Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Filters

1990 - 19973

Settings

Has files: YesSubject: search.filters.subject.Molecular cloning

Search Results

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Cloning and characterisation of the cDNA and gene for sheep liver arginase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North
    (Massey University, 1997) Marshall, Richelle Kaye
    Arginase (arginine amidinohydrolase, EC 3.5.3.1) is a ubiquitous enzyme, notably found in the liver of ureotelic animals. It plays a critical role in the hepatic metabolism of most higher organisms as a cardinal component of the urea cycle (Jenkinson et al., 1996). Arginase has also been identified in numerous organisms and tissues where there is no functioning urea cycle. In animals, many extrahepatic tissues have been shown to contain a second form of arginase. closely related to the hepatic enzyme but encoded by a distinct gene or genes and involved in a host of physiological roles. Recent interest in arginase has been stimulated by it's demonstrated involvement with the metabolism of nitric oxide. Subcloning the sheep hepatic cDNA sequence would allow a ruminant arginase to be compared with other known arginases. Probing a sheep genomic library for the arginase gene could ultimately lead to the characterisation of regulatory elements of the gene. Partial purification of sheep liver arginase was carried out to develop a DNA probe to screen a sheep liver cDNA library for the cDNA sequence but the protein was N-terminally blocked. An attempt was made to electroelute arginase from an SDS-PAGE gel with a view to cleaving the purified protein and sequencing some of the resulting peptides. But arginase could not be purified sufficiently for successful electoelution. Total RNA was isolated from both sheep and rat liver. A product of the expected size was produced by RT-PCR on the rat RNA template, but could not be subcloned into a vector. PCR performed on a sheep cDNA library generated a PCR product which was subcloned and sequenced. The sequence had no similarity with known arginase sequences, and showed that the reverse primer sequence was present at both ends of the PCR product. A region of the human arginase cDNA sequence was PCR amplified from the expression plasmid pTAA12. The PCR product was radiolabelled, and used as a probe to screen a sheep liver cDNA library. No positive clones were identified. Northern blot analysis of RNA isolated from sheep liver was carried out. The blot was probed with a fragment of the human arginase cDNA sequence. Nonspecific binding was observed.
  • Thumbnail Image
    Item
    Molecular cloning and characterization of cellulase genes of Ruminococcus flavefaciens strain 186 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand
    (Massey University, 1990) Huang, Chung-Ming
    A genomic library of Ruminococcus flavefaciens 186 was constructed using the lambda vector λNM1149. It constituted 2.1 x 104 recombinant clones which was large enough to represent the entire genome of this bacterium. From this library, 26 CMC+ clones were identified after screening about 2500 recombinant clones. These CMC+ clones were divided into four groups according to their insertion fragment size. Partial restriction maps of these clones have been achieved. Eight representative clones from these four groups showed different mapping patterns. One of the three 9 kb insert clones (λCM903) was selected for further study based on its ability to express all three types of cellulase activities. The locations of endoglucanase and exoglucanase genes in λCM903 were determined. Two separate fragments from λCM903 were subcloned and named λCMEH1 (CMC+) and λCMEH2 (MUC+). Cross hybridization experiment using celA gene of C. thermocellum and the 26 recombinant clones showed no significant homology. However, different degrees of homology were found among the 26 ruminococcal clones. The difficulty of subcloning the ruminococcal DNA fragments into plasmid vectors was one of the major obstacles in the study of cellulase gene in foreign hosts. Vectors with different functions were tried but all the recombinant plasmid clones showed instability. The cellular location of cellulase enzymes in E. coli cell was determined. Most of the endo- and exo-glucanases were found in the periplasmic space. Partial purification of these cellulase enzymes from E. coli cells using chromatography was then performed and the characterization of these enzymes was achieved. Using ExoIII deletion, the locations of endoglucanase and exoglucanase genes were determined in λCM903. The internal HindIII-HindIII fragment of 7.3 kb from λCM903 was sequenced. Five ORFs were detected using computer software (UWGCG) analyses. The first ORF which coded for an endoglucanase gene (renA) was 2157 bp long with putative 680 amino acid residue. The SD sequence and promoter sequence were present. The best fitting cellulase gene tested was that of the cenA gene of Ce. fimi. An unusual structure of Pro-Thr-Ser rich region, which had 38 out of 42 a.a. residue of proline, threonine or serine, was found in the N-terminal of the putative peptide. The second ORF which was 1821 bp long coded for an exoglucanase gene (rex). The putative amino acid sequence had 572 a.a. residue, also a SD sequence and a promoter sequence were found. A Pro-Thr-Ser rich region, which was highly conserved with PTS of renA, was found at the C-terminal of the putative peptide. Again, the cellulase gene, cex gene of Ce. fimi showed the best similarity. It is suggested that the gene structure of cellulase in R. flavefaciens strain 186 was similar to cellulase genes in family A as described by Ong et al, 1989. The third ORF was found overlapping with ORF1 using the transcriptional second reading frame. This ORF had a putative SD sequence but lacked a promoter sequence. The coding region of this ORF has the characteristics of a β-glucosidase gene which was 1300 bp long (a putative sequence of 443 amino acid residues). The fourth ORF which used the second reading frame was 1300 bp long with a SD sequence 5' upstream of the ATG codon, overlapped with ORF1 gene. This ORF coded for a protease gene. The fifth ORF located closely to the 3' end of the 7.3 kb fragment used the second reading frame. This ORF had both putative SD sequence and promoter sequence. It was 1080 bp long with putative 341 amino acid residues and showed the structure of a xylanase gene. A short PTS region was also found in this ORF.
  • Thumbnail Image
    Item
    Cloning and sequencing of the cDNA for bovine lactoferrin : this thesis is submitted to Massey University as partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry
    (Massey University, 1992) Mead, Paul Evan
    Bovine lactoferrin isolated from colostrum was partially sequenced by tryptic mapping and automated peptide sequencing. Homogeneous lactoferrin was used to raise polyclonal antibodies in rabbits. Specific anti-lactoferrin antibodies were isolated from the total rabbit gamma-globulin fraction by affinity chromatography on bovine lactoferrin Sepharose. These antibodies were used to quantify lactoferrin in various solutions (by electroimmuno-diffusion assay) and to demonstrate the de novo synthesis of lactoferrin in involuting bovine mammary tissue. RNA was isolated from mammary tissue biopsies that were synthesizing lactoferrin. The presence of lactoferrin messenger RNA was verified by northern blot analysis. Complementary DNA (cDNA) was prepared from RNA samples and ligated into either the bacteriophage vector λ-gt11 or the plasmid vector pGEM-2. Recombinant clones with cDNA inserts coding for bovine lactoferrin were identified by hybridisation to radiolabelled human lactoferrin cDNA. Several clones were isolated and characterised by restriction map analysis and DNA sequencing. The overlapping nucleotide sequence from these clones encoded most of the mature protein sequence for bovine lactoferrin. Nucleotide sequence encoding the 5' end of the lactoferrin messenger RNA was isolated by enzymatic amplification of homopolymeric-tailed first strand cDNA. Specific oligonucleotide primers were used to direct the synthesis of lactoferrin-specific sequences by the polymerase chain reaction (PCR). Double-stranded products were produced by the inclusion of an oligonucleotide that would prime DNA synthesis from the homopolymeric tract on the 3' end of the first strand cDNA. The nucleotide sequence of the PCR products overlapped the 5'-most sequence of the cDNA clones and extended to encode the initiation codon for bovine lactoferrin. The combined nucleotide sequence of the cDNA and PCR clones overlapped to encode the entire coding region for bovine lactoferrin and included 5' and 3' untranslated flanking sequences. The deduced amino acid sequence of the mature protein concurred with the amino acid sequence of the tryptic peptides prepared from bovine colostrum lactoferrin.