OpenUCT :: Browsing by Subject "Biochemistry"

Browsing by Subject "Biochemistry"

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Open Access
Binding sites in the nuclear envelope for cytoplasmic glucocorticoid receptor complex
(1983) Smith, Peter John; Von Holt, Claus
Using tritiated triamcinolone acetonide to monitor purification, cytoplasmic triamcinolone acetonide-receptor complex has been purified 3 000 fold from rat liver cytosol. The isolated complex sedimented as a single radioactive peak on a 5 - 20% sucrose gradient. Nuclear envelopes isolated from purified rat liver nuclei were found to contain binding sites for the partially purified cytoplasmic triamcinolone acetonide-receptor complex. The binding constants showed two saturable high affinity binding sites and the envelope bound the complex with a specific activity ten times higher than the plasma membrane and more than three times higher than the two endoplasmic types of membrane. Saturable binding to chromatin was not observed in the concentration range tested. Free steroid hormone did not bind the envelope. Binding sites for steroid hormones or steroid hormone-receptor complexes have been demonstrated both in chromatin and the nuclear protein matrix (Barrack and Coffey, 1980; Spelsberg, 1976). Because the nuclear envelope may be isolated with both these nuclear subfractions, the observed binding sites for steroid hormone-receptor complexes might be due to the presence of envelope components. The extent of association of nuclear envelope or nuclear envelope components with chromatin and the matrix was therefore investigated. Nuclear envelope fragments could be isolated from chromatin purified by centrifugation through 1,7 M sucrose. The binding of triamcinolone acetonide-receptor complex to these fragments was indistinguishable from the binding to purified nuclear envelope. A certain class of saturable chromatin binding sites for steroid hormone-receptor complexes may thus be due to the presence of envelope fragments. Extensive association of nuclear envelope polypeptides with the nuclear protein matrix was also observed. The matrix however, failed to bind triamcinolone acetonide-receptor complex. The nuclear envelope comprises an inner and outer membrane with well defined pore complexes spanning both membranes. In order to identify the location of the binding sites for trimacinolone acetonide-receptor complex in the envelope, fractionation and reconstitution of envelope proteins and lipids was attempted. Envelopes were solubilized in 2 - chloroethanol and protein and lipid components separated by chromatography on Sephadex LH 20. Envelope protein and lipid could be successfully reconstituted from chloroethanol by dialysis against aqueous buffer. Results showed that the receptor complex binds to the protein rather than lipid component of the envelope. This component was extractable by concentrations of the nonionic detergent Triton X-100 which do not extract the pore complex or lamina components of the envelope and is therefore probably a loosely bound membrane protein. The presence of specific binding sites for triamcinolone acetonidereceptor complex on the nuclear envelope may be necessary for the transport of the complex into the nucleus. The possibility that the envelope mediates the glucocorticoid response in ways not linked to transport of the cytoplasmic receptor complex into the nucleus cannot be ruled out.
Open Access
Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylophic yeast Pichia pastoris
(1996) Hüsler, Jennifer; Klump, H H; Brandt, Wolf F; Maeder, Dennis
Squash inhibitors are the smallest natural serine protease inhibitors. Their compact, rigid nature has enabled detailed examination of their 3D structure by NMR and X-ray crystallography. Being of a convenient size to synthesise chemically, the effects on activity of selective substitutions and deletions within the sequence have also been investigated. Thus, this family of inhibitors is considered useful as a model system for the study of protein-protein interactions. Curcrbita maxima trypsin inhibitor I (CMTI I) may be thought of as representative of the squash inhibitors, for which there is detailed structural and functional information available. It is a 29 amino acid protein, with the tri-disulphide bridging pattern common to all squash inhibitors. There are only a few examples of squash inhibitors being produced by recombinant DNA technology. As this technique offers a relatively cheap way of producing large amounts of these proteins, further investigation is required. Problems have been experienced with the expression of disulphide-rich proteins in E. coli, as the cytosol of this microorganism is not conducive to their folding. Furthermore extraction of these proteins from the peri plasmic space is often required, resulting in a reduction in yield. To overcome these shortcomings, the methylotrophic yeast Pichia pastoris was investigated as an alternative means of expression, although at the inception of this work, no disulphide-rich proteins of this size had been expressed in P. pastoris. It was a challenge to investigate the feasibility of producing squash inhibitors in this expression host and to compare the activity of the recombinant inhibitor to that of native CMTI I.
Open Access
Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylotropic yeast Pichia pastoris
(1996) Hüsler, Jennifer; Klump, Horst H; Brandt, Wolf F; Maeder, Dennis
Squash inhibitors are the smallest natural serine protease inhibitors. Their compact, rigid nature has enabled detailed examination of their 3D structure by NMR and X-ray crystallography. Being of a convenient size to synthesise chemically, the effects on activity of selective substitutions and deletions within the sequence have also been investigated. Thus, this family of inhibitors is considered useful as a model system for the study of protein-protein interactions. Cucurbita maxima trypsin inhibitor I (CMTI I) may be thought of as representative of the squash inhibitors, for which there is detailed structural and functional information available. It is a 29 amino acid protein, with the tri-disulphide bridging pattern common to all squash inhibitors. There are only a few examples of squash inhibitors being produced by recombinant DNA technology. As this technique offers a relatively cheap way of producing large amounts of these proteins, further investigation is required. Problems have been experienced with the expression of disulphide-rich proteins in E. coli, as the cytosol of this microorganism is not conducive to their folding. Furthermore extraction of these proteins from the peri plasmic space is often required, resulting in a reduction in yield. To overcome these shortcomings, the methylotrophic yeast Pichia pastoris was investigated as an alternative means of expression, although at the inception of this work, no disulphide-rich proteins of this size had been expressed in P. pastoris. It was a challenge to investigate the feasibility of producing squash inhibitors in this expression host and to compare the activity of the recombinant inhibitor to that of native CMTI I.
Open Access
Chemical synthesis, cloning and expression of a gene encoding systemin, a proteinase inhibitor-inducing factor
(1996) Ma, Pei-yin
Wound-inducible proteinase inhibitors in plants elicit a defence mechanism by inactivating the proteinases of insects. This triggers a feedback mechanism causing overproduction of digestive enzymes together with a decrease in appetite, leading to starvation. System in, a polypeptide proteinase inhibitor-inducing factor, when applied to cut stems of young tomato plants induces the accumulation of inhibitors in a manner similar to the normal wounding response. We designed and synthesised the minus strand oligonucleotide template complementary to the system in DNA sequence using Escherichia coli codon preferences. The double stranded fragment encoding the 18 amino acid residue systemin was cloned into pUCJ 8 for amplification and subcloning into pMAL-pk for expression as a maltose binding-fusion protein. The recombinant systemin was released by enterokinase and isolated by HPLC. After further purification, the physical characteristics including amino acid composition, peptide sequence and molecular weight of r-systemin were determined. When the recombinant peptide was applied to young tomato plants, it induced the accumulation of proteinase inhibitor I messenger RNA.
Open Access
Cloning and expression of a chimeric protease inhibitor encoding gene in Escherichia coli and Pichia pastoris
(1996) Matsebula, Aaron Mfanuzile; Botes, DP; Klump, HH; Maeder, Dennis
Squash family protease inhibitors are small peptides of 27-32 residues, hence they are ideal subjects for structure-function studies. Their small size is within the reach of peptide chemical synthesis, which enables one to produce enough peptide material for experimental purposes within a reasonable time frame.
Open Access
Cloning and expression of a modified oryzacystatin inhibitor gene and an investigation of its inhibitory capabilities
(1997) Haworth, Caroline Joanne; Thomson, Jennifer Ann; Maeder, Dennis
Cysteine proteinase inhibitors have shown potential as biocontrol agents for the protection of plants against insect and pathogen attack. With the advent of protein and genetic engineering such inhibitors can now be modified in order to improve their effectiveness. Because cystatins have already been isolated from plants. they provide a good starting point for developing modifications which may improve their function as biocontrol agents. The purpose of this project, therefore, was to design a potentially improved analogue of the rice cysteine proteinase inhibitor, oryzacystatin I, through molecular modelling studies. The gene sequence for this modified protein was then synthesised and expressed for kinetic analysis and insect trial assays. A prediction of the oryzacystatin I (OC I) tertiary structure was made using Biograf software on an Evans and Sutherland workstation. This structure was based on the known structures of stefin B and chicken cystatin ho se co-ordinates are published in the Brookhaven data files. Chicken cystatin is one of the most potent inhibitors of papain in the cystatin superfamily. This is believed to be due, in part, to an increased binding of the cystatin to papain through its amino-terminal region with the residues Leu7 to Gly9 playing a particularly important role.
Open Access
Competition between the purine and pyrimidine triple helix motifs in an oligonucleotide system
(1998) Mills, Martin George; Klump, Horst
Triple helices are classified into two groups according to the composition and orientation of the third strand, namely the pyrimidine motif and the purine motif. These motifs constitute two separate fields of research. It was proposed, based on the alternative design rules, that the two motifs can in fact lead to competing structures. An oligonucleotide system has been designed to demonstrate this competition. Systematic variation of its components give insight into the requirements for optimal binding of a third strand. A palindromic, homopyrimidine oligonucleotide of 22 bases was designed to form an overlapping 9-base, Watson-Crick (WC), duplex with a partly complementary 22-base purine-rich oligonucleotide. This leaves two free 3' extensions under conditions when only the duplex is stable. If the conditions, however, favour triplex formation the pyrimidine tail can compete with the purine-rich tail as third strand for the duplex forming Hoogsteen or reverse-Hoogsteen hydrogen bonding respectively with the purines in the WC double strand. The underlying triplexes and core duplex were synthesised and characterised as controls.
Open Access
Complementary DNA to sea urchin histone messenger RNA
(1977) Woods, Derek Edward; Fitschen, W
A preparation of 9S RNA was isolated from early blastula sea urchin embryo polyribosomes by phenol extraction followed by repeated sucrose density gradient centrifugation. When incubated in an ascites cell free protein synthesizing system this preparation of RNA was shown to support the synthesis of sea urchin histones confirming that this fraction contained histone mRNA.
Open Access
Correlation between physical and genetic maps of plasmids
(1992) Zubrzycki, Igor Z; Klump, Horst H
The aim of this study is to establish a correlation between the physical maps of plasmid DNA (in the form of calorimetric profiles, thermal denaturation profiles and electron micrographs of partly melted DNA sequences) and genetic maps of these DNAs and thus deal with questions which were not answered by previous researchers. viz: Is there a correlation between base sequence function and a measurable physical property which can be assigned to biologically important units such as promoters or coding sequences? Is there a correlation between the denaturation of gene sequences and cooperative transitions observed in a given temperature interval? To answer these questions, a systematic study was initiated based on two families of plasmids with three different genes incorporated, namely the pGV 403 family which contains a Chloramphenicol resistance gene on one side and the pUC9 family which contains an Ampicillin and a Tetracycline resistance gene on the other side. Three different techniques were used to address the stated problems i.e. differential scanning calorimetry, high resolution thermal denaturation and electron microscopy. The reason for using three techniques instead of only one or two as in previous studies is that each technique gives specific results which can be supplemented by the other techniques and only in this way it will be possible to approach a deeper understanding of changes induced by perturbing the sequence based structural integrity by elevating temperature. In addition to measuring the experimentally observable parameters listed. a theoretical model was developed to predict the changes. This approach is termed local compositional complexity (LCC) analysis. The final goal of this investigation was to establish whether there is any correlation between the local compositional complexity and these selected genetic units. Based on the calorimetric experiments an improved table of thermodynamic data including the stacking energy for ten different combinations of basepairs is presented. The prediction of a melting curve based on primary structure information can be based on the enthalpy individual combination of basepairs[41.50.58]. The tables of the thermodynamic data published in the literature are given in Appendix D. In this thesis a slightly different approach to predict tm's was chosen (cf. p 67). The results obtained by this combined approach showed that there is indeed a correlation between the specific base sequence of a given plasmid DNA and its biologically important units (genes) and thus confirms that there is a semi-empirical correlation between genes and the observed cooperative melting units.
Open Access
Cytoplasmic RNA-dependent RNA-synthesis in maturing chicken erythrocytes.
(1976) Boyd, Malcolm Charles David; Fitschen, W
An RNA-dependent RNA polymerase from ribosomes of maturing chicken erythrocytes was investigated. Concurrent with an increase in globin and RNA synthesis during phenylhydrazine induced anaemia, the total and specific activities of ribosome bound RNA-dependent RNA polymerase increased 40 and 9.4 times respectively, indicating the possible involvement of this enzyme activity in the synthesis or control of synthesis of globin. A partially purified RNA-dependent RNA polymerase was prepared from ribosomes of immature chicken erythrocytes. This preparation was shown to be a predominantly primer dependent enzyme activity, incorporating UTP, CTP and to a lesser extent ATP and GTP into homopoly-ribonucleotide material. The ribosome-bound enzyme preparation demonstrated in addition, the synthesis of heteropolyribonucleotide material. A complementary DNA copy of chicken globin mRNA containing-9S RNP, was prepared. Unlabelled complementary DNA was used to demonstrate that no globin mRNA replicase activity was detectable in the ribosome fraction of immature chicken erythrocytes. The radioactively labelled product obtained after incubation of ribosomes with [³H]-UTP was shown by sucrose density gradient centrifugation to have a sedimentation coefficient of 4S; the ratio of non-terminal to terminal incorporation of UTP was 6.08. The product synthesized after incubation of intact cells in the presence of [³H]-uridine and actinomycin D was shown to be very similar with respect to S-value and the ratio of UMP/uridine incorporated. This similarity suggests the presence of a ribosome bound RNA-dependent RNA polymerase activity in intact cells.
Open Access
Deoxyribonuclease probing on sea urchin embryo chromatin
(1983) Landsman, David
The role that the sea urchin, Parechinus angulosus, embryo and sperm histone variants play in chromatin structure has been investigated. Chromatin structure has been determined at different levels of resolution in sperm and in developing embryos using micrococcal nuclease, pancreatic deoxyribonuclease (DNase I) and restriction endonucleases. Micrococcal nuclease and restriction endonuclease digestions of sea urchin gastrula chromatin have been analysed and it is shown that it is not possible to isolate large polynucleosomal chromatin complexes which are soluble in low ionic strength buffers. The nucleosomal DNA repeat lengths for sea urchin blastula, gastrula and sperm have been determined using micrococcal nuclease. The repeat length for sperm is significantly larger than blastula and gastrula repeat lengths whereas blastula and gastrula repeat lengths are not significantly different. Nucleosomal core particles have been isolated from early blastula, gastrula and sperm of sea urchins. After DNase I digestion of 51-labelled core particles the rate constants of cutting of the DNA at the susceptible sites on these core particles have been determined. The DNase I digestion kinetics of blastula and gastrula core particles are similar whereas sperm core particles are digested at a slower rate, mainly at the sites which are closest to the ends of the core particle DNA. Also, a site, which is 5 bases on the outside of the core particle and which is partially protected from nuclease attack, has been identified. The implications of these findings in relation to the histone variants in embryos and sperm of sea urchins are discussed.
Open Access
The effect of dietary lipids on the serum cholesterol concentration of the rat
(1960) Wilkens, J A
The effect of different dietary fats on the serum cholesterol concentration has been discussed. In a series of experiments, conditions have been created under which rats can be induced to imitate humans in this respect. Using the rat as an experimental animal, the hypocholesterolaemic property of dietary sunflower seed oil has been investigated by the chemical and physical fractionation of this oil. By feeding the saponifiable and unsaponifiable matter of the sunflower seed oil, it was found that the latter fraction was partly responsible for the serum regulating property of this oil. This finding was confirmed by altering the composition of the unsaponifiable matter by the physical fractionation process of liquid propane segregation. Several fractions of oils with similar fatty acid compositions but differing in both quality and quantity of the unsaponifiable matter were found to have different effects on the serum cholesterol level.
Open Access
The effect of negative supercoiling on the formation and positioning of nucleosome cores in vitro
(1991) Patterton, Hugh-George; Von Holt, Claus
The effect of the negative supercoiling of DNA on the formation and positioning of nucleosome cores was investigated in a 1915bp plasmid (pHP2) containing a section of the early H1-H4 histone gene spacer of Psammechinus miliaris, previously shown to position the histone octamer (Retief et al., 1987, Biochemistry, 26, 4449-4453). It is shown for the first time, by determinations of the linking difference of reconstituted supercoiled plasmid following topoisomerase I relaxation and the yield and fragment size distribution of micrococcal nuclease digests of supercoiled and linearized plasmid, that nucleosome core reconstitution by urea/salt and salt dialysis proceeds cooperatively on both linearized and supercoiled plasmids. Evidence is further presented which indicates that the nucleosome core.reconstitutes more efficiently on negatively supercoiled plasmids compared to linearized plasmids. The free energy of supercoiling is shown to be sufficient to account for this difference, and may contribut·e to the observed preferential migration of the octamer to negatively supercoiled plasmid compared to linear fragments. This migration is facilitated by high ionic strength, but not by high concentrations of poly[L-glutamate] or 146bp core DNA. It is further shown for the first time, by DNase I digestion and primer extension, that identical translational and rotational positions are adopted by nucleosome cores on linearized plasmids and circular plasmids in the absence and presence of negative superhelical stress. This conservation of the positioning frames is -shown to persist, irrespective of the precision of the core placement, or alterations of the relative angular orientation of the positioning frames of adjacent cores. This finding suggests that the topological and geometric constraints of chromatin loops may be negligible in the determination of nucleosome positioning in vivo. The positioning of the core incorporating a d(A-G)₁₆.d(C-T)₁₆ stretch, shown not to adopt a H-DNA conformation in the reconstituted supercoiled plasmid, is analyzed in terms of known rotational determinants, and possible translational determinants proposed. The biological significance of the determined position of the core is discussed, and lastly, the conclusions related to other studies.
Open Access
An electrophoretic method for the isolation of isohistones from the embryo of the sea urchin Parechinus Angulosus
(1981) Schwager, Sylva; Von Holt, Claus
It is the aim of this project to isolate sea urchin embryo histone variants by preparative polyacrylamide gel electrophoresis and to prove me identity of the isolated proteins by amino acid composition and partial sequencing. This serves two purposes, namely, the unequivocal identification of chromosomal proteins characterized only by their electrophoretic mobility as histone variants, and secondly the creation of a micropreparative electrophoretic method. These are the prerequisites for later investigations into the specific roles of histones in the process of differentiation.
Open Access
The histone H1 of the sea urchin embryo, partial structures, enzymatic modifactions and developmental programme
(1982) De Groot, Petronella Christina; Von Holt, Claus; Strickland, W N
Developmental biology owes a tremendous debt to sea urchins. These animals have proved to be experimental jewels, and since they are distributed abundantly along the Peninsula coastline, they are readily at hand. Sea urchin embryos have been shown to be extremely well suited for analysis of developmental processes at the ultrastructural, biochemical and molecular levels. This project is a study of the very lysine-rich histone fraction or H1-histone fraction of Parechinus angulosus embryo. The first part deals with the characterization and separation of the H1-variants present in the late gastrula embryo. The second part describes the determination of the partial primary structure of the three chromatographically separated H1-fractions: The amino acid composition and sequences of the H1-histone variants are compared to those of H1-histones from other sea urchin embryo species and from various other sources. The third part is a study of the histone variant synthesis program during the early development. [3H] Lysine incorporation into newly synthesized histones was utilized to examine the histone synthetic program. This part also describes the examination of histone acetylation and phosphorylation occurring during the ninth cell cycle of development. Examination of the modification of the different histone variants and modifications of the histone variants at the different cell stages are discussed.
Open Access
Identification of genes involved in macrophage activation and effector functions against intracellular pathogens
(2006) Schwegmann, Anita Ruth; Brombacher, Frank
This dissertation addressed the hypothesis that macrophages have an alternative killing mechanism that is independent of superoxide and nitric oxide but dependent on IFN-γ, TNF and C/EBPβ. Since the mechanism and the genes involved in this alternative pathway are mostly unknown, the aim of this dissertation was to identify these macrophage effector genes and to functionally characterize their role during infection utilizing gene deficient mouse models. Since mice deficient for C/EBPβ (C/EBPβ-/-) expressed normal levels of IFN-y and TNF during Listeria monocytogenes infection, the macrophage effector genes involved in confinement and killing of L. monocytogenes were postulated to be downstream of C/EBPβ. Furthermore, C/EBPβ-/- mice are highly susceptible L. monocytogenes due to impaired listericidal activity. Comparison of the gene expression profiles of WT and C/EBPβ-/- macrophages infected with L. monocytogenes was postulated to increase the probability of identifying these effector genes, which would be differentially expressed between the two groups. Comparative gene expression profiling by DNA microarrays between L. monocytogenes in infected WT and C/EBPβ-/- macrophages, successfully identified 1268 genes to be differentially expressed between the two groups. A focussed functional clustering strategy reduced the number of candidate genes to 220. PKCδ was selected for further study since it was involved in humoral defense, immune signalling, production of superoxide, regulation of transcription and may be putatively transcriptionally regulated by C/EBPβ. Furthermore, PKCδ was indirectly shown to promote L. monocytogenes escape from the phagosome and to negatively regulate transcription activity of C/EBPβ. In addition, since PKCδ was un-regulated, as shown by microarray and confirmed by RT-PCR, in L. monocytogenes infected C/EBPβ-/- macrophages, it was therefore thought to play a detrimental role during L. monocytogenes. However, since this premise has never been investigated directly, the role PKCδ during innate immunity against L monocytogenes was examined using the PKCδ deficient (PKCδ-/-) mouse model. Data in this dissertation provides new insight into the role of PKCδ during innate immunity to L. monocytogenes. PKCδ-/- mice were highly susceptible to L. monocytogenes due to enhanced listerial escape and impaired listericidal activity. Despite full macrophage activation and production of nitric oxide, PKCδ-/- mice displayed uncontrolled bacterial growth and dissemination of L. monocytogenes, which led to early death of the mice. In contrast, PKCδ-/- mice were able to control Mycobacterium infection as well as WT mice, suggesting that the activity of PKCδ may be negatively regulated by L. monocytogenes. A systems biology approach generated the hypothesis that PKCδ may promote Rab5a activation, which together with localized release of superoxide into the phagosome and activation of C/EBPβ by PKCδ, resulted in the confinement of the L. monocytogenes within the phagosome. Alternatively, PKCδ may act in a separate pathway that confines L monocytogenes within the phagosome, by activating and/or synergizing with unidentified proteins to neutralize that activity of listerial LLO and PI-PLC. Data in this dissertation clearly demonstrates that PKCδ is critical for confinement of L monocytogenes within phagosomes and may be part of a listericidal mechanism that is independent or nitric oxide, superoxide and pro-inflammatory cytokines.
Open Access
Immortalisation, characterisation and differentiation of temperature sensitive cell lines from the Olfactory Neuroepithelium
(1999) Boolay, Sihaam; Illing, Nicola
Embryonic olfactory neuroepithelium provides a useful experimental system for the study of olfactory neurogenesis. As a substrate for experimental neural cell biology, olfactory neuroepithelium is of unique interest since, unlike other neural cells, olfactory neurons are continually replaced - a feature that is dictated by their direct exposure to the damaging external environment. Basal cells in the olfactory placode are the source of this replacement. Each olfactory neuron expresses only one or a few of the many olfactory receptors that are encoded by the large array of olfactory genes. Despite this limited cellular display of receptors, vertebrates are able to distinguish many thousands of different odorants, implying a complicated need for perceptive neurological processing of signals coming from individual olfactory neurons. To study the events that take place during the differentiation of neuronal precursors - a process that sustains a diverse receptor repertoire - I felt that lines of conditionally immortalised cells that could be induced to differentiate would provide useful reagents. In this thesis I describe my successful attempts to immortalise olfactory cell lines from the neuroepithelium of E 10.5 mouse embryos. I used a conditionally immortalising retrovirus that included the coding sequence for the temperature-sensitive SY 40 large T antigen. Integration of this retrovirus into the genome of cells allowed continuous proliferation at the permissive temperature of 33°C. A shift to the nonpermissive temperature of 39°C inactivated the SV40 large T antigen, the cells ceased proliferation and differentiation commenced. Sixty cell lines were derived of which four were chosen for further characterisation. These four cell lines (OP6, OP27, OP47 and OP55) were clonally derived and were immortalised rather than transformed. They continued to express the SV40 large T antigen at 33°C but lost expression at 39°C concomitant with cessation of proliferation. When the OP cells were shifted to 39°C in the absence or presence of the morphogen, retinoic acid, morphological changes ensued that were consistent with the development of neuronal characteristics. The OP6, OP27 and the OP47 cells became phase-bright with neuritic extensions. The OP55 cells were the exception in that they did not develop extensions but instead differentiated to form compact epithelial islands when grown in DM-10 medium but not in RA medium. Differentiation of the OP cells at 39°C was further documented by the induced expression of a number of markers demonstrated by RT-PCR and/or immunocytochemistry. The OP cells differentiated at 39°C in DM-10 and in retinoic acid-containing medium to express olfactory receptor transcripts. Cloning and sequencing showed that each cell line expressed a single receptor type but that different receptors were expressed by different cell lines. Sequencing revealed that the receptors cloned from the OP27 cells were 98% homologous to the mouse-M65 olfactory receptor whereas OP55 had greatest homology to rat-Olf3 olfactory receptor. The transcripts induced in OP6 and the OP47 cells showed greatest homology with Gus58 - a taste receptor homologous to olfactory receptors. Sequences obtained from OP6, OP47 and OP55 cells were not 100% identical to published receptors and could thus represent members of different subfamilies. Interestingly, induced OP55 cells also expressed mRNA for clusterin - a molecule that has no homology with olfactory receptor transcripts but is involved in differentiation during embryogenesis.
Open Access
Immunochemical studies with tryptic peptides of tobacco mosaic virus protein
(1979) Milton, Raymond Cecil de Lisle; Van Regenmortel, M H V
The antigenic determinants of the protein subunit of tobacco mosaic virus CTMV) have been studied by inhibition of complement fixation, inhibition of micro-precipitin and direct binding experiments. The viral subunit has been found to possess six antigenic determinants. Two of these, situated in tryptic peptide I (residues 1-41), were found to be a cryptotope (i.e. an antigenic determinant absent from the outer surface of the assembled viral capsid) and a viral antigenic determinant. Tryptic peptides 4 (residues 62-68) and 8(residues 93-112) also contained cryptotopes which were situated in the region of residues 63-65 and 108-112 respectively. Tryptic peptide 12 (residues 142-158) contained both a cryptotope and a neotope (i.e. an antigenic determinant which is only expressed on the outer surface of the viral capsid), which are situated in the C-terminal region of the polypeptide chain of the TMV protein, residue156 being associated with the cryptotope and residue 158 with the neotope. No antigenic activity could be demonstrated in tryptic peptide I I (residues 135-141 ). When the results are analyzed in terms of the three-dimensional structure of the viral subunit, it appears that all the antigenic reactive regions occupy highly accessible locations on the surface of the protein.
Open Access
The impact of global and local composition on the stability of Triple Helical DNA
(1993) Völker,Jens; Klump, Horst H
It is common practise in antisense technology to view third strand binding to be controlled by the same principles which are found to determine the stability of the double helix. In contrast to this view based on a general consideration of the various forces contributing to the binding energy of the third strand it was proposed that the dominant contributions will originate from electrostatic interactions. These electrostatic contributions can be subdivided into sequence independent repulsive forces between the negatively charged backbones and into sequence dependent attractive forces between the positively charged protonated Hoogsteen cytosines and the backbone phosphates. The observable changes in the stability of triple helices should be a reflection of the number (global composition) and distribution (local composition) of cytosines in the third strand. To this aim two families of 38-mer oligonucleotides were synthesized, which have as a common design feature a linear array of 10 homopurine bases followed by 10 homopyrimidine bases as Watson & Crick complementary strand to the homopurine region and ending in a 10 homopyrimidine residue stretch which binds to the W&C helix via Hoogsteen base-pairing. This arrangement of homopurine and homopyrimidine sections with connecting pyrimidine linkers allows the formation of intramolecular triple helices of predetermined stoichiometry and strand orientation. Physical (UV-spectroscopy, CD-spectroscopy and fluorimetry) and biochemical techniques (P1-nuclease digestion) have been used to show that the oligonucleotides undergo a stepwise folding process from a random coil into a hairpin with 3'dangling tail and then into a intramolecular triple helix. This folding occurs as a function of pH and/or ionic strength. The effect of local and global composition on the stability of the three conformational transitions has been evaluated from a comparison of the melting temperatures and the behavior of the phase boundaries of the different oligonucleotides. As the result of this thesis the following general rules emerge: The stability of the third strand depends on the particular combination of sequence, pH and ionic strength. At physiological conditions (pH 7.1, 150 mM Na⁺) thymines and cytosines contribute equally to the stability (global effect) provided that the cytosines are spaced by more than one thymine. (local effect). Below pH 7.1 (150 mM Na⁺) the stability increases linearly with the number of cytosines and at pH above pH 7.1 ( 150 mM Na⁺) it decreases. At ionic strength below 400 mM Na⁺ (pH 6. 75) the stability increases with the number of cytosine while above 400 mM Na⁺ (pH 6. 75) it decreases. Based on these results a rational approach for the design of oligonucleotide third strands and the choice of appropriate environmental conditions for the formation of a particular triple helix becomes feasible.
Open Access
Investigating the mechanisms of desiccation tolerance in the resurrection plant, myrothamnus flabellifolius (WELW)
(1999) Koonjul, Priyum K; Lindsey, George G; Brandt, Wolf F; Farrant, Jill M
Resurrection plants, including Myrothamnus flabellifolius, grow in shallow soil upon rocky outcrops where they experience regular periods of water stress. Associated with this is light stress. The presence of light under water limiting conditions can result in photo-oxidation which causes damage to plant tissues. M flabellifolius is a homoichlorophyllous plant and thus retains chlorophyll during desiccation. The mechanisms whereby this plant prevents photo-oxidation damage are not known and thus one of the objectives of this study was to characterise the chloroplasts and the changes they undergo during dehydration. It was shown that chloroplasts from M flabellifolius could only be isolated using trehalose gradients (instead of sucrose gradients) and were found to have a higher buoyant density than chloroplasts isolated from another resurrection plant, Craterostigma wilmsii. The latter had the same buoyant density as those isolated from the desiccation sensitive plant Pisum sativum. The increased buoyant density in M flabellifolius was ascribed to the unusual ultrastructure of the thylakoid membranes. The latter have a staggered conformation (staircase arrangement) rather than the discrete granal and intergranal conformation found in most plants.