by Cheung Pok Lap. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 225-251). / Abstracts in English and Chinese. / Abstract --- p.i / 摘錄 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Abbreviations --- p.xiv / Chapter CHAPTER 1 --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Biology of Metals --- p.1 / Chapter 1.1.1 --- Mechanism for Monitoring and Controlling Intracellular Metal Ions --- p.2 / Chapter 1.1.2 --- Metal Ions Homeostasis --- p.5 / Chapter 1.2 --- Metallothionein (MT) --- p.8 / Chapter 1.2.1 --- Classification of MT --- p.8 / Chapter 1.2.2 --- Structure of MT --- p.9 / Chapter 1.2.3 --- Organization of MT Genes --- p.11 / Chapter 1.2.4 --- Biological Functions of MT --- p.13 / Chapter 1.2.5 --- MT as a model of Transcriptional Regulation of Gene Expression --- p.19 / Chapter 1.3 --- Fish MT Genes --- p.24 / Chapter 1.4 --- Aims and Rationale of Present Study --- p.28 / Chapter CHAPTER 2 --- CLONING AND CHARACTERIZATION OF TILAPIA MT (tiMT) GENE PROMOTERS --- p.30 / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- The Biology of Tilapia --- p.30 / Chapter 2.1.2 --- The Study of Tilapia MT --- p.31 / Chapter 2.1.3 --- Fish MT Promoters --- p.35 / Chapter 2.1.4 --- Specific Aims of This Chapter --- p.36 / Chapter 2.2 --- Materials and methods --- p.37 / Chapter 2.2.1 --- Animals --- p.37 / Chapter 2.2.2 --- General Molecular Biology Technique --- p.37 / Chapter 2.2.3 --- PCR Primers Used --- p.40 / Chapter 2.2.4 --- Cloning of tilapia MT Gene 5'-flanking Region Using Inverse PCR --- p.41 / Chapter 2.2.5 --- Cloning of full length of tilapia MT Genes / Chapter 2.2.6 --- Transient Transfection Assay --- p.43 / Chapter 2.3 --- Results --- p.47 / Chapter 2.3.1 --- Tilapia MT Genes --- p.47 / Chapter 2.3.2 --- Functional Analysis of tiMT Gene Promoter by Transient Transfection --- p.49 / Chapter 2.4 --- Discussions --- p.58 / Chapter 2.4.1 --- Tilapia MT Genes --- p.58 / Chapter 2.4.2 --- Functional Analysis of tiMT Gene Promoter by Transient Transfection --- p.63 / Chapter 2.5 --- Conclusion --- p.67 / Chapter CHAPTER 3 --- DETECTION OF MT mRNA LEVELS BY QUANTITATIVE RT-COMPETITIVE PCR --- p.68 / Chapter 3.1 --- Introduction --- p.68 / Chapter 3.1.1 --- Quantitative RT-competitive PCR --- p.69 / Chapter 3.1.2 --- Specific Aims of This Chapter --- p.71 / Chapter 3.2 --- Materials and methods --- p.72 / Chapter 3.2.1 --- Animals --- p.72 / Chapter 3.2.2 --- Isolation of total RNA and preparation of first strand cDNA --- p.72 / Chapter 3.2.3 --- Design of primers for preparation of MT mimic and competitive PCR --- p.73 / Chapter 3.2.4 --- Preparation of MT mimic cDNA --- p.74 / Chapter 3.2.5 --- Optimization PCR cycle number for β-actin and MT amplification --- p.77 / Chapter 3.2.6 --- Quantitative competitive PCR --- p.78 / Chapter 3.2.7 --- Quantitative analysis --- p.81 / Chapter 3.2.8 --- Statistical analysis --- p.82 / Chapter 3.3 --- Results --- p.83 / Chapter 3.3.1 --- Preparation of MT mimic cDNA --- p.83 / Chapter 3.3.2 --- Optimization PCR cycle number for MT and β-actin amplification --- p.84 / Chapter 3.3.3 --- Quantification of the MT cDNA levels by competitive PCR --- p.86 / Chapter 3.4 --- Discussions --- p.154 / Chapter 3.4.1 --- Comparison of MT Gene Expression in both in vivo tilapia liver andin vitro PLHC-1 fish cell line --- p.154 / Chapter 3.4.2 --- Absolute Quantification of mRNA using Real Time RT-PCR --- p.159 / Chapter 3.5 --- Conclusion --- p.160 / Chapter CHAPTER 4 --- TILAPIA MTF-1: PCR-CLONING AND GENE EXPRESSION STUDIES --- p.161 / Chapter 4.1 --- Introduction --- p.161 / Chapter 4.1.1 --- General Features of MTF-1 --- p.164 / Chapter 4.1.2 --- Activation MRE-binding of the MTF-1 --- p.166 / Chapter 4.1.3 --- Possible Models for Heavy-metal Regulated MT Genes Transcription --- p.169 / Chapter 4.1.4 --- Genes Under the Regulation of MTF-1 --- p.175 / Chapter 4.1.5 --- Specific Aims of This Chapter --- p.176 / Chapter 4.2 --- Materials and methods --- p.177 / Chapter 4.2.1 --- Cloning of a partial fragment of MTF-1 in tilapia --- p.177 / Chapter 4.2.2 --- Rapid Amplification of cDNA 5'ends --- p.180 / Chapter 4.2.3 --- Rapid Amplification of cDNA 3'ends --- p.184 / Chapter 4.2.4 --- Cloning of The Full-Length Tilapia MTF-1 cDNA Isoforms --- p.185 / Chapter 4.2.5 --- Northern blot analysis of MTF-1 transcripts in tilapia tissues --- p.186 / Chapter 4.2.6 --- Differential Expression of MTF-1 Isoforms in Tilapia Tissues --- p.187 / Chapter 4.2.7 --- Cotransfection Study on Tilapia MTF-1 cDNAs --- p.189 / Chapter 4.3 --- Results --- p.191 / Chapter 4.3.1 --- PCR Amplification of the Partial Sequence of tiMTF-1 --- p.191 / Chapter 4.3.2 --- Rapid Amplification of cDNA 5' and 3' ends of tiMTF-1 --- p.193 / Chapter 4.3.3 --- Cloning of The Full-Length Tilapia MTF-1 cDNA Isoforms --- p.196 / Chapter 4.3.4 --- Northern Blot Analysis of tilapia MTF-1 isoforms --- p.205 / Chapter 4.3.5 --- Differential Expression of MTF-1 Isoforms in Tilapia Tissues --- p.206 / Chapter 4.3.6 --- Cotransfection Study on Tilapia MTF-1 cDNAs --- p.207 / Chapter 4.4 --- Discussions --- p.212 / Chapter 4.4.1 --- Tilapia MTF-1 --- p.212 / Chapter 4.4.2 --- Biological Activity of MTF-1 --- p.216 / Chapter 4.5 --- Conclusion --- p.218 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.220 / REFERENCES --- p.225

Chan, Wai Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 140-157). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.1 / List of Tables --- p.4 / List of Figures --- p.5 / List of Abbreviations --- p.8 / Chapter 1. --- Introduction --- p.10 / Chapter 1.1 --- Biology of metals --- p.10 / Chapter 1.2 --- Metal detoxification systems --- p.11 / Chapter 1.3 --- Metallothionein --- p.13 / Chapter 1.4 --- Classification of MTs --- p.15 / Chapter 1.5 --- Biological roles of MT --- p.15 / Chapter 1.5.1 --- Homeostasis of essential transition metal ion --- p.15 / Chapter 1.5.2 --- Detoxification of non-essential heavy metal ion --- p.17 / Chapter 1.5.3 --- Protection against oxidative stress --- p.18 / Chapter 1.5.4 --- Role in neurodegenerative diseases --- p.19 / Chapter 1.6 --- Molecular biology of MT --- p.19 / Chapter 1.6.1 --- MT gene structure --- p.19 / Chapter 1.6.2 --- MT gene regulation --- p.21 / Chapter 1.7 --- MRE binding transcription factor-1 (MTF-1) --- p.30 / Chapter 1.8 --- Activation of MTF-1 --- p.31 / Chapter 1.9 --- Target genes of MTF-1 --- p.32 / Chapter 1.10 --- Fish MT gene and MTF-1 --- p.33 / Chapter 1.11 --- Tilapia --- p.39 / Chapter 1.12 --- Study of tilapia MT --- p.41 / Chapter 1.13 --- Aims and rationale of study --- p.43 / Chapter 2. --- Materials and Methods --- p.45 / Chapter 2.1 --- Cloning of tilapia MT gene 5'-flanking region --- p.45 / Chapter 2.1.1 --- Animals --- p.45 / Chapter 2.1.2 --- Preparation of tilapia genomic DNA --- p.45 / Chapter 2.1.3 --- DNA walking --- p.45 / Chapter 2.1.4 --- Amplification of whole tiMT gene --- p.50 / Chapter 2.2 --- Determination of transcription start site --- p.51 / Chapter 2.2.1 --- Total RNA extraction --- p.51 / Chapter 2.2.2 --- Rapid amplification of 5，complementary DNA ends (5' RACE) --- p.52 / Chapter 2.3 --- Transient transfection assay --- p.54 / Chapter 2.3.1 --- Cell culture --- p.54 / Chapter 2.3.2 --- Construction of pGL3-tiMT deletion mutants --- p.54 / Chapter 2.3.3 --- Preparation of heavy metal solutions --- p.56 / Chapter 2.3.4 --- Determination of heavy metal ion toxicities by alamarBlue´ёØ assay --- p.56 / Chapter 2.3.5 --- Transient transfection of plasmids to Hepa-T1 cells --- p.56 / Chapter 2.3.6 --- Metal ions treatment and study of tiMT promoter activities --- p.57 / Chapter 2.3.7 --- Transient gene expression studies of deletion mutants of tiMT promoter --- p.57 / Chapter 2.4 --- Site-directed mutagenesis of tiMT promoter --- p.58 / Chapter 2.4.1 --- Polymerase chain reaction (PCR)-based site-directed mutagenesis --- p.58 / Chapter 2.4.2 --- Transient transfection of plasmids to Hepa-T1 cells and study of tiMT promoter activities --- p.62 / Chapter 2.5 --- Electrophoretic mobility shift assay (EMSA) --- p.63 / Chapter 2.5.1 --- Extract preparation --- p.63 / Chapter 2.5.2 --- Preparation of radiolabeled tiMRE oligonucleotides --- p.63 / Chapter 2.5.3 --- Electrophoretic mobility shift assay (EMSA) --- p.64 / Chapter 3. --- Results --- p.66 / Chapter 3.1 --- "Cloning of tilapia MT (tiMT) gene 5,-flanking region and amplification of whole tiMT gene" --- p.66 / Chapter 3.2 --- Determination of transcription start site --- p.69 / Chapter 3.3 --- Cloning of tiMT promoter fragment into reporter vector --- p.72 / Chapter 3.4 --- Determination of heavy metal ion toxicities by alamarBlue´ёØ assay --- p.72 / Chapter 3.5 --- Study of tiMT promoter activities by heavy metal ions exposure..… --- p.72 / Chapter 3.6 --- Cloning of deletion mutants of tiMT promoter --- p.79 / Chapter 3.7 --- Transient gene expression studies of deletion mutants of tiMT promoter --- p.80 / Chapter 3.8 --- Cloning of mutants with site-directed mutagenesis in tiMT promoter --- p.88 / Chapter 3.9 --- Site-directed mutagenesis of tiMT promoter --- p.92 / Chapter 3.10 --- Electrophoretic Mobility Shift Assay (EMSA) --- p.97 / Chapter 4. --- Discussion --- p.102 / Chapter 4.1 --- Tilapia MT gene --- p.102 / Chapter 4.2 --- Resistance of tilapia to heavy metal ions --- p.107 / Chapter 4.3 --- Functional analysis of tiMT gene promoter by transient transfection --- p.111 / Chapter 4.4 --- DNA binding of metal responsive transcription factor in Hepa-T1 cells --- p.121 / Chapter 4.5 --- Conclusion --- p.138 / References --- p.140

The present study was undertaken to investigate colour and sex determination mechanisms through the application of androgenesis, gynogenesis and controlled breeding programme with the objective of producing all red males in 0. niloticus. The highest yield of androgenetic haploid to pigmentation stage was 24.6±3.5% (relative to controls) with optimal UV irradiation dose of 450Jm"2 for 5 minutes. The highest survival rate of diploid androgens was 0.07±0.07% (relative to controls) to yolk sac stage using a heat shock of 42.5°C for 3 minutes 30 seconds applied at 25 minutes after fertilisation. All paternal inheritance of diploid androgenetic tilapia was verified using DNA fingerprinting. The mean recombination frequency of the red skin colour gene in meiotic gynogens was 0.12±0.04. All maternal inheritance of meiotic gynogens was verified using the isozyme locus ADA*. Analyses of sex ratios of meiotic gynogens suggested that male progenies were produced by an epistatic sex determining locus (SDL-2 with two alleles SR and sr) causing female to male sex reversal in the homozygous phase (srsr) but with limited penetrance. A close linkage was found between a sex determining locus (SDL-2) and the red gene. No significant difference was found between colour genotypes (namely homozygous red, heterozygous red and wild type) in terms of total fecundity, ISI (inter spawning interval), egg size and survival rate. Overall mean ISI was 26.3±1.0 days. Mean total fecundity was 1096 eggs. Fecundity varied over successive spawns but this variation did not appear to be related to spawning periodicity. Hormonal and thermal feminisation were compared on all YY male progeny of 0. niloticus. While similar female percentages of 32.0±5.2 and 33.8±1.5% were produced, significantly higher intersex percentages of 18.5±2.5 and 1.6±0.8 were observed in heat and DES treated groups, respectively. Heat treatment groups showed the lowest survival rate of 62.6±9.8% compared to the survival rates of 97.0±0.9% and 97.3±0.8% in controls and DES treated groups, respectively. YYRR males and YYRR neofemales were produced by integrating existing YYrr males and YYrr neofemales from the Egypt-Swansea-Philippine isolate and YYRR androgenetic males from the Stirling isolate with XXRR females and XYRR males of the Stirling isolate of Egyptian strain 0. niloticus. In summary, this study provides valuable information regarding the colour and sex determination mechanisms of 0. niloticus. The research in this thesis also demonstrated that both YY genotype and red coloration can be combined in a single strain in order to produce all male and stable red coloured 0. niloticus.

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1984. / Bibliography: leaves 103-110. / Microfiche. / x, 110 leaves, bound ill., map 29 cm

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Forming part of a conservation programme, this study was concerned with two species of Cichlid fish (Oreochromis mossambicus and O. ni/oticus), which were brought into contact with each other by unnatural ways. They are now hybridizing to some extent and there is also evidence that the foreign O. ni/oticus may out compete the native O. mossambicus. To cast light on what the current distribution is of both these species and the hybrids in Southern Africa, it is important to identify specimens very accurately. In attempting to find genetic markers to distinguish between two species of Cichlids we tested 20 microsatellite dinucleotide (CAn) repeats during a preliminary study and found five of these promising to exhibit little intra-specific genetic diversity but large genetic variation between species. We amplified these five loci in 145 individuals from 10 populations, which included the two species and their hybrids. Exact sizes of the fragments were determined using an automated DNA sequencer. Between the two species, allele sizes were overlapping, but when data were analyzed by statistical models, the differences could be seen for populations, however on individual level there was overlap between the species. The hybrids were found to be intermediate positioned between the two pure species. Our attempt to assign individuals to populations provided doubtful results. Thus, using this set of markers, populations can be ascribed to one of these species, but not individuals by themselves. / AFRIKAANSE OPSOMMING: As deel van 'n natuurbewarings program, word daar in hierdie studie twee spesies van vis ondersoek was in kontak met mekaar gekom het op onnatuurlike wyse. Hierdie twee visspesies vanuit die CICHLIDAEfamilie (Oreochromis mossambicus en 0. ni/oticus) kan hibridiseer wanneer hul saam voorkom, maar dit is ook bekend dat die uitheemse O. ni/oticus die inheemse O. mossambicus kan bedreig in terme van leefruimte, kos en broeispasie. Om die voorkoms van hibriede tussen die twee spesies te ondersoek in Suider Afrika se varswater opvangsgebiede, is dit baie belangrik om individue baie akkuraat te identifiseer. In hierdie poging om genetiese merkers te vind wat die twee spesies van mekaar onderskei, het ons 20 mikrosateliet di-nulkleotied (CAn) herhalende volgordes op verskillende loci ondersoek. Vyf daarvan het belowend voorgekom om as spesie spesifieke merkers te dien. Die fragmente op die vyf loci is ge-amplifiseer in 145 individue vanuit 10 populasies. Presiese groottes van die fragmente is bepaal met behulp van 'n ge-outomatiseerde DNA volgorde bepaler waarna genotiepes vir elke individu toegeken is. Tussen die twee spesies het alleel groottes oorvleuel, maar wanneer data geanaliseer word met behulp van statistiese metodes, was verskille tussen die spesies duidelik op populasie vlak. Die hibriede het intemediêr tussen die twee spesies voorgekom. Dus met behulp van hierdie stel merkers kan onderskei word tussen die twee spesies op populasie vlak, hoewel individue nie op sig self identifiseer kan word nie.

Thesis (MScAgric)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: DNA micro satellite loci express extensive allelic variation making them convenient markers for research in many fields employing population genetic tools, including aquaculture and conservation genetics. Twelve Oreochromis mossambicus populations from wild, captive and introduced sources in Southern Africa were screened for genetic variation at ten CA repeat micro satellite loci. Three of the loci - UNHI04, UNHlll, and UNH123 - were sufficiently well resolved to screen extensively and were interpreted according to a model of Mendelian inheritance. Data was analyzed in terms of genetic structure and levels of genetic variation, the effect of management regime in captivity through successive generations on genetic diversity, and the nature of phylogenetic relationships present between populations. Exact tests, carried out using Monte Carlo type multiple resampling techniques, and F-Statistics were used to detect and quantify genetic structure among the twelve populations. The Exact test X2 (P < 0.001), a FST of 0.27 (P < 0.001), eST of 0.26, RsT of 0.28, and a <l>ST of 0.17 all indicated significant structuring among the populations. The evident genetic structuring endorsed the practice of maintaining the populations as separate genetic stocks, in separate tanks, in order to preserve unique genetic material for aquaculture strain development. Populations also exhibited some significant deviations from Hardy Weinberg equilibrium characterised by an overall reduced heterozygosity across the loci. In microsatellite studies, null alleles are often suggested as major contributors to heterozygote deficits. To test for null alleles, two controlled crosses of 0. mossambicus were made. The progeny from each cross were examined for expected parental allelic ratios at the UNHI04, UNHlll and UNH123 loci. All three loci presented evidence of possible null alleles. Accelerated inbreeding and genetic drift through successive generations in captivity can reduce heterozygosity and gene diversity. To investigate loss of diversity a sample taken from the Bushmans population in 1999 (N = 25) was compared with a Bushmans 2000 sample (N = 36). The comparison highlighted altered allele frequencies, a significant increase in average observed heterozygosity and a non-significant change in average expected heterozygosity using the UNHI04 and UNH123 loci. Calculation of genetic distances and phylogenetic comparisons between the populations provided insight into the degree of management required in conserving genetic diversity in natural populations of Mozambique tilapia. UPGMA and Neighbour-Joining techniques were used to construct phylogenetic trees using Dm and ({)~)2 distance matrices. Clustering of populations appeared to reflect geographic locality of the source populations, however certain populations were not congruent with geography. Mantel tests were used to expose a possible association between genetic distance matrices generated from each individual locus. An association would support a geographic background to population genetic structure. The Mantel tests did not provide conclusive evidence. Mantel tests for association between the combined locus Dm and (81l)2 genetic distance matrices and a geographic distance matrix were similarly non-significant. Multi-dimensional scaling (MDS) plots of Euclidean distance values for Dm and (81l)2 matrices presented a two-dimensional view of the genetic distance data. The degree of similarity with the UPGMA and Neighbour-Joining tree-clustering pattern was higher for the (81l)2 than for the Dm MDS plots. Scatter plots indicated a reliable non-linear correlation between Euclidean distance and genetic distance for the two-dimensional MDS. The micro satellite markers employed in this research provided molecular information needed for complimenting a co-study on quantitative genetic evaluation of the twelve populations. The quantitative co-study provided measures of average length and weight gain indices for the populations based on progeny growth trials. No significant correlation of average heterozygosity (gene diversity) with either average weight or length gain was found. The significant genetic diversity and structure present between the twelve populations provided rationale for implementing strategies to conserve natural 0. mossambicus populations as genetic resources, and manage captive populations for long term maintenance of genetic diversity. / AFRIKAANSE OPSOMMING: Die verstaffing van groot alleliese variasie deur DNA mikrosateliete maak van hulle gerieflike merkers vir navorsing in 'n verskeidenheid van velde wat gebruik maak van populasie genetiese gereedskap, ingesluit akwakultuur en bewarings genetika. Twaalf 0. mossambicus populasies wat verkry was vanuit die natuur, in gevangeneskap en ingevoerdes, van Suidelike Afrika was getoets vir genetiese variasie by tien verskillende CA-herhalende mikrosateliet loci. Drie van die loci - UNHI04, UNHlll en UNH123 - is op grootskaal getoets en volgens In model van Mendeliese oorerwing geinterpreteer. Die data was ontleed volgens genetiese struktuur en vlakke van genetiese variasie, die effek wat bestuur strategie in gevangeneskap op genetiese diversiteit in opeenvolgende generasies uitgeoefen het, so wel as die aard van die filogenetiese verhoudings wat teenwoordig is tussen die populasies. "Exact" toetse is uitgevoer deur gebruik te maak van Monte Carlo tipe veelvuldige hermonsterinsamelings tegnieke en F-statistieke is gebruik vir die deteksie en kwantifisering van die genetiese struktuur tussen die twaalfpopulasies. Die Exact toets X2 (P < 0.001), 'n FST van 0.27 (P < 0.001), SST van 0.26, RsT van 0.28, en 'n <DST van 0.17 gee almal 'n indikasie van betekenisvolle strukturering tussen die populasies. Die genetiese struktuur bevestig die beleid dat die populasies behou moet word as aparte genetiese voorraad, in aparte tenke, om te verseker dat die unieke genetiese materiaal behoue bly om akwakultuur variante te ontwikkel. Populasies het ook betekenisvolle verskuiwings van die Hardy Weinberg ekwilibrium getoon, wat gekarakteriseer word deur 'n algemene verlaging van heterosigositeit oor die loci. Nul allele word dikwels aanbeveel om in mikrosateliet studies groot bydraes te maak tot hetersigotiese defekte. Om vir nul allele te toets was twee gekontroleerde kruisings van 0. mossambicus gemaak. Die nageslag van elke kruising was getoets vir verwagte ouer alleliese verhoudings by die UNHI04, UNHlll en UNH123 loci. Al drie loci het getoon dat dit moontlike nul allele kan wees. Versnelde inteling en genetiese drywing deur opeenvolgende generasies in gevangeneskap kan die heterosigositeit en diversiteit verminder. Om die vermindering van diversiteit te toets was 'n monster van die Busmans 1999 (N=25) populasie vergelyk met 'n monster van die Bushmans 2000 (N=36) populasie. Die vergelyking het veranderde alleel frekwensies, 'n betekenisvolle vermeerding in gemiddelde waargeneemde heterosigositeit en 'n onbetekenisvolle verandering in gemiddelde verwagte heterosigositeit getoon deur gebruik te maak van die UNHI04 en UNH123 loci. Berekening van genetiese afstande en filogenetiese vergelykings tussen die populasies het nuwe insig gegee oor die graad van bestuur wat nodig is om genetiese diversiteit in die natuurlike populasies van 0. mossambicus tilapia te behou.UPGMA en Neighbour-Joining tegnieke was gebruik om filogenetiese bome op te stel deur gebruik te maak van Dm en (OIl)2 afstand matrikse. Populasie bondeling het geblyk om geografiese lokaliteit van die bron populasies te toon, alhoewel van die populasies nie met die geografiese lokaliteit ooreengestem het nie. Mantel toetse is gebruik om 'n moontlike assosiasie tussen genetiese afstand matrikse wat verkry is van elke loci bloot te stel. 'n Assosiasie sou 'n geografiese agtergrond tot populasie genetiese struktuur steun. Oortuigende bewys is nie deur die Mantel toetse verskaf nie. Mantel toetse vir assosiasie tussen die gekombineerde loci Dm en (OJ..l)2 genetiese afstand matrikse en In geografiese afstand matriks was ook onbetekenisvol. 'n Tweedimensionele beskouing van die genetiese afstand data is voorgestel deur multidimensionele skaal (MDS) grafieke van Euclidean afstand waardes van die Dm en (OJ..l)2 matrikse te teken. Die graad van ooreenstemming met die UPGMA en Neighbour-Joining boom samevoeging patroon was hoër vir die (OJ..l)2 as vir die DmMDS grafieke. Verspreiding grafieke het 'n vertroubare nie-liniêre korrelasie tussen Euclidean afstande en genetiese afstande vir die twee-dimensionele MDS grafieke getoon. Die mikrosateliet merkers wat in die studie gebruik was het molekulêre informasie verskaf wat nodig is vir 'n komplimentêre studie oor die kwantitatiewe genetiese evalueering van dié twaalf populasies. Die kwantitatiewe studie het afmetings van gemiddelde lengte en gewig vermeerdering van die populasies verskaf gebaseer op nageslag proewe. Geen betekenisvolle korrelasie van gemiddelde hetersigositeit (geen diversiteit) was getoon met óf gemiddelde gewig óf lengte vermeerdering. Die betekenisvolle genetiese diversiteit en struktuur teenwoordig tussen die twaalf populasies het rede gegee om strategieë te implimenteer om natuurlike 0. mossambicus populasies te konserveer as genetiese bronne en om populasies in gevangeneskap te bestuur vir langtermyn instandhouding van genetiese diversiteit.

Au, Yee Man Candy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 128-144). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter 1. --- Chapter One Introduction / Chapter 1.1 --- Homeostasis and detoxification of metal ions --- p.1 / Chapter 1.2 --- Biochemistry of metallothionein --- p.3 / Chapter 1.2.1 --- Structure of metallothionein --- p.4 / Chapter 1.2.2 --- Isoforms of metallothionein --- p.5 / Chapter 1.2.3 --- Roles of metallothionein --- p.6 / Chapter 1.2.4 --- Structure of metallothionein gene --- p.9 / Chapter 1.2.5 --- Metal responsive element (MRE) --- p.10 / Chapter 1.2.6 --- Regulation of MT gene --- p.11 / Chapter 1.3 --- Metal responsive element-binding transcription factor 1 (MTF-1) --- p.16 / Chapter 1.3.1 --- Structure of MTF-1 --- p.16 / Chapter 1.3.2 --- Target genes of MTF-1 --- p.18 / Chapter 1.4 --- Teleost MT and MTF-1 --- p.20 / Chapter 1.5 --- Tilapia --- p.26 / Chapter 1.6 --- Tilapia MT and MTF-1 --- p.26 / Chapter 1.7 --- Aims of study --- p.30 / Chapter 2. --- Chapter Two Materials and Methods / Chapter 2.1 --- Quantification of MTF-1 isoforms and MT mRNA levels in tilapia and Hepa-Tl cells by real-time PCR --- p.32 / Chapter 2.1.1 --- Heavy metal exposure on tilapia --- p.32 / Chapter 2.1.1.1 --- Animals --- p.32 / Chapter 2.1.1.2 --- Heavy metal exposure --- p.32 / Chapter 2.1.1.3 --- Total RNA extraction --- p.33 / Chapter 2.1.1.4 --- Reverse Transcription --- p.35 / Chapter 2.1.2 --- Heavy metal exposure on Hepa-Tl cells --- p.36 / Chapter 2.1.2.1 --- Cell Culture --- p.36 / Chapter 2.1.2.2 --- Metal treatment on Hepa-Tl cells --- p.37 / Chapter 2.1.3 --- SYBR green --- p.39 / Chapter 2.1.3.1 --- Primer Design --- p.39 / Chapter 2.1.3.2 --- Validation of cycling condition --- p.41 / Chapter 2.1.3.3 --- Determination of relative amount of target gene present in the samples --- p.43 / Chapter 2.1.3.4 --- Statistical analysis --- p.44 / Chapter 2.1.4 --- TaqMan probes --- p.44 / Chapter 2.1.4.1 --- Primer Design --- p.44 / Chapter 2.1.4.2 --- Validation of cycling condition --- p.45 / Chapter 2.2 --- Localization study of MTF-1 isoforms --- p.46 / Chapter 2.2.1 --- Amplification of the full length tilapia MTF-1 isoforms --- p.46 / Chapter 2.2.2 --- Preparation of Escherichia coli competent cells --- p.48 / Chapter 2.2.3 --- Transformation --- p.49 / Chapter 2.2.4 --- Confirmation of the insert of the ligation products --- p.50 / Chapter 2.2.5 --- Cloning of MTF-1-L and MTF-1-S gene into phrGFPII-1 vector --- p.51 / Chapter 2.2.6 --- Transient transfection of plasmids to Hepa-Tl cells --- p.54 / Chapter 2.2.7 --- Staining of the nucleus by Hoechst 33342 --- p.55 / Chapter 2.2.8 --- Metal treatment on Hepa-Tl cells --- p.55 / Chapter 2.3 --- Electrophoretic mobility shift assay (EMSA) --- p.56 / Chapter 2.3.1 --- Preparation of Hepa-Tl whole-cell protein extract --- p.56 / Chapter 2.3.2 --- In vitro transcription/translation of tilapia MTF-1 isoforms --- p.57 / Chapter 2.3.3 --- Annealing of the tiMREg oligonucleotides --- p.58 / Chapter 2.3.4 --- Labeling of the annealed tiMREg oligonucleotides --- p.58 / Chapter 2.3.5 --- Electrophoretic mobility shift assay --- p.59 / Chapter 3. --- Chapter Three Results / Chapter 3.1 --- Quantification of MTF-1 isoforms and MT mRNA levels in tilapia and Hepa-Tl cells by real-time PCR --- p.62 / Chapter 3.1.1 --- Validation of primers for real-time PCR --- p.62 / Chapter 3.1.2 --- Tissue distribution of MTF-1 isoforms in tilapia and Hepa-Tl cell-line --- p.63 / Chapter 3.1.3 --- Effect of metal treatment on MTF-1 isoforms and MT gene expression level in different tissues of tilapia and Hepa-Tl cell-line --- p.68 / Chapter 3.2 --- Localization study of MTF-1 isoforms --- p.82 / Chapter 3.2.1 --- Cloning of MTF-1 isoforms into phrGFPII-1 vector --- p.82 / Chapter 3.2.2 --- Transient transfection of phrGFPII-1 plasmids to Hepa-Tl cells --- p.82 / Chapter 3.3 --- Electrophoretic mobility shift assay (EMSA) --- p.96 / Chapter 4. --- Chapter Four Discussion / Chapter 4.1 --- Tissue distribution of MTF-1 isoforms --- p.104 / Chapter 4.2 --- Effect of metal stress on the mRNA expression level of MT and MTF-1 isoforms --- p.106 / Chapter 4.3 --- In vitro study of the localization of the MTF-1 isoforms --- p.114 / Chapter 4.4 --- DNA binding of MTF-1 synthesized by in vitro transcription/translation method --- p.121 / Chapter 4.5 --- Conclusion --- p.125 / Chapter 5. --- REFERENCES --- p.128

Lam Kwok Lim. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 107-126). / Abstract also in Chinese. / Acknowledgments --- p.i / Presentations Derived from the Present Thesis Work --- p.ii / Abstract --- p.iv / Abbreviations --- p.vii / Abbreviation Table for Amino Acids --- p.ix / List of Figures --- p.x / List of Tables --- p.xii / Contents --- p.xiii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Metallothionein (MT) --- p.1 / Chapter 1.1.1 --- Classification of MT --- p.1 / Chapter 1.1.2 --- Structure of MT --- p.2 / Chapter 1.1.3. --- Structure of MT Genes --- p.4 / Chapter 1.1.4 --- Function of MT --- p.5 / Chapter 1.1.5 --- Regulation of MT Expression --- p.7 / Chapter 1.1.6 --- Fish MT --- p.9 / Chapter 1.1.7. --- Aims and Rationale of the Present Study --- p.12 / Chapter 2 --- MT mRNA Induction of Tilapia After Intraperitoneal Injection of Metal --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.1.1. --- Specific Aims of This Chapter --- p.19 / Chapter 2.2 --- Materials and Methods --- p.20 / Chapter 2.2.1 --- Regents --- p.20 / Chapter 2.2.1.1 --- Purification of Total RNA --- p.20 / Chapter 2.2.1.2 --- Denaturing Gel and Vacuum Blotting of RNA (Northern Blotting) --- p.20 / Chapter 2.2.1.3 --- Hybridization --- p.21 / Chapter 2.2.2 --- Methods --- p.21 / Chapter 2.2.2.1 --- Purification of Total RNA --- p.21 / Chapter 2.2.2.2 --- Vacuum Blotting of Total RNA (Northern Blotting) --- p.22 / Chapter 2.2.2.3 --- Radioactive Labeling of Nucleic Acid Probes --- p.22 / Chapter 2.2.2.4 --- Hybridization --- p.22 / Chapter 2.2.2.5 --- Densitometric Analysis --- p.23 / Chapter 2.2.2.6 --- Calculation of MT mRNA Levels and Analysis of Results --- p.23 / Chapter 2.2.3 --- Endogenous MT mRNA Expression of Juvenile Tilapia and Carp --- p.23 / Chapter 2.2.4 --- Induction of MT mRNA Juvenile Tilapia and Carp Injected with Metals --- p.24 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Endogenous Levels of MT mRNA in Tilapias in Normal Conditions --- p.25 / Chapter 2.3.2 --- Induction of MT mRNA Levels in Juvenile Tilapia Injected with Metals --- p.25 / Chapter 2.3.1.1 --- Copper Injection --- p.25 / Chapter 2.3.1.2 --- Zinc Injection --- p.25 / Chapter 2.3.1.3 --- Cadmium Injection --- p.26 / Chapter 2.3.3 --- Induction of MT mRNA Levels in Juvenile Carp with Zinc Injection --- p.26 / Chapter 2.4 --- Discussion --- p.26 / Chapter 2.4.1 --- MT mRNA Expression of Tilapia and Carp Injected with Metals --- p.26 / Chapter 2.5 --- Conclusions --- p.29 / Chapter 3 --- Induction Level of MT mRNA in Tilapia After Aqueous Exposure to Metals --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.1.1 --- Specific aims of this chapter --- p.36 / Chapter 3.2 --- Material s and Methods --- p.36 / Chapter 3.2.1 --- 96hours LC-50 values for zinc and copper --- p.36 / Chapter 3.2.2 --- Induction of MT mRNA in Juvenile Tiapias under Metal Aqueous Exposures --- p.37 / Chapter 3.2.3 --- Calculation of Fold Induction of MT mRNA and Analysis of Results --- p.38 / Chapter 3.2.4 --- Metal Analysis --- p.38 / Chapter 3.3 --- Results --- p.38 / Chapter 3.3.1 --- LC-50 values of metals for Juvenile Tilapia --- p.38 / Chapter 3.3.2 --- Induction of MT mRNA in Juvenile Tilapia under Metal Aqueous Exposures --- p.39 / Chapter 3.3.2.1 --- Aqueous Exposure to Copper --- p.39 / Chapter 3.3.2.2 --- Aqueous Exposure to Zinc --- p.40 / Chapter 3.3.2.3 --- Aqueous Exposure to Cadmium --- p.41 / Chapter 3.3.3 --- Induction of MT mRNA in Juvenile Carp after Aqueous Exposures to Metal --- p.41 / Chapter 3.3.3.1 --- Aqueous Exposure to Cadmium --- p.41 / Chapter 3.3.4 --- Metal Concentrations of Water Samples from the Aquaria in the Metal Exposure Test of Tilapia and Carp --- p.42 / Chapter 3.4 --- Discussion --- p.42 / Chapter 3.4.1 --- LC-50 values of Metals for Tilapia --- p.42 / Chapter 3.4.2 --- MT mRNA Expression of Tilapias under Metal Aqueous Exposure --- p.44 / Chapter 3.4.3 --- Normalization of the Signals of Northern Blot Analysis --- p.47 / Chapter 3.5 --- Conclusions --- p.48 / Chapter 4 --- Field Study --- p.58 / Chapter 4.1 --- Introduction --- p.58 / Chapter 4.1.1 --- Specific Aims of this Chapter --- p.59 / Chapter 4.2 --- Materials and Methods --- p.59 / Chapter 4.2.1 --- Sampling Sites --- p.59 / Chapter 4.2.2 --- Data Analysis --- p.60 / Chapter 4.2.3 --- Harvest of Feral Tilapia --- p.60 / Chapter 4.2.4 --- Determination of Metal Concentration of Metal Concentration in the Tissues of Feral Tilapia --- p.60 / Chapter 4.2.5 --- Endogenous MT mRNA Levels Using Northern Blot Analysis --- p.61 / Chapter 4.2.6 --- Calculation of MT mRNA Levels and Analysis of Results --- p.61 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Metal Concentrations in the Tissues of Feral Tilapia --- p.62 / Chapter 4.3.2 --- Comparison of Metal Concentrations Among Different Tissues of Feral Tilapia --- p.62 / Chapter 4.3.3 --- MT mRNA Levels in the Tissues of Feral Tilapia --- p.63 / Chapter 4.3.4 --- Correlation Between Metal Concentrations and Endogenous MT mRNA Levels in the Tissues of Feral Tilapia --- p.63 / Chapter 4.4 --- Discussion --- p.64 / Chapter 4.4.1 --- Bioaccumulation of Metals --- p.64 / Chapter 4.4.2 --- Endogenous Levels of MT mRNA in the Feral Tilapia --- p.67 / Chapter 4.5 --- Conclusions --- p.68 / Chapter 5 --- Cloning of Tilapia MT Genes --- p.86 / Chapter 5.1 --- Specific Aims of This Chapter 、 --- p.86 / Chapter 5.2 --- Materials and Methods --- p.87 / Chapter 5.2.1 --- Regents --- p.87 / Chapter 5.2.1.1 --- Preparation of Plasmid DNA --- p.87 / Chapter 5.2.1.2 --- Preparation of Genomic DNA --- p.87 / Chapter 5.2.1.3 --- Restriction Enzyme Digestion --- p.88 / Chapter 5.2.1.4 --- Vacuum Blotting of DNA (Southern Blotting) --- p.88 / Chapter 5.2.1.5 --- Polymerase Chain Reaction --- p.89 / Chapter 5.2.1.6 --- Transformation of E.coli Competent Cells --- p.89 / Chapter 5.2.1.7 --- Nucleotide Sequence Determination --- p.89 / Chapter 5.2.1.8 --- List of Primers --- p.90 / Chapter 5.2.1.8.1 --- Primers for Nucleotide Sequence Determination --- p.90 / Chapter 5.2.1.8.2 --- Tilapia MT Specific Primers for PCR --- p.90 / Chapter 5.2.2 --- Methods --- p.91 / Chapter 5.2.2.1 --- Preparation of Plasmid --- p.91 / Chapter 5.2.2.2 --- Preparation of Genomic DNA --- p.91 / Chapter 5.2.2.3 --- Preparation of Enzyme Digestion --- p.92 / Chapter 5.2.2.4 --- Vacuum Blotting of Genomic DNA (Southern Blotting) --- p.92 / Chapter 5.2.2.5 --- Radioactive Labeling of Nucleic Acid Probes --- p.92 / Chapter 5.2.2.6 --- Hybridization --- p.93 / Chapter 5.2.2.7 --- Polymerase Chain Reaction --- p.93 / Chapter 5.2.3 --- Southern Blot Analysis of Tilapia Genomic DNA --- p.93 / Chapter 5.2.4 --- Analysis of the Sequences of Tilapia MT Genes --- p.94 / Chapter 5.2.4.1 --- Amplification of MT Genes Using PCR --- p.94 / Chapter 5.2.4.2 --- Cloning of the MT Genes --- p.94 / Chapter 5.2.4.3 --- Transformation of E.coli Competent Cell --- p.94 / Chapter 5.2.4.4 --- Nucleotide Sequence Determination --- p.95 / Chapter 5.3 --- Results --- p.95 / Chapter 5.3.1 --- Southern Blot Analysis of Tilapia Genomic DNA --- p.95 / Chapter 5.3.2 --- Amplification of MT Gene Fragments Using PCR --- p.95 / Chapter 5.3.3 --- Analysis of the Sequences of Tilapia MT Genes --- p.96 / Chapter 5.4 --- Discussion --- p.96 / Chapter 5.4.1 --- Fish MT Genes --- p.96 / Chapter 5.5 --- Conclusions --- p.98 / Chapter 6 --- General Discussion --- p.104 / References --- p.107

Production of all-male tilapia for aquaculture is assuming an increasingly important role. An important pre-requisite to repeated obtainment of monosex tilapia is a clear understanding of the mechanisms underlying sex differentiation. Histological observations on gonadal morphorgonesis and sex differentiation provided basic data for hormonal sex manipulation in four commercially important species. Results indicate that gonadal morphogenesis starts at different times ranging from eight days after hatching in 0. mossambicus to 17 days in 0. niloticus. Sex differentiation followed a similar pattern, and ranged from 22 days in O. mossambicus to 36 days in 0. niloticus. The effects of subjecting fish to different rearing temperatures was assessed. No significant influence was found on sex ratio of treated fish. Observations on offspring sex ratio in intraspecific breeding and interspecific hybridization demonstrated that significant differences between batches are a common occurrance and their regularity cannot be adequately explained on the basis of sex chromosome theory alone. Treating fish with synthetic androgen (17 alpha methyltestosterone) and synthetic oestrogen, (17 alpha ethenylestradiol) resulted in species specific and dosage dependant differences in sex ratios. Results also revealed significant differences in sex ratios of different batches of fish subjected to the same treatment, thus demonstrating that success rate in sex inversion varies not only between species and between stocks, but in sib groups as well. Results of intraspecific and interspecific breeding suggest that sex determination in tilapia is under the influence of multiple factors. Results of hormone treatments indicate variations in inversion rate at batch level, thus demonstrating presence of individual differences in lability. On the basis of results from these four experiments, it is hypothesized that sex in tilapia is influenced by multiple genes and the fishes' propencity to change sex varies in individual fish. Progeny testing oestrogen sex inversed fish indicates that on the basis of the chromosome theory of sex determination, S. galileaus and O. niloticus are female homogametic, while O, macrochir is female heterogametic. The implications of the results obtained in this study for production of all-male tilapia are briefly discussed.

Thesis (PhD (Botany and Zoology))--Stellenbosch University, 2008. / Endocrine disruption is an alteration of the chemical messaging processes in the body. The value of studies‐ and monitoring of endocrine disruption using techniques included in the field of toxicogenomics is undoubtedly supported by scientific literature over the past four decades, as is demonstrated in Chapter 1 where I review relevant literature on the topic. Clearly, well sustained bio‐monitoring will include studies both in vitro and in vivo, and very well on transcriptional and translational levels. Animals are providing good models for in vivo studies to report or monitor endocrine disruption. It is imperative though to first understand such an animal’s biology, especially its endocrine system, and characterize what is considered “normal” for a species before engaging in endocrine disrupting exposures. A multitude of studies report endocrine disruption in relation to reproductive systems, with more recent work illustrating alteration of metabolism related to thyroidogenic disruption within the last decade. It is therefore essential to consider sex determination and ‐differentiation when studying sentinel species. Apart from the obvious academic interest in the matter of sex differentiation, altered patterns of sex differentiation in certain appropriate species provide for a very convincing endpoint in monitoring estrogenic endocrine disruption. As I approach to study a potential sentinel species for the southern African subcontinent, I set forward to study aspects of endocrine disruption influencing the reproductive system in a piece‐meal manner, starting with estrogenic endocrine disruption as this is the best studied facet of the endocrine disruption hypothesis to date. Yet, one learn from vast amounts of literature that in cases where sex is not exclusively determined by the genetic fraction of an individual, a number other characteristics may very well be used to determine estrogenic disruption in ecosystems. Quantitative production of the egg yolk precursor protein (vitellogenin) resides under these characteristics, and in the proposed sentinel, South African tilapiine, Oreochromis mossambicus phenotypic sex can be altered by environmental sex determination. The present study therefore targeted firstly the product most often used in tier I screening processes, vitellogenin (VTG). Specimens of O. mossambicus were cultured for this purpose from wild breeding stock, sampled at 5 day intervals and the transcription levels of vitellogenin gene (vtg) studied in those. Hereby, Chapter 2 describes the cloning of partial vtg gene and subsequent temporal expression of vtg quantitatively in O. mossambicus. To shed light on the state of gonadal differentiation sub‐samples were subjected to histology, illustrated in Chapter 3. In addition the quantitative vtg responses has been described in this study at a transcriptional level, both of adult males and juveniles subjected to low and very high levels of natural estrogens. In addition, a 3 kb 5’ flanking region of vtg was cloned and sequenced, and several putative binding sites identified for transcription factors of vtg, including several estrogen responsive elements (EREs). These indicate the expected regulational process of vtg by estrogens. Subsequently I measured the transcription levels of the only enzyme capable of aromatizing androgens into estrogens, Cytochrome P450 19 (cyp19) as has been characterized in Chapter 3. For stable binding of an estrogen to an ERE, binding of the ligand to its specific nuclear receptor (Estrogen receptor, ESR) is required. Since E2 is known to have different mechanisms of action in vertebrates, the expression levels of the ESRs were evaluated in our sample set after cloning 3 different homologues of ESR in O. mossambicus. The results on this matter is discussed in Chapter 4 and provides in addition to data on vtg and cyp19 a platform of “normal” transcription levels of these candidate genes involved in estrogenic endocrine disruption of O. mossambicus. Ultimately, characterization of those candidate genes involved extensively in phenotypic sex, contribute to our understanding of sex determination and differentiation in this species in a small way.