-O-linked-α-D-galactopyranose and 4-O-linked 3,6-anhydro-β-L-galactopyranose. A moderately thermophilic marine bacteria S3PY (lab code) which was isolated from hot springs in the intertidal zone of Lutao, Taiwan, produce at least 8 different extracellular agarases in molecular weight. That was Gram-negative halophile growing optimally at 0-5％NaCl. The optimal growth temperature range was approximate 50-55℃. The strains tolerated a relatively narrow pH range from 6 to 6.5 and had 64.5 mo1％G + C contents. Several physiology test and 16s rDNA sequence prove that S3PY was identical to the Alterococcus agarlyticus published by Shien in 1998. Partially Sau3A-digested genomic DNA of Alterococcus agarlyticus was cloned into BamHI-digested pUC19 vector and transformed to E.coli DH5α MCR‘. After 40,000-50,000 transformants were isolated, one with agarolytic activity clone was picked out. The pUC19 plasmid with a 5.2 kb insert harbored by the clone was designated as pUC3A. Multiple agarases expressed by the clone exactly match the agarases in S3PY, except 2-3Kda larger in molecular mass. SDS-PAGE containing 6M urea and 5 times of SDS showed those multiple agarases are individual monomer. Single open reading frame of 3,411bp was found and designated as agoI beginning with ATG at nucleotide 3409 (Met1) and ending with TAG at XbaI site of pUC19. Based on BLAST, the putative agarase gene belongs to GH 86; The search of Conserved domain shows that AgoI is consist of several module protein may served as substrate binding or surface adhesion. The functional fragments subcloning by PCR amplification implied those multiple agarases were originated from the single ORF agoI. The ORF subcloned in pET25b expression vector validate the comsuption. To further understand the regulation of Alterococcus Agarolyticus agarases, several hypotheses have been proposed.
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„Molecular cloning of cellulase gene from volvariella volvacea“. Chinese University of Hong Kong, 1995. http://library.cuhk.edu.hk/record=b5888545.
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by Ka-shing Cheung.Thesis (M.Phil.)--Chinese University of Hong Kong, 1995.
Includes bibliographical references (leaves 112-114).
Abstract --- p.i
Acknowledgments --- p.iii
Table of contents --- p.v
Abbreviations --- p.x
List of figures --- p.xi
List of tables --- p.xiii
Chapter 1. --- Introduction
Chapter 1.1 --- General introduction --- p.1
Chapter 1.2 --- Purpose of study --- p.3
Chapter 2. --- Literature review
Chapter 2.1 --- Cellulose: properties and degradation --- p.4
Chapter 2.2 --- Cellulase system
Chapter 2.2.1 --- Definition and substrate specificity --- p.5
Chapter 2.2.2 --- Co-operation of cellulases --- p.5
Chapter 2.2.3 --- Multiplicity of cellulases --- p.6
Chapter 2.2.4 --- Regulation of cellulase synthesis --- p.6
Chapter 2.2.5 --- Architecture of cellulase protein --- p.8
Chapter 2.3 --- Molecular biology of fungal cellulase genes
Chapter 2.3.1 --- Structural organization of fungal cellulase genes --- p.15
Chapter 2.3.1.1 --- Promoter and regulatory sequence --- p.15
Chapter 2.3.1.2 --- Sequence at transcriptional start point (tsp) --- p.16
Chapter 2.3.1.3 --- Signal peptide --- p.18
Chapter 2.3.1.4 --- Intron --- p.18
Chapter 2.3.1.5 --- General sequence homology --- p.21
Chapter 2.3.2 --- Regulation of cellulase production at molecular level --- p.23
Chapter 2.3.3 --- Multiplicity of cellulase gene --- p.24
Chapter 2.3.4 --- Tactics to clone fungal cellulase genes --- p.25
Chapter 2.3.4.1 --- Past experience --- p.25
Chapter 2.3.4.2 --- Present approach --- p.28
Chapter 2.3.5 --- The importance of cellulase gene cloning --- p.29
Chapter 2.4 --- Cellulolytic microorganisms
Chapter 2.4.1 --- Ecological roles and diversity --- p.31
Chapter 2.4.2 --- "Biology of the straw mushroom, Volvariella volvacea" --- p.31
Chapter 3. --- Materials and methods
Chapter 3.1 --- Recipes of media and solutions
Chapter 3.1.1 --- Culture media and microbial-growth related chemicals --- p.34
Chapter 3.1.2 --- Solutions --- p.36
Chapter 3.2 --- Bacterial and fungal strains and the growth and storage of mycelium
Chapter 3.2.1 --- Bacterial and fungal strains --- p.42
Chapter 3.2.2 --- Growth and storage of mycelium --- p.42
Chapter 3.3 --- Extraction of DNA from mycelium --- p.43
Chapter 3.4 --- Degenerate polymerase chain reaction (PCR)
Chapter 3.4.1 --- Primers --- p.45
Chapter 3.4.2 --- Amplification conditions of degenerate PCR --- p.46
Chapter 3.5 --- Cloning of PCR products
Chapter 3.5.1 --- Ligation --- p.47
Chapter 3.5.2 --- Transformation --- p.47
Chapter 3.5.3 --- Screening by blue/white selection --- p.47
Chapter 3.5.4 --- Screening by PCR --- p.48
Chapter 3.6 --- Plasmid extraction by alkaline lysis
Chapter 3.6.1 --- Midi-preparation of plasmid by Qiagen column --- p.51
Chapter 3.6.2 --- Preparation of plasmid using Promega's Wizard minipreps DNA purification system --- p.51
Chapter 3.7 --- Sequencing analysis of cloned PCR products
Chapter 3.7.1 --- Growth and titering of helper phage R408 --- p.53
Chapter 3.7.1.1 --- Plate elution method --- p.53
Chapter 3.7.1.2 --- Liquid culture method --- p.53
Chapter 3.7.1.3 --- Titering of R408 --- p.53
Chapter 3.7.2 --- Rescue of single-stranded DNA from pCR-Script phagemid --- p.54
Chapter 3.7.3 --- Sequencing by chain-termination reaction --- p.54
Chapter 3.7.4 --- Preparation of polyacrylamide gel for DNA sequencing --- p.56
Chapter 3.7.5 --- Running a sequencing gel --- p.57
Chapter 3.7.6 --- "Fixation, exposure and development of sequencing gel and X-ray film" --- p.57
Chapter 3.7.7 --- Sequence analysis --- p.58
Chapter 3.8 --- Digestion of DNA with restriction enzymes --- p.59
Chapter 3.9 --- Agarose gel electrophoresis --- p.60
Chapter 3.10 --- Purification of DNA from agarose gel by Qiaex --- p.61
Chapter 3.11 --- Southern hybridization
Chapter 3.11.1 --- Southern blotting and DNA immobilization --- p.62
Chapter 3.11.2 --- Random-labelling of DNA probe and removal of unincorporated nucleotides --- p.63
Chapter 3.11.3 --- Pre-hybridization and hybridization --- p.63
Chapter 3.11.4 --- Exposure and development --- p.64
Chapter 3.11.5 --- Determination of molecular weight of hybridization signals --- p.65
Chapter 4. --- Results
Chapter 4.1 --- Extraction of DNA from the straw mushroom mycelium --- p.66
Chapter 4.2 --- Amplification of V. volvacea genomic DNA using degenerate primers --- p.70
Chapter 4.3 --- Cloning of PCR products using pCR-Script SK (+) cloning kit
Chapter 4.3.1 --- Screening by blue/white selection --- p.77
Chapter 4.3.2 --- Screening by PCR --- p.77
Chapter 4.4 --- Plasmid extraction by alkaline lysis --- p.80
Chapter 4.5 --- Preparation of single-stranded DNA template for sequencing
Chapter 4.5.1 --- Growth and titering of helper phage R408 --- p.82
Chapter 4.5.2 --- Rescue of single-stranded DNA from pCR-Script phagemid --- p.82
Chapter 4.6 --- Sequencing of cloned PCR products
Chapter 4.6.1 --- The choice of template --- p.84
Chapter 4.6.2 --- DNA and translated amino acid sequence of PCR clones --- p.84
Chapter 4.6.3 --- Alignment of DNA sequences against other fungal cellulase genes --- p.93
Chapter 4.6.4 --- Alignment of translated amino acid sequences against other fungal cellulase --- p.96
Chapter 4.7 --- Purification of DNA from agarose gel by Qiaex --- p.98
Chapter 4.8 --- Southern hybridization
Chapter 4.8.1 --- Restriction digestion of genomic DNA --- p.101
Chapter 4.8.2 --- Hybridization --- p.104
Chapter 5. --- Discussion
Chapter 5.1 --- Extraction of DNA from V. volvacea mycelium --- p.107
Chapter 5.2 --- Rationales of designing degenerate primers from heterologous amino acid sequence --- p.107
Chapter 5.3 --- Amplification of V. volvacea DNA using degenerate primers --- p.110
Chapter 5.4 --- Cloning of PCR products using pCR-Script system --- p.111
Chapter 5.5 --- The precaution of using Qiaex-purified DNA --- p.112
Chapter 5.6 --- Sequencing analysis
Chapter 5.6.1 --- DNA sequence analysis --- p.113
Chapter 5.6.2 --- Protein sequence analysis --- p.114
Chapter 5.7 --- Southern hybridization --- p.116
Chapter 6. --- Conclusion and further analysis --- p.117
Chapter 7. --- References --- p.119
45
HE, XING-FEN, und 何杏芬. „Molecular cloning of a □2 -adrenoceptor subtype gene“. Thesis, 1990. http://ndltd.ncl.edu.tw/handle/69343417531275585558.
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46
iWu, Hsiao-we, und 吳孝薇. „Molecular cloning of lipoxygenase-like gene in rat“. Thesis, 2015. http://ndltd.ncl.edu.tw/handle/08421717447841218332.
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碩士慈濟大學
分子生物暨人類遺傳學系碩士班
103
A novel Lipoxygenase-like (Lipo-like) gene, was identified within the rat IEL rupture QTL region using various bioinformatics tools. Seven primer pairs were designed and RT-PCR from lung or spleen single strand cDNA demonstrated that rat Lipo-like gene encoded 762 amino acid residues with three putative PLAT (plasminogen activator) and one Fascin domains. Lipo-like gene consists of 20 exons with a CDS of 2289 bp, located in rat chromosome 5q11 region covering more than 246-kb. Lipo-like mRNA was highly expressed in lung, spleen, heart, muscle, and kidney; modestly expressed in liver, testis and large intestine; weakly expressed in stomach and small intestine but almost undetectable in brain, white blood cells or abdomental aorta. Two corresponding rat ESTs clones, CB326534.1 and CR464603.1, have been identified from NCBI EST Database. Attempts to order these two clones and sequenced were unsuccessful. Compared with SD rat CDS, BN has three point mutations, namely p. S432T, p. K492N and p. H592D. A homolog search in NCBI EST database further identified two human LIPO-like EST clones, AK316511.1 and AK304443.1 with 82% homology at the DNA level. The human homolog LIPO-like gene was successfully RT-PCR and cloned from H1299 cell line, a metastatic human lung cancer cell. Human LIPO-like gene is located at chromosomal 8q12.1, spanned about 210-kb with 24 exons of an ORF of 916 amino acid residues and a CDS of 2751 bp. Attempts to RT-PCR human LIPO-like in other cancer cells were unsuccessful. As PLAT domain has been implicated in cell migration and tissue remodeling whereas Fascin is a monomeric actin filament bundling protein and binds β-catenin, which suggested that this novel LIPO-like protein may play a role in cell-cell adhesion, cell migration or tissue remodeling.
47
YOU, ZHI-WEN, und 游志文. „Molecular cloning of Chinese hamster metallothionein-I gene“. Thesis, 1992. http://ndltd.ncl.edu.tw/handle/98111551591836149587.
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48
Wen, Hui-Ju, und 溫慧如. „Molecular cloning and expression of zebrafish single minded gene“. Thesis, 2000. http://ndltd.ncl.edu.tw/handle/49529384597426540684.
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49
邵逸昕. „Molecular Cloning of the thdF Gene from Streptococcus Agalactiae“. Thesis, 1998. http://ndltd.ncl.edu.tw/handle/45663417275579734324.
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碩士國立臺灣大學
微生物學研究所
86
In the genus of streptococcus, Streptococcus agalactiae (group B streptococcus, GBS) is the only microorganism expressing hippuricase activity that can degrade hippuric acid into glycine and benzoic acid. In the attempt to isolate hippuricase gene (hipO) of GBS, a pair of primers were designed from the hipO gene nucleotide sequence of Camphylobacter jejuni. Using GBS genomic DNA as template, a 2.0 Kb DNA fragment was obtained by PCR. Then, the PCR product was labeled with digoxigenin-dUTP and used as a probe to screen GBS genomic library constructed with Lambda DASH Ⅱvector. A clone positively reacted with the probe was isolated and purified its DNA. The phage DNA fragment positively reacted with the probe was isolated and subcloned to pUC18 and sequenced. The nucleotide of the inserted DNA fragment was verified containing a partial thiophene degradation gene (thdF)fragment. Comparing amino acid sequence of thdF with that of other bacteria, the GBS gene had high homology with the gene of Escherichia coli, Bacillus subtilis, Borrelia burgdorferi, Haemophilus influenzae, Pseudomonas putida and Mycoplasm gentialium. By PCR study, thdF gene seems to be existed in some but not all GBS strains. Using the partial thdF gene containing recombinant plasmid to transform a thdF mutant strain E.coli NAR 967 for expression assay, none of transfornants was obtained recovering wild strain phenotype. Many questions are discussed in detail.
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Chen, Tsung-Kun, und 陳宗坤. „Molecular cloning of eps8 gene from chicken embryonic cell“. Thesis, 1999. http://ndltd.ncl.edu.tw/handle/51481172423952507360.
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碩士國立成功大學
藥理學研究所
87
Previous studies have indicated that eps8 is one of the EGF receptor substrates and its overexpression can potentiate EGF-induced mitogenic effects. In v-Src transformed murine fibroblasts, two eps8 isoforms ( p97eps8 and p68eps8 ) can be recognized by anti-murine eps8 antibody generated in our laboratory previously. However, in chicken embryonic cells, a 200 kDa protein was detected by this antibody in the Western immunoblot analysis. In an attempt to confirm this 200 kDa protein was a chicken eps8 homologue, We screened a chicken embryonic cDNA library with 32P-labeled murine eps8 cDNA and got several positive clones. Sequence analysis of these chicken eps8-positive cDNA clones demonstrated the absence of the sequences at the 5' region. Utilizing 32P-labeled chicken eps8 cDNA as a probe, we demonstrated the molecular size of chicken eps8 mRNA was 4.7 Kb. And through 5'-RACE, we attempted to obtain the whole sequence of 4.7 kb chicken eps8 cDNA. Sequence comparison showed that eps8 cDNA from these two different species shared strong homology and both encoded 97 KDa protein. Thus, the detected 200 kDa protein in CE cell should represent a protein that shared the same antigen epitope present in mouse eps8. In addition, a rabbit polyclonal antibody specifically recognize chicken eps8 was attempted. Hopefully, with this antibody, we could study the interaction between v-Src and eps8 in CE cell.