1. The work described in this thesis forms part of an investigation of eukaryotic gene control. The system studied was the avian erythroid cell series since it is possible to isolate pure populations of the various cell types which have well-defined biochemical activities. These cells contain an unusual tissue - specific histone H5, which may be involved in the progressive repression of transcription observed as these cells differentiate. Although the gene controlling function of this histone must be at a very gross level, this represents a unique opportunity to investigate one facet of gene control. Probably the most sensitive technique is to assay for specific messenger RNA and gene sequences by hybridisation to an appropriate probe. The aim of this thesis was to prepare such a probe from H5 mRNA and to use it to calculate the reiteration frequency of the H5 gene in the chicken genome. 2. The cells employed were chicken reticulocytes since the only histone made in these cells is H5. Experiments were conducted which demonstrated that H5 mRNA is probably a minor species compared to globin mRNA in these cells. Furthermore, calculations indicate that the two mRNAs are probably of similar molecular weight which may complicate the isolation of H5 mRNA. As a result globin mRNA was first purified and characterised. Properties which may have proved useful in the separation of this mRNA from H5 mRNA are discussed. The globin mRNA was used to optimise techniques for the in vitro translation and identification of chicken mRNAs. This was considered necessary as mRNAs from different sources vary in the conditions required for optimal translation and it was reasoned that mRNAs from the same cell would have similar optima. 3. Total polysomal RNA was fractionated on the basis of size and poly A content. Although large amounts of globin mRNA were present, H5 mRNA could only be detected in the non - poly A containing RNA. Even in this fraction however, there was still a large excess of globin mRNA which was difficult to remove due to the demonstrated similarity of their molecular weights. 4. Since it had proved impossible to isolate the H5 mRNA by conventional techniques, immunological methods of isolating the polysomes producing H5 were investigated. Using immunoadsorbents, mRNA was prepared in small amounts which programmed the synthesis in vitro of more than 70 % H5. The yield and specificity were improved by modifying the procedure to indirect immunoprecipitation followed by oligo ( dT ) - cellulose chromatography. The resulting mRNA programmes the synthesis in vitro of more than 90 % H5. The chemical purity of the mRNA is discussed. 5. The immunologically prepared H5 mRNA was not copied into cDNA by RNA - dependent DNA - polymerase. Since this was probably due to the lack of a 3 ' poly A tract on the mRNA, an enzyme was purified and characterised which would add such a tract. The enzymically modified mRNA could then be copied into cDNA of high specific activity. 6. The H5 cDNA was characterised in terms of size and fidelity of copying. By hybridisation analysis it was demonstrated that the amount of contaminating rRNA and globin mRNA complementary sequences present in the cDNA was insignificant. The complexity of the cDNA was shown to be of the same size as the H5 mRNA and will back hybridise to this mRNA to greater than 75 %. These results are discussed to demonstrate that the cDNA is a faithful copy of H5 mRNA. The possible uses of the resulting probe are also discussed. 7. The H5 cDNA was employed to quantify the number of H5 genes in the chicken genome. The significance of this result is discussed in terms of the known reiteration and organisation of histone genes in other species, and the possible role of H5 as a gene control agent. / Thesis (Ph.D.)--Department of Biochemistry, 1975.

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The effects of various concentrations of 2-chloroethylphosphcnic acid (Ethephon), an ethylene-releasing compound, on the total protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) levels in Scenedesmus quadricauda IU 614 were investigated.

vii, 121 leaves : ill., graphs, tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1981

vii, 136 leaves : ill., graphs, tables, photos ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1979) from the Dept. of Biochemistry, University of Adelaide

Artificial ribonucleases (ARs) are recognised as a potential strategy to selectively target and cleave biologically significant RNA in cells. However, in order to work as true enzymes they must exhibit catalytic turnover. Many of the reported ARs incorporate metal-containing centres (e.g. dysprosium, copper) in order to induce substantial phosphodiester cleavage, which is not amenable to use in vivo. Therefore, new strategic directions employing metal-independent ARs, such as peptidyl-oligonucleotide conjugates (POCs), need to be investigated. Previous work has shown that poor or non-complementary POCs demonstrate catalytic turnover of a HIV-1 substrate; however, sequence specificity is an issue. For POCs to be useful from a therapeutic standpoint they must only cleave specific RNA molecules and do so in a catalytic fashion, therefore removing the requirement for stoichiometric drug delivery and binding. Consequently, novel POC design strategies are required that allow selective RNA targeting but promote dissociation of the POC following phosphodiester cleavage. In this research, three types of different peptidyl-oligonucleotide conjugate designs have been implemented with the attempt to find an appropriate balance between selectivity and catalytic turnover.(i) Selective targeting and quantitative cleavage (97-100%) of a tRNAPhe target was achieved through phosphoramidate attachment of a 17-mer TΨC-targeting oligonucleotide to catalytic amphiphilic peptide sequences containing leucine, arginine and glycine. Although the half-life of tRNAPhe was less than 1 h on exposure to some of these POCs, hybridisation studies reveal that the POCs bind too tightly to their target RNA sequences and thus an excess of POC is required for efficient cleavage activity. The effect of peptide and oligonucleotide sequence variations as well as the role of enhanced conformational freedom via incorporating an abasic deoxyribose linker between the oligonucleotide targeting motif and catalytic peptide is also investigated. (ii) Most of ‘Dual’ peptidyl-oligonucleotide conjugates containing an amphiphilic RNA-cleaving peptide placed between two RNA recognition motifs directed towards the TΨC loop and 3’ acceptor stem of tRNAPhe demonstrate marked RNA binding and cleavage activities. Interestingly, those dual conjugates which showed poor or negligible binding ability in electrophoresis assays, demonstrated sufficient RNA cleavage (70%) within the vicinity of the 65GACAC61 target region. Therefore, weak POC:RNA complexes may exist which could facilitate substrate turnover. (iii) Finally, POCs were designed which induce bulge-loops in their target RNA region upon hybridisation. By introducing regions of non-complementarity into the oligonucleotide sequences, 2- to 5- membered bulges sizes were formed. Via attachment of a catalytic peptide to an internally modified oligonucleotide residue, catalytic peptides were placed directly adjacent to single-stranded RNA regions to promote cleavage by nuclease mimics. Through probing the hybridised complexes with RNase H, the presence of bulges were confirmed for all bulge-loop sizes, which will be followed by cleavage experiments to assess the possibility for reaction catalytic turnover. In conclusion, a variety of POCs have been synthesised, characterised and partially tested for their RNA cleaving and turnover activity. Based on the encouraging results presented POCs could be further developed to target disease specific RNA sequences such as micro- or messenger RNAs.

Masters of Science / Viral genomes consist of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The viral RNA molecules are responsible for two functions, firstly, their sequences contain the genetic code, which encodes the viral proteins, and secondly, they may form structural elements important in the regulation of the viral life-cycle. Using a host of computational and bioinformatics techniques we investigated how predicted secondary structure may influence the evolutionary dynamics of a group of single-stranded RNA viruses from the Picornaviridae family. We detected significant and marginally significant correlations between regions predicted to be structured and synonymous substitution constraints in these regions, suggesting that selection may be acting on those sites to maintain the integrity of certain structures. Additionally, coevolution analysis showed that nucleotides predicted to be base paired, tended to co-evolve with one another in a complimentary fashion in four out of the eleven species examined. Our analyses were then focused on individual structural elements within the genome-wide predicted structures. We ranked the predicted secondary structural elements according to their degree of evolutionary conservation, their associated synonymous substitution rates and the degree to which nucleotides predicted to be base paired coevolved with one another. Top ranking structures coincided with well characterized secondary structures that have been previously described in the literature. We also assessed the impact that genomic secondary structures had on the recombinational dynamics of picornavirus genomes, observing a strong tendency for recombination breakpoints to occur in non-coding regions. However, convincing evidence for the association between the distribution of predicted RNA structural elements and breakpoint clustering was not detected.

>Magister Scientiae - MSc / Insect-derived baculoviruses have been used extensively as a safe and versatile research model for transgenic protein expression. Preclinical studies have revealed the promising potential of Baculoviruses as a delivery vector for a variety of therapeutic applications, including vaccination, tissue engineering and cancer treatments. Coronaviruses are enveloped viruses containing linear, non-segmented ribonucleic acid. Human coronavirus NL63 was first discovered in the Netherlands in January 2004, where a 7-month-old girl presented with an acute respiratory tract infection that was later established to predominantly infect infants, the elderly and immunocompromised individuals. In addition to the known non-structural and structural proteins of coronaviruses, an accessory protein known as open reading frame 3 which is conserved in the Coronaviridae family has not been extensively researched. Open reading frame 3 encodes a putative membrane-bound protein. This study cloned the open reading frame 3 viral gene of 741 base pairs into the baculovirus expression construct via competent bacterial cell lines. Open reading frame 3-Baculovirus particles were generated in Spodoptera frugiperda insect cells. Recombinant cells containing the viral protein gene were used to infect healthy Spodoptera frugiperda 9 cells at varying ratios of multiplicity of infection over a fixed time-course. The open reading frame 3 viral protein was not detected by quantification methods at a molecular weight of 26 kilo Dalton, due to polyclonal antibody degradation.

Master of Science / Department of Plant Pathology / Harold Trick / The object of this study was to find a target sequence for the known Heterodera glycines Y25 sequence that contained no homology to any known Glycine max genes so homologous endogenous soybean gene expression will not be effected. In addition, in attempt to improve the accuracy of SCN bioassays performed in greenhouse settings, applications of a variety of insecticides with differing modes of action were applied to screen for any detectable effects on the SCN populations. The full-length sequence of the Y25 gene was blasted against the G. max genome using the National Center for Biotechnology Information blast database and a portion of the gene was found to contained no homology to the G. max genome. A rapid hairy root assay was used to screen for resistance to H. glycines. The sequence was transformed into Agrobacterium rhizogenes using a modified heat shock method. The transformed A. rhizogenes were used to inoculate soybean seedlings. The inoculated seedlings developed hairy roots expressing the target sequence. Upon finishing the hairy root assay it was discovered that there were no detectable differences across any of the treatments or the controls. It was neither proved nor disproved that the new target sequence containing no homology to the G. max genome was as effective as the original target. Further investigation will need to be conducted to show the level of control for the new target sequence.