Thesis (M.Sc. (Med.))--University of the Witwatersrand, Faculty of Health Sciences, 1991

microRNAs (miRNAs) are small (20-23 nt), non-coding single stranded RNA molecules that play an important role in post-transcriptional regulation of protein-coding genes. miRNAs have been found in all animal lineages, and have been implicated as critical regulators during development in multiple species. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development due to their well-characterized transcriptional gene networks, ease of working with their embryos in the laboratory and phylogenetic position as invertebrate deuterostomes. Literature on miRNAs in echinoderm embryogenesis is limited. It has been shown that RNAi genes are developmentally expressed and regulated in sea urchin embryos, but no study in the sea urchin has examined the expression of miRNAs. The goal of my work has been to study miRNA regulation in echinoderm developmental gene networks. I have identified developmentally regulated miRNAs in sea urchin and sea star embryos, using a combination of computational and wet lab experimental techniques. I developed a probabilistic model (named HHMMiR) based on hierarchical hidden Markov models (HHMMs) to classify genomic hairpins into miRNA precursors and random stem-loop structures. I then extended this model to make an efficient decoder by introduction of explicit state duration densities. We used the Illumina Genome Analyzer to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of S. purpuratus and P. miniata. We developed a computational pipeline for analysis of these miRNAseq data to reveal the miRNA populations in both species, and study their differential expression. We also used northern blots and whole mount in situ hybridization experimental techniques to study the temporal and spatial expression patterns of some of these miRNAs in sea urchin embryos. By knocking down the major components of the miRNA biogenesis pathway, we studied the global effects of miRNAs on embryo morphology and differentiation genes. The biogenesis genes selected for this purpose are the RNAse III enzyme, Dicer and Argonaute. Dicer is necessary for the processing of mature miRNAs from hairpin structures while Ago is a necessary part of the RISC (RNA interference silencing complex) assembly, which is required for the miRNA to hybridize to its target mRNA site. Knocking down these genes hinders normal development of the sea urchin embryo and leads to loss of the larval skeleton, a novel phenotype not seen in sea stars, as well as abnormal gastrulation. Comparison of differentiation gene marker expression between control and Ago knocked down sea urchin embryos shows interesting patterns of expansion and suppression of adjoining some embryonic territories, while ingression of larval skeletogenesis progenitors does not occur.

Components of the South African indigenous flora are disappearing at an alarming rate, due to pressures on land use. The flora is protected by proclamation of reserves and conservation legislation, however these measures can never be wholly successful. For these reasons, methods for propagting Clivia miniata, Gloriosa superba and Sandersonia aurantiaca using in vitro techniques were investigated. The highly sought after Clivia miniata var citrina can be successfully cultured using fruit and floral explants. Use of these explants may limit the number of plants produced in culture due to the seasonal nature of flowering. Gloriosa superba and Sandersonia aurantiaca can be propagated using corm explants, with subsequent in vitro stimulation of cormlet formation. To establish a successful tissue culture procedure an integrated approach to all aspects of the culture is necessary. Sterilization techniques should be empirical and specific for each species and explant. The most critical factor in establishing a culture technique is the choice of a suitable explant. Without a suitable explant the success of the culture procedure may be severely limited. Nutritional and environmental variation may modify the explant response in culture, but initial culture response can be directly related to the origin of the explant, particularly, size, time of the year, age and physiological status. Since the discovery of colchicine in Gloriosa by CLEWER, GREEN and TUTIN (1915) a number of researchers have put forward the idea that Gloriosa would serve as a source of colchicine. The present trend in biochemical production is via artificial synthesis, however many desirable compounds still have to be extracted from plant material for biochemical production. The utilization of plant cells that are cultured in vitro provides a viable alternative to the problems involved in the production of chemical compounds. Levels of colchicine in Gloriosa and Sandersonia are very similar, in the range of ± 0,9%. From evidence presented by BELLET and GAIGNAULT (1985), levels of colchicine in the two study species is much higher than the recorded level (0,62%) of Colchicum. This higher level of the alkaloid makes these two plants a viable source for commercial colchicine production. Levels of colchicine recovered from in vitro grown roots and callus was 10 - 20 times lower than that found in -in -viv-o tissue. Levels of colchicine extracted from plantlets grown in vitro was the same as that normally recorded for parent tissue. Higher levels of colchicine in malformed roots adds to the evidence that differentiation increases colchicine production in Gloriosa tissue in vitro. It has been shown that Gloriosa and Sandersonia tissue can synthesize colchicine in vitro. The extent to which the cells synthetic capacity can be enhanced has yet to be determined. However, research into speedier and more wide ranging methods for metabolite production in culture is receiving attention throughout the world. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1988.