published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

The mop2 gene is required for epigenetic silencing; it was originally defined as a mutation, Mop2-1, which when dominant prevented paramutation at b1. Paramutation is an allele communication that causes a mitotically and meiotically heritable change in gene expression. Mop2-1 was subsequently shown to be involved in maintaining the silenced paramutant state and to prevent dsRNA-mediated transcriptional gene silencing (activities revealed only when the mutation is homozygous). Understanding the product encoded by mop2 will help dissect the underlying mechanisms involved in paramutation and dsRNA-mediated transcriptional silencing. This dissertation describes map-based cloning and candidate gene approaches directed toward the eventual goal of identification of mop2.Initial mapping of mop2 placed it within a region delineated by the markers umc1823 and eks1. On the maize physical map this region contains 21 BAC (Bacteria Artificial Chromosome) clones, representing 2.9 Mb. Skim sequencing identified additional markers for mapping and revealed the gene content. Extensive candidate gene examinations, including gene sequencing, expression profiling with microarrays and RT-PCR, and complementation tests with mutant alleles did not identify any of the four chromatin and RNAi-related genes as mop2.The new markers developed from the skim sequence enabled further mapping and molecular genotyping, which revealed that the Mop2-1 mutation was unstable. Approxi¬mately 10% of phenotypic heterozygous plants were actually genotypic homozygous. Further mapping using only Mop2-1 homozygous plants reduced the mop2 interval to a region of nine BACs, containing 57 genes.The mop2 region is highly syntenic to a rice region of 1.25 Mb on chromosome 4. The gene alignment and repetitive sequence analyses between the syntenic regions in these two species revealed both syntenic and non-syntenic blocks of sequences. Analyses suggested several potential mechanisms for the collinearity breakage, including, but not limited to, tandem duplications of genes in one species but not the other and the presence of gene fragments in maize, but not in rice.The research described herein provides the basis for continued efforts to clone mop2. Fine-structure mapping with new markers and a larger population, as well as candidate gene sequencing in the Mop2-1 BAC library, should be pursued to clone mop2.

Early flowering is important for maize adaptation to short-season growing environments. Dwarfism, by preventing lodging, has the potential to increase grain yield. This thesis investigates three novel mutants of maize. The early flowering mutant (EarlyF) sheds pollen 1 to 5 days earlier than wild type plants. EarlyF, was shorter and developed fewer leaves than wild type plants, suggesting an earlier transition from vegetative to reproductive development. A candidate QTL for EarlyF maps to bin 7.03. The two allelic dwarf mutants, brachytic3-1 and brachytic3-2, have short internodes at maturity, resulting in severely reduced plant height. Despite being short, days to pollen shed and number of leaves were unchanged for both brachytic3-1 and brachytic3-2. brachytic3 maps to a ~ 7 Megabase region of bin 5.04. This thesis characterizes EarlyF, br3-1 and br3-2 and sets the stage for positionally cloning the mutations causing these mutants and has potential to contribute to maize improvement.

No description available.

The technique of restriction fragment length polymorphism (RFLP) analysis, of chloroplastic and genomic DNA, was investigated as a means of identifying eucalypt species and cultivars which are morphologically indistinguishable from one another. In order to resolve chloroplast DNA (cpDNA) RFLPs, a method was developed to extract high yields of intact chloroplasts from Eucalyptus grandis S/N M6. Starch contamination was reduced by incubation of saplings in the dark for 48 h prior to extraction and watering with a solution containing 370 mM Na-phosphate and 296 mM KN03. Optimal chloroplast yields (25 ug chlorophyll/g fresh mass) were obtained by chopping leaf material, using a vertical homogenizer, in a buffer containing 350 mM sorbitol, 50 mM tris-HCL and 5 mM EDTA, 0.1 % (w/v) bovine serum albumin, 0.15 % (w/v) 2-mercaptoethanol, 2 mM L-ascorbic acid and 1 mM MgCI2 followed by washing of leaf pieces in a buffer containing only sorbitol, tris-HCL and EDTA. When these chloroplasts were used in an "in-organelle" DNA digestion procedure, polymorphisms were observed between the cpDNA profiles resolved for E. grandis S/N M6 and that of an outgroup species (spinach). However, the developed chloroplast extraction technique could not be used to obtain chloroplasts from various other eucalypt species, probably as a result of variability in the material at an ultrastructural or biochemical level. For the analysis of genomic DNA RFLPs, a DNA extraction procedure was optimized for use with various eucalypt species and cultivars. This included the development of a purifcation technique during which DNA was ammonium acetate-ethanol precipitated and subjected to mini-dialysis. Following Dra I restriction of DNA, the extract was electrophoresed and Southern blotted onto both nylon and nitrocellulose membranes. These were probed with a Hind-III restricted sample of the multilocus plasmid probe pV47-2. This probe was labelled using 32p as well as a non-radioactive labelling substance digoxygenin (DIG). Hybridization conditions, including the composition of the hybridization buffer, were optimized for use with these labels, and DNA RFLPs (fingerprints) were resolved for the eucalypt species E. grandis and E. macarthurii and cultivars of E. grandis (S/N M6, TAG 5 and TAG 14). An average of 8.5 bands were detected with 32p and 5.0 fragments with DIG. All the species and cultivars fingerprinted with the 32P-label could be distinguished from one another. However, as a result of the reduced sensitivity of the DIG system, two of the E. grandis cultivars, S/N M6 and TAG 5, could not be differentiated. It is concluded that the latter system would be most suitable for incorporation into a routine eucalypt screening programme, although it is suggested that the colourimetric detection assay, used in this study to resolve DNA bands, be replaced by a more sensitive one. / Thesis (M.Sc)-University of Natal, Durban, 1993.

A hybrid vector, pBVl, has been constructed which is capable of regulating expression of cloned genes in both Escherichia coli and in cyanobacterium Synechocystis 6803. Vector pBVl contains the powerful rightward promoter of bacteriophage lambda to ensure that cloned genes are transcibed at a high level. In addition, pBV] also contains a gene, cI857, encoding the lambda temperature sensitive repressor protein.The CAT gene coding for chloramphenicol aceyltransferase (CmActase) was cloned into pBV1 downstream of the lambda regulatory features to make plasmid pTC1. The expression of the CAT gene was quantified spectrophotometrically following addition of chloramphenicol and a shift in the growth temperature of the cell culture. The specific activity of CmActase increased from 540 to 5400 units within 30 minutes. Thus, using the newly constructed hybrid vector, pBVl, temperature regulation of gene expression in E. coli was observed. / Department of Biology

A hybrid vector, pANIC1, was to be constructed which was capable of regulating expression of cloned genes in both Escherichia coli and Anacystis nidulans R2 by controlling the iron content of the growth medium. Plasmid pANIC1 would have origins of replication for E. coli and A. nidulans R2, and a marker gene conferring ampicillin resistance. It would also contain the promoter for the irpA gene which is active only in low iron growth conditions.The first two stages of the construction were successfully completed, but unfortunately the final construction proved to be unstable. Recent information has shown that operator sequences upstream from the irpA gene's promoter result in an unstable message. This may be interfering with the normal functioning of the host cell, resulting in an unstable construction. In future experiments it may be neccessary to alter the growth conditions or remove the upstream sequences in order to stablize the construction. / Department of Biology

The hypervariable locus DXS255 in human Xp11.22 has a heterozygosity exceeding 90% and has therefore facilitated the localization of several disease genes which map to the proximal short arm of the X chromosome, including the immune deficiency Wiskott-Aldrich syndrome and the eye disorders retinitis pigmentosa, congenital stationary night blindness and Aland Island eye disease. In addition, a microdeletion involving DXS255 has been identified in patients suffering from Dent's disease, a familial X-linked renal tubular disorder which is characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis (kidney stones) and eventual renal failure. Two YAC contigs were constructed in Xp11.23-p11.22 in order to aid transcript mapping; the first centred on the DXS255 locus, the second mapping distal to the first and linking the genes GATA, TFE3 and SYP to the OATL1 cluster. Eleven novel markers were generated, one of which contains an exon from a novel calcium channel gene. Four putative CpG islands were detected in the region. Analysis of the microdeletion associated with Dent's disease using markers from the DXS255 contig demonstrated that it is confined to a 370kb interval. A YAC overlapping this deletion was hybridized to a kidney-specific cDNA library to isolate coding sequences that might be implicated in the disease aetiology. The clones thus identified detect a 9.5kb transcript which is expressed predominantly in kidney, and originate from a novel gene (CLCN5) falling within the deleted region. Sequence analysis indicates that the 746 residue protein encoded by this gene is a new member of the C1C family of voltage-gated chloride channels. The coding region of CLCN5 is organized into twelve exons, spanning 25-30kb of genomic DNA. Using the information presented in this thesis, other studies have identified deletions and point mutations which disrupt CLCN5 activity in further patients affected with X-linked hypercalciuric nephrolithiasis, confirming the role of this locus in renal tubular dysfunction.