Computational Methods for Locating and Analyzing Conserved Gene Regulatory DNA Elements

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.

Heat stress effects on grain sorghum productivity-biology and modelling

Heat stress can cause sterility in sorghum and the anticipated increased frequency of high temperature events implies increasing risk to sorghum productivity in Australia. Here we summarise our research on specific varietal attributes associated with heat stress tolerance in sorghum and evaluate how they might affect yield outcomes in production environments by a crop simulation analysis. We have recently conducted a range of controlled environment and field experiments to study the physiology and genetics of high temperature effects on growth and development of sorghum. Sorghum seed set was reduced by high temperature effects (>36-38oC) on pollen germination around flowering, but genotypes differed in their tolerance to high temperature stress. Effects were quantified in a manner that enabled their incorporation into the APSIM sorghum crop model. Simulation analysis indicated that risk of high temperature damage and yield loss depended on sowing date, and variety. While climate trends will exacerbate high temperature effects, avoidance by crop management and genetic tolerance seems possible.

Integrating Rapid Phenotyping and Speed Breeding to Improve Stay-Green and Root Adaptation of Wheat in Changing, Water-Limited, Australian Environments

Temperatures have increased and in-crop rainfall decreased over recent decades in many parts of the Australian wheat cropping region. With these trends set to continue or intensify, improving crop adaptation in the face of climate change is particularly urgent in this, already drought-prone, cropping region. Importantly, improved performance under water-limitation must be achieved while retaining yield potential during more favourable seasons. A multi-trait-based approach to improve wheat yield and yield stability in the face of water-limitation and heat has been instigated in northern Australia using novel phenotyping techniques and a nested association mapping (NAM) approach. An innovative laboratory technique allows rapid root trait screening of hundreds of lines. Using soil grown seedlings, the method offers significant advantages over many other lab-based techniques. Another recently developed method allows novel stay-green traits to be quantified objectively for hundreds of genotypes in standard field trial plots. Field trials in multiple locations and seasons allow evaluation of targeted trait values and identification of superior germplasm. Traits, including yield and yield components are measured for hundreds of NAM lines in rain fed environments under various levels of water-limitation. To rapidly generate lines of interest, the University of Queensland “speed breeding” method is being employed, allowing up to 7 plant generations per annum. A NAM population of over 1000 wheat recombinant inbred lines has been progressed to the F5 generation within 18 months. Genotyping the NAM lines with the genome-wide DArTseq molecular marker system provides up to 40,000 markers. They are now being used for association mapping to validate QTL previously identified in bi-parental populations and to identify novel QTL for stay-green and root traits. We believe that combining the latest techniques in physiology, phenotyping, genetics and breeding will increase genetic progress toward improved adaptation to water-limited environments.

Genetic conservation of Phosphine Resistance in the Rice Weevil Sitophilus oryzae (L.)

High levels of resistance to phosphine in the rice weevil Sitophilus oryzae have been detected in Asian countries including China and Vietnam, however there is limited knowledge of the genetic mechanism of resistance in these strains. We find that the genetic basis of strong phosphine resistance is conserved between strains of S. oryzae from China, Vietnam and Australia. Each of four strongly resistant strains has an identical amino acid variant in the encoded dihydrolipoamide dehydrogenase (DLD) enzyme that was previously identified as a resistance factor in Rhyzopertha dominica and Tribolium castaneum. The unique amino acid substitution, Asparagine > Threonine (N505T) of all strongly resistant S. oryzae corresponds to the position of an Asparagine > Histidine variant (N506H) that was previously reported in strongly resistant R. dominica. Progeny (F16 and F18) from two independent crosses showed absolute linkage of N505T to the strong resistance phenotype, indicating that if N505T was not itself the resistance variant that it resided within 1 or 2 genes of the resistance factor. Non-complementation between the strains confirmed the shared genetic basis of strong resistance, which was supported by the very similar level of resistance between the strains, with LC50 values ranging from 0.20 to 0.36 mgL-1 for a 48 hour exposure at 25°C. Thus, the mechanism of high level resistance to phosphine is strongly conserved between R. dominica, T. castaneum and S. oryzae. A fitness cost associated with strongly resistant allele was observed in segregating populations in the absence of selection.

Inventories and significance of the genetic resources of an African mahogany species (Khaya senegalensis (Desr.) A. Juss.) assembled and further developed in Australia.

The forest tree species Khaya senegalensis (Desr.) A. Juss. occurs in a belt across 20 African countries from Senegal-Guinea to Sudan-Uganda where it is a highly important resource. However, it is listed as Vulnerable (IUCN 2015-3). Since introduction in northern Australia around 1959, the species has been planted widely, yielding high-value products. The total area of plantations of the species in Australia exceeds 15,000 ha, mostly planted in the Northern Territory since 2006, and includes substantial areas across 60-70 woodlots and industrial plantations established in north-eastern Queensland since the early-1990s and during 2005-2007 respectively. Collaborative conservation and tree improvement by governments began in the Northern Territory and Queensland in 2001 based on provenance and other trials of the 1960s-1970s. This work has developed a broad base of germplasm in clonal seed orchards, hedge gardens and trials (clone and progeny). Several of the trials were established collaboratively on private land. Since the mid-2000s, commercial growers have introduced large numbers of provenance-bulk and individual-tree seedlots to establish industrial plantations and trials, several of the latter in collaboration with the Queensland Government. Provenance bulks (>140) and families (>400) from 17 African countries are established in Australia, considered the largest genetic base of the species in a single country outside Africa. Recently the annual rate of industrial planting of the species in Australia has declined, and R&D has been suspended by governments and reduced by the private sector. However, new commercial plantings in the Northern Territory and Queensland are proposed. In domesticating a species, the strategic importance of a broad genetic base is well known. The wide range of first- and advanced-generation germplasm of the species established in northern Australia and documented in this paper provides a sound basis for further domestication and industrial plantation and woodlot expansion, when investment conditions are favourable

Head motion and Inattention/Hyperactivity share common genetic influences: Implications for fMRI studies of ADHD

Head motion (HM) is a well known confound in analyses of functional MRI (fMRI) data. Neuroimaging researchers therefore typically treat HM as a nuisance covariate in their analyses. Even so, it is possible that HM shares a common genetic influence with the trait of interest. Here we investigate the extent to which this relationship is due to shared genetic factors, using HM extracted from resting-state fMRI and maternal and self report measures of Inattention and Hyperactivity-Impulsivity from the Strengths and Weaknesses of ADHD Symptoms and Normal Behaviour (SWAN) scales. Our sample consisted of healthy young adult twins (N = 627 (63% females) including 95 MZ and 144 DZ twin pairs, mean age 22, who had mother-reported SWAN; N = 725 (58% females) including 101 MZ and 156 DZ pairs, mean age 25, with self reported SWAN). This design enabled us to distinguish genetic from environmental factors in the association between head movement and ADHD scales. HM was moderately correlated with maternal reports of Inattention (r = 0.17, p-value = 7.4E-5) and Hyperactivity-Impulsivity (r = 0.16, p-value = 2.9E-4), and these associations were mainly due to pleiotropic genetic factors with genetic correlations [95% CIs] of rg = 0.24 [0.02, 0.43] and rg = 0.23 [0.07, 0.39]. Correlations between self-reports and HM were not significant, due largely to increased measurement error. These results indicate that treating HM as a nuisance covariate in neuroimaging studies of ADHD will likely reduce power to detect between-group effects, as the implicit assumption of independence between HM and Inattention or Hyperactivity-Impulsivity is not warranted. The implications of this finding are problematic for fMRI studies of ADHD, as failing to apply HM correction is known to increase the likelihood of false positives. We discuss two ways to circumvent this problem: censoring the motion contaminated frames of the RS-fMRI scan or explicitly modeling the relationship between HM and Inattention or Hyperactivity-Impulsivity

A model for mammalian male determination based on a passive Y chromosome

A model is suggested for mammalian male determination based on interactions postulated to occur among an autosomal repressor gene, an X-linked male-determining gene termed Tdx, and multiple copies of certain DNA sequences on the Y chromosome that do not code for any protein. The repressor, synthesised in limited amounts, has higher affinity for the Y-linked sequences than for Tdx and its affinity for Tdx is greater than that of RNA polymerase. In XY cells the Y effectively binds all available repressor, permitting transcription of Tdx to occur. In XX cells, since competition from the Y-linked high-affinity sequences is absent, the repressor binds to Tdx and prevents transcription. As a result of this competition between Tdx and the Y-linked high-affinity sites for limiting concentrations of the autosomal repressor, the product of the Tdx gene (TDX) is synthesized in the male but not in the female. It is suggested that in determination of the male sex, the role of the Y chromosome is to serve as a sink for the Tdx repressor. The proposed interactions provide a plausible explanation for the genetic properties of several anomalies of sexual development in mouse, man, and other mammals. The model suggests that the postulated multiple, highaffinity sequences on the Y chromosome of the mouse are included among the DNA sequences referred to as the Sxr-Bkm sequences.

Automatic phonetic transcription of Tamil in Roman script

Simple formalized rules are proposed for automatic phonetic transcription of Tamil words into Roman script. These rules are syntax-directed and require a one-symbol look-ahead facility and hence easily automated in a digital computer. Some suggestions are also put forth for the linearization of Tamil script for handling these by modern machinery.

Lymphatic vessels in health and disease: Role of the VEGF-C/VEGFR-3 pathway and the transcription factor FOXC2

The circulatory system consists of two vessel types, which act in concert but significantly differ from each other in several structural and functional aspects as well as in mechanisms governing their development. The blood vasculature transports oxygen, nutrients and cells to tissues whereas the lymphatic vessels collect extravasated fluid, macromolecules and cells of the immune system and return them back to the blood circulation. Understanding the molecular mechanisms behind the developmental and functional regulation of the lymphatic system long lagged behind that of the blood vasculature. Identification of several markers specific for the lymphatic endothelium, and the discovery of key factors controlling the development and function of the lymphatic vessels have greatly facilitated research in lymphatic biology over the past few years. Recognition of the crucial importance of lymphatic vessels in certain pathological conditions, most importantly in tumor metastasis, lymphedema and inflammation, has increased interest in this vessel type, for so long overshadowed by its blood vascular cousin. VEGF-C (Vascular Endothelial Growth Factor C) and its receptor VEGFR-3 are essential for the development and maintenance of embryonic lymphatic vasculature. Furthermore, VEGF-C has been shown to be upregulated in many tumors and its expression found to positively correlate with lymphatic metastasis. Mutations in the transcription factor FOXC2 result in lymphedema-distichiasis (LD), which suggests a role for FOXC2 in the regulation of lymphatic development or function. This study was undertaken to obtain more information about the role of the VEGF-C/VEGFR-3 pathway and FOXC2 in regulating lymphatic development, growth, function and survival in physiological as well as in pathological conditions. We found that the silk-like carboxyterminal propeptide is not necessary for the lymphangiogenic activity of VEGF-C, but enhances it, and that the aminoterminal propeptide mediates binding of VEGF-C to the neuropilin-2 coreceptor, which we suggest to be involved in VEGF-C signalling via VEGFR-3. Furthermore, we found that overexpression of VEGF-C increases tumor lymphangiogenesis and intralymphatic tumor growth, both of which could be inhibited by a soluble form of VEGFR-3. These results suggest that blocking VEGFR-3 signalling could be used for prevention of lymphatic tumor metastasis. This might prove to be a safe treatment method for human cancer patients, since inhibition of VEGFR-3 activity had no effect on the normal lymphatic vasculature in adult mice, though it did lead to regression of lymphatic vessels in the postnatal period. Interestingly, in contrast to VEGF-C, which induces lymphangiogenesis already during embryonic development, we found that the related VEGF-D promotes lymphatic vessel growth only after birth. These results suggest, that the lymphatic vasculature undergoes postnatal maturation, which renders it independent of ligand induced VEGFR-3 signalling for survival but responsive to VEGF-D for growth. Finally, we show that FOXC2 is necessary for the later stages of lymphatic development by regulating the morphogenesis of lymphatic valves, as well as interactions of the lymphatic endothelium with vascular mural cells, in which it cooperates with VEGFR-3. Furthermore, our study indicates that the absence of lymphatic valves, abnormal association of lymphatic capillaries with mural cells and an increased amount of basement membrane underlie the pathogenesis of LD. These findings have given new insight into the mechanisms of normal lymphatic development, as well as into the pathogenesis of diseases involving the lymphatic vasculature. They also reveal new therapeutic targets for the prevention and treatment of tumor metastasis and lymphatic vascular failure in certain forms of lymphedema. Several interesting questions were posed that still need to be addressed. Most importantly, the mechanism of VEGF-C promoted tumor metastasis and the molecular nature of the postnatal lymphatic vessel maturation remain to be elucidated.

Role of Eda and Troy pathways in ectodermal organ development

Several organs of the embryo develop as appendages of the ectoderm, the outermost layer of the embryo. These organs include hair follicles, teeth and mammary glands, which all develop as a result of reciprocal tissue interactions between the surface epithelium and the underlying mesenchyme. Several signalling molecules regulate ectodermal organogenesis the most important ones being Wnts, fi broblast growth factors (Fgfs), transforming growth factor -βs (Tgf-βs) including bone morphogenetic proteins (Bmps), hedgehogs (Hhs), and tumour necrosis factors (Tnfs). This study focuses on ectodysplasin (EDA), a signalling molecule of the TNF superfamily. The effects of EDA are mediated by its receptor EDAR, an intracellular adapter protein EDARADD, and downstream activation of the transcription factor nuclear factor kappa-B (NF-кB). Mice deficient in Eda (Tabby mice), its receptor Edar (downless mice) or Edaradd (crinkled mice) show identical phenotypes characterised by defective ectodermal organ development. These mouse mutants serve as models for the human syndrome named hypohidrotic ectodermal dysplasia (HED) that is caused by mutations either in Eda, Edar or Edaradd. The purpose of this study was to characterize the ectodermal organ phenotype of transgenic mice overexpressing of Eda (K14-Eda mice), to study the role of Eda in ectodermal organogenesis using both in vivo and in vitro approaches, and to analyze the potential redundancy between the Eda pathway and other Tnf pathways. The results suggest that Eda plays a role during several stages of ectodermal organ development from initiation to differentiation. Eda signalling was shown to regulate the initiation of skin appendage development by promoting appendageal cell fate at the expense of epidermal cell fate. These effects of Eda were shown to be mediated, at least in part, through the transcriptional regulation of genes that antagonized Bmp signalling and stimulated Shh signalling. It was also shown that Eda/Edar signalling functions redundantly with Troy, which encodes a related TNF receptor, during hair development. This work has revealed several novel aspects of the function of the Eda pathway in hair and tooth development, and also suggests a previously unrecognized role for Eda in mammary gland development.

Evaluation of methods and applications for behavioural profiling of transgenic mice

During the last 10-15 years interest in mouse behavioural analysis has evolved considerably. The driving force is development in molecular biological techniques that allow manipulation of the mouse genome by changing the expression of genes. Therefore, with some limitations it is possible to study how genes participate in regulation of physiological functions and to create models explaining genetic contribution to various pathological conditions. The first aim of our study was to establish a framework for behavioural phenotyping of genetically modified mice. We established comprehensive battery of tests for the initial screening of mutant mice. These included tests for exploratory and locomotor activity, emotional behaviour, sensory functions, and cognitive performance. Our interest was in the behavioural patterns of common background strains used for genetic manipulations in mice. Additionally we studied the behavioural effect of sex differences, test history, and individual housing. Our findings highlight the importance of careful consideration of genetic background for analysis of mutant mice. It was evident that some backgrounds may mask or modify the behavioural phenotype of mutants and thereby lead to false positive or negative findings. Moreover, there is no universal strain that is equally suitable for all tests, and using different backgrounds allows one to address possible phenotype modifying factors. We discovered that previous experience affected performance in several tasks. The most sensitive traits were the exploratory and emotional behaviour, as well as motor and nociceptive functions. Therefore, it may be essential to repeat some of the tests in naïve animals for assuring the phenotype. Social isolation for a long time period had strong effects on exploratory behaviour, but also on learning and memory. All experiments revealed significant interactions between strain and environmental factors (test history or housing condition) indicating genotype-dependent effects of environmental manipulations. Several mutant line analyses utilize this information. For example, we studied mice overexpressing as well as those lacking extracellular matrix protein heparin-binding growth-associated molecule (HB-GAM), and mice lacking N-syndecan (a receptor for HB-GAM). All mutant mice appeared to be fertile and healthy, without any apparent neurological or sensory defects. The lack of HB-GAM and N-syndecan, however, significantly reduced the learning capacity of the mice. On the other hand, overexpression of HB-GAM resulted in facilitated learning. Moreover, HB-GAM knockout mice displayed higher anxiety-like behaviour, whereas anxiety was reduced in HB-GAM overexpressing mice. Changes in hippocampal plasticity accompanied the behavioural phenotypes. We conclude that HB-GAM and N-syndecan are involved in the modulation of synaptic plasticity in hippocampus and play a role in regulation of anxiety- and learning-related behaviour.