Academic literature on the topic 'Fields of Research – 270000 Biological Sciences – 270100 Biochemistry and Cell Biology'

The relative contributions of the three main intermediates of indole-3-acetic acid (IAA) biosynthesis from L-tryptophan (L-Trp); indole-3-pyruvate (IPyA), tryptamine (TNH2) and indole-3-acetaldoxime (IAOX), were investigated in vivo in tomato shoots. Initially, L-Trp, D-Trp, IPyA, TNH2 and IAA were purified from shoots, identified by full-scan mass spectrometry and their concentrations measured using gas chromatography with an electron capture detector. High specific activity [5-3H]IAOX and [5-3H]IPyA were synthesized from L-[5-3H]Trp and used as internal standards. Purification of endogenous IPyA was enabled by forming a stable pentafluorobenzyl oxime derivative in the crude plant extract. The respective endogenous concentrations of L-Trp, D-Trp, TNH2, IPyA and IAA were found to be 2,520, 103, 146.3, 5.9 and 8.5 ng g-1 f. wt. However, IAOX could not be identified as a natural constituent of tomato shoots by full-scan GC-MS. Secondly, incubation of tomato shoots for 6, 10 and 21 h in 30% 2H2O was used as a means of labelling IAA and its putative precursors in vivo. L-Trp, D-Trp, TNH2, IPyA and IAA were then extracted and purified and the 2H content measured by combined gas chromatography-mass spectrometry. These indole compounds were labelled rapidly with up to four 2H atoms. Direct comparison of the number and the amount of 2H atoms incorporated (pattern) was obtained from the mass spectral data on the common m/z 130 ion and its isotope peaks. IAA and L-Trp demonstrated an increase in 2H label with up to 17% and 21% of their molecules labelled at 10 h respectively. This was followed by a significant decrease in 2H label at 21 h to 12% for both L-Trp and IAA. This decrease in 2H label was attributed to an increase in protein catabolism, following shoot excision, resulting in the dilution of free L-Trp pool(s) with unlabelled L-Trp from which IAA is biosynthesized. This is reflected in the observed 1.6 to 1.8 fold increase of free L-Trp from 10 to 21 h. In contrast, tryptamine demonstrated a continual increase in 2H label with an average of 8, 20 and 28% of the molecules labelled at 6, 10 and 21 h respectively, suggesting that TNH2 and IAA were synthesized from separate Trp pools. In addition, the relatively slow rate at which 2H is incorporated into tryptamine would not be sufficient to account for the rate at which IAA becomes labelled. However, IPyA demonstrated a rapid increase in 2H with 22% and 37% of its molecules labelled at 6 and 10 h respectively. From the rate at which IPyA was labelled with 2H and the concentration of IPyA in tomato shoots a rate of synthesis for IPyA in tomato shoots was estimated which was sufficient to provide most of the shoot IAA requirements. Furthermore, the extent to which IAA and IPyA were labelled relative to that of total L-Trp would imply that a smaller more rapidly metabolised pool(s) of L-Trp was the precursor of these compounds. The rate and extent that D-Trp was labelled was consistently less than that of IAA precluding it as a possible precursor of IAA. These results indicate that in tomato shoots IAA is biosynthesized from a rapidly metabolized sub-pool(s) of L-trptophan predominantly via IPyA.

Aluminium (Al) toxicity to plants is a significant problem, limiting agricultural production in up to 40% of the world's arable soils. In spite of a large amount of research, there is still no consensus on the physiological mechanisms of Al toxicity in plants. In addition, very little is known about the molecular response of plants to Al stress. This body of research was aimed at identifying the changes in gene expression that occurred in the root tips of plants that had been stressed with Al. A cDNA library made from the root tips of Al-treated wheat (Triticum aestivum L., cultivar Warigal) plants was differentially screened to identify clones whose expression was induced by Al stress. Seven cDNA clones, representing five different genes were identified as being induced in the presence of Al. Initial sequencing and northern analysis revealed that none of the clones isolated were full-length, and that some contained multiple cloning adaptors at their 5' ends. A new cDNA library was then constructed from the root tips of Al-treated Warigal plants, and homologues to each of the original five genes were isolated. These five clones were named wali1 to wali5 (for wheat aluminium induced). Northern analysis showed that wali1, -3 and -5 were induced 24 to 96 h after Al treatment, and their expression declined when the Al was removed. wali4 had a similar pattern of expression with a transient increase in expression also observed after 0.5 h of Al stress. Each of these four genes was induced by inhibitory concentrations of Al in two wheat cultivars - Warigal, an Al-sensitive cultivar, and Waalt, an Al-tolerant cultivar, - and also in two inbred lines of wheat, RR (Al-tolerant) and SS (Al-sensitive). The fifth gene (wali2) had a bimodal pattern of induction, and was induced by Al only in the Al-sensitive Warigal and the Al-tolerant RR. The nucleotide sequence of each of the wali clones was determined, and the databases were searched for homologous sequences. Wali1 was found to be homologous to a group of metallothionein-like proteins (MLPs) from plants, and wali4 was homologous to phenylalanine ammonia-lyase (PAL). wali3 and wali5 encode related, cysteine-rich proteins with homology to Bowman-Birk proteinase inhibitors, and wali2 encodes a novel protein with a repeating motif of cysteine amino acids. The induction of the wali genes was investigated in response to a number of other stresses through northern analysis. The expression of wali1, -3, -4 and -5 was induced in root tips of wheat after 2 d treatments with toxic levels of all other metals tested (Cd, Fe, Zn, Cu, Ga, In and La). The expression levels of wali1, -3, -4, and -5 also increased in the root tips of plants grown in the presence of low levels of Ca (10μM). The transcript levels of wali1, -3 and -5 increased in wounded leaf and root tissue, whereas the transcript levels of wali4 increased only in wounded leaves. The expression of wali2 was greatly reduced by low concentrations of Ca, and showed no induction, or a variable response with most of the other treatments. The site of expression of wali1, -2, -3 and -5 in root tips (and wali1 also in leaf tissue) was identified using in situ hybridisation. Wali1 was expressed predominantly in the meristematic tissue of the root tip, while wali3 and wali5 were expressed predominantly in the cortical tissue of the root. wali2 expression was detected primarily in the epidermis and root cap. Some changes in the site of expression of these genes were evident in the roots of Al-treated plants. In leaf tissue, wali1 expression was found in the mesophyl1 layer of cells. The coding sequences for wali1,-2,-3 and -5 were each cloned into the bacterial expression vector pGEX-2T. The resultant fusion proteins between glutathione S-transferase (GST) and the walis were then successfully purified from E coli. Antibodies were made to the wali1-GST fusion protein and purified by immunoaffinity chromatography. However, when used in western analysis, no specific bands corresponding to the native wali1 protein were identified. The wali2-GST protein was used in a south-western procedure to determine if the protein was capable of binding DNA, but no DNA binding to this protein was detected under the conditions tested. The wali3 and wali5 fusion proteins were tested in proteinase inhibitor assays, where no inhibition of either trypsin or chymotrypsin was detected. It is possible that the native wali3 and wali5 proteins may not function as proteinase inhibitors, or that the lack of activity detected for the fusion proteins may be due to incorrect folding or processing in the bacterial system. This research constitutes the first identification of plant genes whose expression is increased by Al stress. The genes identified are also induced in response to other environmental and nutrient stresses, indicating that they form part of the plant's general response to stress.
Appendix 4 restricted.

This project involved the study of Lewis and related blood group glycosphingolipid isolated from individuals with normal and aberrant Lewis/secretor phenotypes. The objective was to find a biochemical basis for the unusual expression of Lewis and secretor phenotypes in Polynesians and to use this information to shed light on the "normal" expression of Lewis antigens. By using purified glycolipids, presenting them in the cell free environment of thin layer chromatography to Lewis antibodies and by determining structures by mass spectrometry it has been shown that: l. The Lec epitope is a terminal Galβ1-3Gal sequence, and not an internal branch as proposed by Hanfland (Hanfland et a1.,1986). 2. Lec or H-5-1 are present in Lewis negative phenotypes and their consequent consumption by the Le and ,Se transferases resulting in the known Lea and Leb antigens can be seen in the Lewis positives. 3. Phenotypically Le(a-b-) individuals have small amounts of Lewis antigens. This clearly demonstrates that although the Lewis negative phenotype exists at the crude serological level, this phenotype is not an "all-or-nothing" phenomenon at the chemical level. This also allows it to be postulated that the le gene is probably partially active. 4. Le(a+b+) individuals have both Lea and Leb glycolipids in the erythrocyte membrane and in plasma. Observed phenotyping anomalies appear to be related to there being quantitatively less Leb-6 in the Polynesian Le(a+b+) erythrocyte membrane than in the Le(a-b+) membrane. 5. The Le(a+b-) phenotype of Polynesians is actually the Le(a+b+) phenotype but with serologically undetectable Leb. This allows it to be postulated that the nonsecretor gene (se) is absent in Polynesians. 6. Extended structures are present in most of the Polynesian samples which is in support of a postulated weak secretor gene (Sew). It now appears that the difference between the extended Lewis glycolipids of Caucasians and Polynesians is quantitative. The postulated, Sew transferase appears to be inefficient and allows for increased formation of elongated glycoconjugates (polyglycosylceramides) to result. 7. Reduced fucosyltransferase activity allows increased elongation of the precursor chain to occur, which allows it to be postulated that fucosylation of the precursor prevents, or at least markedly reduces, chain elongation. It is speculated that, as almost everyone is either Lewis and/or secretor positive, perhaps the prevention of chain elongation is a biological reason as to why the Lewis and Secretor polymorphisms exist. 8. Differences in ceramide patterns of Lewis active glycolipids suggests that the small intestinal tract is not the only origin of plasma glycolipids, or there is differential absorption. 9. There is no plasma glycolipid-based reason for there being increased H type 2 antigen reactivity in the Polynesian erythrocyte membrane, nor a reason for the H antigen association with the Le(a+b+) phenotype.

Glycoprotein II (GpII) is a glycoprotein isolated from the membranes of chromaffin granules in the adrenal medulla. The chromaffin granules of the adrenal medulla are responsible for the biosynthesis, storage and secretion of catecholamines, neuropeptides and various proteins. The abundance of chromaffin granules makes them an excellent model to further study the organelles specialized in the synthesis and secretion of hormones and neurotransmitters, from both endocrine and synaptic vesicles. When viewed by two-dimensional electrophoresis GpII is a heterogeneoug glycoprotein (80000-100000 daltons) consisting of two components, upper (GpIIa) and lower (GpIIb). Protein and immunological characterisation revealed that the two components of GpII are distinct from each other. Further molecular characterisation of GpIIa showed that it consisted of a 1224 base pair cDNA, encoding a 383 amino acid polypeptide, with a calculated molecular mass of 40500 daltons. This characterisation also revealed 19 potential glycosylation sites, with deglycosylation studies further demonstrating that an estimated 55% of the molecular mass of GpII is asparagine-linked carbohydrate. The presence of poly-N-acetyllactosamine carbohydrate groups on GpII was also demonstrated. The predicted amino acid sequence of GpIIa shares a 72% amino acid identity with the human lamp-l type protein, which belongs to a highly conserved group of lysosomal associated membrane glycoproteins (lamp proteins). As the C-terminal region of GpIIa was identical to the C-terminal region of lamp proteins, a synthetic peptide to the C-terminal region of GpII was used to raise antisera to this region. This antisera was then used to demonstrate that the C-terminal region of GpII wag present on chromaffin granules and facing the cytoplasm. This same C-terminal region on lamp proteins is known to be a cytoplasmic tail that is essential for the intracellular targeting of lamp proteins to the lysosome. A common feature to the pathways of both GpII and lamp proteins is their appearance on the cell surface followed by internalisation via endocytosis. As endocytosis of the chromaffin granule membrane is known to occur, the possibility that GpII was a marker for endocytosis was investigated. The presence of GpII within clathrin coated vesicles was shown, and evidence was presented to demonstrate that the C-terminal region of GpII interacts with adaptor proteins of clathrin coated vesicles. Adaptor proteins are known to mediate between the cytoplasmic domain of proteins internalised by endocytosis, and the outside coat of clathrin. The evidence presented would suggest that the cytoplasmic tail of GpII is a potential marker for endocytosis within the chromaffin granule membrane. The results presented in this study indicate that GpII is the secretory granule and species counterpart to the lamp proteins. This finding however raises some interesting questions regarding intracellular targeting between the lysosomes and secretory granules within the chromaffin cell.

Gonadotrophic hormones are heterodimeric glycoproteins and their N-glycans attached to specific amino acid residues are currently thought to play important roles in hormonal biosynthesis, secretion and function. The studies reported in this thesis aimed at isolation and characterisation of structural properties of the N-glycans on ovine and human luteinizing hormones. Initially, chromatographic methods were developed using reverse-phase HPLC for the analytical separation of the three human pituitary glycoprotein hormones and their subunits. Separation of intact oLH and its subunits was also effected by a single HPLC step. A preparative procedure was developed for the efficient purification of hLH and hTSH from crude human pituitary extracts using hydrophobic chromatography which gave highly purified hormones in good yields and with high biological activities. This method did not significantly influence the hormones' extensive charge heterogeneity and it offered potential advantages in the characterisation of their carbohydrate structures. A preparative scheme was developed for the isolation of the N-linked oligosaccharides from each glycosylation site of o- and hLH. Charge heterogeneity of oligosaccharides, which were released by hydrazinolysis from subunits and glycopeptides, was characterised by anion-exchange HPLC. 1H-NMR analysis showed that the structures of all three N-glycans on hLH were highly heterogeneous but mainly diantennary complex-type, with site-specific patterns of terminal sialylation and sulphation as well as core-fucosylation. Sulphated/sialylated and/or disialylated oligosaccharides were the major components at each site. A set of new mono- and disialylated oligosaccharides with the terminal sequence NeuAcα2-6GalNAcβ1-4G1-cNAcβ1-2Manα1-3 was identified. This finding suggested unique site-specific terminal sialylation of oligosaccharides at Asn 78 (hLHα) by an unknown α2-6 sialyltransferase(s) in the human pituitary gonadotroph cell. Each glycosylation site in oLH had a distinct set of oligosaccharides ranging from mainly monosulphated hybrid-type at the two sites of oLHα to predominantly disulphated diantennary complex-type on oLHβ. Core-fucosylation also differed at each site. These results suggested that processing of the oligosaccharides of the α- and β-subunits by α-mannosidase II and α1-6 fucosyltransferase was differently regulated by protein structure in oLH. Whereas hCG, hLH and oLH share similar biological activities, no apparent relationship between their N-glycan structures was found, which suggested that specific branching and peripheral structures of N-glycans on LH and hCG may not be essential for biological function, although the N-glycan nearer the N-terminus of the α-subunit of hCG has been implicated in hormonal activity.

Research in this thesis describes the characterisation of mRNA sequences coding for actinidin, a cysteine protease found in abundance in the fruit of kiwifruit (Actinidia deliciosa). The first step in the characterisation required the isolation of mRNA from ripe kiwifruit tissue. The suitability of a number of RNA extraction procedures was investigated. The method finally adopted differed from that used for unripe fruit tissue, and was chosen as a result of the nature of the polysaccharide that contaminated nucleic acids prepared from extracts of kiwifruit fruit tissue. RNA extracted from ripe fruit was used to synthesis a partial cDNA library and clones for actinidin were isolated. A number of these cDNA clones were sequenced; three clones were almost full-length. The actinidin cDNA clones obtained fall into two broad sequence classes. The majority of them encode acidic proteins (pI˜4.7), with 97% homology to the published amino acid sequence of actinidin. The second class encode basic proteins (pI˜8.1), with 83% homology to the published amino acid sequence of actinidin. Both classes of actinidin cDNA sequence encode zymogens, which contain N- and C-terminal extensions not present in the mature form of the enzyme. The N-terminal extension of both sequence classes includes a putative signal peptide. Northern hybridization analysis was used to investigate the tissue specificity of actinidin mRNA expression, and the expression of mRNA for the two actinidin sequence classes during fruit ripening. Both actinidin sequence classes were expressed differentially during the latter stages of kiwifruit fruit development and through post-harvest fruit ripening. The expression of both sequence classes increased from just prior to fruit maturity through ripening and reached a maximum as fruit attained the stage of 'eating' ripeness. The level of expression of the sequences encoding acidic actinidin reached a plateau at this point, while the expression of the sequence encoding basic actinidin appeared to decrease slightly as fruit continued to ripen. The sequences encoding acidic actinidin were expressed during ripening at a much higher level than those encoding basic actinidin. No actinidin mRNA was detected in other tissues except for very low levels of the acidic form in kiwifruit leaf, and low levels of the basic form in senescing petals. A full-length, acidic, actinidin cDNA sequence was introduced into tobacco (Nicotiana tabacum) plants via Agrobacterium tumefaciens-mediated transformation. Using the binary vector pGA643, the sequence was introduced in both the sense and antisense orientation relative to the cauliflower mosaic virus 35S promoter and transgenic plants were obtained for both sequence orientations. The presence of the T-DNA cassette (containing the actinidin sequence) in the plant genomes was determined using PCR analysis, and confirmed by Southern hybridization. A number of the transgenic plants contained multiple insertions of the actinidin sequence, and most plants contained at least one intact copy of the T-DNA cassette. The transcription of the introduced actinidin sequence was investigated by Northern hybridization analysis. All of the plants containing actinidin in the sense orientation, and some of those incorporating the antisense construct, transcribed the actinidin sequence. Attempts to detect actinidin protein in the transgenic plants were unsuccessful. Acidic actinidin was identified as one of the most abundant bands in the total protein profile from ripe kiwifruit fruit tissue. The identity of the protein was confirmed by N-terminal sequence analysis. The electrophoretic mobility of actinidin, both in the total cell homogenate and when partially purified, suggested that the first step in post-translational processing of the zymogen may be the removal of the N-terminal extension. Actinidin was also partially purified and used to raise antibodies. Poor specificity of the antibody for actinidin led to preliminary evidence for the glycosylation of actinidin.

The work in this Thesis concerns the replication control system of the P307 plasmid RepFIB replicon. The basic replicon occupies ≈ 1.6kb of DNA and contains a single large open reading frame (repA) flanked on either side by a series DNA repeat elements. The organisational structure of fie replicon has placed RepFIB into the Step function class of replicons. The placement of RepFIB within this group, as well as a strong homology between RepFIB and mini-P1, has resulted in a series of predictions concerning the control elements of RepFIB replication. The aim of this work was to test some of these predictions and to characterise the fundamental control elements utilised by the replicon. This Thesis describes three different active promoter elements found embedded within the repeat elements flanking repA. Although the functional significance of two of the promoter is unknown (orip and EFp), the third is responsible for the expression of RepA and has been designated 'repAp'. All three promoters are sensitive to RepA in trans, demonstrating that repA is autoregulated and that RepA is a DNA-binding protein capable of recognising copies of the repeat elements. RepA DNA-binding has also been demonstrated in vitro using a modification of the Western analysis technique (referred to a 'Western-DNA') in order to complement the in vivo experimental results. Although the coding region of repA had been determined in earlier work, the identification of the translational start codon was uncertain. This uncertainty has been resolved by limited N-terminal sequence analysis of a RepA:β-galactosidase fusion protein which has demonstrated that translation begins from a CTG codon located upstream of the predicted start sites. Finally, a series of genetic experiments have been used to determine the functional significance of RepA binding to the repeat elements. The repeat group upstream of repA are involved in autoregulation and also form part of the origin of replication, whilst the downstream repeats appear to be involved in the sensing and setting of plasmid copy number. Although the work presented in this Thesis does not directly test the applicability for RepFIB of various control models proposed to explain the behaviour of Step function replicons, the nature and type of control elements identified in RepFIB support the placement of RepFIB within the Step function class. As a result of this work, it is clear that RepFIB is confronted by the same kind of control paradox faced by replicons such as mini-P1 and mini-F. All three replicons use autoregulation and titration to control the supply of initiator protein required for replication. However, concurrent autoregulation and titration appear to be incompatible in current control models, and the identification of both mechanisms in these replicons has lead to a control paradox. Some of the results presented here suggest potentially valuable avenues of future research which may help resolve the paradox faced by RepFIB and other Step function replicons.

A structural relationship between gonadotropic hormones and certain types of enzymes has been suggested in previous studies and an investigation into the possibility of enzymatic activity associated with the gonadotropic hormones has been the primary focus of the research presented in this thesis. Partial sequence homology between human chorionic gonadotropin (hCG) and α-chymotrypsin prompted the recent proposal of a tertiary structure of hCG using α-chymotrypsin as a folding template, which suggested the possibility of intrinsic peptidase activity associated with hCG. Highly purified hCG (CR127) was assayed for enzymatic activity against a range of synthetic peptide substrates and was found to exhibit Arg-specific peptidase activity. This activity was almost completely inhibited by diisopropylfluorophosphate (DFP), soybean trypsin inhibitor (STI), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and to a lesser extent by N-α-p-tosyl-L-lysine chloromethyl ketone (TLCK), which indicated that the observed protease activity was serine protease-like. To establish whether this activity was intrinsic to the hormone or due to contaminants, extensive purification procedures were carried out. Hydrophobic interaction chromatography (HIC) and soybean trypsin inhibitor-affinity chromatography were found to effectively separate the protease activity from the hormone, indicating the presence of exogenous protease contaminants in the highly purified preparation of hCG. Further analysis by [3H]-DFP labelling of hCG and SDS-PAGE of the isolated contaminants revealed the presence of possible serine proteases with apparent molecular masses of 60 and 20 kD. Because serine proteases are known to stimulate cAMP production in the same target cells, it was necessary to determine the effects of the contaminating proteases on the receptor binding of hCG and cAMP production. The presence of these contaminants was found to have no apparent effect on the receptor binding capability of hCG, however the in vitro biological activity of hCG as determined by maximal cAMP production was decreased after HIC-HPLC purification of the hormone. These observations suggested that the serine protease-like contaminants contributed to the total cAMP production, thereby introducing significant error in biological assays that use hCG (CR127). The possible intrinsic enzymatic activity of hCG against its receptor as a natural substrate was further investigated. A membrane-bound receptor preparation was isolated from porcine ovaries and a receptor binding assay successfully established. The effects of hCG binding upon the membrane-bound receptor were studied and receptor proteolysis was observed. However, this proteolysis could not be definitively attributed to the actions of hCG. A purified receptor was subsequently prepared by hCG-affinity chromatography and analysed by SDS-PAGE with detection by autoradiography and silver staining. The purified receptor was found to have undergone proteolysis during the purification procedure, presumably following incubation with the hCG affinity matrix. Recent reports of the presence of homologous amino acid sequences in the active site of thioredoxin and the β-subunit of the gonadotropic hormones luteinizing hormone (LH) and follicle stimulating hormone (FSH), and subsequent demonstration of thioredoxin-like activity associated with these hormones, prompted an investigation into the possibility of thioredoxin-like activity associated with hCG. LH, FSH and hCG were all assayed for their ability to promote reactivation of reduced and denatured RNase. Although LH was shown to be capable of reactivating reduced RNase, the level of activity detected was significantly lower than that previously reported, whereas FSH and hCG were not found to be capable of this thioredoxin-like activity. These results suggested that the previously reported thioredoxin-like activity may be due to contamination of the hormone preparation, by the ubiquitous enzyme thioredoxin. The possibility of LH possessing intrinsic dithiol-disulphide interchange activity was investigated further using [3H]-iodoacetic acid. RNase/LH were incubated in an attempt to quench a dithiol intermediate. Preliminary results suggested that the presence of LH in this reaction increased the amount of protein radiolabelled, however, the isolation of a radiolabelled dithiol intermediate which could be conclusively identified as LH was not forthcoming. Furthermore the lack of RNase reactivation activity in hCG, suggests that the putative thioredoxin-like activity of LH, if intrinsic, may not be involved in receptor activation and/or signal transduction, as hCG and LH share the same receptor and should therefore have a similar mechanism of activation.

A system has been developed to aid in the establishment of Agrobacterium-mediated transformation for new plant species. A series of binary vectors have been constructed that express a chimaeric β-D-glucuronidase (GUS) gene in plants cells but not in bacterial cells. This feature allows GUS activity from transformed plant cells to be assayed in the presence of Agrobacterium. Preliminary experiments examined the expression of these chimaeric GUS genes in transformed petunia leaf discs. GUS expression was detectable 2 days after inoculation, peaked at 3 – 4 days and then declined; if selection was imposed expression increased again after 10 - 14 days. The amount of expression observed 4 days after inoculation correlated well with stable integration as measured by kanamycin resistance, hormone independence, and gall formation. Histochemical staining of inoculated leaf discs confirmed the transient peak of GUS expression 3 - 4 days after inoculation. Surprisingly, GUS expression was concentrated in localized zones on the circumference of the disc; within these zones essentially all the cells appeared to be expressing GUS. These results suggest that the frequency of gene transfer from Agrobacterium is extremely high within localized regions of the petunia leaf explants, but that the frequency of stable integration is several orders of magnitude lower. A reliable Agrobacterium-mediated transformation system has been established for kiwifruit (Actinidia deliciosa var. deliciosa cv. Hayward) by using transient expression of GUS to monitor gene transfer frequencies. In vitro culture of kiwifruit plants and conditions for regeneration of plants from leaf discs have been established. Several factors were found to improve gene transfer frequencies in kiwifruit: (i) healthy actively growing source tissue; (ii) the use of Agrobacterium strain A281; (iii) the presence of a layer of moistened filter paper between the leaf explants and the cocultivation media; and (iv) the presence of 20 μM acetosyringone in both the bacterial culture media and in the cocultivation media. Pre-culture of leaf explants significantly inhibited gene transfer, particularly at the cut edge of the explants. Using the optimized transformation system, at least one transgenic plant can be regenerated from each leaf inoculated. Stable transformation frequencies have been shown to vary significantly between different binary vectors. Phenotypic, PCR, and Southern analysis has confirmed the presence of stably integrated T-DNA in several transgenic kiwifruit plants.

Lens transparency is primarily maintained by the anaerobic metabolism of glucose. Glucose is transported from the aqueous humuor to the lens epithelial cells however, it has not yet been established how glucose penetrates to the inner part of the lens. The core of the lens is acidic, approximately pH 6.5, as an effect of the accumulation of lactate, the end-product of glycolysis. This confirms that glucose is drawn deep into the core of the lens. Until recently it was assumed that glucose was transported to the core via a gap junction-mediated route by cell-cell diffusion. However, passive diffusion is limited in capacity and is unlikely to be sufficient for nutrient transport especially for larger lenses. Instead, an active circulation system has been proposed that has the potential to transport glucose deep into the lens via an extracellular route. This would imply that fibre cells may have evolved their own glucose uptake system, yet no direct evidence to this effect has been available. My thesis describes new molecular evidence that both epithelial and fibre cells have evolved their own glucose uptake system. The rat lens expresses the facilitative glucose transporters GLUTI and GLUT3 differentially. GLUTI is predominantly expressed in the epithelium while GLUT3 is predominantly expressed in the fibre cells of the lens. In the normal lens, this makes good physiological sense. GLUTI has a high Km suitable for situations of high glucose concentrations, as is the case for the epithelium where the aqueous humour mirrors glucose concentrations found in blood. GLUT3 has a lower Km and is particularly suitable for the fibre cells, where the supply of glucose from the tortuous extracellular space is limited. The discovery of GLUT3 in the fibre cells lends strong support for the existence of an active circulation system in the lens and makes it seem unlikely that glucose is transported into the core via gap junction-mediated diffusion. In the diabetic state, sorbitol - a product of glucose metabolism, occurs at elevated levels of about 30 times more than that of the normal, suggesting a significant increase in glucose uptake. This imposes an osmotic stress on the lens, which can be countered by regulated cell volume decrease only in the outer but not in the inner cortex. As a consequence inner cortex tissue breaks down and opacities result. My studies of the diabetic rat lens shows that the situation is made worse by an apparent up-regulation of GLUT3 in the fibre cells. Quantitative RT-PCR shows that the GLUT3 transcript is up-regulated six-fold during the initial weeks of diabetic insult in the streptozotocin rat model. The up-regulated GLUT3 protein is detected in the region where maximum tissue damage occurs. These results suggest a new mechanism for the initial tissue damage in the diabetic lens, whereby increased uptake of glucose leads to an over-production of sorbitol which causes osmotic stress on the fibre cells that is beyond their defense capability of regulated cell volume decrease. While my results described above have revolutionized our view of nutrient transport in the lens and its potential role in the early stages of diabetic cataract, the picture is only complete when the functionality of GLUT3 can be demonstrated. For this purpose, vesicles were prepared from isolated lens fibre cells and subjected to quantitative uptake studies using a fluorescent glucose derivative. Uptake was greatly reduced using the GLUT-specific inhibitor phloretin demonstrating that GLUT3 of the rat lens fibre cells is indeed fully functional. In summary, my results add a new dimension to our understanding of how the lens maintains homeostasis and tissue transparency. The lens has evolved an ingenious system to supply, nutrients to the core region to compensate for the absence of a vasculature. However, by achieving this, it has rendered itself unable to defend itself against the adverse effects of high glucose. My results also contribute towards developing new strategies of rational drug design to prevent or delay cataract.