Department of Biotechnology

Permanent URI for this community

https://hdl.handle.net/10566/13203

Browse

Now showing 1 - 20 of 368

A study of the problems and possibilities of using the marine intertidal zone for teaching principles of ecology in senior secondary schools: A survey of biology teachers in the western cape.
(University of the Western Cape, 1994) Reddy, Chris; Keats, D.W.
In this study I investigate the attitudes of a group biology teachers in the Western Cape, to using the marine intertidal zone for teaching principles of ecology in senior secondary schools, by exploring the problems experienced and solutions envisaged. The study investigates the problems perceived/experienced by means of a semistructured interview and seeks solutions via teacher workshops and an excursion to the seashore. Teachers mentioned many constraints and school based problems such as time-tab1ing, teacher attitudes, lack of ethos lack of funds, Iarge numbers in classes and collectively found useful answers which reduced the emphasis of many of the problems mentioned. Problems such ds, the lack of knowledge of the marine environment, limited experience of fieldwork technique and management, could only be solved by pre and in-service teacher education programmes. The workshops produced useful solutions and suggestions for implementation by teachers, education departments and governmental and non-governmental agencies that would assist in making this a reality. These include resource development, teacher networking, peer teaching, in-service and pre-service programmes with a marine emphasis, and funding of appropriate programmes
Molecular detection and genetic manipulation of the Black Queen Cell Virus
(University of the Western Cape, 2002) Benjeddou, Mongi; Davison, Sean; Dept. of Biotechnology; Faculty of Science
The South African isolate of the Black Queen-Cell Virus (BQCV), a honeybee virus, was previously found to have an 8550 nucleotide genome excluding the poly(A) tail. Its genome contained two ORFs, a 5'-proximal ORF encoding a putative replicase protein and a 3'-proximal ORF encoding a capsid polyprotein.A reverse transcriptase PCR (RT -PCR) assay was developed for the detection of BQCV and acute bee-paralysis virus (ABPV). Complete genomes sequences w ere used to design unique PCR primers within a l-kb region from the 3' end of both genomes to amplify a fragment of 70.0 bp from BQCV and 900 bp from ABPV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing BQCV and ABPV. Sensitivities were of the order of 130 genome equivalents of purified BQCV and 1600 genome equivalents of ABPV.
The molecular characterization of trichoplusia ni single nucleocapsid nucleopolyhedrovirus: a study on early regulatory features
(University of the Western Cape, 2003) Wang, Weizhou; Davison, S
With the development of biological insecticides, many research efforts have been made in baculoviruses to investigate fundamental molecular aspects of these viruses, such as the function and regulation of genes, genome organization, mode of entry, DNA replication and virus factors that determine the host range and virulence. Previously, a South African Trichoplusia ni single capsid nuclear polyhedrosis virus (TnSNPV) isolate was partially characterized as a novel baculovirus. During the process of the characterization, a few late genes of the virus were identified. This thesis describes a molecular characterization of the TnSNPV early genes to gain insight into the functional roles of these genes, their unique features and further determination of the placement of TnSNPV in baculovirus phylogeny.
Human testis angiotensin-converting enzyme: crystal structure of a glycosylation mutant and investigation of a putative hinge-mechanism by normal mode analysis
(University of the Western Cape, 2004) Watermeyer, Jean Margaret; Sewell, Bryan Trevor; Sturrock, E.D.; Dept. of Biotechnology; Faculty of Science
Human angiotensin-converting enzyme (ACE) is a key enzyme in the regulation of blood pressure via the renin-angiotensin and kallikrein-kinin systems. A number of orally active drugs have been developed over the years that target somatic ACE, for the treatment of hypertension, myocardial infarction and congestive heart failure. Protein structural information about ACE is an important key for the understanding of the mechanism and substrate-specificity of the enzyme. However, this information has only begun to be elucidated in the past year, with the solution of crystal structures of human testis ACE (tACE), and homologues Drosophila AnCE and human ACE2. tACE is identical to the C-terminal domain of somatic ACE, which consists of two homologous domains, each having a slightly different substrate-specificity. This thesis describes the purification, crystallisation and X-ray crystal structure-determination of a glycosylation-deficient mutant of tACE, tACEG1,3, to 2.9 Å. The structure of tACE-G1,3 aligns closely with that of native tACE, indicating that the mutations did not alter the conformation. The ability to achieve minimal glycosylation of tACE for crystallisation purposes via mutation, rather than using expensive glycosidase inhibitors, iii should prove advantageous for further structural studies, such as the study of the binding of novel inhibitors. In all of the tACE structures thus far observed, the active site is closed off from the external medium in a deep cleft, so that it is unclear how a large substrate molecule could gain access. However, a hinge motion that opens this cleft has been observed in the structures of ACE2. Temperature factor and sequence comparison between tACE, tACE-G1,3, AnCE and ACE2 suggests the functional conservation of three flexible loop regions, as well as the sequence conservation of three constrained regions, involved in the hinge. Normal mode analysis reveals the intrinsic flexibility of tACE, and further suggests that a putative open form of tACE would behave similarly to the open form of ACE2. Based on these indications, a conservation of the ACE2 hinge-bending mechanism is proposed. Temperature factor analysis also reveals that subdomain II, containing bound chloride ions, is more structurally rigid than subdomain I, in all structures considered. Based on these results, lines of investigation are suggested that should yield insight into the mechanisms of action of ACE and its association with various substrates and inhibitors, ideally aiding in the development of novel drugs for the treatment of cardiac disease.
Investigation into the role of DWNN in cell death
(University of the Western Cape, 2004) Seameco, Tumelo; Rees, D.J.G.; Dept. of Biotechnology; Faculty of Science
Many genes are activated to influence the self-destruction programme of the cell. This programme entails synchronised instigation and implementation of numerous subprograms. The arrival of gene targeting aided in the determining of the functions of novel genes. Such genes may have been sequenced, but not functionally characterised. The fulfillment of this requirement through gene targeting technology has swiftly developed. The mode by which DWNN operate in organisms in which it is thought to be covalently linked to some other proteins, which have a definite role in apoptosis, is not yet unraveled. This study attempted the functional characterisation of DWNN in light to the hypothesis that it may be involved in Cytotoxic T lymphocyte killing and apoptosis.
The prevalence and survival of Campylobacter, Salmonella and Listeria species in poultry processing plant
(University of the Western Cape, 2004) Mabogo, Rudzani David Lesly; Gouws, P.A.; Dept. of Biotechnology; Faculty of Science
The organisms in this study were chosen due to their associations with foods and their potential as food borne pathogens. Food borne diseases are an import public health problem in most countries. Bacteria of the genera Campylobacter, Salmonella and Listeria can be transported by poultry and poultry products to humans. Gastroenteritis, typhoid fever, diarrhea, dysentery may originate from the infection. This study was undertaken to determine the incidence of pathogens in a poultry processing plant using polymerase chain reaction and conventional tests and to determine the formation and survival of biofilm cells of food pathogens in trisodium phosphate.
Recombinant expression of the pRb- and p53-interacting domains from the human RBBP6 protein for in vitro binding studies
(University of the Western Cape, 2004) Ndabambi, Nonkululeko; Pugh, David J.R.; Dept. of Biotechnology; Faculty of Science
This thesis describes the cloning and recombinant expression of domains from the human RBBP6 protein for future in vitro binding studies with pRb and p53. RBBP6 is a splicing-associated protein that is known to interact with both p53 and the Retinoblastoma gene product (pRb), and has recently been shown to be highly upregulated in oesophageal cancer. The pRb binding domain (RbBD) and the p53 binding domain (p53BD) were each expressed using the glutathione-S-transferase (GST) tag affinity system, and affinity purified using a glutathione-linked agarose column. Purified fusion proteins were cleaved to separate the target protein from GST using PreScission™ Protease, for which there is a recognition sequence located immediately upstream of the multiple cloning site on the pGEX-6P series of plasmids. The pRb binding and p53 binding domains were further purified using cation exchange chromatography. Mass spectrometry confirmed that the RbBD was expressed as a single species of the expected molecular weight. However preliminary NMR analysis suggested that the domain was not fully folded. A total yield of 8 mg of protein was achieved from 1l of culture, which make it feasible to express 15N and 12C labelled samples for NMR. The p53BD was found to be expressed at lower levels and subject to C-terminal degradation, which suggest that the C-terminus is unstructured most likely due to the presence of poly-lysine tail. Human pRb protein was also successfully expressed and purified using the GST affinity system. Human p53 protein was expressed but was found to be insoluble and attempts to purify it were not pursued. Attempts to confirm the interactions between human RBBP6 and p53 and pRb proteins are on-going but fall outside the scope of this thesis. Expression constructs for the RING and zinc finger domains from human RBBP6 were also cloned into the pGEX system for future structural studies using NMR. Both domains were found to be expressed as soluble fusion proteins in preliminary expression studies.
Characterization of two Arabidopsis thaliana genes with roles in plant homeostasis
(University of the Western Cape, 2004) Ludidi, Ndomelele Ndiko; Gehring, C. A.
Plants are continuously exposed to varying conditions in their environment, to which they have to adapt by manipulating various cellular processes. Environmental (abiotic) and pathogen (biotic) stress are challenges against which plants have to defend themselves. Many plant responses to stress stimuli are a result of cellular processes that can be divided into three sequential steps; namely signal perception, signal transduction m1d execution of a response. Stress signal perception is, in most of these cases, facilitated by cell surface or intracellular receptors that act to recognize molecules presented to the cell. In several cases, hormones are synthesized in response to stress signals and in turn these hormones are perceived by cellular receptors that trigger signal transduction cascades. Propagation of signal transduction cascades is a complex process that results from activation of various signaling molecules within the cell. Second messengers like calcium (Ca2+) and guanosine 3', 5'-cyclic monophosphate (cGMP) play a vital role in mediating many signal transduction processes. The result of these signal transduction cascades is, in most instances, expression of genes that contribute to the plant's ability to cope with the challenges presented to it. Plant natriuretic peptides (PNPs) are novel plant hormones that regulate water and salt homeostasis via cGMP-dependent signaling pathways that involve deployment of Ca2+. The aim of this study is to partially characterize a PNP and a guanylyl cyclase, both from Arabidopsis thaliana. Guanylyl cyclases synthesize cGMP from the hydrolysis of guanosine 5' -triphosphate (GTP) in the cell. The study also aims to investigate the effect of drought and salinity on cGMP levels in plants, using sorbitol to mimic the osmolarity/dehydration effect of drought and NaCl as a source of salinity stress and thus link NaCl and sorbitol responses to both AtPNP-A and cGMP up-regulation.
Ancient Genes in Cancer Gene Expression?
(University of the Western Cape, 2004) Panji, Sumir; Hide, Winston
Backsround: The Cancer/testis (CT) antigens are a division of germ cell specific genes not expressed in somatic cells, exceptions being placental cells and 20Vo - 4OVo of cancer types. The aptitude of CT antigens to elicit humoral immune responses, their restricted expression profile, absence of major histocompatability complex expression in male germline cells have contributed to the emergent attraction of CT antigens as ideal, prospective cancer vaccination candidates. Motivation: Presently there are M CT gene families containing a total of 97 gene products and isoforms. Due to the promulgation in sensitivity and specificity of rapid serological immunodetection assays e.g. serial analysis of recombinant cDNA expression libraries (SEREX), the magnitude of novel CT genes and gene families will increase. Hence, characteization of this unique subset of CT genes is fundamental to our erudition of this rapidly emerging novel subset of genes. Obiectives: The sequencing of the human genome provides a useful biological framework for the categoization and systematization of rapidly accumulating biological information. A genomic approach was used to ascertain the locations of the CT genes in the human genome and determine if the genomic locations of the CT genes is nonrandom. An in-silico expression study was conducted for the CT genes with the aim of establishing if CT gene expression is restricted to the testis. A portion of the human genome housing the largest proportion of the CT genes was selected for analysis in order to determine if the surrounding genomic architecture influences CT gene expression. A comparative genomics approach was used in determining if the CT genes are "ancient genes.
Microbial diversity of Antarctic Dry Valley mineral soil
(University of the Western Cape, 2004) Moodley, Kamini; Cowan, D.; Dept. of Biotechnology; Faculty of Science
Antarctica provides some of the most extreme environments on earth. Low temperatures, low water availability and nutrient deficiency are contributing factors to the limited colonisation of Antarctic biotopes, particularly in the continental Dry Valleys. The survival of microorganisms in this harsh continent provides the basis for the significance of this study. This study aimed to explore microbial phylotypic diversity across a 500 m altitudinal transect in the Miers Dry Valley, Ross Desert, East Antarctica. The study also attempted to infer from phylogenetic data, the possible presence of indicative phenotypes which might contribute to a functional microbial community.
Isolation and identification of PNP-A homologues from Xerophyta viscosa Baker
(University of the Western Cape, 2004) Mabuto, S; Gehring, C
Maintenance of growth, development and normal physiological processes remains crucial for the survival of plants. Plant hormones are described as integrators of external signal and internal developmental process in plants. They also attribute as mediators of physiological and developmental adaptation throughout the plant life cycle (Wilkinson et al.,1997). It has been well over 50 years since Went and Thimann (1937) published their classical book Phytohorrnones. At that time, the term phytohorrnone was synonymous to auxin, although compounds like cell division factors were anticipated to be phytohormones based on physiological experiments. Since then a number of plant hormones were discovered and a series of studies concluded that plant hormones are at least partly responsible for plant growth, development, cell elongation, cell division, differentiation and stomatal movement in response to environmental stimuli. Plant hormones are categorized into three groups, known as classical, non-classical and peptide hormones. Here, Plant Natriuretic Peptide (PNP) will be reviewed as a proposed additional peptide hormone.
Recombinant expression of the pRb- and p53-interacting domains from the human RBBP6 protein for in vitro binding studies
(University of the Western Cape, 2004) Ndabambi, Nonkululeko; Pugh, David
This thesis describes the cloning and recombinant expression of domains from the human RBBP6 protein for future in vitro binding studies with pRb and p53. RBBP6 is a splicing-associated protein that is known to interact with both p53 and the Retinoblastoma gene product (pRb), and has recently been shown to be highly upregulated in oesophageal cancer. The pRb binding domain (RbBD) and the p53 binding domain (p53BD) were each expressed using the glutathione-S-transferase (GST) tag affinity system, and affinity purified using a glutathione-linked agarose column. Purified fusion proteins were cleaved to separate the target protein from GST using PreScission ™ Protease, for which there is a recognition sequence located immediately upstream of the multiple cloning site on the pGEX-6P series of plasmids. The pRb binding and p53 binding domains were further purified using cation exchange chromatography. Mass spectrometry confirmed that the RbBD was expressed as a single species of the expected molecular weight. However preliminary NMR analysis suggested that the domain was not fully folded. A total yield of 8 mg of protein was achieved from 11 of culture, which make it feasible to express 15Nand 12Clabelled samples for NMR. The p53BD was found to be expressed at lower levels and subject to C-terminal degradation, which suggest that the C-terminus is unstructured most likely due to the presence of polylysine tail. Human pRb protein was also successfully expressed and purified using the GST affinity system. Human p53 protein was expressed but was found to be insoluble and attempts to purify it were not pursued. Attempts to confirm the interactions between human RBBP6 and p53 and pRb proteins are on-going but fall outside the scope of this thesis. Expression constructs for the RING and zinc finger domains from human RBBP6 were also cloned into the pGEX system for future structural studies using NMR. Both domains were found to be expressed as soluble fusion proteins in preliminary expression studies.
An investigation of the plants used medicinally In self-care in the Bredasdorp / Elim region
(University of the Western Cape, 2004) Thring, T; Weitz, F
Much of the traditional medicinal plant knowledge is to a large extent known by the older generations. This knowledge is at risk of disappearing due to not being passed down to younger members of the respective families and communities. The Bredasdorp / Elim area in the Southern Overberg has many individuals who possess such knowledge. The aims of this study were to identify what plants were in use in the area, to document this knowledge and to choose certain plants to test in antimicrobial bioassays.
The genease activity of mung bean nuclease: fact or fiction?
(University of the Western Cape, 2004) Kula, Nothemba; Hide, Winston; Dept. of Biotechnology; Faculty of Science
The action of Mung Bean Nuclease (MBN) on DNA makes it possible to clone intact gene fragments from genes of the malaria parasite, Plasmodium. This “genease” activity has provided a foundation for further investigation of the coding elements of the Plasmodium genome. MBN has been reported to cleave genomic DNA of Plasmodium preferentially at positions before and after genes, but not within gene coding regions. This mechanism has overcome the difficulty encountered in obtaining genes with low expression levels because the cleavage mechanism of the enzyme yields sequences of genes from genomic DNA rather than mRNA. However, as potentially useful as MBN may be, evidence to support its genease activity comes from analysis of a limited number of genes. It is not clear whether this mechanism is specific to certain genes or species of Plasmodia or whether it is a general cleavage mechanism for Plasmodium DNA .There have also been some projects (Nomura et al., 2001;van Lin, Janse, and Waters, 2000) which have identified MBN generated fragments which contain fragments of genes with both introns and exons, rather than the intact genes expected from MBN-digestion of genomic DNA, which raises concerns about the efficiency of the MBN mechanism in generating complete genes.Using a large-scale, whole genome mapping approach, 7242 MBN generated genome survey sequences (GSSs) have been mapped to determine their position relative to coding sequences within the complete genome sequences of the human malaria parasite Plasmodium falciparum and the incomplete genome of a rodent malaria parasite Plasmodium berghei. The location of MBN cleavage sites was determined with respect to coding regions in orthologous genes, non-coding intergenic regions and exon-intron boundaries in these two species of Plasmodium. The survey illustrates that for P. falciparum 79% of GSSs had at least one terminal mapping within an ortholog coding sequence and 85% of GSSs which overlapped coding sequence boundaries mapped within 50 bp of the start or end of the gene. Similarly, despite the partial nature of P.berghei genome sequence information, 73% of P.berghei GSSs had at least one terminal mapping within an ortholog coding sequence and 37% of these mapped between 0-50 bp of the start or end of the gene. This indicates that a larger percentage of cleavage sites in both P.falciparum and P.berghei were found proximal to coding regions. Furthermore, 86% of P.falciparum GSSs had at least one terminal mapping within a coding exon and 85% of GSSs which overlapped exon-intron boundaries mapped within 50bp of the exon start and end site. The fact that 11% of GSSs mapped completely to intronic regions, suggests that some introns contain specific cleavage sites sensitive to cleavage and this also indicates that MBN cleavage of Plasmodium DNA does not always yield complete exons. Finally, the results presented herein were obtained from analysis of several thousand Plasmodium genes which have different coding sequences, in different locations on individual chromosomes/contigs in two different species of Plasmodium. Therefore it appears that the MBN mechanism is neither species specific nor is it limited to specific genes.
Identification and characterisation of a novel gene, DWNN, isolated from promoter-trapped Chinese hamster ovary cells
(University of the Western Cape, 2005) Skepu, Amanda; Rees, D.J.G.; Dept. of Biotechnology; Faculty of Science
The process of cytotoxic T lymphocyte (CTL) killing involves the recognition and destruction of foreign antigens by cytotoxic T cells and is of crucial importance to the defence of the organism against viral infections. Defects in this process can lead to various autoimmune diseases and cancer. The aim of this study was to identify more genes involved in the cell death pathway and to link CTL killing, apoptosis and cancer.
Cloning, expression and characterization of Novel Lipase and Esterases from Burkholderia multivorans UWC10
(2005) Rashamuse, Konanani J; Cowan, Don A
An esterase and lipase producing Burkholderia multivorans strain was isolated by culture enrichment strategies. A shotgun library of Burkholderia multivorans genomic DNA (prepared in E. coli/pUC18) was screened for lipase and esterase activities. Three positive recombinant clones, pTEND5, pHOLA6 and pRASHI4, conferring esterolytic and lipolytic phenotypes respectively, were identified. Full-length sequencing of DNA inserts was performed using subeloning and "primer-walking" strategies. Nucleotide sequence analysis revealed that the pRASH14 plasmid DNA consisted of two open reading frames (ORPI and ORP2) encoding 356 and 350 amino acids, respectively. Database searches revealed that ORPI and ORP2 were homologous to lipases and chaperones from subfamily I.2. In the pTEND5 sequence, an open reading frame consisting of 978 bp, encoding 326 amino acids, was identified. Database searches revealed that this open reading frame was homologous to family Vesterases. Nucleotide sequence analysis revealed that pHOLA6, plasmid DNA consisted of 1194 bp encoding 398 amino acids and showed homology to family VIII esterases. The primary structures of LipA, EstEFH5 and EstBL from pRASHI4, pTEND5 and pHOLA6, respectively, showed a classical GxSxG motif, which is conserved in many serine hydrolases. In addition, EstBL also showed a consensus SxxK motif, the serine of which acts as a catalytic nucleophile in class C B-lactames and some peptidases.
Characterization and engineering of Bacillus megaterium AS-35, for use in biodegradation of processed olive wastewater
(University of Western Cape, 2005) Van Schalkwyk, Antoinette; Cowan, Don A
The popularization and health benefits associated with the "Mediterranean diet" saw a world wide increase in the production and consumption of processed olives and olive oil. During the brining of table olives large quantities of processed olive waste water is seasonally generated. This blackish-brown, malodours liquid is rich in organic and phenolic compounds, which cause environmental problems upon discarding. Currently, processed wastewater is discarded into large evaporation ponds where it poses serious environmental risks. The biodegradation of organic substrates present in the olive wastewater is inhibited by the high concentrations of phenolic compounds.
The evaluation of Y-STR loci for use in forensics
(University of the Western Cape, 2005) Ehrenreich, Liezle Suzette; Leat, Neil; Davison, Sean; Dept. of Biotechnology; Faculty of Science
The aim of this study was to investigate the forensic usefulness of various Y-chromosome short tandem repeat loci among South African sub-populations. Three different sets of Y-chromosome short tandem repeat loci were chosen for investigation.
Structure, enzymology and genetic engineering of Bacillus sp. RAPc8 nitrile hydratase
(University of the Western Cape, 2005) Tsekoa, Tsepo L.; Cowan, Don A.; Dept. of Biotechnology; Faculty of Science
Microbial nitrile hydratases (NHases) are important industrial enzymes that catalyse the conversion of nitriles to the corresponding amides. A thermostable, cobalt-type Bacillus sp. RAPc8 NHase was previously cloned and expressed in E. coli. In this study, the primary aim was to determine the molecular structure of Bacillus sp. RAPc8 NHase. The heterotetrameric enzyme was purified to near homogeneity using heatpurification, hydrophobic interaction chromatography and ion exchange chromatography. Purified NHase was crystallised using the hanging-drop vapourdiffusion method. Crystals produced in the presence of 30% PEG 400, 0.1M MES pH 6.5 and 0.1M magnesium chloride were selected for X-ray diffraction studies. These crystals diffracted well, with diffraction spots visible beyond 2.4Å, with little mosaicity. At 2.5Å, the data were 93% complete. The crystal structure of Bacillus sp. RAPc8 NHase was solved via molecular replacement using the crystal structure of Pseudonocardia thermophila NHase as a search model. The final refined structure had good refinement statistics and geometry. The overall fold was very similar to that of previously determined NHase structures. Bacillus sp. RAPc8 NHase was most similar to Bacillus smithii NHase (0.355År.m.s.d.) and least similar to Rhodococcus sp. R312 NHase (1.191Å r.m.s.d.). One cobalt atom per heterodimer was bound to a typical NHase metal-binding motif, with post-translationally modified cysteine residues among the ligands to the metal. The substrate-binding and catalytic cavity of Bacillus sp. RAPc8 NHase was identified and described in detail. Surface representation of the structure revealed an extended, curved solvent accessible channel with access to bulk solvent from two locations in the heterodimer. The amino-acid residues forming the channel were identified and the geometric dimensions measured. Enzyme inhibition kinetics indicated that benzonitrile was a potent uncompetitive inhibitor of NHase. This information was used to aid the genetic engineering of aromatic substrate specificity into Bacillus sp. RAPc8 NHase. Site-directed mutants of NHase were prepared using the Quickchange mutagenesis procedure. Mutant W76G showed a two to three fold decrease in benzonitrile inhibition compared with the wild-type. Analysis of the substrate channel of this mutant NHase showed an 11% increase in volume and a 20% increase in inner surface area compared to that of the wild-type NHase. Due to the lack of other significant differences between the two structures (an r.m.s.d. of only 0.101Å was observed), this difference was thought to be responsible for the decrease in benzonitrile inhibition. A structure-modelling based approach for assessing the likely structural differences that may result as a result of a specific mutation was suggested and tested. This approach may be of value in future mutagenesis work.
Exploring diversity and ecology of nonarchaea in hydrothermal biotopes
(University of the Western Cape, 2005) Galada, Ncebakazi; Cowan, Donald A.; Dept. of Biotechnology; Faculty of Science
The Nanoarchaeota were proposed as the fourth archaeal sub-division in 2002, and the only fully characterized nanoarchaeon was found to exist in a symbiotic association with the crenarchaeote, Ignicoccus sp. This nanoarchaeote, named Nanoarchaeum equitans could not be detected with “universal” archaeal 16S PCR primers and could only be amplified using specifically designed primers. In order to identify and access a wide diversity of archaeal phylotypes a new set of “universal” archaeal primers A571F (5’-GCYTAA AGSRIC CGT AGC-3’) and UA1204R (5’-TTM GGG GCA TRCIKA CCT-3’) was designed, that could amplify the 16S rRNA genes of all four archaeal sub-divisions. Using these primers community DNA was amplified from Chinese and New Zealand hydrothermaystems. Upon sequencing of amplicons it was discovered that Chinese and. New Zealandsamples contained novel nanoarchaeal phylotypes. The preliminary nanoarchaeal phylotypes were used to design nanoarchaeal-specific primer N989R (5'-GGT TTC CGG TGT CAG TTC-3'), which was coupled with A571F and used in screening of nanoarchaeotes. The nanoarchaeal phylotypes identified with these primers were further screened by amplified ribosomal DNA restriction analysis (ARDRA), which was used to explore the diversity of these phylotypes. The novel nanoarchaeotes cluster into 9 cosely related clades which may represent separate species. Three of the New Zealand phylotypes form one separate clade which is closely related to the published nanoarchaeotes. The following nanoarchaeal sequences were submitted to the GenBank, TC9F (AY572420), TC11-5 (AY571283), TC11-B6 (AY727890), TC11-B7 (AY727887), TC11-C4 (AY727886), TC11-C6 (AY727889), TC11-C8 (AY727888), AND TC11-D4 (AY727891). Fluorescence in situhybridization was also used to simultaneously visualize, identify and localize nanoarchaeotes.

Browse

Browsing by Issue Date

Results Per Page

Sort Options