Evolution and gene regulation of the genomic imprinting mechanism

Genomic imprinting describes an epigenetic mechanism by which genes are active or silent depending on their parental origin. Imprinting exists in plants and mammals, but how this monoallelic expression mechanism has evolved is not understood at the molecular level. Here I describe the mapping, sequencing and analysis of vertebrate orthologous imprinted regions spanning 11.5 Mb of genomic sequence from species with and without genomic imprinting. In eutherian (placental) mammals, imprinting can be regulated by differential DNA methylation, non-coding RNAs, enhancers and insulator elements. The systematic sequence comparison of the IGF2-H19 imprinting cluster, in eutherians and marsupials (tammar wallaby and opossum), has revealed the presence of the enigmatic non-coding RNA H19 in marsupials. Furthermore, we have characterised the marsupial H19 expression status and identified key regulatory elements required for the germline imprinting of the neighbouring IGF2 gene. All the major hallmarks of the imprinting mechanism of the IGF2-H19 locus were found to be conserved in therian mammals. In mammals, this imprinting system is therefore the most conserved germline derived epigenetic mechanism discovered so far. The high-quality genomic sequences have provided early glimpses of the genomic landscapes for species such as the monotreme platypus and marsupial tammar wallaby for which little was previously known. Comparative sequence analysis was used to identify candidate regulatory elements in the neighbouring imprinting centre 1 and 2 regions of human chromosome 11p15.5. Nine novel enhancer elements were identified following in vitro gene-reporter assays and correlation of conserved sequences with recent ENCODE data revealed probable functions for a further 24 elements. This project has led to the formation of the Sequence Analysis of Vertebrate Orthologous Imprinted Regions (SAVOIR) consortium and resources developed here are being used by the imprinting community to further our knowledge of the evolution of the genomic imprinting mechanism

The Genetic basis of resistance and susceptibility in the Albugo laibachii-Arabidopsis thaliana pathosystem

Albugo is a genus of biotrophic plant pathogens that can infect an extensive range of hosts including many Brassicaceae crop species. Little is known about the molecular mechanisms by which Albugo species can suppress host immunity and the mechanisms by which plants can resist Albugo infection. Albugo laibachii (Al) is a specialized pathogen of Arabidopsis thaliana (At). It can colonize ~90% of At accessions and suppress effector-triggered-immunity to other pathogens. It is postulated that Al secretes effector proteins. Analysis of the A. laibachii genome by Kemen et al, (2011, PLoS Biology) revealed a potential class of effectors with a ‘CHXC’ motif in their N-terminus that can mediate translocation into host cells. However, there are only ~35 CHXC effectors in A. laibachii, suggesting that they might not represent its entire effector complement. I took a traditional method to identify Al effectors: clone “avirulence (Avr) genes”. These typically encode effectors that are recognized and trigger a strong response by the immune system of some host accessions. I identified and sequenced four Al isolates from field samples. Using differential phenotype information to guide a genome-­‐ ide analysis, and my expectations of the allelic diversity of Avr genes, I identified two novel recognized effectors. These effectors, short secreted proteins named “SSP16” and “SSP18”, are recognized by the Arabidopsis accessions HR-5 and Ksk-1 respectively. I used classical and Illumina-­‐based genetic mapping to identify the locus conferring SSP16 recognition in HR-5, Resistance to A. laibachii 4 (RAL4). This locus contains three putative CC-NB-LRR class Resistance protein-encoding genes with similarity to Resistance to Peronospora parasitica 7 (RPP7). I demonstrated the utility of combined genomics approaches to identify recognized effectors without known motifs. The identification of the first Avr-Resistance gene pair will pave the way for further dissection of the molecular interactions in this pathosystem

Characterisation of VMO1 in Human Tissues

The Vitelline membrane outer layer protein 1 (VMO1) was first identified in the Vitelline membrane of the chicken egg. This membrane almost entirely protein that separates the egg white from the yolk in amniotic eggs and acts as the last defensive barrier between pathogens and the egg yolk with the developing embryo. Since its discovery in the vitelline membrane, VMO1 has been isolated from a number of different tissues and animals. The one that would appear to have the largest impact on human health would be its discovery in the Reissner’s membrane of mice, whose auditory system has high structural homology to humans. The Reissner’s membrane maintains the electrolyte level in the compartments of the inner ear as well as the resting electrochemical potential which are both critical for correct hearing function. The main aims of the research undertaken in this thesis was to 1) determine the tissues in which VMO1 is or is not expressed in humans and 2) create a genetically modified expression clone of human VMO1 to use in further downstream protein-protein interaction studies. Our hypothesis is that we can amplify VMO1 from human cell lines based on available bioinformatics data. The first objective was to determine the gene expression of VMO1 in multiple human cell lines. RNA was extracted from three commercial cell lines (list) and the integrity of RNA was confirmed by agarose gel electrophoresis by the observation of two rRNA bands. From this cDNA Abstract iii was made, and PCR analysis performed to identify the expression, or not, of VMO1 in these tissues. Successful amplification of VMO1 from lung cells (A549) lead to the creation of a human VMO1 expression clone, by transforming competent Escherichia coli cells with ligated VMO-1 insert/pPLUG vector. White colonies were selected for DNA extraction and confirmed as positive using colony PCR and agarose gel electrophoresis. DNA seuqencing of the positive clone confirmed the nucleotide sequence as VMO1. The second objective was to validate the commercial antibody for human VMO1 using physiological and immunohistochemical methodology. The immunohistochemistry data suggests that the VMO1 protein is a secreted protein since signal was detected in the P5 mouse inner ear and mouse adult lung. However, multiple bands were observed in the western blot. Futher investigation is required to validate the VMO1 antibody to demonstrate that it is indeed specific to recognising its target epitope. The production of recombinant VMO1 protein would be benefical to address this question