Determination of the Existence and Distribution of HIV-I Chemokine Co-Receptor 5 Polymorphism in a Sampled Population from Kenya

Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) is a major public health problem, socio-economic burden and a serious threat to development. Entry of human immunodeficiency virus type 1 (HIV-1) into target cells requires the binding of the external envelope glycoprotein gp 120 to both the CD4 molecule and one of several chemokine receptors, recently discovered to function as co-receptors. T –cell line tropic HIV-1 strains utilize the a-chemokine receptor CXCR4, whereas the b-chemokine receptor 5 (CCR5), which is expressed on monocytes/macrophages, T cells and granulocyte precursors, is the key co-factor for macrophage-tropic HIV-1 strains, which predominate during the asymptomatic phase of infection. A thirty two–base pair (bp) deletion mutation (? 32) within the second extra cellular loop-encoding region of the CCR5 gene, which results in a truncated, non-functional protein, has been associated with relative resistance to HIV -1 infection and slower progression to acquired immunodeficiency syndrome (AIDS). Specifically, ?32/?32 homozygotes are protected against acquisition of HIV-1 by the mucosal route despite high risk exposure, whereas disease progression among CCR5/?32 heterozygote occurs more slowly. In this study, the status of the CCR5 gene polymorphism in Kenyan population was investigated in an attempt to explain the differences in HIV prevalence in different parts of the country. To determine this, 200 samples were collected from the 8 provinces of Kenya, that is, 25 samples per province, some of which were positive for HIV-1. Twenty-five samples were randomly selected from a batch of 250 per province, that is, every tenth sample. The samples were collected from HIV screening centers, district and provincial hospitals. Peripheral blood mononuclear cells (PBMC) were extracted from whole blood. Genomic deoxyribonucleic acid (DNA) was then extracted from PBMC. A targeted region of the CCR5 gene flanking the 32bp deletion was amplified by polymerase chain reaction (PCR) using CCR5 specific primers. All the PCR amplicons were then analyzed by gel electrophoresis. The results showed that CCR5-D 32 mutations do not exist in the Kenyan population. Samples were then randomly selected 4 samples per province and sequenced. This was done to determine the genotype of the PCR products that were amplified. After ClustalW analysis of the sequences generated, it was seen that CCR5 gene is not highly conserved in the Kenyan population, as there were amino acid differences between the sequences analyzed suggesting that CCR5 gene in Kenyan population is highly polymorphic. From this study, it was concluded that CCR5-D 32 mutations do not play any role in HIV-1 susceptibility in the Kenyan population. This is because this mutation does not exist in the Kenyan population as per the samples analyzed. The differences in prevalence of HIV in different parts of the country may be due to cultural practices, religious backgrounds, socio-economic status and other intrinsic genetic factors. Keywords: HIV/AIDS, chemokine receptor

Agrobacterium Mediated Transformation of Selected Maize Inbred Lines with pPZP200 towards Enhancement of Lysine and Methionine Content

Maize (Zea mays (L.) is one of the most important cereals used both for human and animal consumption in the world. Despite its importance, maize is not a suitable single source of nutrition because it does not provide the essential amino acids lysine and methionine in sufficient quantities to meet the nutritional needs of humans and other animals. Lysine is a necessary building block for protein in the body while methionine is the body’s primary source of sulphur. Strategies to improve the nutritional quality of maize for high lysine and methionine have involved both genetic engineering (GE) and non-genetic engineering approaches such as marker assisted selection. Breeding is however laborious, lengthy and carries along undesired alleles. The objective of this work was to manipulate maize inbred lines towards enhancement of lysine and methionine content in the endosperms through Agrobacterium mediated transformation. Maize kernels mainly store proteins as ?, ?, ? and ? zeins. The immature embryos of three tropical maize inbred lines (TL18, CML216 and CML144) and a temperate line (A188) were transformed using Agrobacterium tumefaciens strain EHA101 carrying an expression cassette designed to up-regulate the Z10 protein for methionine enhancement as well as down-regulate the ? zein storage protein by RNAi. The T-DNA also contained P-zp22/6 as the promoter and the phosphinothricin acetyltransfarase gene (bar) used for selection of transformed tissue. Putative transformants were tested for presence of the transgene by PCR designed to amplify the P-zp22/6 promoter sequence. Calli survival frequencies were calculated as a percentage number of surviving calli in relation to the total number of embryos infected. These ranged from 2.89 % for TL18 to 9.11 % for A188. This data did not detect any significant difference (p>0.05) among the genotypes on the percentage of calli which survived. Transformation efficiency was calculated as a percentage of the number of PCR positive plants divided by the total number of embryos infected. This ranged from 0% for TL18 to 1.83% for A188. The data suggest the possibility of manipulating storage proteins and regenerating normal transgenic maize with normal kernels. Further work should involve gene expression assays for accumulation of ?, ? and ? prolamins in the kernels and southern blot analysis to confirm stable integration and the copy numbers of P-zp22/6 gene in the PCR positive plants. Keywords: RNAi, Z10, Lysine, Methionine, pPZP200