Use of the wound-inducible NtQPT2 promoter from Nicotiana tabacum for production of a plant-made vaccine

The wound-inducible quinolinate phosphoribosyl transferase promoter from Nicotiana tabacum (NtQPT2) was assessed for its capacity to produce B-subunit of the heat-labile toxin (LTB) from enterotoxigenic Escherichia coli in transgenic plant tissues. Comparisons were made with the widely used and constitutive Cauliflower Mosaic Virus 35S (CaMV35S) promoter. The NtQPT2 promoter produced somewhat lower average concentrations of LTB protein per unit weight of hairy root tissue but allowed better growth thereby producing similar or higher overall average yields of LTB per culture batch. Transgenic tobacco plants containing the NtQPT2-LTB construct contained LTB protein in roots but not leaves. Moreover, wounding NtQPT2-LTB transgenic plants, by removal of apices, resulted in an approximate 500% increase in LTB levels in roots when analysed several days later. CaMV35S-LTB transgenic plants contained LTB protein in leaves and roots but wounding made no difference to their LTB content.

Genetic structure and sex-biased dispersal of a declining cooperative-breeder, the Grey-crowned Babbler, Pomatostomus temporalis, at the southern edge of its range

Loss and fragmentation of habitat can disrupt genetic exchange between populations, which is reflected in changes to the genetic structure of populations. The Grey-crowned Babbler (Pomatostomus temporalis) is a cooperatively breeding woodland bird, once common and widespread in south-eastern Australia. The species has suffered population declines of >90% across its southern distribution as a result of loss and fragmentation of habitat. We investigated patterns of genetic diversity and population structure of Grey-crowned Babblers in fragmented habitats at the southernmost extent of its range. We sampled blood from 135 individual Babblers from 39 groups stratified into six subpopulations in three regions. Genotypic data were used to estimate genetic diversity, population substructure, local relatedness and dispersal patterns. Individuals showed high heterozygosity within regions, and varying numbers of private alleles among regions suggested differences in levels of connectivity between regions. Four genetic clusters revealed population substructure consistent with treeless landscapes acting as strong barriers to gene flow. In contrast to previous studies,we identified a male-biased dispersal pattern and significant isolation-by-distance patterns for females at fine spatial scales. We recommend that conservation plans for this species incorporate opportunities to increase and enhance corridor areas to facilitate genetic exchange among subpopulations.

Variation in the gene coding for the M5 muscarinic receptor (CHRM5) influences cigarette dose but is not associated with dependence to drugs of addiction : evidence from a prospective population based cohort study of young adults

Background: The mesolimbic structures of the brain are important in the anticipation and perception of reward. Moreover, many drugs of addiction elicit their response in these structures. The M5 muscarinic receptor (M5R) is expressed in dopamine-containing neurones of the substantia nigra pars compacta and ventral tegmental area, and regulates the release of mesolimbic dopamine. Mice lacking M5R show a substantial reduction in both reward and withdrawal responses to morphine and cocaine. The CHRM5, the gene that codes for the M5R, is a strong biological candidate for a role in human addiction. We screened the coding and core promoter sequences of CHRM5 using denaturing high performance liquid chromatography to identify common polymorphisms. Additional polymorphisms within the coding and core promoter regions that were identified through dbSNP were validated in the test population. We investigated whether these polymorphisms influence substance dependence and dose in a cohort of 1947 young Australians.

Results: Analysis was performed on 815 participants of European ancestry who were interviewed at wave 8 of the cohort study and provided DNA. We observed a 26.8% increase in cigarette consumption in carriers of the rs7162140 T-allele, equating to 20.1 cigarettes per week (p=0.01). Carriers of the rs7162140 T-allele were also found to have nearly a 3-fold increased risk of developing cannabis dependence (OR=2.9 (95%CI 1.1-7.4); p=0.03).

Conclusion: Our data suggest that variation within the CHRM5 locus may play an important role in tobacco and cannabis but not alcohol addiction in European ancestry populations. This is the first study to show an association between CHRM5 and substance use in humans. These data support the further investigation of this gene as a risk factor in substance use and dependence.

Altered expression of two zinc transporters, SLC30A5 and SLC30A6, underlies a mammary gland disorder of reduced zinc secretion into milk

Two cases of zinc deficiency in breastfed neonates were investigated where zinc levels in the mothers' milk were reduced by more than 75 % compared to normal. The objective of this study was to find the molecular basis of the maternal zinc deficiency condition. Significant reductions in mRNA expression and protein levels of the zinc transporters SLC30A5 and SLC30A6 were found in maternal tissue, suggesting a causal link to the zinc-deficient milk. Novel splice variants of the SLC30A6 transcript were detected. No modifications were found in coding regions, or in transcription binding sites of promoter regions or in 5' and 3' untranslated regions of both transporters in lymphoblasts and fibroblasts isolated from both mothers. Altered DNA methylation in SLC30A5 at two CpG sites was detected and may account for the reduced levels of SLC30A5 mRNA and protein in lymphoblasts. Reduced SLC30A6 mRNA and protein levels in lymphoblasts may be secondary to reduced SLC30A5 expression, as they function as a heterodimer in zinc transport. In conclusion, two cases of zinc deficiency are linked to low levels of the SLC30A5 and SLC30A6 zinc transporters. These two zinc transporters have not been previously associated with zinc deficiency in milk.

Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes

Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians.

Genetic variation and obesity in Australian women: A prospective study.

Objective: A number of candidate genes have been implicated in the pathogenesis of obesity in humans. This study examines associations between longitudinal changes in body mass and composition and the presence of polymorphisms in the ß-3 adrenergic receptor, tumor necrosis factor-α, leptin, and leptin receptor (Lepr) in a cohort of Australian women.

Research Methods and Procedures: Healthy white Australian women (n = 335) were randomly selected from the Barwon region of Victoria and underwent baseline anthropometry and double-energy X-ray absorptiometry for assessment of body mass and adiposity. These measurements were repeated again at 2-year follow-up. Genomic DNA was extracted and used for polymerase chain reaction-based genotyping of all polymorphisms.

Results: The Pro1019Pro Lepr polymorphism was associated with longitudinal increases in body weight (p = 0.02), fat mass (p = 0.05), and body mass index (p = 0.01) in this study, and individuals homozygous for the A allele at this locus had a greater propensity to gain body fat over time. The largest effects on body composition seemed to be in individuals already obese at baseline. Changes in body weight, fat mass, percent body fat, and body mass index over a 2-year period were not associated with genetic variation in the ß-3 adrenergic receptor (Trp64Arg), tumor necrosis factor-α promoter, or leptin genes in non-obese or obese women.

Discussion: These results suggest that a Lepr polymorphism is involved in the regulation of body mass and adiposity in obese Australian white women, which may have implications for the treatment of obesity in this population.

Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs

Background
The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.
Results
To develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene.
Conclusion
We have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.

Association of genetic vatiation within UBL5 with phenotypes of metabolic syndrome

The BEACON gene was initially identified using the differential display polymerase chain reaction on hypothalamic mRNA samples collected from lean and obese Psammomys obesus, a polygenic animal model of obesity. Hypothalamic BEACON gene expression was positively correlated with percentage of body fat, and intracerebroventricular infusion of the Beacon protein resulted in a dose-dependent increase in food intake and body weight. The human homolog of BEACON, UBL5, is located on chromosome 19p in a region previously linked to quantitative traits related to obesity. Our previous studies showed a statistically significant association between UBL5 sequence variation and several obesity- and diabetes-related quantitative physiological measures in Asian Indian and Micronesian cohorts. Here we undertake a replication study in a Mexican American cohort where the original linkage signal was first detected. We exhaustively resequenced the complete gene plus the putative promoter region for genetic variation in 55 individuals and identified five single nucleotide polymorphisms (SNPs), one of which was novel. These SNPs were genotyped in a Mexican American cohort of 900 individuals from 40 families. Using a quantitative trait linkage disequilibrium test, we found significant associations between UBL5 genetic variants and waist-to-hip ratio (p = 0.027), and the circulating concentrations of insulin (p = 0.018) and total cholesterol (p = 0.023) in fasted individuals. These data are consistent with our earlier published studies and further support a functional role for the UBL5 gene in influencing physiological traits that underpin the development of metabolic syndrome.

Genetic variation in selenoprotein S influences inflammatory response

Chronic inflammation has a pathological role in many common diseases and is influenced by both genetic and environmental factors. Here we assess the role of genetic variation in selenoprotein S (SEPS1, also called SELS or SELENOS), a gene involved in stress response in the endoplasmic reticulum and inflammation control. After resequencing SEPS1, we genotyped 13 SNPs in 522 individuals from 92 families. As inflammation biomarkers, we measured plasma levels of IL-6, IL-1b and TNF-a. Bayesian quantitative trait nucleotide analysis identified associations between SEPS1 polymorphisms and all three proinflammatory
cytokines. One promoter variant, 105G-A, showed strong evidence for an association with each cytokine (multivariate P = 0.0000002). Functional analysis of this polymorphism showed that the A variant significantly impaired SEPS1 expression after exposure to endoplasmic reticulum stress agents (P = 0.00006). Furthermore, suppression of SEPS1 by short interfering RNA in macrophage cells increased the release of IL-6 and TNF-a. To investigate further the significance of the observed associations, we genotyped 105G-A in 419 Mexican American individuals from 23 families for replication. This analysis confirmed a significant
association with both TNF-a (P = 0.0049) and IL-1b (P = 0.0101). These results provide a direct mechanistic link between SEPS1 and the production of inflammatory cytokines and suggest that SEPS1 has a role in mediating inflammation.

A pHMM-ANN based discriminative approach to promoter identification in prokaryote genomic contexts

The computational approach for identifying promoters on increasingly large genomic sequences has led to many false positives. The biological significance of promoter identification lies in the ability to locate true promoters with and without prior sequence contextual knowledge. Prior approaches to promoter modelling have involved artificial neural networks (ANNs) or hidden Markov models (HMMs), each producing adequate results on small scale identification tasks, i.e. narrow upstream regions. In this work, we present an architecture to support prokaryote promoter identification on large scale genomic sequences, i.e. not limited to narrow upstream regions. The significant contribution involved the hybrid formed via aggregation of the profile HMM with the ANN, via Viterbi scoring optimizations. The benefit obtained using this architecture includes the modelling ability of the profile HMM with the ability of the ANN to associate elements composing the promoter. We present the high effectiveness of the hybrid approach in comparison to profile HMMs and ANNs when used separately. The contribution of Viterbi optimizations is also highlighted for supporting the hybrid architecture in which gains in sensitivity (+0.3), specificity (+0.65) and precision (+0.54) are achieved over existing approaches.

Cystic fibrosis in children of the eastern Arabian peninsula : a clinical, spatial and genetic study

Aim: The aim of this thesis is to describe the process by which the inherited disease, cystic fibrosis, (CF) was recognised as an important clinical entity in the United Arab Emirates (UAE) and the Sultanate of Oman (Oman). It examines the clinical presentation of the first patients and assesses their degree of severity. Further, it describes the first studies carried out to determine the underlying CF mutations associated with the disease in the UAE and Oman. An estimate is offered of the birth frequency of the condition. Overall, the cultural, geographical and historical aspect of the societies in which the disease occurs is stressed. Methods: An initial literature search was carried out using Medline of any literature pertaining to the Arab World and CF. this was read and classified into the relevance to Arabs in general, the Middle East and then specifically the Arab (Persian) Gulf societies. Thereafter, a clinic was established at Tawam Hospital, Al Ain, UAE, for children presenting With chronic respiratory disease that could serve as a national referral centre. It was run by the Author as a service of the Paediatric Department of the UAE University Medical School. I sent a letter to every Paediatrician working in the UAE informing them of our clinic and offering our services for the diagnosis and management of chronic respiratory disease in children. This was based on the author's experience as a respiratory paediatrician in Australia and New Zealand and as the Professor of Paediatrics in the UAE. No such service then existed in the UAE. Funding was sought to establish a research programme and develop a molecular genetics laboratory in the UAE Medical School. A series of successful research applications provided the grants to commence the investigations. Once a small number of children had been identified as having CF from those referred to the respiratory clinic, the initial project was to assess and report their clinical presentation. Following this an early start was made on the identification of the mutations responsible. Once these were established an attempt was made to estimate the frequency of the condition at birth. Additional clinical studies revolved around assessing the severity of the condition that was associated with the main mutations that were identified. A clinical comparison was made with those with the mutation AF508 and the other main mutation, despite the obvious limitation of small numbers then available. Radiological assessment was made to evaluate the progression of the disease. The final aspect of the study was to assess patients from Oman and compare their findings and mutations with the neighbouring UAE. Based on information gained hypotheses are proposed regarding the spread of the gene mutation by population drift. Thesis outline: A literature review is presented in the form of a critique on the disease and a resume of the relevant aspects of the genetics of CF. Additionally, facts about the two countries' geography and history are presented. Finally, knowledge about CF mutations and population origins from other areas is presented. The second main section deals with the clinical features of the disorder as it presents in the UAE. Molecular findings are then presented and details of the common mutation found in Bedouin Arabs. Hypotheses are then presented based on the information gathered. Results: CF is not a rare disease in the Arab children of the UAE and Oman. These findings refute previous reports of CF being a rare or non-existent disease in Arabs. The condition presents with a severe clinical picture, with early colonisation of the respiratory tract with staphylococcus, haemophilus and pseudomonas organisms, even with conventional CF management practices in place. The CF mutation S549R is prevalent in Arabs of Bedouin stock, while AF508 is found in those of Baluch origin. The former may be descendants of Arabs who left southern Arabia and travelled to the Trucial Coast at the time of the destruction of the great dam at Marib. The origins of this mutation may lie in the area that corresponds to the modern Republic of Yemen. The latter groups are descendants of those who came originally from Baluchistan. It is hypothesised also that the ancestral home of the AF508 mutation may be in the geographical area now known as Baluchistan, that spans three separate modern political territories. The evidence presented supports the concept that the S549R mutation may be associated with a severe, if not the severest, clinical pattern recognised. It equates with that seen with the homozygous AF508 genotype. The absence of an additional mutation in the promoter region accounts for the different clinical pattern seen in previously described patients. Conclusions: There needs to be a major awareness of the presence of CF as a severe clinical disease in the children of the Gulf States. The clinical presentation and findings support the concept of under recognition of the disease. Climatic conditions put the children at special risk of hyponatraemia and electrolyte imbalance. The absence of surviving adults with the disease suggests premature deaths have occurred, but the high fertility rates have maintained the gene pool for this recessive disorder.

Is life history a barrier to dispersal? Contrasting patterns of genetic differentiation along an oceanographically complex coast

Extreme variation in early life-history strategies is considered a moderately good predictor of genetic subdivision and hence dispersal for a range of marine species. In reality, however, a good deal of population differentiation must reflect historical effects, more subtle variation in life histories, and, particularly, the interaction of larvae with oceanographic processes. Using a combination of allozyme and microsatellite markers, we show that the large-scale genetic structure of populations of three species (direct and planktonically developing cushion stars and a planktonic developing sea anemone that is also asexually viviparous) varies consistently, in line with the predicted capacity for dispersal within three geographic regions. We detected high levels of genetic subdivision for the direct developing cushion star (FST = 0.6), low levels for the planktonically developing cushion star (FST = 0.009), and intermediate levels for the sexual/asexual sea anmone (FST = 0.19). These patterns are exhibited despite the highly variable patterns of current movement and the presence of biogeographic barriers. Our results suggest that, although there is large scale genetic differentiation for two species, patterns of population connectivity are remarkably consistent within major regions and do not reflect variation in major oceanographic processes or genetic discontinuity coincident with biogeographic boundaries.

Genetic variation in PARL influences mitochondrian content

Given their involvement in processes necessary for life, mitochondrial damage and subsequent dysfunction can lead to a wide range of human diseases. Previous studies of both animal models and humans have suggested that presenilins-associated rhomboid-like protein (PARL) is a key regulator of mitochondrial integrity and function, and plays a role in cellular apoptosis. As a surrogate measure of mitochondrial integrity, we previously measured mitochondrial content in a Caucasian population consisting of large extended pedigrees, with results highlighting a substantial genetic component to this trait. To assess the inXuence of variation in the PARL gene on mitochondrial content, we re-sequenced 6.5 kb of the gene, identifying 16 SNPs and genotyped these in 1,086 Caucasian individuals, distributed across 170 families. Statistical genetic analysis revealed that one promoter variant, T-191C, exhibited signiWcant eVects (after correction for multiple testing) on mitochondrial content levels. Comparison of the transcription factor binding characteristics of the T-191C promoter SNP by EMSA indicates preferential binding of nuclear factors to the T allele, suggesting functional variation in PARL expression. These results suggest that genetic variation within PARL inXuences mitochondrial abundance and integrity.

A coarse-grained parallel genetic algorithm employing cluster analysis for multi-modal numerical optimisation

This paper describes a technique for improving the performance of parallel genetic algorithms on multi-modal numerical optimisation problems. It employs a cluster analysis algorithm to identify regions of the search space in which more than one sub-population is sampling. Overlapping clusters are merged in one sub-population whilst a simple derating function is applied to samples in all other sub-populations to discourage them from further sampling in that region. This approach leads to a better distribution of the search effort across multiple subpopulations and helps to prevent premature convergence. On the test problems used, significant performance improvements over the traditional island model implementation are realised.