Real-time detection of DNA interactions with long-period fiber-grating-based biosensor

Using an optical biosensor based on a dual-peak long-period fiber grating, we have demonstrated the detection of interactions between biomolecules in real time. Silanization of the grating surface was successfully realized for the covalent immobilization of probe DNA, which was subsequently hybridized with the complementary target DNA sequence. It is interesting to note that the DNA biosensor was reusable after being stripped off the hybridized target DNA from the grating surface, demonstrating a function of multiple usability.

The development of a model system and high throughput assay for the study of interactions between zinc finger proteins and DNA

It has been recognised for some time that a full code of amino acid-based recognition of DNA sequences would be useful. Several approaches, which utilise small DNA binding motifs called zinc fingers, are presently employed. None of the current approaches successfully combine a combinatorial approach to the elucidation of a code with a single stage high throughput screening assay. The work outlined here describes the development of a model system for the study of DNA protein interactions and the development of a high throughput assay for detection of such interactions. A zinc finger protein was designed which will bind with high affinity and specificity to a known DNA sequence. For future work it is possible to mutate the region of the zinc finger responsible for the specificity of binding, in order to observe the effect on the DNA / protein interactions. The zinc finger protein was initially synthesised as a His tagged product. It was not possible however to develop a high throughput assay using the His tagged zinc finger protein. The gene encoding the zinc finger protein was altered and the protein synthesised as a Glutathione S-Transferase (GST) fusion product. A successful assay was developed using the GST protein and Scintillation Proximity Assay technology (Amersham Pharmacia Biotech). The scintillation proximity assay is a dynamic assay that allows the DNA protein interactions to be studied in "real time". This assay not only provides a high throughput method of screening zinc finger proteins for potential ligands but also allows the effect of addition of reagents or competitor ligands to be monitored.

Discovering properties of new DNA-binding activity of proteins

Protein-DNA interactions are an essential feature in the genetic activities of life, and the ability to predict and manipulate such interactions has applications in a wide range of fields. This Thesis presents the methods of modelling the properties of protein-DNA interactions. In particular, it investigates the methods of visualising and predicting the specificity of DNA-binding Cys2His2 zinc finger interaction. The Cys2His2 zinc finger proteins interact via their individual fingers to base pair subsites on the target DNA. Four key residue positions on the a- helix of the zinc fingers make non-covalent interactions with the DNA with sequence specificity. Mutating these key residues generates combinatorial possibilities that could potentially bind to any DNA segment of interest. Many attempts have been made to predict the binding interaction using structural and chemical information, but with only limited success. The most important contribution of the thesis is that the developed model allows for the binding properties of a given protein-DNA binding to be visualised in relation to other protein-DNA combinations without having to explicitly physically model the specific protein molecule and specific DNA sequence. To prove this, various databases were generated, including a synthetic database which includes all possible combinations of the DNA-binding Cys2His2 zinc finger interactions. NeuroScale, a topographic visualisation technique, is exploited to represent the geometric structures of the protein-DNA interactions by measuring dissimilarity between the data points. In order to verify the effect of visualisation on understanding the binding properties of the DNA-binding Cys2His2 zinc finger interaction, various prediction models are constructed by using both the high dimensional original data and the represented data in low dimensional feature space. Finally, novel data sets are studied through the selected visualisation models based on the experimental DNA-zinc finger protein database. The result of the NeuroScale projection shows that different dissimilarity representations give distinctive structural groupings, but clustering in biologically-interesting ways. This method can be used to forecast the physiochemical properties of the novel proteins which may be beneficial for therapeutic purposes involving genome targeting in general.

Real-time detection of DNA interactions with long-period fiber-grating-based biosensor

Using an optical biosensor based on a dual-peak long-period fiber grating, we have demonstrated the detection of interactions between biomolecules in real time. Silanization of the grating surface was successfully realized for the covalent immobilization of probe DNA, which was subsequently hybridized with the complementary target DNA sequence. It is interesting to note that the DNA biosensor was reusable after being stripped off the hybridized target DNA from the grating surface, demonstrating a function of multiple usability. © 2007 Optical Society of America.

Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation

Cells and organisms respond to nutrient deprivation by decreasing global rates of transcription, translation and DNA replication. To what extent such changes can be reversed is largely unknown. We examined the effect of maternal dietary restriction on RNA synthesis in the offspring. Low protein diet fed either throughout gestation or for the preimplantation period alone reduced cellular RNA content across fetal somatic tissues during challenge and increased it beyond controls in fetal and adult tissues after challenge release. Changes in transcription of ribosomal RNA, the major component of cellular RNA, were responsible for this phenotype as evidenced by matching alterations in RNA polymerase I density and DNA methylation at ribosomal DNA loci. Cellular levels of the ribosomal transcription factor Rrn3 mirrored the rRNA expression pattern. In cell culture experiments, Rrn3 overexpression reduced rDNA methylation and increased rRNA expression; the converse occurred after inhibition of Rrn3 activity. These observations define novel mechanism where poor nutrition before implantation irreversibly alters basal rates of rRNA transcription thereafter in a process mediated by rDNA methylation and Rrn3 factor.

The SEF14 fimbrial antigen of Salmonella enterica serovar Enteritidis is encoded within a pathogenicity islet

The DNA sequence of the chromosomal gene cluster encoding the SEF14 fimbriae of Salmonella enterica serovar Enteritidis was determined. Five contiguous open reading frames, sefABCDE, were identified. The sefE gene shared significant homology with araC-like positive regulators. Serovar-associated virulence plasmid (SAP) genes orf7,8,9 and pefI were identified immediately adjacent to the sef operon. The pefI gene encoded a putative regulator of the Plasmid-encoded fimbrial antigen (PEF) expression. The entire sef--pef region, flanked by two IS-like elements, was inserted adjacent to leuX that encoded a transfer RNA molecule. The organisation of this region was suggestive of a classic pathogenicity islet. Southern hybridisation confirmed two copies of the SAP derived orf7,8,9 and pefI region in S. Enteritidis, one in the chromosome and one on the SAP. Of other group D Salmonella, only S. Blegdam and S. Moscow harboured both chromosomal and plasmid copies of pefI--orf9 region although polymorphism was evident.

Studies on the mechanism of action on antitumour imidazotetrazinones

The imidazotetrazinones are clinically active antitumour agents, temozolomide currently proving successful in the treatment of melanomas and gliomas. The exact nature of the biological processes underlying response are as yet unclear.This thesis attempts to identify the cellular targets important to the cytotoxicity of imidazotetrazinones, to elucidate the pathways by which this damage leads to cell death, and to identify mechanisms by which tumour cells may circumvent this action. The levels of the DNA repair enzymes O6-alkylguanine-DNA-alkyltransferase (O6-AGAT) and 3-methyladenine-DNA-glycosylase (3MAG) have been examined in a range of murine and human cell lines with differential sensitivity to temozolomide. All the cell lines were proficient in 3MAG despite there being 40-fold difference in sensitivity to temozolomide. This suggests that while 3-methyladenine is a major product of temozolomide alkylation of DNA it is unlikely to be a cytotoxic lesion. In contrast, there was a 20-fold variation in O6-AGAT levels and the concentration of this repair enzyme correlated with variations in cytotoxicity. Furthermore, depletion of this enzyme in a resistant, O6-AGAT proficient cell line (Raji), by pre-treatment with the free base O6-methylguanine resulted in 54% sensitisation to the effects of temozolomide. These observations have been extended to 3 glioma cell lines; results that support the view that the cytotoxicity of temozolomide is related to alkylation at the O6-position of guanine and that resistance to this drug is determined by efficient repair of this lesion. It is clear, however, the other factors may influence tumour response since temozolomide showed little differential activity towards 3 established solid murine tumours in vivo, despite different tumour O6-AGAT levels. Unlike mitozolomide, temozolomide is incapable of cross-linking DNA and a mechanism by which O6-methylguanine may exert lethality is unclear. The cytotoxicity of the methyl group may be due to its disruption of DNA-protein interactions, or alternatively cell death may not be a direct result of the alkyl group itself, but manifested by DNA single-strand breaks. Enhanced alkaline elution rates were found for the DNA of Raji cells treated with temozolomide following alkyltransferase depletion, suggesting a relationship between O6-methylguanine and the induction single-strand breaks. Such breaks can activate poly(ADP-ribose) synthetase (ADPRT) an enzyme capable of rapid and lethal depletion of cellular NAD levels. However, at concentrations of temozolomlde relevant in vivo little change in adenine nucleotides was detected in cell lines, although this enzyme would appear important in modulating DNA repair since inhibition of ADPRT potentiated temozolomide cytotoxicity in Raji cells but not O6-AGAT deficient GM892A cells. Cell lines have been reported that are O6-AGAT deficient yet resistant to methylating agents. Thus, resistance to temozolomide may arise not only by removal of the methyl group from the O6-position of guanine, but also from another mechanism involving caffeine-sensitive post-replication repair or mismatch repair activity. A modification of the standard Maxam Gilbert sequencing technique was used to determine the sequence specificity of guanine-N7 alkylation. Temozolomide preferentially alkylated runs of guanines with the intensity of reaction increasing with the number of adjacent guanines in the DNA sequence. Comparable results were obtained with a polymerase-stop assay, although neither technique elucidates the sequence specificity of O6-guanine alkylation. The importance of such specificity to cytotoxicity is uncertain, although guanine-rich sequences are common to the promoter regions of oncogenes. Expression of a plasmid reporter gene under the control of the Ha-ras proto~oncogene promoter was inhibited by alkylation with temozolomide when transfected into cancer cell lines, However, this inhibition did not appear to be related to O6~guanine alkylation and therefore would seem unimportant to the chemotherapeutic activity of temozolomide.