Electron paramagnetic resonance studies of spin-labelled ethidium bromide DNA interactions

Spin-Labelled Ethidium Bromide (SLEB) was prepared in order to study its interactions with natural DNA in the form of fibres . The technique of electron paramagnetic resonance was used in this thesis. Knowledge of the conformational transition pathway of natural DNA for given counterion concentration as a function of relative humidity was utilised in the study of effect DNA confomation on the binding of SLEB. To aid interpretation of the results the relevant background material was reviewed. In order to attempt to extract geometric information on binding computer ERR lineshape simulations were used. To facilitate this a microcomputer spectrometer control system was designed and implemented. This allowed spectra to be acquired in digital form and transfered to the mainframe computer. Two schemes for magnetic field control were investigated, one based on a commercial NMR magnetometer, and a superior pulsed NMR field locking magnetometer developed in this laboratory. In order to obtain lineshapes undistorted by dipolar broadening it is advantageous to use fibres with a high phosphate to drug ratio (P/D), however spectrometer sensitivity becomes a limiting factor. A review of noise in spectrometer systems is included. The use of a microwave low-noise preamplifer to reduce the system noise figure was investigated. An attempt to construct a loop-gap resonator was made and justified theoretically. A 35GHz spectrometer was constructed and a cavity designed and built to allow the humidity to be varied. The system was made compatible with the control system. Spectra recorded and simulated at this frequency should help confirm those obtained at 9GHz. The results obtained from P/D«70 fibres with a 0.5mM NaCl concentration show the SLEB is in a disordered state from 33% to 75% relative humidity. Spectral changes occur in the range 75% to 98% consistant with intercalation. In this humidity range a transition to the B-form is expected

Analysis of a Human Transfer RNA Gene Cluster and Characterization of the Transcription Unit and Two Processed Pseudogenes of Chimpanzee Triosephosphate Isomerase

An 18.5-kb human DNA segment was selected from a human XCharon-4A library by hybridization to mammalian valine tRNAiAc and found to encompass a cluster of three tRNA genes. Two valine tRNA genes with anticodons of AAC and CAC, encoding the major and minor cytoplasmic valine tRNA isoacceptors, respectively, and a lysine tRNAcuu gene were identified by Southern blot hybridization and DNA sequence analysis of a 7.1-kb region of the human DNA insert. At least nine Alu family members were found interspersed throughout the human DNA fragment. The tRNA genes are accurately transcribed by RNA polymerase III in a HeLa cell extract, since the RNase Ti fingerprints of the mature-sized tRNA transcription products are consistent with the DNA sequences of the structural genes. Three members of the chimpanzee triosephosphate isomerase (TPI) gene family, the functional transcription unit and two processed pseudogenes, were characterized by genomic blotting and DNA sequence analysis. The bona fide TPI gene spans 3.5 kb with seven exons and six introns, and is the first complete hominoid TPI gene sequenced. The gene exhibits a very high identity with the human and rhesus TPI genes. In particular, the polypeptides of 248 amino acids encoded by the chimpanzee and human TPI genes are identical, although the two coding regions differ in the third codon wobble positions for five amino acids. An Alu member occurs upstream from one of the processed pseudogenes, whereas an isolated endogenous retroviral long terminal repeat (HERV-K) occurs within the structural region of the other processed pseudogene. The ages of the processed pseudogenes were estimated to be 2.6 and 10.4 million years, implying that one was inserted into the genome before the divergence of the chimpanzee and human lineages, and the other inserted into the chimpanzee genome after the divergence

NaCl-Regulated gene expression in Distichlis

NaCl-induced and -repressed cDNA clones had previously been isolated by differential screening of a cDNA library, prepared from poly(A(^+)) RNA isolated from Distichlis spicata (salt grass) cell cultures grown in the presence of 260 mM NaCl (Zhao, et al., 1989). Eight of these cDNA clones have now been subcloned and/or sequenced and the predicted polypeptides compared with owl sequence data base. Three clones pDZ6.2, pDZVIII 1.2.1 and pDZIX 3.1 encode proline rich proteins, containing an amino acid repeat [PPKKDH(H)Y(Y)]. They have similar amino acid usage to proline-rich cell wall proteins, being rich in P, K, H and Y. The first 20 amino acid residues encode a putative leader sequence, supporting the proposed extracellular role as a cell wall protein. This N-terminal sequence (MPLLVALLLVLAVVAAAGAD) shares some similarity with die leader sequence of a soyabean proline-rich cell wall protein precursor and other extracellular proteins (the conserved residues are underlined). There is an increase in abundance of transcripts hybridising to the inserts from pDZ6.2 and pDZVUI 1.2.1 in response to either 520 mM NaCl or 100 µM ABA, but a decrease in response to 5 mM exogenous proline. It is suggested that the corresponding gene(s) are regulated at the level of either transcription or transcript stability, in response to elevated NaCl, with ABA as a mediator of (or part of) tills response. pDZ6.2 and pDZXI 3.1 have identical nucleotide sequences, whilst pDZVni 1.2.1 differs in three base paks within the putative open reading frame, suggesting that there may be at least two members of a multi gene family. A 68 bp OA repeat has been found in the 5' untranslated region of pDZ6.2 and a corresponding transcript identified by northern analysis using this OA sequence as a probe. Such nucleotide repeats can form triplexes (DNA) or hakpin loops (RNA), which is dependent on pH and ionic conditions. Therefore this OA repeat may play a role in the regulation of the gene corresponding to pDZ6.2 at the level of transcription or translation, possibly by attenuation of these processes, either by the formation of triplexes or hah-pins, or the binding of a protein to this GA region, at low ionic strength. However initial in vitro ttanscription experiments, to compare the transcriptional activity of pDZ6.2 and pDZVin 5.1.1 at different ionic strengths, proved inconclusive. An attempt was also made to identify the corresponding genomic region from D. spicata by anchored PGR.A fourth clone pDZ2.8L encodes a histone 2B protein, having 97.9% similarity to a wheat histone 2B. Its transcript abundance decreased in response to either 520 mM NaCl, 5 mM proline or 100 µM ABA. The sequences of the remaining clones either revealed no significant similarity to any known sequences or were assigned as being cloning artefacts .D. spicata cells accumulate proline within eight hours of exposure to 260 mM NaCl (Heyser, et al., 1989b). An unsuccessful attempt was also made to isolate a pyrroline-5- carboxylate reductase gene homologue from D. spicata, by heterologous probing of Southern blots with a soyabean cDNA pProCl and PCR

Studies on the expression of a cloned Staphylococcus aureus protein A gene in Escherichia coli

The gene coding for staphylococcal protein A has been cloned and the nucleotide sequence coding for the structural gene and its 5' flanking region determined. The DNA sequence showed the gene to be composed of a series of repetitive sequences, with five 120 nucleotide repeats comprising the 5' part of the structural gene and ten 24 nucleotide repeats in a continuous sequence at the 3’ terminus. Analysis of codon usage of the gene and comparison with other S. aureus genes revealed that spa showed a higher degree of homology with S. aureus plasmid genes than with chromosomally- located genes. Comparison with the reported codon preference in E.coli showed spa to have a similar bias, except for the leu codon. An open reading frame of the spa structural gene showed 508 a.a residues, with a predicted protein molecular weight of 55,426. The N-terminal signal peptide comprised the first 36 amino acids. A fifth N-terminal domain (E) was shown to be homologous to the four IgG-binding domains previously reported to constitute the N-terminal portion of SpA. The C-terminal part of SpA was shown to consist of two structurally different regions: an N-terminal repetitive region (Xr) composed of ten octapeptide repeats which are hydrophilic and have been proposed to span the Gram-positive cell wall, followed by a single domain (Xc), 58 a.a in length, whose C-terminal portion contains 20 hydrophobic residues, suggesting anchorage on the cell membrane. Enhanced production of SpA in E. coli has been achieved by placing the E. coli lac promoter immediately upstream of the spa gene. This has allowed yields as high as 1.5g of SpA per litre of culture to be obtained from 150-litre batch fermenters. Problems were encountered during large-scale fermentation, as the recombinant clones were found to be extremely fragile when producing high levels of SpA. Characterisation of the recombinant SpA indicated a molecular weight of 54,000 for the full length protein, compared with 56,000 for SpA produced in S. aureus. SDS-PAGE analysis of the IgG-binding proteins revealed extensive proteolysis of the recombinant SpA. Comparitive analysis of the IgG-binding polypeptides encoded by a truncated spa gene (lacking 69 a.a residues from the C-terminus), together with the determination of the N-terminal amino acid sequence, indicated that proteolysis occurred at the N- terminal portion of the Xc region. Cell fractionation studies and immunoelectronmicroscopy localised the SpA to the periplasm in E. coli. A possible regulatory control region for the spa gene has been localised upstream of the structural gene. The E.coli alkaline phosphatase gene was isolated, cloned and its sequence determined to facilitate the production of SpA-alkaline phosphatase fusion proteins

The cloning and characterisation of an endoglucanase and an endoxylanase from Clostridium acetobutylicum in Escherichia coli

Bibliography: pages 215-244.Clostridium acetobutylicum P262 is an endospore forming Gram-positive obligate anaerobe which has been used for the industrial production of acetone and butanol. Strains of C. acetobutylicum have been reported to exhibit some activity towards cellulosic and hemicellulosic substrates. The aim of this thesis was to establish a genebank of C. acetobutylicum P262 DNA in Escherichia coli and to isolate and characterise genes encoding enzymes which show activity towards hemicellulose and cellulose

The role of the C-terminus in defining the efficiency and specificity of G-protein coupling

In combination with conferring resistance to ADP-ribosylation by Pertussis toxin, the substitution of a conserved cysteine residue (C351) four amino acids from the C-terminus of Gialpha1 has been shown to modulate the efficiency of coupling to the α2A adrenoceptor. Investigation of this phenomenon through systematic substitution of this cysteine residue for all other amino acids highlighted a relationship between the hydrophobicity of the substituted residue and the capacity of the a-subunit to functionally couple to the α2A adrenoceptor. From the results of this investigation, it was noted that wild type Giα1 did not display optimal coupling at this receptor. Relative to wild type Giα1, coupling was enhanced by the substitution of a more hydrophobic residue at position C351, but diminished upon the substitution of a more hydrophilic residue. In contrast to this, substitution of proline or a charged residue at this position essentially attenuated functional coupling with the α2A adrenoceptor. Similarly, pEC50 values of the mutants also showed a high degree of correlation with the hydrophobic nature of the substituted residue, with more hydrophobic residues reducing pEC50 values and more hydrophilic residues increasing pEC50 values respectively relative to wild type Giαl. This change in coupling efficiency could not be attributed to a change in the affinity for nucleotides at the a-subunit or to a change in the rate of basal guanine nucleotide exchange. These data indicate that functional coupling of the Giαl subunit to the α2A adrenoceptor is in part modulated by the physiochemical properties of residue 351 and that a relationship exists between the hydrophobicity of residue 351 and the capacity of the α-subunit to functionally couple to the α2A adrenoceptor. A series of fusion constructs composed of the α2A adrenoceptor covalently linked to selected Giαl C351 mutants were used to assess the effects of substituting residue C351 in Giαl on agonist intrinsic activity at the α2A adrenoceptor. The agonist UK 14304 was shown to elicit a spectrum of responses at the fusion constructs, closely mirroring the order of coupling efficiency previously determined in the separately expressed components. While UK14304 essentially acted as a full agonist compared to adrenaline at the fusion construct composed of wild type Giαl (C351 Giαl), it was shown to act as a partial agonist at an equivalent construct containing a glycine residue at position 351 in the Giαl moiety. In contrast to this, relative to the wild type Giαl fusion, UK14304 displayed greater relative intrinsic activity at the fusion construct containing an isoleucine residue at position 351 in the Giαl moiety. Analysis of a more extensive range of partial agonists demonstrated that the order of agonist relative intrinsic activity at the respective fusion constructs was conserved regardless of the agonist assayed. This discrepancy of intrinsic activity at the fusion proteins could not be attributed to a change in the pharmacological profile of the receptor moiety or to a modification of its affinity for agonist. These data indicate that the intrinsic activity of partial agonists, relative to adrenaline, can be modulated by the physiochemical properties of residue 351 in the Giαl moiety. The capacity of a series of chimeras containing substitutions of the last 6 C- terminal residues of Giαl for those of Gsα, Gqα and G16α were assessed for functional coupling with a range of non Gi-linked receptors. Functional coupling was demonstrated at the Gi/Gs chimera with the V2 vasopressin receptor and α2 adrenoceptor. Similarly, the Gi/Gq chimera was shown to functionally couple with the P2Y4, and TRH, receptors. No functional coupling was detected for the Gi/G16 chimera. The inability of the Gi/G16 chimera to functionally couple to any of the receptors analysed was independent from its capacity to basally exchange guanine nucleotides, which was shown to be unchanged relative to the Gi/Gs, Gi/Gq chimeras and wild type Giαl. The substitution of the 6 C-terminal residues of Giαl for those of Gsα also conferred resistance to ADP-ribosylation by both pertussis and cholera toxins. This was demonstrated by functional coupling of the Gi/Gs chimera to a FLAG(TM) -tagged version of the IP prostanoid receptor following treatment with both bacterial toxins. The GiαlGsα chimera was seen to couple more efficiently at the IP prostanoid receptor in the presence of these toxins than in non toxin treated samples, indicating that coupling efficiency of this chimera at the IP prostanoid receptor was not optimal. These findings indicate that the C-terminus of the G-protein α-subunit is an important determinant in defining G-protein/receptor coupling and that additional determinants, not present in the C-terminus of the α-subunit, are required for optimal coupling efficiency

X-ray diffraction and molecular modelbuilding studies on the deoxyribonucleic acid double helix

The known nucleic acid conformations and the methods by which they are determined using X-ray fibre diffraction are reviewed and discussed. A new stacking scheme for Watson-Crick base-pairs is described and a left-handed model of B-DNA which incorporates this scheme is presented and evaluated. This model is less successful than the conventional B-DNA model in explaining the observed diffraction pattern. The side-by-side of B-DNA is criticised in detail and its predicted diffraction pattern is found to compare unfavourably with that predicted by the double helix. Two forms of Patterson function have been applied to several data sets. The results suggest both that the accepted A-DNA indexing of Fuller et al (1965) is preferable to a new scheme proposed by Saslsekharan, Bansal and Gupta (1981) and also that a left-handed model of D-DNA may be in better agreement with the observed diffraction pattern than is the right-handed model of Arnott et al (1974) but neither function is found to be sufficiently robust to enable reliable conclusions to be drawn concerning molecular conformation. Expressions are derived which describe the effect on the Patterson functions of baseline errors 1n the measurement of Intensities in diffuse patterns. The conformation and transitions of a bacteriophage DNA have been studied and a model is presented which explains the observed behaviour 1n terms of a groove-bridging putrescinyl linkage. Several models (incuding a preliminary coiled-coil model) of the structure of DNA under mechanical tension are described and compared with the observed diffraction patterns

The stoichiometry of interaction between receptors, G-proteins and effector species

G-proteins have been demonstrated to be necessary to allow transduction of information from hormone-activated cell surface receptors to a variety of effector systems. This study has focused on their expression and regulation in response to agonists at receptors which produce stimulation of adenylyl cyclase or stimulation of phospholipase C activity. To understand receptor regulation of cyclic AMP generation, it is important to know the relative or absolute levels of expression of each component (receptor, G- protein (Gs) and adenylyl cyclase) in individual cells, their cellular disposition and how alteration in levels of each component might alter the effectiveness of cellular signalling. In chapter 3, neuroblastoma x glioma hybrid, NG108-15, cells are shown to express Gsalpha in a substantial molar excess over its effector adenylyl cyclase. Sustained exposure of a cell to an agonist for a G-protein coupled receptor can lead to the down-regulation of levels of the receptor and the G-protein and this process can play a substantial part in the regulation of cellular sensitivity to the presence of agonist. Regulation of agonist access to the receptor population by pretreatment of NG108-15 cells transfected to express the human beta2-adrenoceptor with varying concentrations of an irreversible beta-adrenoceptor antagonist (BAAM) demonstrated that the extent of agonist-mediated Gsalpha down-regulation was dependent upon the availability of receptor to agonist and that the levels of receptor expression defines the intrinsic activity and potency of agonists. As noted above, however, the availability of sufficient Gsalpha to interact with the total cellular population of adenylyl cyclase suggests that the adenylyl cyclase is likely to be the limiting component for information transfer. As a means to examine quantitative aspects of the expression of adenylyl cyclase, human beta2-adrenoceptor expressing NG108-15 cells were further transfected to overexpress adenylyl cyclase type 2. The results demonstrated that receptor-mediated maximal output of the stimulatory arm of the adenylyl cyclase cascade can be increased by increasing total levels of adenylyl cyclase but this did not result in any significant alteration in the concentration-effect curves for stimulation of adenylyl cyclase activity produced by either the transfected human beta2-adrenoceptor or any of the endogenously expressed (IP prostanoid, A2 adenosine or secretin) Gsalpha-linked receptors. In chapter 4, human embryonic kidney (HEK-293) cells which express the long splice variant form of the rat thyrotropin-releasing hormone (TRH) receptor (clone E2) were examined to address whether both Gqa and/or G11alpha and Gsalpha are involved in TRH-stimulation of phospholipase C beta1 and/or adenylyl cyclase or whether different splice variants of the receptor selectively interact with different G- proteins to regulate different signal transduction cascades. Activation of this receptor with TRH caused only a large stimulation of production of inositol phosphates in a manner which was mediated by the pertussis-toxin-insensitive phospholipase C-linked Gq and G11 proteins despite the fact that TRH has been reported to stimulate adenylyl cyclase activity via activation of Gsalpha. In addition, with development of a 6 M urea containing SDS-PAGE (10% (w/v) acrylamide, 0.0625% (w/v) bisacrylamide) system which allowed resolution of Gqa and G11alpha I assessed whether a single receptor type could utilise species variants of the same G-protein equivalently. Treatment of clone E2M11 (which expresses the long isoform of the rat TRH receptor and both the endogenous human and exogenous introduced murine G11alpha) with TRH resulted in a qualitatively similar pattern of cellular redistribution and down-regulation of G11alpha isoforms. Quantitative analysis indicated, however, that the murine isoform of G11alpha was less effectively regulated by the long isoform of the rat TRH receptor than the endogenous human form of G11alpha

An association study of PITX2 polymorphism in a cohort of patients with primary open angle glaucoma and considerations on the genetics of glaucoma

Background: Glaucoma is a major cause of blindness world-wide. There is a need for methods to identify individuals at risk of developing glaucoma, so that early treatment can prevent visual loss. Genetic screening tests offer the prospect of pre-symptomatic diagnosis of at risk individuals. There is now strong evidence that a number of different genes are associated with glaucoma susceptibility. Mutations in the PITX2 homeobox transcription factor gene disrupt normal development of the anterior segment and cause overt structural abnormalities. It is possible that, as yet undetected mutations/polymorphisms in PITX2 may produce subtle and undetected abnormalities in anterior segment structure and function that could predispose to glaucoma. Purpose: The aim of this thesis is two fold: 1. Screening for the presence of single nucleotide polymorphisms in PITX2 gene in a cohort of 100 unrelated primary open angle glaucoma/ ocular hypertension patients, 10 Posterior embryotoxon subjects and 100 age and ethnically matched controls to establish the mutation spectrum. 2. Identification, phenotyping and recruitment for genetic studies of primary open angle glaucoma patients with strong family history of glaucoma. Materials and methods: 1. 100 primary open angle glaucoma patients and 60 age and ethnically matched controls were enrolled in the study. Patients and controls were phenotyped and a blood sample for DNA extraction collected. PITX2 exon-specific primers were used to PCR amplify patient and control DNA. Direct sequencing was used to screen for sequence alterations in the entire coding sequence of PITX2 gene. Concurrently, polymorphic sites reported in the PITX2 gene were identified from the NCBI and Ensemble databases and the frequency of polymorphic sites was investigated. The SHEsis and UNPHASED software packages were used for statistical analysis. 2. Patients diagnosed with glaucoma and strong family history were identified from Glaucoma Unit at Sunderland Eye Infirmary, phenotyped and enrolled in the study. The pedigrees were constructed and interested relatives enrolled in the study and phenotyped. A sample of blood for DNA extraction was collected from all people enrolled in the study. Results: 1. Direct sequencing did not identify any sequence variation in the coding region. 26 PITX2 polymorphic sites were identified from the internet databases, including five in the coding sequence. Sixteen non coding SNPs were confirmed within our study group and SNP frequencies were examined. None of the coding sequence SNPs was identified in our cohort, demonstrating a high degree of sequence conservation. Also, none of the SNPs confirmed in this study group showed an increased frequency in the primary open angle glaucoma group compared with the control group. 2. Thirty-three pedigrees were identified with strong family of glaucoma during the time allowed for patient recruitment. Of these, twenty-two agreed to take part in the study. Thirteen pedigrees are presented in this study, mostly demonstrating autosomal dominant inheritance. Conclusion: There is ample evidence to suggest that genetics play an important role in unravelling the pathogenesis of glaucoma. Identification and recruitment of patients for genetic studies is an essential step and the role of the clinician in this process is paramount. Also, developmental glaucoma genes are an important group of genes to be screened in primary open angle glaucoma/ocular hypertension patients, as they may play a role in the pathogenesis of this preventable blinding disease.EThOS - Electronic Theses Online ServiceRoyal College of Surgeons, Edinburgh : Glaucoma Research and Development at Sunderland (GRAD@S) fund : Pfizer OphthalmicGBUnited Kingdo