Mauritz Venter

Characterization of the genetic profile of CYP2C19 in two South African populations

AIMS This study was aimed at elucidating the common sequence variation present in the CYP2C19 gene within the South African Xhosa population and comparing it with the Cape Mixed Ancestry (CMA) population for possible future pharmacogenetic applications. MATERIALS & METHODS Common sequence variation was identified through the resequencing of 15 Xhosa individuals. The detected variants were prioritized for genotyping in an additional 85 Xhosa and 75 CMA individuals, while 5 -upstream variants were analyzed using dual luciferase reporter assays. RESULTS Resequencing of the Xhosa population revealed 30 variants, including the novel CYP2C19*27 and CYP2C19*28 alleles. CYP2C19*27, characterized by -1041G>A, caused a twofold decrease in luciferase activity, while CYP2C19*28 is characterized by the nonsynonymous V374I variant. In addition, the previously characterized variants, CYP2C19*2, CYP2C19*9 and CYP2C19*17, were present in both populations, while CYP2C19*3 was only observed in the CMA population. CONCLUSION Our data demonstrate that both the Xhosa and CMA populations exhibit unique genetic profiles that could influence the outcome of drug therapy in these populations.

Modified Cetyltrimethylammonium bromide method improves robustness and versatility: The benchmark for plant RNA extraction

A wide range of plant RNA extraction methods are available; however, many of these are limited in their application for a diverse range of plant species. With special emphasis on robustness and versatility, we have improved the cetyltrimethylammonium bromide (CTAB) method and isolated high-quality RNA from 16 different plant species. The major modifications made to the protocol described here were a reduction of sample treatment steps and an increase in beta-mercaptoethanol concentration (to 3%) resulting in a robust, rapid and reproducible plant RNA extraction protocol that can be used for a broad range of plant species and tissue types.

A pharmacogenetic study of CD4 recovery in response to HIV antiretroviral therapy in two South African population groups

South Africa, like many other Southern African countries, has one of the highest HIV infection rates in the world and many individuals consequently receive antiretroviral therapy (ART). However, knowledge regarding (i) the prevalence of functional single nucleotide polymorphisms (SNPs) in pharmacologically relevant genes, and (ii) variance in pharmacotherapy both within and between different populations and ethnic groups is limited. The aim of this study was to determine whether selected polymorphisms in cytochrome P450 (CYP) genes (CYP2B6 and CYP3A4) and the multidrug-resistance 1 (ABCB1) gene underlie altered antiretroviral (ARV) drug response in two South African populations. DNA samples from 182 HIV-positive individuals of Mixed-Ancestry and Xhosa ethnicity on ART were genotyped for the A-392G SNP in CYP3A4, the G516T and A785G SNPs in CYP2B6, and the T-129C, C1236T, G2677T/A and C3435T SNPs in ABCB1. Univariate two-way analysis of variance (ANOVA) testing revealed no apparent effect of ethnicity on immune recovery (in terms of CD4-cell count) in response to ART. Univariate one-way ANOVA testing revealed a discernible effect of genotype on immune recovery in the cases of the T-129C (P=0.03) and G2677A (P<0.01) polymorphisms in the ABCB1 gene. This study serves as a basis for better understanding and possible prediction of pharmacogenetic risk profiles and drug response in individuals and ethnic groups in South Africa.

The reallocation of carbon in P deficient lupins affects biological nitrogen fixation.

It is not known how phosphate (P) deficiency affects the allocation of carbon (C) to biological nitrogen fixation (BNF) in legumes. The alteration of the respiratory and photosynthetic C costs of BNF was investigated under P deficiency. Although BNF can impose considerable sink stimulation on host respiratory and photosynthetic C, it is not known how the change in the C and energy allocation during P deficiency may affect BNF. Nodulated Lupinus luteus plants were grown in sand culture, using a modified Long Ashton nutrient solution containing no nitrogen (N) for ca. four weeks, after which one set was exposed to a P-deficient nutrient medium, while the other set continued growing on a P-sufficient nutrient medium. Phosphorus stress was measured at 20 days after onset of P-starvation. During P stress the decline in nodular P levels was associated with lower BNF and nodule growth. There was also a shift in the balance of photosynthetic and respiratory C toward a loss of C during P stress. Below-ground respiration declined under limiting P conditions. However, during this decline there was also a shift in the proportion of respiratory energy from maintenance toward growth respiration. Under P stress, there was an increased allocation of C toward root growth, thereby decreasing the amount of C available for maintenance respiration. It is therefore possible that the decline in BNF under P deficiency may be due to this change in resource allocation away from respiration associated with direct nutrient uptake, but rather toward a long term nutrient acquisition strategy of increased root growth.

Sequence analysis and transcriptional profiling of two vacuolar H + -pyrophosphatase isoforms in Vitis vinifera

Gene expression of grapevine vacuolar H+-pyrophosphatase (V-PPase EC 3.6.1.1.) during fruit ripening has previously been reported. Here we report on putative multiple V-PPase isoforms in grapevine. In this study a full-length cDNA sequence with an open reading frame of 2,295 nucleotides encoding a V-PPase gene (vpp2: acc. nr. AJ557256) was cloned. Sequence analyses of the deduced amino acid residues and RT-PCR experiments indicated that Vitis vinifera L. has at least two distinct isoforms of the V-PPase gene. Bioinformatic analyses of 13 V-PPase protein sequences revealed two highly conserved motifs associated with pyrophosphate (PPi) binding and response to stress, respectively. Both V-PPase isoforms were expressed at higher levels in the late post-véraison stage of grape berry ripening. Results also showed that the expression of grapevine V-PPase was induced by cold stress.

In silico promoters: modelling of cis-regulatory context facilitates target predictio.

•  Introduction •  Combined in silico strategies contribute to accurate deciphering •  Systematic module analysis leads to target discovery •  Applications of in silico promoters based on conserved regulatory context •  Conclusion

Role of PEPc Isoforms in Phosphate-Deficient Nodules

In soils, the concentration of available P for plants is normally very low, because most of the P combines with iron, aluminium, and calcium to form relatively insoluble compounds (Aono et al., 2001). Phosphate (Pi) deficiency is thought to be one of the limiting factors for nitrogen fixation (Aono et al., 2001) due to the high energy requirement of plants performing nitrogen fixation for nitrogenase function (Al Niemi et al., 1997). Pi deficiency has important implications for the metabolic Pi and adenylate pools of plants, which influence respiration and nitrogen fixation (Theodorou and Plaxton, 1995). An alternative route of pyruvate supply during Pi stress has been proposed (Theodorou and Plaxton, 1995), which involves the combined activities of phosphoenolpyruvate carboxylase (PEPc), malate dehydrogenase and NAD-malic enzyme supplying pyruvate to the mitochondrion during Pi stress. Marczewski (1989) purified three isoforms of PEPc from lupin nodules and roots, with two forms being nodule specific. The aim of this project was to determine the possible roles of PEPc isoforms in L upinus luteus nodules under Pi stress.