The identification and characterisation of a novel Apoptotic Gene,Snama, in drosophila melanogaster

Student Number : 9105022E - PhD thesis - School of Molecular and Cell Biology - Faculty of ScienceSNAMA is the Drosophila melanogaster homologue of a group of proteins that are known to bind p53 and the retinoblastoma protein (Rb). This multi domain protein consists of a conserved N-terminal domain called Domain With No Name (DWNN), a zinc finger, a cysteine rich RING finger-like domain, a probable p53 binding region, and a glutamic acid-rich and lysine-rich region. These associated domains indicate that SNAMA plays an important regulatory role in the cell and may function in RNA processing and in apoptosis. The DWNN domain was first identified in Cytotoxic T-cell resistant Chinese hamster ovary (CHO) cells using promoter trap mutagenesis to screen for genes involved in apoptosis. Subsequently, this domain was identified in other eukaryotic organisms including animals and plants. The SNAMA transcriptional unit consists of 9 exons and 8 introns that code for a 1231 amino acid protein with the 76 residue N-terminal DWNN domain. The DWNN domain has a 23.5% sequence identity to the ubiquitin protein and a predicted folded structure similar to ubiquitin. Western blots identified multiple bands indicative of ubiquitin tagged proteins. Taken together this suggests a role in the ubiquitin pathway either as an ubiquitin domain protein or the DWNN domain of SNAMA tagging other proteins. The cysteine rich RING finger-like domain has a histidine to serine substitution at the fourth position of the putative RING finger and represents a distinct class of RING finger-like proteins that could have ubiquitin ligase activity. Northern blot analysis identified a single 4.6 kbp transcript expressed abundantly throughout development early in embryogenesis but reduced in older embryos and in adult male and females. SNAMA probably interacts with Dmp53 as a suppressor of apoptosis or a negative regulator of an activator of apoptosis. It is a vital gene required for development, as the mutant P-element insertion line in which the Pelement is inserted in the first intron of SNAMA is lethal when homozygous. Acridine orange staining of these mutant flies showed a direct correlation between the presence of SNAMA and apoptosis. An increase in the levels of apoptosis occurred in embryos with relatively low levels of SNAMA expression. The mode of this action is either direct, or via other proteins that are involved in the apoptotic pathway

Identification of genes coding for B cell antigens of Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) by using phage display

<p>Abstract</p> <p>Background</p> <p>Contagious bovine pleuropneumonia (CBPP) is a mycoplasmal disease caused by <it>Mycoplasma mycoides </it>subsp. <it>mycoides </it>SC (<it>Mmm</it>SC). Since the disease is a serious problem that can affect cattle production in parts of Africa, there is a need for an effective and economical vaccine. Identifying which of the causative agent's proteins trigger potentially protective immune responses is an important step towards developing a subunit vaccine. Accordingly, the purpose of this study was to determine whether phage display combined with bioinformatics could be used to narrow the search for genes that code for potentially immunogenic proteins of <it>Mmm</it>SC. Since the production of IgG2 and IgA are associated with a Th₁cellular immune response which is implicated in protection against CBPP, antigens which elicit these immunoglobulin subclasses may be useful in developing a subunit vaccine.</p> <p>Results</p> <p>A filamentous phage library displaying a repertoire of peptides expressed by fragments of the genome of <it>MmmSC </it>was constructed. It was subjected to selection using antibodies from naturally- and experimentally-infected cattle. Mycoplasmal genes were identified by matching the nucleotide sequences of DNA from immunoselected phage particles with the mycoplasmal genome. This allowed a catalogue of genes coding for the proteins that elicited an immune response to be compiled. Using this method together with computer algorithms designed to score parameters that influence surface accessibility and hence potential antigenicity, five genes (<it>abc, gapN, glpO, lppB </it>and <it>ptsG</it>) were chosen to be expressed in <it>Escherichia coli</it>. After appropriate site-directed mutagenesis, polypeptides representing portions of each of these proteins were tested for immunoreactivity. Of these five, polypeptides representing expression products of <it>abc </it>and <it>lppB </it>were recognised on immunoblots by sera obtained from cattle during a natural outbreak of the disease.</p> <p>Conclusion</p> <p>Since phage display physically couples phenotype with genotype, it was used to compile a list of sequences that code for <it>Mmm</it>SC proteins bearing epitopes which were recognised by antibodies in the serum of infected animals. Together with the appropriate bioinformatic analyses, this approach provided several potentially useful vaccine or diagnostic leads. The phage display step empirically identified sequences by their interaction with antibodies which accordingly reduced the number of ORFs that had to be expressed for testing. This is a particular advantage when working with <it>Mmm</it>SC since the mycoplasmal codon for tryptophan needs to be mutated to prevent it from being translated as a stop in <it>E. coli</it>.</p

Five diseases, one vaccine : a boost for emerging livestock farmers in South Africa

Livestock are essential to the economic, nutritional and social wellbeing of African farmers. Besides providing food, clothing and other products, they are a measure of wealth and social standing; they are used for barter, as lobola (bride price) at traditional weddings, and also as a ‘bank’, whereby animals can be sold to pay for emergency needs, such as funerals. Given the diverse uses of livestock and their socio-economic importance in farming communities, the loss of even a single animal has a significant, and sometimes crippling effect on a family

Potential link of single nucleotide polymorphisms (SNPs) to virulence of vaccine‐associated field strains of lumpy skin disease virus in South Africa

South Africa is endemic for lumpy skin disease and is therefore reliant on various live attenuated vaccines for the control and prevention of the disease. In recent years, wide‐spread outbreaks of vaccine‐like strains of lumpy skin disease virus (LSDV) were reported internationally, leading to an increase in the generation of full genome sequences from field isolates. In this study, the complete genomes of six LSDVs submitted during active outbreaks in the 1990’s in South Africa were generated. Based on phylogenetic analysis, the six viruses clustered with vaccine strains in LSDV Subgroup 1.1 and are subsequently referred to as vaccine‐associated. The genetic differences between the phenotypically distinct vaccine and vaccine‐associated strains were 67 single nucleotides polymorphisms (SNPs). This study characterised the location and possible importance of each of these SNPs in their role during virulence and host specificity

Peste des petits ruminants virus tissue tropism and pathogenesis in sheep and goats following experimental infection

Peste des petits ruminants (PPR) is a viral disease which primarily affects small ruminants, causing significant economic losses for the livestock industry in developing countries. It is endemic in Saharan and sub-Saharan Africa, the Middle East and the Indian sub-continent. The primary hosts for peste des petits ruminants virus (PPRV) are goats and sheep; however recent models studying the pathology, disease progression and viremia of PPRV have focused primarily on goat models. This study evaluates the tissue tropism and pathogenesis of PPR following experimental infection of sheep and goats using a quantitative time-course study. Upon infection with a virulent strain of PPRV, both sheep and goats developed clinical signs and lesions typical of PPR, although sheep displayed milder clinical disease compared to goats. Tissue tropism of PPRV was evaluated by real-time RT-PCR and immunohistochemistry. Lymph nodes, lymphoid tissue and digestive tract organs were the predominant sites of virus replication. The results presented in this study provide models for the comparative evaluation of PPRV pathogenesis and tissue tropism in both sheep and goats. These models are suitable for the establishment of experimental parameters necessary for the evaluation of vaccines, as well as further studies into PPRV-host interactions.A Canadian International Food Security Research Fund (CIFSRF) grant (no. 106930: Livestock vaccines against viral diseases for sub-Saharan Africa) by the Canadian International Development Research Centre (IDRC) and Canadian International Development Agency (CIDA).http://www.plosone.orgam201

New livestock vaccines - a boost for emerging farmers in Africa

This work was carried out with the aid of a grant from Canada’s International Development Research Centre (IDRC), and with financial support from the Government of Canada, provided through Global Affairs Canada (GAC)Although commercial farmers generally make good use of livestock vaccines, emerging livestock farmers face major challenges in effective vaccine acquisition and use. Given the diverse uses of livestock and their socio-economic importance in farming communities, the loss of even a single animal has significant and sometimes crippling effects on a family. To ensure the relevance of newly developed vaccines to emerging farmers, this project uses an integrative and gender responsive approach, linking vaccine development with education, economics and social science. The researchers believe that the new vaccines will have the potential to control six important African livestock diseases

Gender, small-scale livestock farming and food security : policy implications in the South African context

This work was carried out with the aid of a grant from Canada’s International Development Research Centre (IDRC), and with financial support from the Government of Canada, provided through Global Affairs Canada (GAC)Drawing on insights from multiple studies, this policy brief addresses the importance of gender considerations for small-scale livestock farming communities relative to food security in the South African context. The brief examines some key elements of gender issues in relation to small-scale livestock farming, asks how some of these elements align with current policies and practices, and suggests a number of focused policy recommendations. Two thirds of the world’s 600 million poor livestock keepers are rural women. Within the international agricultural development agenda, women are increasingly identified as key to the eradication of global hunger