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The major histocompatibility complex (MHC) is one of the most variable regions in the vertebrate genome. Many genes within the MHC play important roles in the development of an immune response, including the response to pathogens, by presenting pathogen fragments to T cells. Pathogen-mediated balancing selection is thought to be important in maintaining the high levels of allelic variation at these loci, though the precise mechanism remains unclear. The number of studies of MHC diversity in non-model organisms has increased dramatically in recent years as genotype data have become cheaper and easier to generate; however, key limitations in many such studies remain a lack of high quality MHC genotypes and associated phenotype data. Many studies focus on a single MHC locus, assuming that one locus will represent the full range of variation within each MHC haplotype. Alternatively, the products of different loci may co-amplify, preventing locus-specific genotypes and hence heterozygosity being accurately determined. Non-model systems are also often limited by small sample sizes and limited recording of associated host and pathogen measures, which, combined with high levels of allelic variation at MHC loci, can limit statistical power. Finally, few MHC studies control for the general effect of relatedness in explaining host traits before testing for MHC effects. With so many methodological impediments, it is challenging to identify robust associations between MHC variation and host phenotypes, such as parasite burden or fitness, and to draw conclusions about the mechanisms underpinning the maintenance of diversity at MHC loci. In this thesis, I address these problems by developing a SNP-based haplotyping system for a population of unmanaged Soay sheep (Ovis aries) on Hirta, St. Kilda, for which data is available on pedigree, phenotypic traits and fitness and its components over a 30- year study period. The ovine MHC consists of four classes of loci, within which loci are tightly clustered and show reduced recombination rates compared to the genome average. Although the mammalian MHC is usually highly variable, one would expect that the number of haplotypes within an MHC class in an island population of sheep with no immigration to be limited. The class IIa region of the ovine MHC includes the classical class II loci which are typically thought to be involved in the presentation of peptides derived from extracellular pathogens, including gastrointestinal helminths, in sheep and other mammals. In chapters 2 to 4, I describe the characterisation of class IIa haplotypic diversity in the Soay sheep using direct Sanger sequencing of PCR amplified fragments, which, in combination with cloning, revealed eight distinct haplotypes. With this knowledge of haplotypic diversity, and genotypes for a sample of Soay sheep typed on the Ovine Infinium HD chip (approximately 600K SNPs), I developed a panel of 13 SNPs which could be used to impute the class IIa haplotypes. This panel was genotyped by KASP (Kompetitive Allele Specific PCR) in 6034 samples and used to impute the class IIa haplotypes. After quality control measures, class IIa haplotypes were successfully imputed for 5349 individuals. Evidence of balancing selection was identified using the Ewens-Watterson test at different life history stages and within the standing population each year between 1985 and 2012, showing that allele frequencies were more even than would be expected under neutrality. However, there was no evidence of deviation from Hardy-Weinberg equilibrium identified at different life stages or in the standing population in any year. In chapter 5, I investigate associations between the MHC class IIa haplotypes and individual-level data on host phenotypes - body weight, plasma immunoglobulin levels (measured as anti-Teladorsagia circumcincta third larval stage IgA, IgE and IgG) and strongyle faecal egg counts (FEC). Associations were tested within mixed effects models which were used to account for repeated measures and control for fixed effects known to affect the response variables, as well as within an animal model framework to account for relatedness between individuals. Haplotype heterozygosity was unrelated to any of the traits investigated, suggesting a general heterozygote advantage is unlikely to be operating within the Soay sheep. Six of the eight class IIa haplotypes were associated with multiple traits in different age-sex classes, although many of these associations were removed after inclusion within animal models. The evidence of balancing selection and associations between class IIa haplotypes and phenotypes related to health offers a promising glimpse into the evolutionary mechanisms which may be operating to maintain diversity within this region.

Genetic and functional studies have demonstrated that KIR gene polymorphism, including different haplotypes, allelic polymorphisms and different expression levels of KIRs, may all play a part in the association with HIV-1 infection outcome. Currently, there are very few studies focusing on the association between KIR and HIV in the Chinese population. In this project, we started to look at the polymorphism of KIRs in a unique chronic HIV-1 infected cohort (SM cohort), evaluating the impact of KIR and KIR-HLA interactions in terms of HIV-1 infection progression. The SM cohort is unique because the major factors such as viral strain, transmission route and timing of infection, which could affect the natural history of HIV-1, have been narrowly controlled. Through comparison with a healthy control population, some genetic associations were identified. The frequency of KIR2DL3 was lower in the “slow progressors” group; the compound genotype of KIR3DS1+ Bw4 homozygotes was significantly lower in the “slow progressors” group; additionally, group B genotypes (multiple activating genes) were shown to be likely to mount a greater immune pressure on HIV-1. In terms of KIR footprints, several amino acid positions were identified for which the substitution of an amino acid may be ascribed to the immune response from KIR-modulated NK cells rather than from HLA restricted CTL immune pressure. In Chapter 4, we report a novel method to sequence the entire locus of KIR3DL1/S1. Two specific pairs of primers have been successfully designed and tested to amplify KIR3DL1 and KIR3DS1 exclusively. Using this novel sequencing method, we showed the polymorphism of this locus at a 6-digit level. 8 new KIR3DS1 alleles, 12 new KIR3DL1 alleles, 1 new KIR3DL1 gene and 1 new KIR3DS1 gene have been identified in this study. In Chapter 5, we used a valuable acute HIV-1 infection cohort to further study associations between KIRs and the clinical outcomes. It was interesting to find that the frequency of KIR3DS1 was significantly lower in the slow progressors group (31%) than in the acute group (40.7%), which implies that KIR3DS1 plays a role in HIV-1 disease progression. There are two other trends demonstrated in this study. One trend was that positive KIR2DL2 and/or KIR2DS2 (they are in strong linkage disequilibrium with each other) were associated with a higher set point viral load (at 3 month) (p=0.06). Another trend was that KIR3DS1 might have an association with disease progression (p=0.057). Overall, in this study, the role of KIRs and KIR/HLA interactions were evaluated in acute and chronic HIV-1 infection, which has provided important information for further study.

Since the proposal of the first genetic algorithm (GA) many recombination operators have been proposed. Some are problem specific and require a great deal of knowledge about the problem being solved, resulting in good but highly specialised operators. Other recombination operators have been proposed for more general use. One advantage for such operators is the little knowledge required about the problem being solved; however, the synergy of these operators, the problem being solved and other GA parameters does not always yield optimum performance from the GA. More recently, adaptive recombination operators have been proposed to bridge the gap between general and specialised recombination operators. This thesis presents a novel adaptive recombination operator, namely "Selective Crossover", for use with a genetic algorithm. Selective crossover was designed with three properties that make it a viable strategy to use when little or no knowledge is available about the problem being optimised. The first property is the identification of allele changes made to the candidate solution during recombination. The second property is the use of correlations between parental and offspring fitnesses to discover beneficial alleles. The third property is the preservation of alleles at each locus, during recombination, according to their previous contributions to beneficial changes in fitness. This thesis makes six contributions. The first is the design and implementation of selective crossover. The second is a measurement and comparison of the performance of selective crossover and two traditional recombination operators on a number of different problems. The third is an empirical analysis of the adaptive properties in selective crossover. The fourth is an identification and analysis of four key biases inherent in selective crossover and a demonstration of the existence of these biases in two other similar operators. The fifth is an analysis and comparison of schema propagation in selective crossover and two traditional recombination operators. The final contribution is a construction of a schema survival probability for selective crossover.

Fv1 is a gene of mice known to restrict the replication of Murine Leukaemia virus (MLV) by blocking integration by an unknown mechanism. The gene itself is retroviral in origin, and is located on the distal part of chromosome 4. The sequence of markers known to flank Fv1 in the mouse was used to identify sequence from the human homologues of these 2 genes. The construction of primers to these sequences permitted the screening of 2 YAC and a PAC human genomic libraries for clones containing either of these genes. The YAC libraries were negative for both markers. The human region was finally cloned as 2 overlapping PAC clones. This region was sequenced, and the comparison of this sequence and the analogous region in rat to the Fv1 region in mice allowed the determination of what sequence had been lost and gained during the formation of the gene. The Fv1 ORF of mice from across the Mus genus was PCR-amplified, cloned and sequenced. The analysis of this sequence has shown how Fv1 has evolved during the speciation of the Mus genus. By combining this data with what is known of the distribution of endogenous MLV and Fv1 activity among the Mus genus, a scheme of how and why Fv1 has evolved activity has been proposed. The mouse genome was screened for a 'progenitor' sequence(s) that may have given rise to Fv1 during its germline infection of Mus. A mouse 129/SvJ genomic library was screened by hybridisation for the sequences bearing homology to Fv1. Sequences obtained were shown to be the Fv1 gene itself, members of the murine endogenous retroviral-L (MERV-L) family, or had no known sequence homology as determined by BLAST searching. A polymorphism at an EcoR1 site was identified in the Fv1 gene of the 129 mouse that appears to be responsible for the presence of 2 bands that hybridise with an Fv1-specific probe in a 129 genomic southern blot. Fv1null cell lines expressing mutant Fv1 ORFs were established and assayed for Fv1 activity in an assay using pseudotyped MLV. This allowed the contribution of the 3 changes known to be important between the 2 main alleles to be assessed. The first amino acid change in the Fv1 ORF was found to be the major determinant of Fv1 phenotype, with the difference in the C-terminus contributing to a lesser extent. Fv1d allele, a less common allele of Fv1, was shown to be mediated by a single amino add change and not by a change in the level of Fv1 expression.

The subject of this thesis is the interaction of the MHC class II encoded protein HLA-DM with conventional MHC class II proteins such as HLA-DR and HLA-DQ. MHC class II molecules in DM-negative cells cannot present antigenic peptides but remain loaded with the class II-associated invariant chain derived peptide (CLIP). In order to study the involvement of HLA-DM in the generation of this phenotype an insect cell baculovirus expression system was set up with the goal of producing soluble DM protein. The recombinant HLA-DM was purified and used in peptide exchange assays using CLIP-loaded DR3 molecules from the DM-negative cell line T2.DR3. Recombinant HLA-DM facilitated the exchange of CLIP for antigenic peptides in a catalytic, pH-dependent manner. In addition to CLIP, all those peptides that did not have an allele-specific binding motif were released by DM. Thus, HLA-DM acts a peptide editor, facilitating selection of peptides that stably bind to class II molecules for eventual presentation to the immune system from the pool of available peptides. Certain alleles of another MHC class II isotype, HLA-DQ, are highly associated with autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM). I set out to explore whether an abnormal interaction of certain HLA-DQ alleles with HLA-DM might lead to an altered peptide repertoire. To this end an antiserum recognizing the conserved C-terminus of the DQα chain was generated and the association of different HLA-DQ alleles with DM was assessed using co-immunoprecipitations. No gross differences between alleles were observed. In order to establish a more sensitive system to study the interaction of HLA-DM with DQ, I tried to transfect MHC class II-negative T2 cells with different DQ alleles and generated soluble DQ molecules using the system that was developed for the production of HLA-DM. Soluble DQβ appeared to be unstable indicating putative structural deficiencies of certain DQ alleles.

Apomixis is an asexual way of reproduction leading to the creation of clones of the mother plant. It is controlled by two independent loci in Pilosella piloselloides subsp. Praealta (Hawkweed): LOA (Loss of apomeiosis) and LOP (Loss of parthenogenesis), which are dominant. Apomictic plants develop one extremely competitive genotype and spread it in a colonial strategy. Only a few crops are apomictic; however, the natural creation of clones could considerably speed up the process of plant breeding and quickly stabilize new varieties adapted to climate change. Pilosella is one of the model plants to study apomixis. The purpose of this work was to investigate the LOP locus on a genetic and genomic level to better understand the mechanism of apomictic plants in the hope of applying it to crops. Using a panel of deletion mutants, this study made it possible to define the size of the LOP locus to about 650kb. A sequence of acceptable quality was checked for this locus using BAC isolates. Finally, the PAR gene contained in the locus for parthenogenesis in Taraxacum (dandelion) (Underwood et al., 2020) was sequenced in Pilosella and several species from the Asteraceae family, indicating the level of conservation of the dominant allele compared to the recessive alleles.

The killer-cell immunoglobulin-like receptor (KIR) gene complex, located in human chromosomal region 19q13.42, and the complement component 4 (C4) gene complex, located in human chromosomal region 6p21.33, encode for proteins that have vital roles in immune system function. Component genes of these complexes exhibit copy number variation (CNV), extensive nucleotide polymorphisms, and high sequence similarity with other genes of their complex. Next generation sequencing (NGS) has transformed the world of genomics, offering a high-throughput, high-fidelity and cost-effective sequencing method, however, NGS analysis of the KIR and C4 regions has been thwarted due to the bioinformatics challenges imposed by their complex variation. In this work, the researcher presents the bioinformatics pipelines, PING, developed for KIR sequence analysis, and C4Investigator, developed for C4 sequence analysis. These bioinformatics pipelines provide comprehensive, high-throughput characterization of human KIR and C4 sequence variation from NGS data. These pipelines take in paired-end short-read sequencing data and output gene copy number for both genomic regions, high-resolution genotypes for the KIR complex, and high-resolution mapping of single nucleotide variants (SNVs) for the C4 region. The performance of PING was evaluated by real-world and synthetic datasets, while the performance of C4Investigator was evaluated by real-world datasets and comparison to existing methods. Both PING and C4Investigator showed high performance for copy number determination and SNV characterization. To demonstrate the utility of the C4Investigator pipeline, the researcher applied C4Investigator to whole genome sequencing (WGS) data from the 1000 Genomes Project (1KGP) cohort (N=3199), characterizing C4 copy number and sequence variation for the first time in this dataset. To demonstrate the utility of the PING pipeline, the researched applied PING to targeted sequencing datasets from divergent populations (European N=363, Khoesan N=104), in addition to WGS data from the 1KGP cohort (N=215). To the best of our knowledge, PING and C4Investigator are the only bioinformatics workflows currently available for assessment of KIR and C4 full genomic sequence variation from NGS data.

Rapid DNA biometric identification applications are becoming more essential and widely used in human identity validation processes. Despite their powerful identification capabilities, processing a sample to generate a forensic DNA profile still takes longer compared with other rapid biometric technologies. Methods used to speed up the analysis could lead to signal artifacts similar to those arising from low copy or degraded DNA samples, making the electropherogram unsuitable for forensic interpretation and analysis. The goal of this research effort is to apply biometrics and mathematical approaches to forensic STR (Short Tandem Repeat) profiles. To accomplish this goal, a multi-function software tool was developed to evaluate STR profiles in the form of electropherograms. This tool is capable of generating degraded and non-degraded STR profiles based on allele statistics from the human population using MATLAB. The software also acts as an interface to apply a previously developed signal processing method to recover alleles in electropherograms produced from degraded DNA samples. The user interface offers the capability of visualizing and comparing those discovered peaks with the allelic ladder to confirm recovery or a rejection. The software is demonstrated on both artificial and real degraded STR electropherograms, indicating a higher allele recovery rate when compared with commercial GeneMapper IDx software. Finally, the software produces a match score based on the number of matching alleles when comparing two or more DNA profiles based on the number of existing and recovered allele peaks in the electropherogram.

Breeding for genetic resilience in farm animals is becoming more important as a consequence of the increasing awareness of pathogen resistance to antibiotics and the need to build more resilient and sustainable agricultural production systems. In this context, the important role the Major Histocompatibility Complex (MHC) proteins play in immune responses would suggest that improved understanding of the MHC genes is vital to understanding and manipulating genetic resilience to infectious diseases. The genes of MHC are highly polymorphic, and studies have suggested that the large variation existing within the MHC region may be attributed to response to selective pressure from pathogens. It has therefore been suggested that the alleles of MHC genes carried by an individual animal may be influenced by the type of pathogens in its environment. An understanding of the allelic variation in MHC genes across multiple geographic regions and breeds could therefore assist us to better understand the influence of selective forces and to better characterise distinct populations of cattle. In this study, four breeds of cattle including White Fulani (n =24), Red Bororo (n =5) and Holstein-White x Fulani-cross (n = 11) from Nigeria, and New Zealand Holstein-Friesian x Jersey-cross (n = 40) were genotyped for variation in exon 2 of BoLA-DQA1. In addition: Yak (n= 47); 13 breeds of cattle including White Fulani (n = 30), Red Bororo (n= 20), and White Fulani × Holstein-cross (n = 16) from Nigeria; Holstein-Friesian × Jersey Cross (n = 22), Hereford (n = 20), Murray Grey (n = 20), Red poll (n = 20), Shorthorn (n =22), Angus (n = 20), Simmental (n = 20), Charolais (n = 20), South Devon (n =20) from New Zealand; and Marchigiana (n = 16) from Italy, were genotyped for variation in exon 2 of BoLA-DRA. A Single Strand Conformation Polymorphism approach was used to genotype the animals and selected cattle were subsequently subjected to direct sequencing of the regions of the genes investigated to reveal nucleotide sequence differences. A total of 10 alleles were identified from all samples investigated for BoLA-DQA1 variation. Allele BoLA-DQA*001:02 had the highest frequency of occurrence (17.5%) and BoLA-DQA*010:03 was the least common with a frequency of 6.9%. A total of 12.60% of all nucleotide positions analysed were revealed to be variable. Two novel BoLA-DQA1 alleles are reported in this study for the first time, but no new sequences were revealed for BoLA-DRA. In total, three BoLA-DRA allelic variants were observed in the cattle studied, namely 01011, 01013 and 01014. The New Zealand Holstein Friesian x Jersey (HF X J)-cross cattle and the Yak had only two BoLA-DRA alleles (BoLA-DRA*01011 and BoLA-DRA*01013), while the other breeds studied had the BoLA-DRA*01011, BoLA-DRA*01013 and BoLA-DRA*01014 alleles. This study is helpful in shaping our understanding of the bovine MHC genes. It may also be relevant to understanding how different breeds respond to pathogen challenges, thus determining their adaptability to their regions. Identifying genomic variation is however, only a first step in developing breeding programmes that enhance livestock genetic resilience to disease.

Summary: The main objective was to evaluate the association between SNPs and haplotypes of the FABP1-4 genes and type 2 diabetes, as well as its interaction with fat intake, in one general Spanish population. The association was replicated in a second population in which HOMA index was also evaluated. Methods: 1217 unrelated individuals were selected from a population-based study [Hortega study: 605 women; mean age 54 y; 7.8% with type 2 diabetes]. The replication population included 805 subjects from Segovia, a neighboring region of Spain (446 females; mean age 52 y; 10.3% with type 2 diabetes). DM2 mellitus was defined in a similar way in both studies. Fifteen SNPs previously associated with metabolic traits or with potential influence in the gene expression within the FABP1- 4 genes were genotyped with SNPlex and tested. Age, sex and BMI were used as covariates in the logistic regression model. Results:One polymorphism (rs2197076) and two haplotypes of the FABP-1 showed a strong association with the risk of DM2 in the original population. This association was further confirmed in the second population as well as in the pooled sample. None of the other analyzed variants in FABP2, FABP3 and FABP4 genes were associated. There was not a formal interaction between rs2197076 and fat intake. A significant association between the rs2197076 and the haplotypes of the FABP1 and HOMA-IR was also present in the replication population. Conclusions: The study supports the role of common variants of the FABP-1 gene in the development of type 2 diabetes in Caucasians.

Antimicrobial peptides (host defense peptides) are an evolutionarily conserved component of the innate immune response, and have been shown to kill or inhibit the growth of microbes such as bacteria, fungi, viruses, or parasites. Snakin-1 (SN1) and Snakin-2 (SN2) are low-molecular weight antimicrobial peptides produced in potato tubers. They share common structural features with more than 500 antimicrobial peptides discovered in plants and animals, such as an N-terminal putative signal sequence, a highly divergent intermediate region and a conserved cysteine-rich 60 amino-acid carboxyl-terminal domain. These proteins are thought to play important roles in the innate defense against invading microbes. SN1 and SN2 genes were isolated and sequenced from a range of potato cultivars/clones (Solanum tuberosum), as well as a diverse range of other Solanaceous species. The sequences were aligned with reference sequences of the StSN1 gene (AJ320185) and the StSN2 gene (AJ312424) using Vector NTI Advance 10 software. Allelic variation both within and between genotypes and/or species was observed for both SN1 (four alleles) and SN2 (six alleles). Most of the variation occurred within introns, with relatively few nucleotide substitutions in exons. The predicted amino acid sequence was highly conserved; identical for all SN1 alleles, with only minor amino acid substitutions observed in SN2 alleles present in different Solanum species. Overexpression of the SN genes in potato was achieved using Agrobacterium-mediated gene transfer. Plant expression vectors with both sense and antisense orientations of SN1 (a1 allele) and SN2 (b1 allele) genes were constructed under the regulatory controls of the potato light inducible Lhca3 gene. Potato plants, cultivar Iwa, were transformed via Agrobacterium tumefaciens harboring binary vector containing the intragenic SN cassettes. The resulting plants were confirmed as being transgenic by PCR, then analysed at the molecular level for expression of the transgenes. Quantitative Reverse Transcriptase-PCR (qRT-PCR) analysis demonstrated that the majority of the transgenic potato lines overexpressed the SN genes at RNA level. Based on qRT-PCR of each SN gene, seven and eight lines respectively were selected from each of the Lhca3-SN1 and Lhca3-SN2 transgenic plant populations for further characterisation. Overexpression of the SN genes in leaves, stems, roots and tubers was determined by qRT-PCR analysis and these selected transgenic lines were evaluated in bioassays for resistance to Pectobacterium atrosepticum (formally Erwinia carotovora subsp. atroseptica) causal agent of blackleg in potatoes. Results from the pathogenicity bioassays showed that overexpression of either the SN1 gene or the SN2 gene from the intragenic cassettes conferred resistance to P. atrosepticum blackleg disease. This result has established the functional role of SN genes in plant defense against pathogens in potato. Antisense transformants were difficult to recover and failed to regenerate plants. This indicates that these highly conserved Snakin proteins may also play an important role in cellular metabolism other than just defense against invading microorganism.

In summary, this study investigated the role of novel polymorphisms and rare variants of the RUNX2 gene in influencing BMD, fracture and markers of bone turnover. Two common polymorphisms were identified within the polyA repeat: an 18 base pair deletion and a synonymous alanine codon polymorphism with alleles, A and G. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture within a Geelong population. To verify these findings the RUNX2 alleles were genotyped in 992 women from a Scottish cohort. The magnitude and the direction of the effect of the A allele was maintained in the Scottish cohort. Interestingly, the A allele was shown to exert a menopause specific effect, with postmenopausal women showing the strongest effect. On re-analysis of the GOS data the post-menopausal women were found to drive the significance identified in the cohort. The magnitude of the effect of the A allele on BMD was greater in overweight/obese postmenopausal women indicating a gene-weight interaction for RUNX2. The RUNX2 11Ala allele showed a significant relationship with decreased LS BMD in overweight/obese Scottish women. The 11Ala allele was also associated with higher levels of urinary PYD and DPD in women treated with HRT, indicating higher levels of bone turnover in carriers of the 11Ala allele. In contrast to the Scottish cohort, no significant association with heterozygous carriers of 11Ala was observed in GOS, although a significant association was detected for homozygous carriers and LS BMAD. The 11 Ala RUNX2 allele was significantly associated with decreased serum osteocalcin levels and the A allele was significantly associated with TNF in OA patients. Glutamine variants and an alanine insertion were identified within Geelong and Scottish cohorts, which showed low Z and T scores suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis. Polymorphism of the polyQ/polyA region of RUNX2 were identified within this study were shown to associate with significant differences in BMD. The A allele showed a significant association with increased BMD in postmenopausal women from a Geelong and Scottish cohort, with a decreased frequency of the A allele observed in Colles’ fracture patients from Geelong. The 11Ala deletion allele was significantly associated with decreased LS BMD and increases in markers of bone turnover in the Scottish cohort. A significant decrease in OC serum levels was observed in OA patients suggesting a direct effect of the allele on the transactivation of the RUNX2 gene. Rare variants of RUNX2 were identified which showed low BMD. These studies have provided insight into the role of RUNX2 in influencing BMD, further studies are required to verify the role of the A allele on BMD and fracture, the role of the rare variants and to identify the precise mechanisms behind the observed changes in BMD.

This study describes the development and validation of mutagenized cloned DNA constructs, which correspond to the polymorphic regions of the class II region of the HLA complex. The constructs were used to verify the allelic specificity of primers and probes in polymerase chain reaction (PCR)-based HLA typing assays such as Sequence Specific Primers (SSP) and Sequence Specific Oligonucleotide Probes (SSOP). The constructs consisted of the entire polymorphic region of exon 2 of class II HLA allele sequences that included primer annealing sites or probe hybridization sites. An HLA allele sequence was inserted into a plasmid, cloned, then mutagenized to match a specific HLA allele, and finally, the correct clone was verified by bidirectional sequencing of the insert. Thus, the construct created a cloned reference DNA sample for any specific allele, and can be used to validate the accuracy of various molecular methodologies.

Sub-populations inhabiting the length of a stream may be subject to a number of substantially different spatial and temporal environmental influences. To be able to monitor the effects of localised disturbance throughout a catchment is of prime import, not only to the study of evolutionary processes, but also to the assessment of human intervention in catchment dynamics. To understand the ultimate effects of localised disturbance, either natural or man-maAe, on the evolution of sub-divided populations, it is essential to determine the patterns of genetic differentiation, the levels of genetic exchange between sub-populations, and the potential for natural selection or genetic drift associated with a particular habitat type or disturbance. The research outlined in this thesis, aimed at determining how disturbance over part of a species range interacts with gene flow to alter the evolutionary processes in each deme of a sub-divided population. These aims involved assessing the relative conthbütion of gene flow, natural selection and genetic drift, to the genetic structure of Mosquitofish Gambusia holbrook! populations at particular times during their yearly breeding cycle. Spatial and temporal variation in Pgi allozyme frequencies were examined relative to distance, flow velocity, and bathers to dispersal, for Gambgsia hoibrooki populations inhabiting an intennitant stream in South-eastern Queensland. Isolation by distance explained only a small amount of the genetic variation between sites. Similarly, levels of differentiation could not be explained by a simple relationship between distance and flow velocity common to both catchments examined. Barriers to dispersal allowed movement only in the downstream direction during peak flooding. Different alleles occurred in high frequencies in the headwaters of each catchment. The dominance of a single allele in headwater populations is attributed to the lack of upstream movement, and the ephemeral nature of these populations. This same allele was maintained as the most common within each catchment by downstream emigration. Inter-catchment differences were therefore attributed to the random fixation of different alleles in the headwaters of each catchment, and the dispersal of these alleles downstream during peak flood events. Significant allele frequency differences between populations in one catchment occurred despite immigration. Differentiation fluctuated over a yearly cycle, and was dependent on the level of immigration entering the lower site from alternative upstream sources. Immigration from source populations was in turn related to flow velocities in the tributaries which converged above this site. All alleles were maintained in the lower reaches of both catchments despite the input of a single allele from headwater populations. Given the mechanism by which frequency differences between sites in the lower western catchment were maintained, and severe restrictions only on upstream movement, it is hypothesised that the less common alleles are preserved in the lower reaches of each catchment, by immigration originating in tributaries where alternative alleles have drifted to relatively high frequencies. Natural selection associated with saltwater inundations and the preferential movement of Ga,nbusia size classes created and maintained genetic differences between populations in the lower western catchment, not only while these populations were isolated, but also during periods of low flow. Preferential movement also enabled selection effects to be carried into adjacent populations, causing them to differentiate from populations not receiving immigrants. Selection effects were not consistent, ocurring in some years and not others. When selection took place allele frequency differences could be maintained at the site of selection for up to a year. The influence of selection at an adjacent site receiving immigrants only maintained differences between this and other sites for a period of about one month. No selection effect could compete with gene flow during high flow velocities each year. The maintenance of genetic diversity in lower reach populations was not dependent on selection associated with habitat heterogeneity in this region of the creek, but on the input of different alleles from tributaries converging upstream. Selection would only have a significant effect on the maintenance of diversity following high flow, if it had produced allele frequency differences in the upper reaches of each catchment. The restriction of upstream movement, and the small effective population sizes associated with the ephemoral nature of headwater populations, suggests genetic drift was more important in determining the final allele frequencies, and maintaining diversity in this system.

Cathepsin B is a cysteine protease which has been implicated in the development and progression of various cancers. It is often redistributed to the plasma membrane, and inactive forms of the enzyme are commonly found in tumour tissue exudates. The secretion of cathepsin B from cancer cells is thought to correlate with their invasive potential. The purpose of this study was to identify and characterise polymorphism in the human cathepsin B gene. PCR was used in the amplification of exon 7 and intron 7 of the human cathepsin B gene, using primers designed from the reported bovine and human cathepsin B genes. The two resulting amplimers were of different size, revealing a potential polymorphism. 10 human volunteers were screened for this variation, and sequencing of the variant amplimers identified two previously unreported alleles of the human cathepsin B gene. One allele is thought to confer increased stability to the primary transcript for cathepsin B, and hence may alter gene expression in homozygous carriers.

The objective of this investigation was to determine whether polymorphism in the HL-DQα and HLA-DQβ1 alleles, and the combination of different alleles, is related to the age of onset and susceptibility to IDDM. Patients from Canterbury, New Zealand, diagnosed with IDDM between 1990 and 1996, were used for analysis. Of the 142 subjects in the trial, 20 were non diabetic controls from the Canterbury region, 16 were over the age of 25 at diagnosis of IDDM, and the remaining 106 were individuals who developed IDDM under the age of 20. The entire trial group was HL-DQα1 typed using PCR followed by hybridisation techniques. As a result of complications and time constraints, only 2 subjects were HLA-DQβ1 typed by PCR amplification, restriction digestion and visualisation by gel electrophoresis. HLA-DQα1 0102 and 0201 alleles were significantly higher in the controls compared to IDDM patients (p

Cathepsin B is a lysosomal cysteine protease whose activity has been implicated in a variety of physiological and pathological states including the development of lung disease and the progression of cancers. Cathepsin B has been implicated in the pathogenesis such lung diseases as emphysema and chronic bronchitis. Although no direct associations between cathepsin B and cystic fibrosis are evident, cathepsin B may be implicated to the disease progression through the diseases associated elevation in neutrophil elastase activity. The expression, post translational processing and targeting of cathepsin B is frequently altered in transformed and malignant cancer cells. Associations between malignancy and cathepsin B have been reported for human colon, breast, prostrate and bladder tumours (Keppler and Sloane, 1996). Cathepsin B could have a role in the detachment of cancerous cells from the tumour, and lead hence into metastasis. MacKenzie (1995) identified a 19 nucleotide insertion within intron 7 of the human cathepsin B gene. Sequencing of a B allele also revealed a nucleotide substitution of G to C at position 319 in intron 7. This study reports that this substitution occurs in all B allele homozygotes tested and that polymorphic linkage is present between the absence of the 19 nucleotide insertion and the nucleotide substitution within the B allele. This study also reports the screening of 19 normal DNA samples, 30 DNA samples from cystic fibrosis patients, and 22 colon carcinoma tissue DNA samples (all samples unrelated). A allele frequencies were determined to be 0.62, 0.65, & 0.64 respectively. Chi-square analysis revealed no significant (p=0.99) difference between normal, cystic fibrosis and colon cancer sample populations. The two alleles have been shown to conform to Hardy-Weinberg population distribution within each sample population so are thought to be stably inherited according to Mendalian segregation. Studies have indicated that cathepsin B has multiple forms generated by different cell processing of RNA and glycosylation of the precursor protein. These polymorphisms have been shown to introduce a potential alternative splicing site within intron 7 which, if functional would produce larger sized mRNA. These polymorphisms also have the potential to interfere with the existing intron 7/exon 8 splicing site. This study reports the development of a RNA extraction technique from human blood, and the design and partial optimisation of reverse transcriptase-PCR CRT-PCR). These techniques will be used in the future to study mRNA derived from homozygote individuals blood, and observe any differences in the length or amount of mRNA between the 2 alleles.

The study of complex genetic phenomena can be greatly facilitated by the use of model organisms. The recessive murine mutation ocular retardation (or) is characterized by a small, incompletely developed retina, lack of optic nerve, severely reduced eye size, and corresponding blindness. Genetic mapping with microsatellite markers, followed by candidate gene analysis, led to the identification of a premature stop codon within the Chx10 gene as the causative mutation in $or\sp{J}.$ The ocular retardation phenotype appears to be both allele-specific and dependent on genetic background, making it an excellent model system for the study of genetic complexity. Analysis of backcross offspring ((129/Sv-$or\sp{J}/or\sp{J}\ \times$ CASA/Rk)F1 $\times$ 129/Sv-$or\sp{J}/or\sp{J}\rbrack$ revealed a modifying effect of the CASA/Rk genome on the mutant $or\sp{J}$ phenotype. After six generations of sib-mating modified animals, we developed two inbred strains producing only modified offspring; the retinal phenotype of adult mice from the modified strains is greatly ameliorated as compared to 129-strain $or\sp{J}$'s, presenting an optic nerve, pupillar response, and ERG signaling. These findings demonstrate that the role of Chx10 in retinal development is dependent on genetic context. Results from a genome scan comparing modified vs. unmodified backcross offspring provide evidence for the presence of modifier loci in three genomic regions, on chromosomes 2, 6, and 14 respectively. Candidate genes in the identified regions responsible for mediating phenotype modification are expected to have some redundant role with Chx10 in retinal development. Homozygotes of a second allele of ocular retardation, $or\sp{2J}$, are both blind and sterile, yet sequencing revealed no mutations in the promoter, exons, or exon-intron boundaries of Chx10. Pulsed-field gel electrophoresis has allowed us to detect a 75-100 kb rearrangement $>$10 kb upstream of the Chx10 ATG in $or\sp{2J}$ mice. The extent of this rearrangement suggests the involvement of a second gene effecting the sterility phenotype in the $or\sp{2J}$ mutant, as well as either the existence of an unidentified upstream exon of Chx10, or an upstream regulatory element involved in transcriptional control.

This project investigated the genetic composition of natural populations of Anadara trapezia in Australia at three spatial scales : i) microgeographic (within an estuary, 50 metres to ~ 6 kilometres); ii) microgeographic (within populations, less than 50 metres); and, iii) macrogeographic (hundreds of kilometres along the coast of Australia). Allozyme polymorphism surveys using cellulose acetate strips have revealed, from 43 enzymes screened, 18 putative polymorphic loci. Comparisons of levels of heterozygosity among enzyme structural groups showed no significant differences, however, monomers were significantly more variable as a group than multimers. Significant differences in the level and distribution of polymorphism among functional groups of enzymes were observed. It appears that selection may be acting at the molecular level, not only on a particular locus, but on a group of functionally similar loci. At the macrogeographic scale, significant departures from random mating were observed in most populations. Significant differences in allele distribution among populations of A. trapezia along the east coast of Australia were found. At the macrogeographic scale, heterogeneity of allele frequencies may depend upon the distance separating the populations and surface water currents. Differentiation among population groups in this study is attributed to changes in the direction of the East Australian Current combined with onshore countercurrents. The systematic status of the disjunct western Australian population of A. trapezia was also evaluated as compared with the east coast populations. No evidence of genetic, hence evolutionary divergence was found. The results have serious implications in the management of fisheries as erroneous assumptions in fisheries management models may lead to depletion and near extinction of marine species. The research stresses the necessity of sampling at multiple scales and replication strategies. It also highlighted the complexities researchers are faced with in studies of marine bivalves, such as the presence of null alleles, deficiencies of heterozygotes, apparent inbreeding and the small geographic scales governing population structure.