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.

No description available.

Bibliography: leaves 93-98. / Histone lysine methylation is a post synthetic modification that occurs on the N-terminal tails of histones H3 and H4. This modification occurs at very specific residues, which have been highly conserved throughout evolution. It has been postulated that histone methylation may be involved in the pre-mitotic condensation of chromatin. Others have speculated that it may be involved in transcription. This modification seems to be a general feature of chromatin and occurs in all eukaryotic organisms ranging from mammals to yeast. The functional significance of histone methylation and of proteins in general is still at this stage not very well understood in the first part of this project the levels of histone lysine methylation in various tissues were investigated in order to establish a correlation between the methyl content and cell division, age and differentiation. It was found that the levels of histone lysine methylation were low in the two tissue culture lines that were investigated correlating with the rapid growth rate of these cells. An increase in the histone lysine methylation content was observed when these cells were induced to differentiate. The highest levels of histone lysine methylation were found in brain tissue. Methylation levels in the histones isolated from the mouse and bovine brain tissue are very similar. It was concluded from these results that histone lysine methylation may be linked to the cessation of replication and differentiation of cells. Previous studies on the rate of methylation in rat brain (Lee and Duerre, 1974) have shown that the rate of histone methylation decreases with age of the tissue. It was therefore decided to investigate the rate of histone methylation in nuclei isolated from mouse erythroleukemia (MEL) cells at different stages of differentiation. These cells can be induced to differentiate upon which they cease to replicate and mature into cells that are analogous to red blood cells. The rate of incorporation of radioactive methyl groups into the histones was determined in nuclei isolated from rapidly growing cells and cells that had been induced to differentiate for one and four days. It was established that only histones H3 and H4 are methylated at significant levels. The highest rate of histone methylation occurred in histone H3, and to a much lower degree, H4. This rate was maintained for the first day and decreased significantly after the fourth day after induction. Even after the fourth day after induction, the rate of histone H3 methylation remained significantly higher than that of the other histones. It was concluded that histone methylation proceeds for a substantial amount of time after all DNA replication has ceased. In the final part of this project the effect of the level of histone methylation on the stability of core particles, chromatin and nuclei was investigated. The core particles and chromatin, isolated from tissues with different levels of histone lysine methylation, were 'melted' using UV thermal denaturation. No differences were found. It was therefore concluded that histone lysine methylation did not increase the stability of the DNA of core particles or of soluble chromatin from these samples. Differential scanning calorimetry was used to study the denaturation of nuclei isolated from mouse brain and MEL cells that have high and low levels of histone lysine methylation respectively. The "melting profiles" of the two types of nuclei seem to indicate that structural differences exist. Due to the complex structure of a nucleus it is uncertain whether these differences could be ascribed to the differing levels of histone lysine methylation.

Bibliography: leaves 48-53. / LEA proteins are plant proteins that are characteristically hydrophilic and soluble at elevated temperature. The consistent correlation between desiccation tolerance in orthodox seed tissue and an accumulation of LEA proteins suggests that these proteins play an important role in protecting cells from desiccation induced damage. Yeast (Saccharomyces cerevisiae) has been known to desiccate as part of its normal growth cycle and to remain viable after long periods in the desiccated state. As a result of these properties this project was designed to investigate the presence of LEA-like proteins in yeast. A protein was isolated from baker’s yeast that fulfils the requirements for being a LEA protein. This protein, with a molecular mass of 11 kDa, was found to be the most prevalent heat soluble protein in the yeast extract. Antibodies raised against LEA group I proteins recognised this 11 kDa yeast protein in the total extract but failed to recognise the protein after heat treatment at 80°C for 10 min. Amino acid analysis showed that the ll kDa protein was highly hydrophilic - a characteristic of LEA proteins. The protein was partially sequenced (10 cycles) after CNBr digestion and the sequence obtained was compared with the sequence of known proteins in the Stanford databank. Only one protein, HSP 12, was identified to be 100 % homologous to the obtained sequence without the introduction of gaps. Despite a previous report that HSP 12 is a heat shock protein, HSP 12 was present in a reduced concentration in yeast grown at 37 °C compared with yeast grown at 30 °C. HSP 12 was found to increase in concentration after entry into stationary phase - a time when nutrients are limiting and the yeast is preparing to reduce its water content and sporulate. This might be considered equivalent to plant seed maturation - the stage when LEA proteins are synthesised. Moreover, growth conditions that have been reported to stimulate LEA protein biosynthesis in plants also stimulated HSP 12 synthesis in yeast. Purified HSP 12 was shown to inhibit thermal denaturation of yeast alcohol dehydrogenase (ADH) at elevated temperatures. This is a functional property of the pea seed p11 LEA group I protein. From the above results, it was therefore concluded that HSP 12 should be identified as a LEA-like protein rather than as a heat shock protein.

Bibliography: leaves 53-57. / This thesis investigates cell wall proteins, the presence of which increased in concentration as a result of stress. Two such proteins were found, phosphoglycerate mutase and Hsp 12. Studies on these proteins are reported in chapters 2 (phosphoglycerate mutase) and chapter 3 (Hsp 12).

Bibliography: leaves 116-120. / 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.

Includes bibliographical references. / 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.

Bibliography: p. 86-109. / 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.

No description available.

Biliography : leaves 162-184. / 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.