Browsing by Author "Von Holt, Claus"
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- ItemOpen AccessThe amino acid sequence of chicken histone F3(1973) Brandt, Wolf Friedrich; Von Holt, ClausHistone F3 (III) from chicken erythrocytes was isolated by selective extraction from nucleoprotein with ethanolic-HCl and purified by a single gel filtration step. This protein was found to be homogeneous by the following criteria: gel filtration, electrophoretic mobility, N- and C-terminal amino acid residues and amino acid analysis. The primary structure of this histone was established without resorting to the use of overlapping sequences. This has been achieved with specific chemical cleavages rather than enzymatic degradations chosen and applied, first to the original protein chain, and subsequently to the generated polypeptides, to yield sets of not more than 3 peptides in any single cleavage. Their relative position in the protein or polypeptides became evident after comparison of the N- and C-terminal amino acids in the cleavage products and the uncleaved starting material. The simplicity of the peptide mixture after each cleavage, resulting in easy separation of the peptides, together with the highly efficient Edman degradation of automatic sequencing, allowed a rapid and relatively nonlaborious primary structure determination. Finally, the amino acid sequence is compared with those of protamines and other histones. The evolution and the structure of this protein in relation to DNA is briefly considered.
- ItemOpen AccessBinding sites in the nuclear envelope for cytoplasmic glucocorticoid receptor complex(1983) Smith, Peter John; Von Holt, ClausUsing 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.
- ItemOpen AccessThe effect of negative supercoiling on the formation and positioning of nucleosome cores in vitro(1991) Patterton, Hugh-George; Von Holt, ClausThe 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.
- ItemOpen AccessAn electrophoretic method for the isolation of isohistones from the embryo of the sea urchin Parechinus Angulosus(1981) Schwager, Sylva; Von Holt, ClausIt 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.
- ItemOpen AccessThe histone H1 of the sea urchin embryo, partial structures, enzymatic modifactions and developmental programme(1982) De Groot, Petronella Christina; Von Holt, Claus; Strickland, W NDevelopmental 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.
- ItemOpen AccessInsulin, added to nuclei, stimulates transcription of specific genes(1990) Stickells, Brenda Jane; Von Holt, ClausInsulin regulates cellular gene expression and modulates specific mRNA levels in liver cells. As yet, the mechanism of this control is still unclear. The effects are initiated following the binding of insulin to the plasma membrane receptor. Although several mediators of the signal from the plasma membrane to the nucleus have been proposed, none has proved capable of eliciting all of the effects of insulin on gene expression. Therefore, the possibility that insulin itself may directly regulate transcription at the level of the nucleus, was investigated.
- ItemOpen AccessIsolation and characterisation of histone transacetylases(1977) Thwaits, Bruce Hellier; Von Holt, ClausAcetylation, one of the post-synthetic modifications of histones, weakens histone-DNA interactions and may play a regulatory role in gene control of eukaryotes. The literature available on histone acetylation as well as other post-synthetic modifications of histone has been reviewed. Histone acetylation is catalysed by an enzyme(s) which transfers acetyl groups from a donor molecule to histones. A crude histone transacetylase preparation was isolated from nuclei and the optimal conditions for the acetylation of histones were determined. This enzyme(s) was shown to be specific for histones with protamine displaced histone complex being the best substrate. Using this histone transacetylase preparation ³H-acetyl total histone was prepared in sufficient yield and with a high enough specific activity to enable sequential Edman degradation of the histone sub-fractions isolated from the total histone complex to be undertaken. Histones H3 and H4 were isolated from the acetylated total histone as they exhibited the highest degree of acetylation. Histone H4 peptides were generated by chymotryptic and tryptic digestion as the intact histone H4 polypeptide chain is blocked at its N-terminus. The Edman degradations of histone H3 and H4 showed that the acetylation sites that have been determined under in vitro conditions are the same as those undergoing acetylation in vivo. All of the acetylation was found in the N-terminal region of histones H3 and H4 with histone H4 showing a gradient of decreasing acetylation from the N- to the C-terminus, in contrast to histone H3 where the first two possible acetylation sites are acetylated to a minor degree only.
- ItemOpen AccessThe isolation and characterization of a P. Angulosus homeobox(1991) Pfeffer, Peter Lance; Von Holt, ClausThe aim of this thesis was to isolate and characterize a homeobox-containing gene of the South African sea urchin Parechinus angulosus. This was achieved by constructing a genomic library of several individuals and screening this library using a probe containing the Antennapedia homeobox. Eight clones were isolated and shown to represent different alleles of the same gene. One clone was sequenced, revealing a homeobox which was termed PaHboxl. This homeobox was compared to published homeobox sequences and shown to be a member of the Antp (Hoxl.l) subclass (table 1.1). A splice donor site was identified 23 bp upstream of the homeobox and the observation confirmed by RNAase mapping. PaHboxl is situated in a genomic area showing a significantly higher degree of restriction fragment polymorphism than expected. This was shown by a statistical analysis which should be of general value in the interpretation of such polymorphisms. The expression of PaHboxl was examined by RNAase protection assays and Northern blotting. Two distinct phases of expression were observed - during embryogenesis PaHboxl is expressed transiently at low levels in 11,5 hr mesenchyme blastula stage embryos (44 ± 8 transcripts per embryo) with levels 3-5 fold lower 2,5 hr before and after this stage. Expression is observed again at up to 160 fold higher levels in the adult with maximal expression in testis (11 transcripts per 10 pg total RNA), and increasingly lower levels in intestines, ovary and Aristotle's lantern. Two transcripts of size 5,2 and 5,7 kbp were observed. Expression in Aristotle's lantern and embryonic stages could not be detected by Northern analysis.
- ItemOpen AccessPhysical mapping of an early sea urchin gene battery from Parenchinus angulosus(1988) Lawson, T N; Sewell, Bryan Trevor; Von Holt, ClausThe aim of this project was to characterise an early histone gene battery isolated from Parenchinus angulosus. An early histone gene battery (named H27) which was believed to have been isolated from Parenchinus angulosus, appeared by restriction enzyme mapping and partial sequencing to be identical to H22, an early histone gene battery isolated from Psammechinus miliaris. (This latter gene was obtained from M. Birnstiel.) This was further confirmed by electron microscopy, and proved to be a convenient testing ground for the electron microscopic techniques of denaturation mapping and heteroduplex anlysis. Another gene battery (named SU1) isolated fromParenchinus angulosus, was then characterised using the techniques developed whilst studying H27. The restriction enzyme map of this clone is different to that of H22, indicating that differences do indeed exist between these two early histone gene batteries. SU1 also showed the expected order of the five histone genes, as determined by hybridization against the coding regions of H22. The denaturation map of SU1 showed AT rich spacer regions and GC rich coding regions. Heteroduplex analysis indicated that the spacer regions between the Hl and H2A, the H2A and H3, and the H3 and the H2B gene coding areas are essentially nonhomologous. The H4 structural gene and corresponding spacer regions were not included in this analysis. Because it is known that all the five histones are coded for on the same strand of DNA in H22, and that each of the genes is transcribed in the same direction, it follows that, the same holds for, at least, the Hl, H2A, H3 and H2B genes of SU1.
- ItemOpen AccessPoly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris(1986) Retief, Jacques D.; Von Holt, ClausThis thesis investigates whether DNA and histones contain sufficient information to direct nucleosome cores into specific positions. The "in vitro" assembly of nucleosome cores promoted by poly(glutamic acid) has been optimized with respect to rate and yield. This was achieved by paying attention to the purity of the core constituents and in particular by the use of histones in their octameric form. The suitability of a number of octamer purification protocols, to produce pure undenatured histone octamers, has been investigated and the methodology improved. The particles assembled on random DNA have been found to be indistinguishable from native nucleosome cores by the following criteria: Their S value on sucrose gradient centrifugation, resistance to Micrococcal nuclease digestion, DNase I digestion patterns, DNase I digestion kinetics at the susceptible sites, electronmicroscopic appearance, hi stone content and electrophoretic mobility. Cores were also assembled on unique DNA, namely the intact h22 histone quintet of Psammechinus miliaris. Low resolution mapping, by indirect endlabelling of polycores assembled on the quintet, did not reveal any preferred sites of assembly. To investigate the core associated DNA at single base pair resolution, a series of fragments, excised from the H2A-Hl and the Hl-H4 spacer areas, were inserted into pGV403 plasmids. These plasmids can be strand specifically end-labelled with the Klenow fragment at the two different Tth 111 I excision sites utilised to isolate the propagated insert. On the free linearised DNA a complex digestion pattern is produced due to the sequence specificities of Micrococcal nuclease and DNase I. When cores are assembled on this DNA the digestion pattern is changed. This pattern reveals two preferential frames of assembly and indicates that in the remainder of the fragments cores are assembled, randomly, or in a number of overlapping frames. It is concluded that the DNA fragments investigated and the hi stone octamer contain enough structural information to influence the positions occupied by some nucleosome cores. The implications of these findings are discussed.
- ItemOpen AccessThe primary structure of histones H2B from sperm of the sea urchins Parechinus angulosus and Psammechinus miliaris(1978) Strickland, Marie Shields; Von Holt, ClausHistones H2B have been purified from sperm of the sea urchin Parechinus angulosus and Psammechinus miliaris. Three H2B variants have been completely sequenced from P. angulosus and two H2B variants from P. miliaris have been partially sequenced. The sequences of the sperm histones H2B are compared to the known sequences Of other H2B histones. The sea urchin sperm histones H2B are all considerably more arginine rich than other histones H2B and contain an extended amino-terminal region containing reiterated pentapeptides.
- ItemOpen AccessRat liver nuclear envelope insulin binding and its effects on endogenous protein kinases(1992) Sabbatini, G P; Sabbatini, G P; von Holt, C; Von Holt, ClausThe postulated model for the insulin - stimulated induction of mRNA efflux (Purrello et al., 1983) is based on the demonstrated binding of insulin to intracellular membrane structures (see chapter 2, section 2.2.1), and the in vitro effect of insulin on nuclear envelope phosphorylation, NTPase activity, and mRNA efflux (see chapter 5, section 5.1). These independent observations have led to the development of a model for the direct induction by insulin, at the level of the nucleus, of mRNA efflux (figure 1.1). However, the specific intracellular insulin binding has been inf erred from kinetic or morphological studies which have not identified a discrete membrane - bound polypeptide(s) as an insulin docking molecule in situ (Goldfine, 1981). Also, the stimulation of NTPase activity has only been established by monitoring the level of general ATP hydrolysis of nuclear envelope fractions in the presence and absence of insulin (Purrello et al., 1983). The scope of this thesis has been to further the understanding of this mechanism by attempting to a) unequivocally identify a specific nuclear envelope - associated insulin docking polypeptide in situ and b) to demonstrate that insulin directly affects the ATP - binding of nuclear envelope ATP - binding proteins. The latter would demonstrate a primary effect of insulin i.e. the modulation of the ATP - binding capacity of identified NTPases / protein kinases (or their release from some inactive storage form), and not a general phenomenon such as elevated ATP.
- ItemOpen AccessReceptor mediated targeting of liposomes(1990) Friede, M H; Von Holt, ClausThe targeting of liposomes to cells and the delivery of the liposomal contents into the cells have been investigated using either α-melanocyte stimulating hormone or Ricin-B-chain as ligands for promoting the binding of liposomes to cells. α-melanocyte stimulating hormone has been conjugated to liposomes and to Ricin-A-chain via the Lys₁₁ residue without significant loss of biological activity. The resulting conjugates were found to bind to Bl6 melanoma cells which express receptors for the hormone. Hormone targeted ricin was shown to be toxic to the cells, indicating receptor mediated internalisation of the conjugate. The hormone targeted liposomes however were unable to mediate the delivery of cytotoxic levels of methotrexate. Ricin-B-chain, a lectin which mediates membrane translocation of the toxic ricin-A-chain, was examined for its applicability for targeting of liposomes to cells. This lectin was shown to promote the binding of liposomes to cells and to mediate the delivery of cytotoxic concentrations of methotrexate. Further evidence of functional ricin-B-chain mediated intracellular delivery of the liposomal contents was shown by liposome mediated transformation of cells, and delivery of nuclease into the cell resultin in digestion of genomic DNA. The study demonstrates that α-melanocyte stimulating hormone is unsuitable as a ligand by which to achieve delivery of large quantities of material into cells, although cell-specific targeting can be achieved. Ricin-B-chain is however ideally suited for this task, though is less cell-specific. This finding may be of use in studies in which investigators wish to achieve intracellular delivery of compounds.
- ItemOpen AccessThe reconstitution of the histone octamer(1987) Greyling, H J; Von Holt, Claus; Sewell, Bryan TrevorThis thesis describes methodology for the reconstitution of the chicken erythrocyte octamer from acid-denatured histones or the natural H3-H4 tetramer and H2A-H2B dimers. Oligomeric properties of reconstituted octamers were elucidated during column chromatographic and chemical cross-linking studies. The conformational identity of the natural and reconstituted octamers was demonstrated by the ability of all preparations to crystallise as helical octamer tubes. The application of the reconstitution methodology in addressing fundamental problems of chromatin research, was demonstrated during subsequent studies, namely (i) The reconstitution of hybrid histone octamers containing a structural variant of a specific histone. These studies were undertaken to study the effect on histone-histone interactions in hybrid octamers of which erythrocyte H2B was substituted for by sea urchin sperm H2B(l) or erythrocyte H3 and H4 were substituted for by dethiolated H3 and sea urchin sperm H4 respectively. (ii) The reconstitution of an octamer suitable for the sitespecific derivatisation of a specific histone, or covalently labelled with aurothiomalate in a specific histone complex. These studies were concluded to represent general labelling strategies which may be of use in crystallographic or physico-chemical studies of nucleosome structure.
- ItemOpen AccessTAXI : a new vehicle for the transfer of genes into monocotyledonous plants(1996) Chen, Wusi; Von Holt, Claus; Thomson, Jennifer Ann; Klump, HHThe transfer of foreign genes into cereals followed by their correct expression in a tissue specific, developmentally regulated manner has become an important research focus. Based on the mechanism of Agrobacterium tumefaciens mediated transformation in dicots, a modified method to transform the monocotyledonous rye ( Secale cereale L. ) via A.tumefaciens mediated transformation was attempted. The induced bacterium culture was injected into rye seedlings, the transferred reporter gene, uid.A, was detected by PCR, and the expression of the gene was tested by histochemical assays. However, successful transformation and integration of the transgenes remained doubtful, because the frequency of kanamycin resistance in the progenies ( Rl , R2 and R3 ) did not increase. To achieve a real transformation and heritable transgenic rye, a new vehicle for gene transfer to plants was developed. A macromolecular complex, termed the TAXI, consisted of histone HI-protected single stranded DNA, containing a selectable marker gene (npt JI), linked either to a reporter gene (uidA) or a glutenin gene. The constructs were transferred by injection of rye seedlings. Molecular analyses demonstrated that all three genes were integrated and expressed in transformed rye and their progenies ( Rl and R2 ). TAXI mediated gene transfer to rye revealed an important advantage in that single or low numbers of transgenes were inserted into the transformed plant genome. However, the method of TAXI delivery to plants was not efficient. To improve this, a new approach, combining TAXI transformation and the biolistic process, was developed. A rapid regenerable callus line of a grass species, Digitaria sanguinalis, was established as a test system. TAXI coated gold particles, carrying a selectable marker gene (bar) and a reporter gene (uid.A), were used in bombardment experiments. The results of herbicide resistance and molecular analyses demonstrated that single copies or low numbers of the bar gene were inserted and expressed in regenerated transformed D. sanguinalis. Mendelian segregation in the Rl population was observed in four out of five transgenic lines.
- ItemOpen AccessThe primary structure of histone H2B from the mollusc Patella granatina(1978) Van Helden, Paul David; Von Holt, ClausHistones H2B were isolated from the gonads of a mollusc (Patella granatina) and from chicken (Gallus domesticus), crocodile (Crocodylus niloticus) and amphibian (Xenopus laevis) erythrocytes. The H2B's were purified by ion-exchange and gel exclusion chromatography. The complete primary structure of the mollusc hi stone H2B t 11 has been deduced from the sequences established of pa e a adjoining and overlapping peptides by the Edman degradation procedure. The partial structure of H2B from chicken erythrocytes (87 residues), crocodile erythrocytes (75 residues) and Xenopus erythrocytes (63 residues) was also established. The amino acid sequences are compared to those of other histones H2B. The effect of mutations on the predicted secondary structure of histone H2B is considered.