The nucleotide sequence of the 3' terminus of soybean mosaic virus (SMV) VA/G1 RNA has been determined by dideoxynucleotide sequencing of oligo(dT) primed cDNA cloned into a pUC19 cloning vector via EcoR1 linkers. One recombinant plasmid, pSMV49, identified by colony and dot-blot hybridization to ¹²⁵I-SMV RNA, contained an insert of 1443 nucleotides and had an open reading frame of 1119 nucleotides terminating 224 nucleotides from the 3' terminal poly(A) tract. The coat protein cistron identified by a glutamine:serine dipeptide cleavage site 792 nucleotides upstream from the termination sequence could potentially code for a 29.8 kDa protein. The amino acid sequence predicted from the nucleotide sequence of this cistron contains regions identical to other potyvirus coat proteins. / Master of Science

Epigenetics involves heritable changes in an individual’s traits resulting from variations in gene expression without alterations to the DNA sequence. In eukaryotes, genomic DNA is usually folded with histones into chromatin. Post-translational modifications (PTMs) on histones not only play crucial roles in regulating various biological processes, including gene expression, but also store the majority of epigenetic information. A fundamental question in this field is how cells transmit these PTMs to their progeny. Before I began my thesis research, a well-established dogma in the field was that parental histones containing PTMs are symmetrically distributed to daughter DNA strands during DNA replication. These modified histones serve as templates for PTM duplication, thereby restoring the original chromatin states on both daughter strands. Several histone chaperones have been identified as regulators of parental histone segregation. However, their impact on epigenetic inheritance is controversial, which I reasoned is due to the lack of proper systems to examine epigenetic inheritance. This prompted me to use the unique characteristics of fission yeast heterochromatin as a model of epigenetic inheritance. In this organism, heterochromatin formation involves two distinct steps: establishment and inheritance. Reporter systems have been established to allow precise examination of heterochromatin inheritance. However, parental histone segregation pathways have not been characterized in this organism, and their impact on heterochromatin inheritance is unknown. My thesis work investigates the role of parental histone chaperones in regulating parental histone segregation and epigenetic inheritance in fission yeast. It comprises 5 chapters: Chapter 1 introduces epigenetics, with a focus on chromatin-based epigenetic inheritance. It also highlights the unique features of fission yeast heterochromatin that make it an excellent model for studying epigenetic inheritance.Chapter 2 is the focus of my thesis work. I employed inheritance-specific reporters in fission yeast to investigate the roles of three parental histone chaperones on epigenetic inheritance. In addition, in collaboration with Dr. Zhiguo Zhang’s lab, I adapted the Enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN) method, a recently developed technique designed to quantify the bias of specific proteins at replication forks, to examine parental histone segregation in fission yeast. My analyses demonstrated a critical role for parental histone segregation in epigenetic inheritance. Moreover, I discovered that both the symmetric segregation of parental histones and their density on daughter strands are critical for this process. Chapter 3 uncovers a novel function of a DNA replication protein Mrc1 in regulating epigenetic inheritance, distinct from its established roles in DNA replication checkpoint activation and replication speed control. I demonstrated the critical role of Mrc1 in regulating the symmetrical transfer of parental histone and the proper inheritance of heterochromatin. These results provide essential mechanistic insights into the function of Mrc1. Chapter 4 explores the function of an additional DNA replication protein and histone chaperone, Swi7 (Pol alpha). I have found that mutations in Swi7 lead to defects in parental histone segregation and heterochromatin inheritance, laying a strong foundation to further investigate its mechanism of action. Chapter 5 discusses potential future research directions that can build upon my thesis work.In conclusion, my thesis represents a thorough examination of parental histone chaperones in regulating epigenetic inheritance in fission yeast. By combining innovative genetic assays and advanced methodologies such as eSPAN, I have provided critical insights into the molecular mechanisms of epigenetic inheritance. In addition, the assays that I have developed during my thesis work also pave the way for future studies aimed at elucidating the mechanism of epigenetic inheritance in this important model organism.

Single-cell sequencing techniques, such as single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq), have revolutionized our understanding of cellular diversity and function. Genetic and epigenetic factors influence phenotypic heterogeneity in ways that are just beginning to be understood. In this work, we develop methods for inferring mechanisms of biological heterogeneity in single-cell data, with particular applications to cancer biology. First, we develop a kernel archetype analysis method for overcoming noise and sparsity in single-cell data by aggregating single cells into high-resolution cell states. We show that the proposed approach captures robust and biologically meaningful cell states and enables the inference of epigenetic regulation of phenotypic heterogeneity. In the second part of this thesis, we develop methods for linking genotypic and phenotypic information, first by using aggregated single-cell RNA sequencing and a hidden Markov model to infer copy number variation. We demonstrate that aggregation improves copy number inference over existing approaches. We then integrate DNA sequencing with single-cell RNA sequencing to infer copy number profiles in a rapid autopsy of a patient with metastatic pancreatic cancer. We develop a scalable algorithm for inferring phylogenetic relationships between cells from noisy copy number profiles. We show that our approach more accurately recovers phylogenetic relationships between cells and apply it to understand the relationship between genotype and phenotype in metastatic cancer. Finally, we develop a metric for quantifying the extent to which genotype determines phenotype in lineage tracing data. We show that it more accurately quantifies phenotypic plasticity compared to existing approaches. Altogether, these methods can be used to help uncover the mechanisms underlying phenotypic heterogeneity in biological systems.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The Cape Floristic Region (CFR) is situated at the southern tip of Africa and possesses a flora that is unique amongst the floras of the rest of the world, both in terms of its incredibly high species richness, and its high levels of endemism. Proteaceae, the family to which Serruria belongs, is widely distributed amongst the landmasses of the southern hemisphere, with its centres of diversity occurring in Australia and southern Africa. Previous molecular and morphological analyses performed on the South African subfamily Proteoideae have shown Serruria, a CFR endemic, to form a well-supported monophyletic group. Based upon the strong monophyly of Serruria, DNA sequence data were collected for 53 of the 55 species from the plastid (rps16 intron, atpB-rbcL intergenic spacer, trnL-F region and psbA-trnH intergenic spacer) and nuclear (internal transcribed spacer region or ITS) genomes in order to investigate evolutionary relationships within the genus. Spatalla taxa were used as the outgroup. Both parsimony and Bayesian analyses were carried out on each of these data sets. The resulting trees were reasonably well resolved. All the Serruria taxa grouped together in a well-supported clade, except for S. f1ava, which emerged well within the Serruria clade in the analyses of the nuclear genome, but outside the clade in the plastid analyses. It was therefore proposed that this taxon represents a hybrid. Apart from this case, there was widespread agreement between the trees reconstructed using data from the two genomes. The plastid and nuclear data were therefore combined in order to analyse the data sets together. The molecular data does not support most of the groupings proposed by previous authors based on morphological data. Additionally, in some cases, multiple representatives of species do not group together. These specimens probably do not represent monophyletic taxa. Current ideas about relationships within Serruria are based predominantly on floral characters, and it is suggested that pollinator pressures have led to plasticity in the floral characters. Consequently, it is evident from this study that relationships within Serruria need to be re-examined in order to determine the patterns of evolution within the genus. / AFRIKAANSE OPSOMMING: Die Kaapse Floristiese Streek is aan die suiderpunt van Afrika geleë, en beskik oor 'n unieke flora relatief tot ander wêreldfloras, beide ten opsigte van die ongelooflike hoë spesie diversiteit en die hoë vlakke van endemisme. Proteaceae, die familie waaraan Serruria behoort, kom wydverspreid tussen die vastelande van die Suidelike Halfrond voor, en het diversiteitsentrums in Australië en suider Afrika. Vorige molekulêre sowel as morfologiese analises wat op die Suid-Afrikaanse subfamilie Proteoideae uitgevoer is, dui aan dat Serruria (wat endemies is tot die Kaapse Floristiese Streek) 'n goed ondersteunde monofiletiese groep is. Gebaseer op die sterk monofilie van Serruria, is DNA-volgorde-data vir 53 van die 55 spesies vanuit die plastied (rps16 intron, atpB-rbcL intergeniese spasie, trnL-F area en psbA-trnH intergeniese spasie) en kern (intern getranskribeerde spasie area, ook ITS genoem) ingewin om die evolusionêre verwantskappe binne die genus te ondersoek. Spatalla is as die buitegroep gebruik. Beide parsimonie en Bayesian analises is op elk van hierdie datastelle uitgevoer. Die resulterende bome het redelike hoë resolusie getroon. AI die Serruria-taxa het in 'n goed ondersteunde klade saam gegroepeer, behalwe vir S. f1ava, wat binne die Serruria klade val vir die kern genoom, maar buite die klade vir die plastied analise. Dit is dus voorgestel dat hierdie taxon as 'n hibried beskou mag word. Behalwe vir hierdie geval, was daar wydverspreide ooreenstemming tussen die bome wat verkry is vanaf data van die twee genome. Die plastied- en kern-data is derhalwe gekombineer om die datastelle saam te kan analiseer. Die molekulêre data ondersteun nie die meerderheid van morfologiese groeperings wat deur verskeie outeurs voorgestel is nie. Verder, in sommige gevalle, groepeer verskillende monsters van dieselfde spesies nie bymekaar nie. Dit is derhalwe voorgestel dat hierdia taxa nie monofileties is nie. Huidige idees omtrent die verwantskappe binne Serruria is grotendeels op blommorfologiese kenmerke gebaseer, en dit word voorgestel dat bestuiwing-druk gelei het tot plastisiteit van die blommorfologiese kenmerke. Verskille tussen die bome wat uit plastied- en kern-data gerekonstrueer is word aan vroeëre hibridisasie gebeure toegeskryf. Op grond van hierdie studie is dit duidelik dat die verhoudings binne Serruria verder ondersoek moet word om die patrone van evolusie binne die genus te bepaal.

published_or_final_version / abstract / Computer Science and Information Systems / Master / Master of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

TOL plasmids of Pseudomonas putida encode enzymes for the degradation of toluene and related aromatics. These genes are organized into two operons regulated by the Xy1R and Xy1S transcriptional activators. Previous analysis of the TOL pDK1 catechol-2,3-dioxygenase gene (xy1E) and a comparison of this gene to xy1E from the related TOL plasmid pWW0, revealed the existance of a substantial level of sequence homology (82%).

A single plaque-pure lambda clone designated λBA84 that hybridized to a ˆ32P-labeled bovine arginine tRNA was isolated from a bovine genomic library harbored in a lambda bacteriophage vector. A 2.3-kilobase segment of this clone was found to contain an arginine transfer RNAccg gene by Southern blot hybridization analysis and dideoxyribonucleotide DNA sequencing. This gene contains the characteristic RNA polymerase III split promoter sequence found in all eukaryotic tRNAs and a potential RNA polymerase III termination site, consisting of four consecutive thymine residues, in the 3'-flanking region. Several possible cis-acting promoter elements were found within the 5'-flanking region of the sequenced gene. The function of these elements, if any, is unknown.

The TOL plasmids of Pseudomonas putida encode enzymes required for the oxidation of toluene and other related aromatic compounds. These genes are organized into two operons, the xylUWCMABN operon (upper), and the xylXYZLTEGFJQKIH operon (lower). Here we report the nucleotide sequence of a 7107 bp segment of the TOL pDK1 plasmid encoding the region just upstream of the "upper" operon through the genes encoding xylUWCMA. Sequence analysis, comparison of base-usage patterns, codon-usage patterns, and intergenic distances between genes help support the idea that the "upper" and "lower" operons have evolved independently in different genetic backgrounds and have only more recently been brought together in TOL and related catabolic plasmids.

Student Number : 0314429G - MSc research report - School of Molecular and Cell Biology - Faculty of Science / Tomato (Lycopersicon esculentum) is a horticultural commodity of great economic importance in many parts of the world, including South Africa. A previous study identified a new begomovirus, Tomato curly stunt virus (ToCSV), as the causative virus of a new and potentially devastating disease of tomatoes in South Africa. In this study, symptomatic plants, suspected of infection with an uncharacterized ToCSV isolate (01/2521) were collected for screening from Pietermaritzburg, South Africa. A host range study was conducted with the original ToCSV isolate (99/0631). Two small DNA molecules (1449 nts and 755 nts) were found associated with ToCSV [01/2521] using near-full length primers AL1c2745 and PAR1v32 specific for ToCSV. A single small DNA molecule (842 nts) was also found in association with the original ToCSV isolate. Nucleotide sequence analysis revealed that the two small DNA molecules (1449bp and 755bp) have no significant nucleotide sequence identity (less than 20%) with any known begomovirus. The 842bp molecule has the most significant nucleotide sequence identity (48%) to that of ToCSV (AF261885), while less than 20% nucleotide sequence identities were found when compared with other begomoviruses. Nucleotide sequence alignment of the 842bp DNA molecule to the ToCSV sequence, showed that this small DNA molecule is a chimeric molecule that could have arisen through recombination, partly from the coding regions of the ToCSV genome, but the rest of the molecule is of unknown origin. All three small DNA molecules identified in this study were compared to some known begomovirus associated subgenomic molecules and satellite molecules, and sequence identities of less than 20% were found. To our knowledge, this is the first report of a small DNA molecule found associated with the ToCSV genome. The complete genome sequence of ToCSV [01/2521] was not determined. Based on the results we obtained from the host range study, all the chosen test plants are not susceptible to ToCSV infection. The infectivity of all the small molecules identified in this study, is currently being investigated.