Bibliography: leaves 89-96. / The Actinomycete organism, Streptomyces alboflavus, produces the antibiotic oxytetracycline. Treatment with curing agents resulted in S. alboflavus losing the ability to produce oxytetracycline. However, the loss of oxytetracyc1ine production was reversible, and non-producing colonies regained the ability to produce oxytetracycline when the curing agent was removed. Therefore the loss of oxytetracyc1ine production was not due to the irreversible loss of genetic material specifying oxytetracycline production, but was possibly due to genetic instability. Two extrachromosomal DNA species were isolated from S. alboflavus, (SAP1 and SAP2). SAP1 was approximately 8 - 10 x 10â ¶ da1tons, and appeared to be linear and heterogenous. SAP2 was 20 - 25 x 10â ¶ daltons and appeared to be covalently closed circular. The functions of SAP1 and SAP2 are unknown, although transformation experiments with SAP1 suggested that it may play a role in the production of an antibiotic-like substance, possibly by acting as a promotor of the genes coding for the antibiotic-like substance.

Bibliography: pages 147-162. / The initial aim of this work was to develop a laboratory system for the study of the ABE fermentation under laboratory conditions. The development of defined and simple laboratory inoculation and build-up procedures for the ABE process was investigated. A defined spore preparation in distilled water gave solvent yields comparable to the yields obtained in the commercial ABE process. A laboratory inoculation procedure was developed which avoided the lengthy culture build-up procedures presently utilised. An investigation into solvent production by Clostridium acetobutylicum in clostridial basal medium (CBM) , reinforced clostridial medium (RCM) , Leung and Robson media was undertaken with the aim of developing a partially defined laboratory medium which produced solvent yields comparable to the molasses fermentation medium (MFM). The solvent yields obtained in the partially defined laboratory media were substantially lower than those obtained in MFM. It became apparent that the initial aim of trying to identify and manipulate a few key factors to give better solvent yields would not be easily attained. Both the solvent levels and the overall pattern of cell development were markedly different in the various laboratory systems. In view of these differences, a more detailed investigation of the growth patterns, morphological and physiological changes were undertaken.

1. 195 IgM, 75 IgM, and IgG were purified from the serum of Trypanosoma eguiperdum infected rabbits. The following antisera were also prepared for the quantitation of rabbit IgM and IgG: i. goat anti rabbit IgM antiserum; ii. goat anti rabbit IgG antiserum; iii. goat antiserum to rabbit serum proteins. 2. The serum levels of 195 IgM, measured by gel diffusion and rocket immunoelectrophoresis were shown to increase approximately ten times at the peak of the Trypanosoma eguiperdum infections in the rabbit. The serum IgG. concentrations, measured by gel diffusion increased more than seven times.aver the infection period. Low molecular weight 75 IgM was also detected in the serum by rocket immunoelectraphoresis and quantitative gel diffusion, but accurate concentration determinations could not be made. 3. Rheumatoid factors were not detected by passive haemagglutination and latex fixation tests, despite the use of a variety of sensitizing globulins. These results failed to confirm earlier reports of the presence of rheumatoid factors in infected rabbits. 4. After separation of the 75 and 195 fractions by density gradient centrifugation, both fractions were found by complement fixation to contain anti trypanosome antibodies. These antibodies were also detected by immunofluorescence in the serum and 75 fractions, and by agglutination in the 195 fractions. 5. The 195 fractions also contained complement fixing antibodies reacting with the tissue antigens of rabbit liver, heart and kidney. Maximum titres of 1/80 - 1 /160 were found at the peak of the infection. Absorption of the rabbit sera with trypanosomes indicated that the trypanosomal and tissue antibodies were distinct.

Bibliography : pages 218-230. / Maize plants collected in three geographically distinct regions of South Africa were found to be doubly infected with maize dwarf mosaic (MDMV) and cucumber mosaic virus (CMV). A mixed infection of these two viruses could be maintained in maize plants grown under laboratory conditions. The possibility of synergism or of an interference mechanism between MDMV and CMV in dual infections was investigated and it was found that prior infection with CMV interfered with subsequent infection by MDMV. MDMV and CMV were shown to be non-persistently transmitted by Myzus persicae, Rhopalosiphum padi and Rhopalosipbum maidis aphids. Protoplasts were isolated from maize seedlings and could be viably maintained for up to 66 hours. The maize protoplasts were infected with CMV and MDMV either singly, or together as a mixed inoculum. Infection curves for each virus were plotted. The presence of CMV in a mixed inoculum appeared to prevent infection of the protoplasts by MDMV. Protoplasts were isolated from plants systemically infected with CMV and/or MDMV. Superinfection of protoplasts prepared from CMV infected seedlings with MDMV was not possible. As a possible vehicle for virus infection of protoplasts liposomes were produced. Initially fluorescent dyes were incorporated in them. These were fused to the maize protoplasts. Attempts were made to encapsulate virus particles in the liposomes and fuse them to maize protoplasts but this was not successful.

Bibliography: pages 158-164. / Gamma linolenic acid (GLA) is an essential fatty acid which has been shown to be beneficial for a large number of diseases. At present the fatty acid is marketed as a health product and the prime source of GLA is the evening primrose flower. There are several problems associated with producing the oil and as there is a growing demand for GLA for pharmaceutical and clinical applications, alternative sources are being sought. Choanephora curcubitarum is known to produce GLA and the production of GLA by fermentation of this organism has been successfully achieved by National Chemical Products. The ultimate aim of this project is to genetically manipulate this organism in order to increase the yields of GLA. Tn this thesis the basic groundwork needed to develop a genetic system in C. curcubitarum has been covered.

Bibliography: pages 125-135. / Aerated cultures of Vibrio alginolyticus produced histidase at 30°C but production of histidase was repressed by either incubation at 37°C or a lack of oxygen. A similar regulation system by temperature and oxygen has been reported for collagenase and prntease production by V. alginolyticus (Hare et al., 1981). V. alginolyticus had identical growth rates at 30 and 37°C. The histidine-utilization (hut) enzymes were coordinately induced by histidine. The inducible nitrogen catabolic enzymes arginase, alanine dehydrogenase and histidase were not subject to nitrogen catabolite repression. Various amino acids and ammonium ions stimulated the production of histidase and arginase. Urocanase and formiminoglutamate hydrolase were repressed by nitrogen-containing compounds. Tryptophan, glutamine and isoleucine either repressed or had little effect on the production of histidase and urocanase. The hut enzymes and alanine dehydrogenase were sensitive to catabolite repression by glucose. The addition of (NH₄)₂SO₄ stimulated histidase production. Cyclic AMP did npt relieve repression by glucose. Catabolite repression by glucose of collagenase and protease production in V. alginolyticus was also not relieved by cyclic AMP (Reid, 1981; Long et al., 1981).

Bibliography: pages 80-90. / The aim of this project was to construct a high level plant expression vector from the RNA 3 of cucumber mosaic virus strain Y (CMV Y). The 5'- and 3'-untranslated regions (UTRs) of this genome segment were reverse transcribed, cloned and sequenced. The chloramphenicol acetyl transferase gene (CAT) was inserted between the two UTRs. This artificial viral cDNA (5'cat3') was cloned immediately downstream of the cauliflower mosaic virus 35 S promoter at the transcription initiation site to make a DNA vector. An RNA vector construct was made by placing the 5'cat3' segment under the control of a T7 RNA promoter sequence. In vitro transcripts, as well as linearised DNA vector constructs were inoculated onto CMV infected plants. Inoculated plants were monitored for CAT expression. No CAT could be detected in total protein extracts of inoculated plants. No CAT mRNA could be detected in northern blots of total RNA extracted from inoculated plants. The vector constructed from the 5' - and 3' -UTR of the RNA 3 of CMV Y did not appear to contain all the necessary attributes for a viral expression vector. To study the expression of a foreign antigen in tobacco, the L 1 capsid protein of human papillomavirus type 16 was cloned into Agrobacterium tumefaciens and used to make transgenic Nicotiana tabacum. Kanamycin resistant tobacco plants were shown to carry the L 1 capsid gene using PCR screening, but western blots on total protein extracts of the transformed plants were indeterminate. Further studies are needed to determine whether the antigen is produced and if it is correctly processed.

The aim of this investigation was to determine the suitability of single carbon organic compounds as hydrogen donors for denitrification. Although methanol has been used for this purpose with some success, methane has apparently been disregarded as a possible carbon source. A denitrifying unit was operated with methanol as a carbon source, in order to determine the influence of methanol on rates of denitrification with several different nitrate concentrations. With methanol as an additive, nitrate reduction was of the order of 90%, while less than 40% denitrification was achieved with the supernatant from settled domestic sewage as the sole carbon source. Isolates were taken from this unit for identification. A similar unit was constructed, receiving methane as the sole carbon source. Bacteria were isolated from this unit and shown to be able to use nitrate as a terminal electron acceptor and as a source of cell nitrogen. These bacteria were identified and their specificity to methane tested. In addition to methane they were found to be able to use several other organic compounds for growth and denitrification. Isolates from the methanol unit were found to be able to use methane for denitrification.

Includes bibliographical references. / Geminiviruses replicate via a rolling circle mechanism, which initiates at the origin of replication located within the long intergenic region (LIR). The viral replication associated-protein (Rep) in conjunction with the host's DNA replication machinery is responsible for the initiation and termination of the replication cycle from a stem-loop structure, located within the LIR and conserved throughout the three genera of Geminiviridae. The specific interaction between the Rep protein with sequences within the intergenic region has been well characterised for the begomoviruses and to some extent the curtoviruses; however, this interaction in the mastreviruses, and in particular maize streak virus (MSV), has yet to be fully explored. A theoretical model has been proposed based on sequence data and informed by the current understanding of replication specificity in begomoviruses. Due to the lack of conservation of the stem sequence of the stem-loop structure amongst mastreviruses, the model implicates a pair of nucleotide sequence repeats called iterons. These are located within the stem structure, and on the complementary sense side of the LIR. The former is the putative site of Rep interaction with the LIR. These iterons would therefore potentially act as the determinants of replication specificity amongst mastreviruses.

Bibliography: pages 163-176. / The immunospecific inactivation of bacteriophage is one of the most sensitive methods available for the detection of very low concentrations of antibody. By chemically modifying the phage coat-protein, this sensitivity can be extended to antibodies against a wide variety of haptens and proteins. Phage particles that have been modified by attaching some molecule onto their surface are sensitive to antibodies directed against the coupled chemical moiety. Furthermore, the inactivation of the modified phage by antibody can be inhibited by free antigen, and this provides a sensitive assay for small quantities of antigen. Antibody and antigens have been detected at the nanogram level by this technique. The modified phage technique can also be used to distinguish antibodies of different specificities and to discriminate between closely related antigens. This technique has not yet been applied to the immunochemical study of viral components and the present work represents such an attempt. Tobacco mosaic virus (TMV) was chosen as a model system since it permits the study of numerous inununological phenomena (Rappaport, 1965; van Regenmortel, 1966). A series of preliminary experiments were performed to obtain experience in the methodology of the technique. These included the inactivation of native T4 phage by phage antiserum and anti phage IgG, the chemical modification of phage with DNP and the attachment of lysozyme to phage under a variety of conditions. The success of the covalent binding of protein to phage particles depends on finding conditions under which a proportion of the phage remains viable and, at the same time, can still be neutralized by anti-protein sera. To this end, different proportions of reactants and three different bifunctional reagents were tested. To prevent aggregation of tobacco mosaic virus protein (TMVP) at the high concentration used, the protein was treated with N-bromosuccinimide. TMVP-phage conjugates which were sensitive to antiserum were prepared using bis-diazobenzidine as the bifunctional reagent.