<p>Metagenomics has been successfully used to discover novel enzymes from uncultured microorganisms in the environment. In this study, metagenomic DNA from a Malawian hot spring soil sample was used to construct a fosmid library. This metagenomic library comprised of more than 10000 clones with an average insert size of 30 kb, representing more than 3.0 x 108 bp of metagenomic DNA (equivalent to approximately 100 bacterial genomes). The library was screened for cellulase activity using a Congo red plate assay to detect zones of carboxymethylcellulose hydrolysis. This yielded 15 positive fosmid clones, of which five were further characterised for activity and thermostability using the 3, 5-dinitrosalicylic assay. Two of the five fosmids (XP008C2 and XP026G5) were selected for DNA pyrosequencing. The full sequence of the XP008C2 (29800bp) fosmid insert is presented in this study and genes thereon were chosen for further study.</p>

As a consequence of safety issues encountered by the use of viral vectors in gene therapy, there has been a steady increase in the development and application of non-viral vectors, especially liposomes. Cationic liposome mediated delivery is one of the most promising nonviral delivery methods. These liposomes are prepared from synthetic lipids, are positively charged and interact favourably with DNA through electrostatic interactions. Cationic liposomes have also shown immense potential in the targeting of specific cell types such as HepG2 (hepatocellular carcinoma) cells, a model in vitro gene delivery system for the study of hepatocyte function. However, these liposomes also have a number of limitations in vivo. In an attempt to overcome these restrictions, a hydrophilic polymer, polyethylene glycol (PEG) is incorporated into the cationic liposome. This covalent attachment of (PEG) to the liposomal surface is thought to sterically stabilise liposomes, promote biological stability, inhibit aggregation, decrease toxicity and immunogenicity, prevent interaction with serum proteins and complement and thus prolonging the circulation time of liposomes in vivo. The versatility and simplicity of cationic liposomes have made them vitally significant non-viral gene delivery vehicles for human gene therapy. In this investigation novel untargeted and targeted glycosylated liposomes with and without PEG were synthesised to evaluate their gene transfer activities in vitro to potentially develop a suitable gene delivery system for future in vivo applications. A constant molar quantity of the cationic cholesterol derivative, 3 [N-(N’, N’-dimethylaminopropane)-carbamoyl] cholesterol (CHOL-T) was mixed with dioleoylphosphatidylethanolamine (DOPE) and a galactose/glucose derivative to produce targeted cationic liposomes. PEG liposomes were prepared in the same way with the addition of distearoylphosphoethanolamine polyethylene glycol 2000 (DPSE-PEG2000), 2% on a molar basis. Supported by transmission electron microscopy characterisation, we present evidence that the pegylation of liposomes affects the DNA binding capability and transfection efficiencies of the cationic liposomes in addition to protecting the plasmid DNA in lipoplexes from serum nuclease degradation. Optimal DNA : liposome binding ratios were obtained from gel retardation studies and confirmed by ethidium bromide intercalation assays. These complexes were then tested on the human hepatoma cell line, HepG2, to determine toxicity and assess transfection efficiencies. From results obtained in this study, it appears that both cationic and pegylated cationic liposomes are well tolerated by cells in vitro. The results further suggest that targeting by use of glycolipids incorporated into the structure of the liposome increases transfection, while pegylation of cationic liposomes marginally decreases the transfection efficiency of the lipoplexes to HepG2 cells in vitro. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2009.

Currently the breeding of desirable traits in orchid flowers is a lengthy and unpredictable process. A shortened breeding time and a more direct method of introducing specific genetic characteristics could be achieved if more information were available on the specific genes responsible for flower characteristics. In order to identify some of these genes, the genetic relationships between a hybrid, Dendrobium Gatton 'Sunray', and the parent species bred to produce it, D. chrysotoxum Lindley and D. pu/che//um Lindley were examined.Ball State UniversityMuncie, IN 47306These results were supported by Restriction Fragment Length Polymorphisms (RFLPs) observed following amplification of the Internal Transcribed Spacer (ITS) regions of the rDNAs.In order to clone genes responsible for specific flower characteristics, mRNA differential display was performed using total RNA isolated from the leaves, immature flowers, and mature flowers of the hybrid orchid and its two parents. Bands unique to D. Gatton 'Sunray' flower tissue, which were common to the hybrid and a single parent, were excised from a denaturing acrylamide gel. Four of the bands, which represented expressed genes determining inherited flower characteristics, were re-amplified, cloned, and three were sequenced. Partial sequence information obtained for two of the clones was used to search the GenBank database for homologous genes. One of the clones had sequence homology to plant 26S ribosomal genes and the other clone was homologous to sequences encoding regulatory proteins active during development (for example, the human retinoblastoma susceptibility gene or the Caenorhabditis e/egans cosmid R06F6 containing a serine/threonine protein kinase gene). / Department of Biology

The major digestive enzymes of larval Diabrotica undecimpunctata howardi, the southern corn rcotworm (SCR), have been investigated. A number of proteases have been identified, the majority cysteine proteases, some aspartic acid proteases are also present. In vitro assays of cysteine proteases showed that almost all activity could be arrested by E64 or chicken egg-white cystatin. This activity was also affected by TLCK, CpTI and thaumatin. Two inhibitory activities were demonstrated in protein extracts from rice seed. The first, oryzacystatin-I, caused marked inhibition of both the insect cysteine proteases and papain. The second produced strong inhibition of insect cysteine proteases but left papain virtually unaffected. Amino acid sequence data for this novel inhibitor was obtained, and significant homology demonstrated to the rice allergenic proteins (Izumi et a].,1992; Adachi et al., 1993). Oryzacystatin-I was expressed as a fully functional fusion protein (Rozc) in Escherchia coli, isolated, characterised and employed in feeding trials with larval SCR, a significant rise in mortality was demonstrated. Other protease inhibitors were also assayed in vivo, but none exhibited the efficacy of Rozc. A single iso-form of a-araylase was identified and characterised. In vitro assays with amylase inhibitors demonstrated the effectiveness of WAAI and the weak effect of BAAI. WAAI, employed in bioassay, produced a significant decrease in survival. Five lectins were tested by bioassay. WGA and GNA caused marked alterations in larval development, GNA was most effective. Saporin caused little effect when incorporated into artificial diet. WAAI, CpTI and GNA were employed in combination bioassays. An enhanced level of effectiveness was demonstrated with the double and triple combinations assayed. While further work is necessary, especially assaying protein efficacy in planta and establishing mechanisms of action, this project has successfully demonstrated the clear potential of plant antimetabolic proteins for use in the enhancement of inherent resistance of crop plants to insects, and of employing a number of proteins in a multi-mechanistic defence.

No description available.

Potato is a major part of the human diet in many countries of the world, providing substantial levels of carbohydrate, protein, and vitamins. This study examined the tuber protein content. In the first part of the research, total soluble protein (TSP) and patatin concentration were determined in periderm, cortex, and pith, in tubers of 20 important potato cultivars. TSP concentration was greater in periderm and lesser in cortex and pith tissues. Patatin was present in all tuber tissues but with the opposite pattern, less in periderm and greater in cortex and pith tissues. For intercultivar comparisons, a means of converting the specific tissue-based TSP and patatin data (dry weight) into a uniform weight whole tuber basis was developed. This relied on conversion factor values that were generated from percent weight tissue proportion and percent dry matter for each tissue layer. Cultivars with relatively more or less TSP and patatin in each tissue layer, and on a whole tuber basis, were identified. In the second part of the study, disassembly of chimeral (Russet Burbank) and putatively chimeral (Alpha, Bintje, Red Gold) tubers into their component genotypes was evaluated as a strategy for the production of intraclones with altered protein content. Explants were selected from tissue with greater or lesser protein levels and somatic embryogenesis was used to produce regenerants from each tissue source. Russeting was used as a phenotypic marker and TSP as a biochemical marker. Russet Burbank was confirmed as a periclinal chimera, although chimeral instability was evident, since some non-chimeral regenerants showed displacement of LI tunic cells with the russeting mutation into the pith. Red Gold was "uncovered" as an LII periclinal chimera (Red-Gold-Red). The value of chimeral disassembly in explaining an important component of somatic variation was clearly seen with this cultivar. The inconsistent TSP distribution in Russet Burbank intraclones proved that TSP was not distributed in a periclinal chimeral manner, as initially hypothesized. However, there was clear variation in protein content in the tubers of non-chimeral regenerants. Periclinal chimeral disassembly and somatic embryogenesis are potentially useful technologies for the production of improved intraclones of potato.

Thesis (M.S.)--University of Hawaii at Manoa, 2007. / Includes bibliographical references (leaves 78-80). / viii, 82 leaves, bound ill. 29 cm

Bananas are an extremely important crop, serving as both a staple food in developing countries and as a dessert fruit in Western society. Two of the most devastating pathogens currently affecting both commercial and subsistence banana production are Fusarium oxysporum (Foc; causal agent of Fusarium wilt) and Mycosphaerella species (causal agent of black and yellow Sigatoka). Conventional breeding programs designed to improve the disease resistance characteristics of the commercially elite Cavendish cultivar have, thus far, been largely unsuccessful. Genetic engineering is now regarded as the most promising method to generate enhanced disease resistance in banana. In other crops and model species, strategies to enhance disease resistance have included the transgenic expression of defense-related genes such as; disease resistance genes (R genes), downstream signaling genes (eg. NPR1, non-pathogenesis related) and antimicrobial peptides (AMPs). The overall aims of this research were to amplify and compare the nucleotide binding site (NBS) domains of potential disease resistance genes from disease resistant and disease susceptible banana cultivars. To isolate and compare complete R gene sequences from these cultivars. To generate transgenic Lady Finger banana plants expressing the D4E1 antimicrobial peptide under the control of two different promoters and finally to assess extracts from these plants for their ability to inhibit the growth of Foc Race1. Using degenerate primers, the NBS domains of six resistance gene candidate (RGC) sequences were amplified from the disease resistant cultivar Calcutta 4 (C4) and the disease susceptible cultivar Cavendish (Cav). The RGC 1, 2, 5 and 6 sequences showed similarity to previously characterized R gene sequences isolated from monocotyledonous plant species, while RGCs 3 and 4 showed similarity to R genes which form part of the Fusarium wilt resistance locus isolated from the dicotyledonous species, Lycopersicon esculentum; as well as other monocotyledonous R genes. RGCs 1-4 and 6 were present and transcriptionally active in both C4 and Cav, whereas RGC-5 was present in Cav only and was not transcribed. The transcripts could not be detected by Northern analysis, which is consistent with previous reports that R genes are constitutively transcriptionally active at only low levels. The NBS domains of RGCs 1-6 showed less than 65% similarity (amino acid level) to one another but when each individual RGC isolated from the C4 and Cav gDNA and cDNA templates was compared the sequences showed greater than 97% similarity (amino acid level). Comparative sequence analysis revealed amino acid positions that were consistently different between the C4 and Cav clones. Southern analysis revealed that RGC 1-5 were present in both the C4 and Cav genomes in only low copy number (1-2 gene copies with 1-3 alleles), whereas RGC-6 showed high copy number in both cultivars. Complete RGC sequences were subsequently amplified by RNA-ligase-mediated (RLM) -RACE and 3'-RACE using specific primers designed to each of the RGC 1-4 NBS domains. Amplicons for each RGC were assembled to form potentially complete RGC sequences. Analysis of the sequences revealed the presence of coiled coil (CC) motifs in two of the amino terminal sequences while leucine rich repeats (LRRs) were identified at the carboxy terminal of all sequences. Multiple 3'-RACE products were amplified for each RGC sequence. Although the polyadenylated products were of different lengths, the sequences were greater than 98% identical at the amino acid level (except an RGC 3 clone which was 91-95% identical to the other RGC 3 clones due to a 37 amino acid deletion). Specific primers used to amplify each complete RGC sequence from both C4 and Cav DNA revealed that: RGC 1 (3.53 kbp) could be amplified from both C4 and Cav; RGCs 2 (2.99 kbp) and 4 (4.44 kbp) could be amplified from only Cav, however, the proposed truncations of these sequences (RGC 2: 1.3 kbp, RGC 4: 2.8 kbp and 2.9 kbp) could be amplified from both cultivars; RGC 3 (4.57 kbp) could not be amplified from either C4 or Cav, however, the three shorter sequences (1.96 kbp, 1.34 kbp and 1.28 kbp) could be amplified from both templates. The functional significance of the truncated sequences is currently unknown, however, truncated sequences have been detected in a number of R gene families isolated from other crops. No major sequence differences, such as deletions/insertions or early stop codons, were identified between the RGC sequences amplified from C4 as compared to Cav (greater than 91% amino acid similarity) and no sequence was identified as being present in the susceptible but absent from the resistant cultivar. However, comparative analysis of multiple clones isolated from C4 and Cav did reveal amino acid residues that were consistently different between the two cultivars. These differences may result in differing resistance capabilities, functional genomics studies would need to be undertaken to determine this. It has been proposed that CC-NBS-LRR type R genes employ NDR1/HIN1-like (NHL) proteins, after pathogen invasion is detected, in the signaling process that ultimately leads to the elaboration of a defense response. A NHL partial sequence (420 bp) was amplified from the C4 banana cultivar. The complete sequence of this gene (termed NHL-1) was isolated using RLM and 3'-RACE technologies (576 bp and 535 bp amplicons, respectively) and subsequently the 1.106 kbp sequence was PCR amplified from both the C4 and Cav cultivars. The banana NHL-1 gene contained conserved motifs/domains previously identified within other NHL-type gene sequences. These included a signal peptide motif, a transmembrane domain and three previously identified conserved motifs. Based on current research into NHL type genes, the banana NHL-1 sequence may not be useful as a transgene to enhance disease resistance in elite cultivars. However, it potentially plays an important role in the defense response signal transduction pathway and therefore will further our understanding of plant-pathogen interactions in banana. Transgenic Lady Finger banana plants expressing the D4E1 antimicrobial peptide under the control of either the maize polyubiquitin (Ubi) or banana bunchy top virus (BBTV) DNA-6 (Bt6.1) promoters were generated. These plants were subsequently assessed for the ability of their crude protein extracts to inhibit the germination of Fusarium oxysporum f.sp. cubense Race1 conidia in vitro. These anti-fungal bioassays revealed that fungal colony growth was reduced by 37-100% using extracts from the pUbi-D4E1 transgenic lines and 89-99% using extracts from the pBt6.1-D4E1 transgenic lines. The transgenic lines are currently undergoing multiplication in preparation for glasshouse and small plant challenge trials for resistance to Fusarium wilt. These preliminary results suggest that D4E1 may be useful in enhancing disease resistance in banana.

No description available.

No description available.