Ashley Pretorius | University of the Western Cape (original) (raw)

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Books by Ashley Pretorius

Functional analysis of the mouse RBBP6 gene using interference RNA (A. Pretorius) ABSTRACT A no... more Functional analysis of the mouse RBBP6 gene using interference RNA (A. Pretorius)

ABSTRACT

A novel hamster gene homologous to the human cDNA clone 21C4 was identified in a genetic screen to identify new genes involved in the MHC class I antigen processing and presentation pathway. The identified gene however showed no significant matches to sequences in the Genbank Database and was subsequently named the Domain Without No name (DWNN). The corresponding human gene was found to be located on chromosome 16p12.2, upstream of the previously identified RBBP6/PACT/P2P-R gene. Analysis of cDNA sequences showed that the sequence coded for the previously unidentified N-terminus of the RBBP6 protein (Dlamini et al, in prep), which was named the DWNN domain.

RBBP6 is one of the few proteins identified that has been shown to interact with both p53 and Rb, suggesting a possible model for the integration of the regulation of transcription, cell cycle control and apoptosis. Over-expression of the mouse homolog P2P-R has been shown to lead to cell cycle arrest and apoptosis. The conserved mechanisms of programmed cell death play a fundamentally important role in tissue homeostasis, embryogenesis and cellular defense and had only recently been subjected to molecular analysis.

The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene.

Genetic analysis showed the presence of two promoters for RBBP6 namely Promoter 0 (P0) and Promoter 1 (P1) both being responsible for the different RBBP6 transcripts. The Enhanced Green Fluorescent Protein (EGFP) and the Red Fluorescent Protein (DsRed1) were both placed under the transcriptional control of P0 and P1. Promoter activities were measured using FACS and Real-Time qRT-PCR. The results showed P0 to have a higher level of transcriptional activity than P1 before and after camptothecin-induced apoptosis.

RNAi was used to target the expression of the RBBP6 gene. Several small interference RNA (siRNA) constructs were designed that would result in the expression of two distinct siRNA oligonucleotides designated DWNN-A and DWNN-B targeting different regions of the gene. Two stable siRNA-expressing cell lines were established, namely RU6A and GU6B expressing DWNN-A and DWNN-B respectively. Fluorescence microscopy and Real-Time qRT-PCR analysis were used to evaluate the effect on RBBP6 expression following (i) transient transfections of the siRNA constructs into the parental cell line NIH 3T3 cell line (ii) as well as expression in the stable cell lines RU6A and GU6B. The silencing effect of the DWNN-A oligonucleotide appeared to be more potent than that exerted by DWNN-B.

Both stable lines were used in assays to determine the effect of RBBP6 silencing on apoptosis. The stable cell lines proved to be significantly more resistant to apoptosis induced by camptothecin compared to the parental NIH 3T3 cell line. Furthermore complementing the stable cell lines with the full-length DWNN-200 cDNA, restored sensitivity to camptothecin and the degree of cell death observed in the parental cell line. In addition, over-expression of the RBBP6 protein showed an increase in the apoptotic population following camptothecin-induced apoptosis. Apoptosis mediated through RBBP6 was shown to be dependent on p53 expression and possibly follows an intrinsic pathway. Finally, the siRNA stable expressing cell lines RU6A and GU6B were also shown to be restricted in the G1 phase of the cell cycle implicating the RBBP6 gene in cell cycle progression.

From the study the RBBP6 Promoters showed an increase in transcriptional activity following the induction of apoptosis. Furthermore the involvement of the RBBP6 was implicated in the processes of apoptosis and the cell cycle although the exact mechanisms have not been fully elucidated.

Keywords:
Apoptosis
Real-Time qRT-PCR
Cell cycle
Interference RNA (RNAi)
Homologous recombination
Promoter
p53
Retinoblastoma
PACT
RBBP6

Papers by Ashley Pretorius

The demand for antimicrobial peptides (AMPs) is rising because of the increased occurrence of pat... more The demand for antimicrobial peptides (AMPs) is rising because of the increased occurrence of pathogens that are tolerant or resistant to conventional antibiotics. Since naturally occurring AMPs could serve as templates for the development of new anti-infectious agents to which pathogens are not resistant, a resource that contains relevant information on AMP is of great interest. To that extent, we developed the Dragon Antimicrobial Peptide Database (DAMPD, http://apps.sanbi.ac.za/dampd) that contains 1232 manually curated AMPs. DAMPD is an update and a replacement of the ANTIMIC database. In DAMPD an integrated interface allows in a simple fashion querying based on taxonomy, species, AMP family, citation, keywords and a combination of search terms and fields (Advanced Search). A number of tools such as Blast, ClustalW, HMMER, Hydrocalculator, SignalP, AMP predictor, as well as a number of other resources that provide additional information about the results are also provided and integrated into DAMPD to augment biological analysis of AMPs.

Abstract OBJECTIVE: The objective of this study was to evaluate the association between the in... more Abstract

OBJECTIVE:

The objective of this study was to evaluate the association between the interleukin-1 composite gene polymorphism and the severity of periodontal disease in the Xhosa population of South Africa.
BACKGROUND:

Periodontitis is a bacterially-induced chronic inflammatory disease that destroys the tooth supporting tissues. A specific pattern of interleukin-1 polymorphisms (known as the composite IL-1 genotype) has been found to influence the severity of chronic periodontitis in some ethnic groups.

METHODS:

Ninety-nine subjects, 35-60 years of age, of Xhosa descent, who were non-smokers and free of systemic disease, were enrolled in a case-control study depending on their periodontal status (healthy to mild vs. moderate to severe disease). A buccal smear was obtained from each subject; the DNA was isolated then amplified using the polymerase chain reaction (PCR). Allele identification was either by real-time PCR or by size fractionation following restriction digestion and separation on a polyacrylamide gel.

RESULTS:

The prevalence of the composite genotype was only 6% in the 99 subjects of the study population, which occurred more frequently in "cases" (8.2%) than in "controls" (4%). The frequency of IL-1A +4845 allele 2 genotype was 47% in cases and 22% in controls (p = 0.009), and that for IL-1B +3954 was 14.3% in cases and 20% in controls (p = 0.595).
CONCLUSIONS:

This study demonstrated that the IL-1 composite polymorphism occurred among only few subjects in the Xhosa population of South Africa, and so was not significantly associated with the severity of chronic periodontitis in this population.

African Journal of …, Jan 1, 2010

Culture has always been recognized as the gold standard for detecting oral anaerobes in dental pl... more Culture has always been recognized as the gold standard for detecting oral anaerobes in dental plaque samples. However, many of the bacterial morphotypes observed by microscopy are difficult to culture, thus necessitating the need for alternative methods of detection. The objective of this study was to evaluate the sensitivity and specificity of BANA (N-benzoyl-DL-arginine-B-naphthylamide) and PCR (Polymerase Chain Reaction) as reliable detection methods for detecting oral anaerobes such as Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola (often referred to as the "red complex") in subgingival dental plaque. Of the 372 samples analysed, 7.25% tested positive for the BANA test and 36.29% yielded a positive PCR test. This study showed that PCR was more sensitive than BANA in detecting members of the "red complex" in plaque samples.

International journal of …, Jan 1, 2005

In this study we investigate the spoilage of ultra high temperature UHT mango juice as well as a ... more In this study we investigate the spoilage of ultra high temperature UHT mango juice as well as a carbonated fruit juice blend to identify organisms contributing to the spoilage. The mango concentrate, the final product, as well as the other ingredients used during manufacturing, were tested for the presence of Alicyclobacillus by polymerase chain reaction (PCR) and sequencing analyses. Microbiological examination of the mango pureeá nd spoiled fruit juices, using YSG agar [yeast extract 2 g, glucose 1 g, soluble starch 2 g, pH 3.7 (adjust with 2N H 2 SO 4 ), H 2 O 1000 mL, bacto agar 15 g] incubated at 55°C, detected sporeforming, acid dependent and thermotolerant bacteria. The hyper variable region of the 16S rDNA was amplified. The nucleotide sequence of the PCR fragments was determined using the ABI Prism 310 automated DNA sequencer and the collected sequencing data were analysed and compared with the non-redundant database using NCBI-BLAST. Alicyclobacillus acidocaldarius were isolated and identified by 16S rDNA gene sequences analyses. The results indicated that the mango pure`e, as well as the final product of mango juice and the fruit juice blend, were positive for Alicyclobacillus. The preventative measures of low pH, pasteurization of mango juice and the subsequent use of aseptic packaging were not regarded as sufficient to prevent the outgrowth of Alicyclobacillus spoilage organisms.