Browsing by Author "Klump, Horst H"

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    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.
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    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.
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    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.
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    Restoration of native folding of single-stranded DNA sequences through reverse mutations: an indication of a new epigenetic mechanism
    (Elsevier, 2006) Shepherd, Dionne N; Martin, Darren P; Varsani, Arvind; Thomson, Jennifer A; Rybicki, Edward P; Klump, Horst H
    We used in vivo (biological), in silico (computational structure prediction), and in vitro (model sequence folding) analyses of singlestranded DNA sequences to show that nucleic acid folding conservation is the selective principle behind a high-frequency single-nucleotide reversion observed in a three-nucleotide mutated motif of the Maize streak virus replication associated protein (Rep) gene. In silico and in vitro studies showed that the three-nucleotide mutation adversely aVected Rep nucleic acid folding, and that the single-nucleotide reversion [C(601)A] restored wild-type-like folding. In vivo support came from infecting maize with mutant viruses: those with Rep genes containing nucleotide changes predicted to restore a wild-type-like fold [A(601)/G(601)] preferentially accumulated over those predicted to fold diVerently [C(601)/T(601)], which frequently reverted to A(601) and displaced the original population. We propose that the selection of native nucleic acid folding is an epigenetic eVect, which might have broad implications in the evolution of plants and their viruses.
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    The triple-helical DNA four-way junction
    (1999) Makube, Neo; Klump, Horst H
    This Thesis will show that third strands can be incorporated into the four-way junctions combining the properties of the triple helices and those of branched structures within one system without compromising either of the two. It is now known that folding into secondary and tertiary structures by nucleic acids is crucial for their biological functions. However, what remain to be clarified are the mechanisms involved in the folding of nucleic acids into -noncanonical structures. It requires a thorough understanding of the chemical and physical properties of the structure in question. This in tum will improve the design of new secondary and tertiary structures that may add to the DNA nanotechnology. With this aim, thermodynamic and structural properties of two triple-helical DNA fourway junctions (JTIT3 and JT2T4) are reported and discussed. JTIT3 and JT2T4 differ only in the polarity of the third strands (and/or position of the loops). Both junctions contain the same Watson-Crick double-helical four-way junction, named Js, as a core structure. Js was constructed from four 20-mer oligomers, two of which consists of purines and the other two strands pyrimidines. Extending each of the pyrimidine strands of Js at the 3' end by four cytosines followed by twenty pyrimidine bases results in JTIT3. Similarly, JT2T4 is formed by extending the same pyrimidine strands at the 5' end. The junctions are named according to the position of the C4-loops. JTIT3 and JT2T4 are further simplified into JT1, JT2, JT3 and JT4. Lowering the pH from 12 to 2 allows the oligomers to fold sequentially from random coil into the double-helical four-way junction, Js, and finally into the triple-helical four-way junction. The analysis of the structures discussed is based on the biochemical methods such as native polyacrylamide gel electrophoresis and chemical footprinting using osmium tertroxide as a probe. The analysis is also based on the physical methods, UV spectroscopy and DSC. The native polyacrylamide gel electrophoresis has been used to verify the formation of the complete four-way junctions. Chemical footprinting has been used to detect the formation of the junctions as well as to indicate the conformations these junctions assume under different pH and/or salt conditions. The phase diagrams enthalpies and entropies of the structures are determined mainly by DSC. The results indicate that all the junctions are highly sensitive to salt concentrations and/or pH. The Tm vs. [Na+] results show that above 0.4M Na+, the structures adopt a conformation that suggests that the junctions are folded into stacked helix structures. The differences in thermal stabilities of the junctions JT1, JT2, JT3 and JT are due to the sequence composition of the arms and not the loops. JT1, JT2, JT3 and JT4 are thermally more stable than the underlying double-helical junction, Js. Similarly; the complete triple-helical four-way junctions JTIT3 and JT2T4 are thermally more stable than their substructures JT1, JT2, JT3 and JT4, The compiled transition enthalpies obtained for the individual arms of Js, JT1, JT2, JT3 and JT4 are less than the transition enthalpies associated with the melting of the complete junctions. The higher calorimetric enthalpies of the structures are due in part to the contribution from the single strands resulting from the partly unfolded arms. The overall results show that third strands have a stabilizing effect on the structure of the four-way junction.