Dissertations / Theses on the topic 'Fields of Research – 270000 Biological Sciences – 270200 Genetics'
To see the other types of publications on this topic, follow the link: Fields of Research – 270000 Biological Sciences – 270200 Genetics.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Fields of Research – 270000 Biological Sciences – 270200 Genetics.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Zhang, Liangtao. "Identification of Hordeum vulgare-H bulbosum recombinants using cytological and molecular methods." Thesis, University of Auckland, 2000. http://hdl.handle.net/2292/2355.
Full textAbstract:
Barley (Hordeum vulgare L. subsp. vulgare) is an important crop and ranks fourth in overall production of the major cereal crops in the world. Like other cereal crops, barley suffers from a narrowing of its genetic base and susceptibility to diseases, pests and environmental stresses. H. bulbosum is a possible source of desirable genes for introgressing into barley to restore genetic diversity and improve current cultivars. Sexual hybridisation between barley and H. bulbosum is the main method for interspecific gene transfer in barley breeding but there are several barriers to overcome. Two of these are reduced recombination and the ability to identify recombinants quickly and efficiently. The aim in this thesis was to gain a better understanding of meiotic chromosomal behaviour in the two species and their hybrids and to improve the characterisation of recombinants from the hybrids. To study the events during meiosis, synaptonemal complex (SC) analysis was carried out on the two species and two H. vulgare - H. bulbosum hybrids. The results indicated that there were interspecific and intraspecific variations in SC length. Mean SC length was positively correlated with recombination frequency but not related to genome size. This suggests that the ratios of mean SC length to genome size (SC/DNA) show divergence among these Hordeum examples. An hypothesis based on the conformation of chromatin associated with axial element, which is dependent on SC/DNA ratio, was presented to explain the relationship between SC length and recombination frequency. Chromosome pairing in the two hybrids was determined by observation at pachytene and metaphase I (MI). Mean percentages of synapses were similar but there were different frequencies of MI pairing between these two hybrids, indicating that different mechanisms may regulate synapsis and MI pairing in the hybrids. To investigate meiotic recombination, genomic in situ hybridisation (GISH) was performed on the two hybrids at MI and anaphase I (AI). It was observed that intergenomic pairing and recombination events occur in distal chromosome segments. A great discrepancy between mean pairing and recombination frequencies was observed in both hybrids and several possible reasons for this discrepancy were discussed. Hybrid 102C2 with high MI pairing had a significantly higher recombination frequency than the low pairing 103K5, suggesting that high MI pairing appears to be associated with high recombination in the hybrids. An interesting finding is that the ratio of recombination to MI pairing in 103K5 (l:8.9) is twice as high compared with 102C2 (l:17). However, the mechanism for this difference in the ratio between the two hybrids remains unknown. Sequential fluorescence in situ hybridisation (FISH) and GISH were used successfully to localise the introgressions in selfed progeny from a tetraploid hybrid derived from chromosome-doubled 102C2 (102C2/colch). This procedure is fast, cheap and can efficiently detect and locate introgressions. Several disease-resistant recombinants were analysed in more details and leaf rust and powdery mildew resistance was associated with distal introgressions on chromosomes 2HS and 2HL (leaf rust) and 2HS (powdery mildew). It is possible that the leaf rust and powdery mildew resistances were closely linked in the distal region of 2HS. A considerable variation in introgression size was observed at similar chromosomal sites among the different recombinants, which will provide useful information for map-based cloning of genes.
2
Pontin, David R. "Factors influencing the occurrence of stinging jellyfish (Physalia spp.) at New Zealand beaches." Lincoln University, 2009. http://hdl.handle.net/10182/1580.
Full textAbstract:
Individuals of the cnidarian genus Physalia are a common sight at New Zealand beaches and are the primary cause of jellyfish stings to beachgoers each year. The identity of the species and the environmental factors that determine its presence are unknown. Lack of knowledge of many marine species is not unusual, as pelagic invertebrates often lack detailed taxonomic descriptions as well as information about their dispersal mechanisms such that meaningful patterns of distribution and dispersal are almost impossible to determine. Molecular systematics has proven to be a powerful tool for species identification and for determining geographical distributions. However, other techniques are needed to indicate the causal mechanisms that may result in a particular species distribution. The aim of this study was to apply molecular techniques to the cnidarian genus Physalia to establish which species occur in coastal New Zealand, and to apply models to attempt to forecast its occurrence and infer some mechanisms of dispersal. Physalia specimens were collected from New Zealand, Australia and Hawaii and sequenced for Cytochrome c oxidase I (COI) and the Internal transcribed spacer 1 (ITS1). Three clans were found: a Pacific-wide clan, an Australasian clan and New Zealand endemic clan with a distribution confined to the Bay of Plenty and the East Coast of the North Island. Forecasting Physalia occurrence directly from presence data using artificial neural networks (ANN) proved unsuccessful and it was necessary to pre-process the presence data using a variable sliding window to reduce noise and improve accuracy. This modelling approach outperformed the time lagged based networks giving improved forecasts in both regions that were assessed. The ANN models were able to indicated significant trends in the data but would require more data at higher resolution to give more accurate forecasts of Physalia occurrence suitable for decision making on New Zealand beaches. To determine possible causal mechanisms of recorded occurrences and to identify possible origins of Physalia the presence and absence of Physalia on swimming beaches throughout the summer season was modelled using ANN and Naϊve Bayesian Classifier (NBC). Both models were trained on the same data consisting of oceanographic variables. The modelling carried out in this study detected two dynamic systems, which matched the distribution of the molecular clans. One system was centralised in the Bay of Plenty matching the New Zealand endemic clan. The other involved a dynamic system that encompassed four other regions on both coasts of the country that matched the distribution of the other clans. By combining the results it was possible to propose a framework for Physalia distribution including a mechanism that has driven clan divergence. Moreover, potential blooming areas that are notoriously hard to establish for jellyfish were hypothesised for further study and/or validation.
3
Eaton, Carla Jane. "Investigation of signalling involved in maintaining the mutually beneficial association between Epichloe festucae and perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand." Massey University, 2009. http://hdl.handle.net/10179/1179.
Full textAbstract:
Content removed from thesis due to copyright restrictions: Eaton, C. J., I. Jourdain, et al. (2008). "Functional analysis of a fungal endophyte stress-activated MAP kinase." Current Genetics 53(3): 163-174. Scott, B. and C. J. Eaton (2008). "Role of reactive oxygen species in fungal cellular differentiations." Current Opinion in Microbiology 11(6): 488-493.In the mutually beneficial association between the fungal endophyte Epichloë festucae and perennial ryegrass, fungal growth is highly regulated and coordinated with that of the host. This implies there must be signalling between the fungus and its host to maintain this close association. Recent work has shown a novel role for reactive oxygen species (ROS) in this symbiotic maintenance, with multiple components of the superoxideproducing NADPH oxidase (Nox) complex being essential for normal association. However, the mechanism by which the Nox complex is regulated is unclear. To identify potential regulators of the E. festucae Nox complex, comparisons were made with well-characterised mammalian systems. This search identified three candidate regulators: a stress activated MAP kinase, sakA, and the p21-activated kinases, pakA and pakB. To investigate if these genes were involved in symbiotic maintenance, replacement mutants were generated by homologous recombination. In culture analysis revealed that the ?sakA mutant was hypersensitive to a range of stresses, whereas the pak mutants were hypersensitive to cell wall stress-inducing agents and displayed altered growth and morphology. Examination of perennial ryegrass infected with these mutants revealed drastically altered plant interaction phenotypes for the ?sakA and ?pakA mutants in comparison to the wild-type strain. ?sakA-infected plants were stunted and displayed striking changes in development, with the base of tillers showing loss of anthocyanin pigmentation and disorganisation of host cells below the meristem, resulting in swollen bases. Plants infected with the ?pakA mutant were severely stunted, had no more than two tillers and senesced soon after planting. In contrast, plants infected with the ?pakB mutant were similar to wild-type, with only slight deregulation of growth in planta. Examination of ROS in culture revealed that ?sakA and ?pakA displayed elevated levels of both superoxide and hydrogen peroxide. ROS levels were also elevated around ?sakA hyphae in planta. These results support roles for SakA and PakA in Nox regulation. This work highlights the fine balance between mutualism and antagonism, and provides insight into the molecular basis for mutualism.
4
Gallie, Jenna. "Evolutionary and molecular origins of a phenotypic switch in Pseudomonas fluorescens SBW25 : a thesis submitted in partial fulfilment of the requirements for the degree of Ph.D. in Evolutionary Genetics at Massey University, Auckland, New Zealand." Massey University, 2010. http://hdl.handle.net/10179/1215.
Full textAbstract:
Survival in the face of unpredictable environments is a challenge faced by all organisms. One solution is the evolution of mechanisms that cause stochastic switching between phenotypic states. Despite the wide range of switching strategies found in nature, their evolutionary origins and adaptive significance remain poorly understood. Recently in the Rainey laboratory, a long-term evolution experiment performed with populations of the bacterium Pseudomonas fluorescens SBW25 saw the de novo evolution of a phenotypic switching strategy. This provided an unprecedented opportunity to gain insight into the evolution and maintenance of switching strategies. The derived ‘switcher’ genotype was detected through colony level phenotypic dimorphism. Further microscopic examination revealed the cellular basis of phenotypic switching as the bistable (ON/OFF) expression of a capsule. Transposon mutagenesis demonstrated that the structural basis of the capsule was a colanic acid-like polymer encoded by the Pflu3656-wzb locus. Subsequently, whole genome re-sequencing enabled elucidation of the series of mutational events underlying the evolution of capsule bistability: nine mutations were identified in the switcher. Present in both forms of the switcher, the final mutation – a point mutation in a central metabolic pathway – was shown to be the sole mechanistic cause of capsule switching; it ‘set the stage’ for a series of molecular events directly responsible for bistability. Two models were proposed to explain capsule switching at the molecular level: the genetic amplification-reduction model, and the epigenetic feedback model. Collective results of biochemical and genetic assays proved consistent with the epigenetic model, whereby a decrease in flux through the pyrimidine biosynthetic pathway activates an already-present feedback loop. Subsequent analysis of a second switcher (evolved independently of and in parallel with the first) revealed a radically different genetic route leading to phenotypically and mechanistically similar capsule switching. In addition to providing the first empirical insight into the evolutionary bases of switching strategies, the work presented in this thesis demonstrates the power of natural selection – operating on even the simplest of organisms – to forge adaptive solutions to evolutionary challenges; in a single evolutionary step, selection took advantage of inherent intracellular stochasticity to generate an extraordinarily flexible phenotype.
5
Yeoman, Carl. "The auxiliary replicons of Butyrivibrio proteoclasticus : a thesis presented in fulfilment of the Doctorate of Philosophy degree at Massey University, Palmerston North, New Zealand." Massey University, 2009. http://hdl.handle.net/10179/983.
Full textAbstract:
Butyrivibrio proteoclasticus B316T is the most recently described species of the Butyrivibrio / Pseudobutyrivibrio assemblage and now the first to have its genome sequenced. The genome of this organism was found to be spread across four replicons: a 3.5 Mb major chromosome and three additional large replicons: 186, 302 and 361 Kb in size. This thesis describes the sequencing, analysis, annotation and initial characterisation of all three B. proteoclasticus auxiliary replicons. Most significantly, these analyses revealed that the 302-Kb replicon is a second chromosome. This small chromosome, named BPc2, encodes essential systems for the uptake and/or biosynthesis of biotin and nicotinamide adenine mononucleotide, as well as the enzymes required for utilisation of fumarate as the terminal electron acceptor during anaerobic respiration, none of which are found on the main chromosome. In addition, BPc2 contains two complete rRNA operons, a large number of enzymes involved in the metabolism of carbohydrates, nitrogen and fatty acids. In contrast to BPc2, both megaplasmids appear largely cryptic, collectively encoding 421 genes not previously described in public databases. Nevertheless, only the 186-Kb, but not 361-Kb megaplasmid, could be cured from Butyrivibrio proteoclasticus B316T. The largest megaplasmid has a copy number of 5, while all other replicons are present at a copy number of 1. %GC content and codon usage analyses strongly suggests that all three auxiliary replicons have co-resided with the major chromosome for a significant evolutionary period. Moreover, the replication machineries of these three replicons are conserved. Interestingly, a survey of a number of Butyrivibrio / Pseudobutyrivibrio species revealed that the megaplasmids are widespread in this assemblage, however these other large plasmids do not show concordance with their 16S rRNA phylogeny and appear distinct to those of B. proteoclasticus B316T. A microarray analysis of gene expression in a co-culture experiment between B. proteoclasticus and the important ruminal methanogen, Methanobrevibacter ruminantium M1, revealed a potentially mutualistic interspecies interaction. In this relationship M. ruminantium appears to provide B. proteoclasticus with glutamate, essential to the final step of NAD+ biosynthesis, while B. proteoclasticus appears to provide M. ruminantium with formate, hydrogen and carbon dioxide, each important substrate for methanogenesis.
6
Brown, Samuel David James. "Molecular systematics and colour variation of Carpophilus species (Coleoptera: Nitidulidae) of the South Pacific." Diss., Lincoln University, 2009. http://hdl.handle.net/10182/1430.
Full textAbstract:
The sap beetle genus Carpophilus Stephens (Coleoptera: Nitidulidae) is a large genus consisting of over 200 species and are found worldwide. Several species are important pests of crops and stored products, and are frequently intercepted as part of biosecurity operations. The genus is poorly known taxonomically, and there are several species groups that are challenging to identify by morphological methods. In particular, two species found across the Pacific, C. maculatus Murray and C. oculatus Murray are frequently confused with each other. These two species are similar in size and colour, but differ primarily by the shape of the colour pattern on their elytra. However, this colour pattern is highly variable within both species, leading to ambiguity in the indentification of these species. Within C. oculatus, three subspecies have been described based on differences in the male genitalia and pronotal punctation: C. o. oculatus and C. o. gilloglyi Dobson are distributed widely across the Pacific, while C. o. cheesmani Dobson is known only from Vanuatu. A search of literature records and specimen collections revealed 32 species of Carpophilus recorded from the Pacific region. In addition there remain several unidentified specimens representing at least four species, two of which will be described subsequent to this research. A number of species recorded in the literature may have been misidentified, and these require further field collections and inspection of museum specimens to confirm their presence in the Pacific. To test the validity of the subspecies of C. oculatus, and its distinctiveness from C. maculatus, a phylogeny of available specimens of Carpophilus was inferred from one mitochondrial gene (cytochrome c oxidase subunit I (COI)), and two nuclear genes (28S ribsomal RNA (28S) and the internal transcribed spacer 2 (ITS2)). These data show large genetic distances between the three subspecies of C. oculatus of 7-12%. Given these distances are similar to those between other species in the genus, this indicates these subspecies may be elevated to full species. The data also consistently support a monophyletic relationship between C. o. oculatus and C. o. gilloglyi. Nuclear genes also support C. o. cheesmani as part of a clade with the other subspecies, but these relationships are unresolved in COI. Carpophilus maculatus was not supported as being the sister taxon of the C. o. oculatus and C. o. gilloglyi clade. Other relationships within Carpophilus were unresolved, possibly due to a combination of incomplete taxon sampling, and saturation of substitutions within the COI gene. Phylogeographic analysis of specimens collected from several localities within the range of C. oculatus showed that, with only one exception, there were no shared haplotypes between archipelagoes. This result suggests it may be possible to determine the provenence of intercepted specimens, providing further information regarding potential invasion pathways. A degree of geographic structuring was also present within C. o. gilloglyi, being separated into a western clade found in Fiji and Rotuma and an eastern clade distributed from the Kermadec Islands and Tonga to French Polynesia. This separation was most profound in COI data, with a mean pairwise distance between the clades of 7%. ITS2 data also demonstrates a degree of differentiation between the two clades, based on differences in the insertions and deletions between the clades. The variability in the shape and colour of the elytral pattern of C. oculatus was also investigated. Colour was quantified using a method based on Red-Green-Blue (RGB) colour values derived from digital photographs, while an outline analysis of the elytral pattern was conducted using elliptic Fourier analysis (EFA). Principal Components Analysis of the RGB values and EFA coefficients showed no clear separation between subspecies, nor were any trends correlated with host fruit or collection localities. Variation at all levels and all measures studied in this thesis show that this geographic region and this genus of beetles offer intruiging insights into speciation, biogeography and biological invasions. There is much scope for further research on the causes and consequences of this variation and the lives of these interesting insects.
7
Brasell, Emma. "Identification of genes regulating the plant-specific expression of the ItmM gene in Epichloe festucae : this thesis is presented as a partial fulfillment of the requirements for the degree of Master of Science (Msc) in Genetics at Massey University, Palmerston North, New Zealand." Massey University, 2008. http://hdl.handle.net/10179/1106.
Full textAbstract:
The fungal endophyte Epichloë festucae forms a largely mutualistic association with the ryegrass species Lolium perenne. E. festucae produces a range of bio-protective alkaloids that protect the host grass from herbivory by both mammals and insects. One such alkaloid, Lolitrem B, is a potent mycotoxin and the causative agent of ryegrass staggers in livestock. Ten genes required for biosynthesis of lolitrem B are encoded in the ltm gene cluster. The ltm genes are expressed in a plant-specific manner, with high levels of expression in planta and very low levels of expression in culture. The mechanism regulating ltm gene expression is unknown but it is predicted to involve signalling from the host plant. The ltmM gene was chosen for use in the investigation of ltm gene regulation because the flanking regions do not contain retrotransposon sequence, which surrounds much of the ltm gene cluster. To identify fungal genes involved in the plant-induced expression of ltmM, a mutagenesis and screening system was developed using a PltmM-gusA ‘knock-in’ construct to detect expression from the ltmM promoter. Agrobacterium tumefaciens-mediated T-DNA mutagenesis was used to create a set of mutants with random insertions in the genome. Mutants were then screened for altered PltmM-gusA expression, both in culture and in planta. Three mutants were identified with increased PltmM-gusA expression in culture, however, no mutants were identified with loss of PltmM-gusA expression in planta. This indicates that a mechanism of repression is involved in the plant-induced expression of ltmM, either directly or indirectly. TM mutants of interest were also observed for altered symbiosis phenotypes. Mutants were identified with reduced colonisation rates and altered hyphal growth in planta. Integration sites were identified for two colonisation mutants and the disrupted genes are predicted to be the CTP:cholinephosphate cytidylyltransferase (CCT) gene PCT1 and the mitogen-activated protein kinsase kinase (MAPKK) gene mkk2.
8
McGaughran, Angela. "Polar eveolution: molecular genetic and physiological parameters of Antarctic arthropod populations : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biosciences at the Allan Wilson Centre of Molecular Ecology and Evolution, Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand." Massey University, 2009. http://hdl.handle.net/10179/1163.
Full textAbstract:
This thesis is presented as a collection of research papers synthesising knowledge gained during the period of candidacy. Its underlying focus is the examination of evolution from a variety of perspectives for terrestrial arthropods (springtails) in an Antarctic setting. These perspectives include investigation of the ways in which springtail populations respond both physiologically and genetically to environmental variability over historical and contemporary time-scales. While the physiological and genetic may seem two worlds apart, this thesis recognises that, in reality the two are inextricably linked. Thus, when genetic differentiation between populations of the same species can be demonstrated, physiological differentiation of these populations may also be predicted (and vice versa). Therefore, across several locations and springtail species, physiological and genetic parameters of individuals and populations are examined both separately and, where possible, in concert. The physiological aspect of this thesis focuses on the springtail Gomphiocephalus hodgsoni from continental Antarctica. In addition to providing the first metabolic rate data for a continental Antarctic springtail, seasonal variation in metabolic rates is examined across multiple temporal and spatial scales to evaluate the ways in which individuals and populations respond to environmental variability. Metabolic activity in this species is intricately linked to a variety of factors, both intrinsic and extrinsic. These include biological function, temperature profiles in the local microclimate, and body mass and genetic differences among populations. In the genetically-focused aspect of this thesis, population genetic patterns of G. hodgsoni from several continental locations and Cryptopygus antarcticus antarcticus from locations across the Antarctica Peninsula are compared. Here, the importance of differing evolutionary histories in influencing patterns of contemporary genetic population structure is highlighted. While both species have been similarly affected genetically by Pleistocene (2 Ma – present) glacial cycling, it is clear that differences in timing of colonisation events and subsequent population expansions have left distinct genetic signatures in each species. In a separate molecular study, phylogenetic analyses are employed to study members of the circum-Antarctic springtail family Isotomidae. Thesis Abstract The genetic ancestry among these closely related species is shown to reflect a diverse evolutionary origin in the Miocene (23 – 5 Ma), subsequent to which both vicariant and dispersal processes have been important. Phylogenetic re-constructions tease out the relationships among sister species, and the identification of several genetically distant lineages suggests that a revision of current species designations is required. Finally, two studies that integrate the physiological and molecular genetic are presented. First, metabolic rate variation across several locations on sub-Antarctic Marion Island in the springtail Cryptopygus antarcticus travei is examined. This variation is related to the genetic structure of populations to show that historical and contemporary environmental characteristics have left their trace in the expression of both genetic and physiological variability of these populations. Second, the perceived association between metabolic rate and genetic (mutation) rate is investigated more closely - a sophisticated Bayesian correlation analysis detects that there is an indirect relationship between metabolic rate and underlying species phylogeny in C. a. travei. Thus, the physiological and molecular genetic elements of this thesis test or advance important hypotheses within their own fields, and the integrated approach applied is a new step in interpreting evidence of physiological adaptation in Antarctic species. In its multi-faceted approach to evolutionary studies, this thesis enhances understanding of the current picture of springtail evolution in polar environments.
9
Gibb, Gillian Claire. "Birds in a tree : a journey through avian phylogeny, with particular emphasis on the birds of New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics." Massey University, 2010. http://hdl.handle.net/10179/1329.
Full textAbstract:
Two main themes to the avian research presented in this thesis are, 1. Deep resolution of birds generally, and 2. Investigation of specific aspects of the New Zealand avifauna. More specifically, this thesis covers phylogeny, and predictions about palaeognaths, pigeons, pelecaniforms and passerines. Significant progress is made in resolving the basal branches of Neoaves. This thesis examines whether the six-way basal Neoavian split of Cracraft (2001) is, in principle, resolvable. New mitochondrial genomes are added to improve taxon sampling, break up long branches, and allow testing of the prior assumptions of six Neoavian groups. This research shows the six-way split is resolvable, although more work is required for specific details. From a life-history perspective, it is interesting that the two bird-of-prey groups (falcons and buzzards) are very divergent, and may not be sister groups. Molecular dating supports major diversification of at least 12 Neoavian lineages in the Late Cretaceous. Additionally, novel avian mitochondrial gene orders are investigated and a hypothesis put forward suggesting gene conversion and stable intermediate forms allows an apparently rare event (gene rearrangement) to occur multiple times within Neoaves. One of Cracraft’s six groups, informally called the ‘Conglomerati’, is particularly difficult to resolve. The pigeons (Columbiformes) lie within the ‘Conglomerati’, and this chapter examines two aspects along the continuum of pigeon evolution. Firstly the large South Pacific fruit pigeon radiation is examined with mid-length mitochondrial sequences. This clade contains a third of all pigeon species, and has been very successful in island colonisation throughout South East Asia and the Pacific. Secondly, candidates for the closest relative of pigeons are tested using analysis of whole mitochondrial genomes. Highest support was found for the grouping of sandgrouse and pigeon, although they are clearly very divergent. Also within the ‘Conglomerati’ is the traditional order Pelecaniformes, and their close allies the Ciconiiformes. These orders (the P&C) are part of an adaptive radiation of seabird water-carnivores, including loons, penguins, petrels and albatrosses. This group is separate from the large shorebird water-carnivore group; although both appear to have begun radiating abut 70 million years ago. The tropicbird represents a separate, convergent life history and is not part of the Pelecaniformes, nor within the larger seabird water-carnivore group. Resolution of the basal phylogeny of oscine passerines is important for interpreting the radiation of this group out of the Australasian region. Many endemic New Zealand oscine passerines belong to ‘basal corvid’ lineages, but have not previously been investigated with mitochondrial DNA. This chapter shows that many ‘basal corvid’ lineages are actually ‘basal passerine’ lineages, and there is a discrepancy between nuclear Rag-1 phylogenies (the most commonly used gene in passerine phylogenetics) and other phylogenies, including mitochondrial, that requires further investigation. Taken as a whole, this thesis adds significantly to our understanding of the evolution of birds, and provides a foundation for future research, not only of phylogenetic relationships, but also of avian life history, long-term niche stability and macroevolution.
10
Guo, Yanan. "Identification and characterization of Dothistromin biosynthetic genes in the peanut pathogen Passalora arachidicola : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand." Massey University, 2008. http://hdl.handle.net/10179/894.
Full textAbstract:
Dothistromin (DOTH) is a secondary metabolite produced by the fungal peanut pathogen Passalora arachidicola and pine needle pathogen Dothistroma septosporum. The chemical structure of DOTH is similar to a precursor of aflatoxin (AF) and sterigmatocystin (ST), which are secondary metabolites produced by Aspergillus species. A size fractionated genomic library was made and 11 putative DOTH genes were identified in P. arachidicola. The DOTH genes in P. arachidicola were compared to DOTH genes in D. septosporum as well as to AF and ST genes in Aspergillus species. The DOTH gene products in P. arachidicola showed 73 - 96% amino acid identity to DOTH genes in D. septosporum and 50 - 69% amino acid identity to AF/ST genes in Aspergillus. The DOTH biosynthesis genes in P. arachidicola had similar gene organization and direction of transcription to DOTH biosynthesis genes in D. septosporum and is similar in that 11 putative DOTH genes are separated into three mini-clusters. This differs from the AF/ST clusters in which 25 AF/ST genes are tightly clustered in a 70 kb region. Identification of transcription factor binding sites upstream of DOTH genes in P. arachidicola and D. septosporum suggested similar co-regulation of DOTH gene expression in P. arachidicola and D. septosporum. Tandem and inverted repeat sequences were identified in intergenic regions in the P. arachidicola DOTH gene cluster, but the distribution of those repeats appears to be random. This suggests that the fragmentation of the DOTH biosynthesis gene cluster is not due to retrotransposon activity or recombination between repeat sequences. The DOTH biosynthesis gene clusters in P. arachidicola and D. septosporum could be ancestral to AF/ST biosynthesis clusters in Aspergillus species.
11
Pratt, Renae. "Patterns and processes in animal evolution : molecular phylogenetics of Southern Hemisphere fauna : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics." Massey University, 2008. http://hdl.handle.net/10179/958.
Full textAbstract:
Three kinds of processes are known to modify the geographical spatial arrangement of organisms: dispersal, extinction and vicariance. The Southern Hemisphere has an intriguing and complicated geological history that provides an ideal backdrop to study these processes. This thesis focuses on three historical events that illustrate these processes: the proposed marine inundation of New Zealand in the Oligocene, the asteroid impact at the K – Pg boundary, and the continental breakup of Gondwana. It investigates what impact these events had on species diversification by studying the phylogenetic relationships of two groups of taxa – the family Anostostomatidae (insects), and Neoaves (birds). Anostostomatidae were studied in relation to the Oligocene drowning and the break up of Gondwana as they have a wide southern distribution, found on all “Gondwanan” fragments with the exception of Antarctica, and are thought represent an ancient lineage that predates the Gondwanan breakup. Birds, in particular Neoaves, were studied in relation to the asteroid impact at the K – Pg boundary. Although birds are mobile and many circumnavigate the globe between seasons, they are suggested to have originated in the Southern Hemisphere in Gondwanan times, and subsequently undergone range expansion and diversification around the world. In order to address the relationship (if any) between modern biotic diversity and historical geological events, phylogenetic relationships were determined and where possible, molecular clock analysis carried out. Timing information provided by molecular clock analysis is important as it enables distinction between opposing hypotheses such as vicariance and dispersal. In Chapter Two, the phylogenetic relationships within the family Anostostomatidae are investigated. One of the most controversial times in New Zealand’s geological history is during the Oligocene. Some suggest that the lack of fossils and evidence for recent dispersal of numerous taxa support the notion that all modern biota reached the region during the last 25 million years. Anostostomatidae were chosen as they represent a group of insects that are thought to be ancient and there is little published data in the literature. Previous studies focused on the relationships within Hemideina and Deinacrida suggesting that these groups diversified in the early Miocene. The data presented here are from mitochondrial (COI and 12S) and nuclear (18S and 28S) sequences. Molecular dating using a relaxed clock as implemented in BEAST suggest that in fact some lineages were present at or shortly after continental breakup and could have survived throughout this turbulent time. As there were no definitive fossils to use for calibration points, geological events were used as calibration points for the molecular clock. Mutation rates obtained from the different analyses were compared to those published for other insects in an attempt to identify the most likely model. Both maximum likelihood and Bayesian analyses support the presence of three distinct ecological groups in New Zealand; Hemiandrus (ground weta), Anisoura/Motuweta (tusked weta) and Hemideina–Deinacrida (tree–giant weta). With regards to their Australasian relatives (taxa from Australia and New Caledonia) it appears that the family is divided with the most northern New Zealand taxa (tusked weta) more closely related to New Caledonian taxa while all other New Zealand taxa are more closely related to Australian taxa. There does not appear to be any link between the Australian and New Caledonian taxa studied here. Results should be viewed with caution however as an increased mutation rate was observed in the New Caledonian-tusked weta lineage, something future studies will have to address. Chapter Three presents new sequence data and phylogenetic analyses that go towards resolving the apparent basal polytomy of neoavian birds. This chapter includes analyses carried out on previously published data with the addition of nine new mitochondrial genomes. My contribution to this larger project was to perform the phylogenetic analysis and to sequence three of the nine mitochondrial genomes. The genomes I sequenced were the Southern Hemisphere species: dollar bird (Eurystomus orientalis), Owlet nightjar (Aegotheles cristatus cristatus) and great potoo (Nyctibius grandis). The inclusion of these nine new genomes allows assessment and comparison of the six hypothesised groups reported in Cracraft (2001). First an improved conditional down-weighting technique is described reducing noise relative to signal, which is important for resolving deeper divergences. Second, a formula is presented for calculating probabilities of finding predefined groupings in the optimal tree. Maximum likelihood and Bayesian based phylogenetic analyses were carried out and in addition, dating using a relaxed molecular clock was performed in BEAST. Results suggested that the six groups suggested by Cracraft (2001) represent robust lineages. The results suggested that one group, the owls, are more closely related to other raptors, particularly accipitrids (buzzards/eagles) and the osprey rather than the Caprimulgiformes, which could indicate morphological convergent evolution. In addition, a group termed shorebirds appears to be distinct from the large group referred to as ‘Conglomerati’ to which previous publications have suggested they belong. The ‘Conglomerati’ is the least well studied group and may actually comprise of at least three subgroups (as suggested by Cracraft). Within the three suggested groups, Cracraft grouped shorebirds with pigeons and sandgrouse, neither of which (pigeons or sandgrouse) were analysed here. So although the shorebirds are at least close to the ‘Conglomerati’ and may be within that group, their exact position is still not clear. The molecular dating reported here utilised two fossil calibrations (Vegavis and Waimanu), for which there is relatively little dispute as to age or the lineage to which they belong. Calibrations resulting from BEAST analyses suggest that at least 12 distinct lineages were present prior to the K – Pg boundary, a finding supported by previous studies. Robust phylogenies will allow future studies to investigate not only the relationships within Neoaves, but look more closely at the biological and ecological evolution of the group. Chapter Four for the first time investigates whether the phylogenetic relationships within the family Anostostomatidae follow the conventionally accepted order and timing of Gondwanan breakup. Following the initial restults for taxa studied in Australasia (Chapter Two) an attempt to resolve family relationships in a wider spatial (geographic) context was carried out to determine if Australasian taxa are monophyletic when other members of the family are included. Again both maximum likelihood and Bayesian phylogenetic analyses were carried out on both mitochondrial (COI and 12S) and nuclear (18S and 28S) sequences. In this chapter, datasets included samples from across the geographic range of Anostostomatidae (South Africa, Madagascar, South America, Australia, New Caledonia and New Zealand), and two clades were observed, congruent with earlier findings. Sequence divergence within geographic regions was found to be relatively high in the mitochondrial genes (COI and 12S) while low in the nuclear ribosomal RNA genes (18S and 28S) as expected given their relative mutation rates. Under the vicariance paradigm, phylogenetic relationships should follow the order of continental breakup, but this was not found. Further, if dispersal and colonisation were continuous, no geographic substructure is expected, however distinct geographic substructure within clades was consistently observed. This interesting phylogenetic pattern may be a case of convergent evolution or paraphyletic sampling which highlights taxonomic issues of the group. Future studies need to include not only molecular data but information on morphology, ecology and behaviour along with the implementation of biogeographic programs that can test alternative hypotheses (such as dispersal and vicariance) directly. Also, the inclusion of the recently reported fossil from the subfamily Euclydesinae (Martins-Neto 2007) should allow for more accurate date estimates within the family. Taken as a whole the results presented in this thesis suggest that microevolutionary processes are sufficient to explain modern diversity without the need to invoke abiotic events. The three cases investigated here - marine inundation, asteroid impact and continental drift - all appear to have had only a limited effect on the diversity of taxa studied. To reach even stronger conclusions future studies should incorporate different data (for instance nuclear genes, intron position, and genome structure) and use biogeographic software capable of including ecological, morphological and habitat information.
12
Stepper, Judith. "The molecular and cellular characterisation of the first glycocin, plantaricin KW30 : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand [Ph. D] EMBARGOED." Massey University, 2010. http://hdl.handle.net/10179/1184.
Full text
13
Vargas, Mariana L. "Host-parasite coevolution in New Zealand: how has Odontacarus, a mite with a free-living stage in its life-cycle, coevolved with its skink host?" Lincoln University, 2006. http://hdl.handle.net/10182/1072.
Full textAbstract:
The effect of a free-living stage in host-parasite coevolution: a skink mite phylogenetic study in New Zealand. During the last decade, phylogenetic trees have even been used to compare ecologically related taxa such as parasites and their hosts, and are used to determine their level of coevolution or reciprocal adaptation in time. Diverse coevolutionary events have been detected for this ecological association, where generally the parasite has been regarded as one that feeds exclusively on the host and is likely to cospeciate with it. A different coevolutionary pattern might occur when the parasite has a free-living stage in its life cycle, in which the parasite may have the opportunity to abandon its host and successfully colonise a new species (host-switching) making cospeciation less likely. Many New Zealand skinks are infested with a parasitic mite, Odontacarus sp. (Prostigmata: Leeuwenhoekiidae), which becomes free-living as an adult. The genetic variation of these mites found on four hosts was analyzed for host- parasite coevolutionary events. The hosts were the McCann’s skink and the common skink in coastal Birdling Flat, Canterbury, plus these species and the Grand and Otago skinks in Macraes Flat, Central Otago, South Island, New Zealand. The genetic variation of fast evolving nuclear Internal Transcribed Spacers 2 and mitochondrial Cytochrome c Oxidase I in Odontacarus mites found on these hosts was determined by PCR and DNA sequencing and phylogenetic trees were built using the computer programs PAUP*4 and MrBayes 3. The results show that mite haplotypes only had a significant geographical division and no host-related differences. In Birdling Flat, the COI haplotypes were represented in two groups that infested both regional hosts and had 5.7 % divergence. The same individual mites belonged to a single ITS 2 haplotype, thus indicating a historical geographical division between two populations that now interbreed successfully. The Macraes Flat mites were divided into two COI haplotypes with 2.4% divergence and internal nodes, which showed greater genetic variability than the Birdling Flat populations. The Macraes Flat mites formed two ITS 2 haplotypes with 6% divergence. This greater geographical structure of the Otago mites is probably due to the older age of the mainland area compared to the recently exposed coastal locality of Birdling Flat. The COI haplotypes from the two different regions had a mean distance of 15.5%, with an earlier divergence time than that known for the hosts. For both genes, the haplotypes from different regions had 100% bootstrap support and the parasite showed no host specificity. Mites of the different COI and ITS haplotypes were found on most of the host species that were sampled in Canterbury and Otago. The results of this study suggest that a free-living stage in a parasite’s life cycle can favour coevolutionary events such as inertia (failure to speciate) and host-switching, probably as a result of resource-tracking of the parasite. NB: Electronic files contained on CD to accompany print copy are not included with this version of the thesis.
14
Dalebout, Merel Louise. "Species identity, genetic diversity, and molecular systematic relationships among the Ziphiidae (beaked whales)." Thesis, University of Auckland, 2002. http://wwwlib.umi.com/dissertations/fullcit/3083930.
Full textAbstract:
Beaked whales (family Ziphiidae) are one of the least known of all mammalian groups. The majority of species have been described from only a handful of specimens. Found in deep ocean waters, these species are widespread and often sexually dimorphic. Little is known of intra-specific variation in morphology, and many species are very similar in external appearance. A reference database of mitochondrial DNA sequences was compiled for all 20 recognised ziphiid species to aid in species identification. All reference sequences were derived from validated specimens, which were often represented only by bone or teeth. DNA was obtained from this ‘historic’ material using ‘ancient’ DNA methods. For three species, holotypes were sampled. Phylogenetic analyses using this database led to the discovery of a new, previously unrecognised species of beaked whale (Mesoplodon perrini), new specimens of Longman's beaked whale (Indopacetus pacificus), a species known previously from only two partial skulls and the synonymy of a third (M. traversii = M. bahamondi). Phylogenetic reconstructions based on sequence data from three mitochondrial and two nuclear loci (total, 2815 bp) using neighbour joining, parsimony, and maximum likelihood methods, resolved many of the sister-species relationships in this group. Inferred relationships among Mesoplodon beaked whales indicated that cranial and tooth morphology may be far more variable between closely related species than previously assumed. No support was found for a linear-progression of tooth form as suggested by Moore (1968) in his phenetic evaluation of relationships among the Ziphiidae. The geographic distribution of Mesoplodon species with similar or divergent tooth morphology is likely due to a combination of sexual selection and selection for species recognition. Both hypotheses predict similar patterns, such as dissimilar tooth morphology among species with sympatric or parapatric distributions. However, only sexual selection appears to offer an explanation for why there are so many Mesoplodon beaked whales. Investigation of mtDNA diversity among a number of beaked whale species indicated that nucleotide diversity was generally lower in this group than in other wide-ranging oceanic cetaceans. The cause of this low diversity was not clear but may be indicative of overall low abundance. Particularly low levels of diversity were found in Baird's beaked whale Berardius bairdii , Arnoux's beaked whale B. arnuxii and the northern bottlenose whale Hyperoodon ampullatus. Strong geographic structure in haplotype frequencies was observed among a worldwide sample of Cuvier's beaked whales Ziphius cavirostris.Subscription resource available via Digital Dissertations only.
15
Jenkins, Toni E. "Introgression of genes from rape to wild turnip." Lincoln University, 2005. http://hdl.handle.net/10182/1844.
Full textAbstract:
Introgression of genes from crops into ruderal populations is a multi-step process requiring sympatry, synchronous flowering, chromosomal compatibility, successful pollination and development of the zygote, germination, establishment and reproduction of hybrid progeny. The goal of this thesis was to generate data on as many steps in this process as possible and integrate them into a predictive statistical model to estimate the likelihood of successful introgression under a range of scenarios. Rape (Brassica napus) and wild turnip (B. rapa var. oleifera) were used as a model system. A homozygous dominant mutation in the rape genome conferring herbicide resistance provided a convenient marker for the study of introgression. Potential differences between wild turnip populations from a wide range of geographic locations in New Zealand were examined. Hand pollination established the genetic compatibility of rape and wild turnip and a high potential for gene introgression from rape to wild turnip. Interspecific hybrids were easily generated using wild turnip as the maternal plant, with some minor differences between wild turnip populations. The frequency of successful hybridisation between the two species was higher on the lower raceme. However, the upper raceme produced more dormant interspecific hybrid seed. Field trials, designed to imitate rare rape crop escapes into the ruderal environment, examined the ability of rare rape plants to pollinate wild turnip plants over four summers. At a ratio of 1 rape plant for every 400 wild turnip plants, the incidence of interspecific hybridisation was consistently low (<0.1 to 2.1 % of total seed on wild turnip plants). There was a significant year effect with the first season producing significantly more seed and a greater frequency of interspecific hybrid progeny than the other years. The frequency of interspecific hybrid progeny increases when the ratio of rape: wild turnip plant numbers increases. The relative importance of anemophily and entomophily in the production of interspecific hybrids was examined. Wild turnip plants produced twice as many seeds with bee pollination relative to wind pollination. However, the frequency of interspecific hybrids under wind pollination was nearly twice that for bee pollination. Light reflectance patterns under UV light revealed a marked difference between wild turnip and rape flowers compared to near identical appearance under visible light. The data indicates that bees are able to distinguish between rape and wild turnip flowers and exhibit floral constancy when foraging among populations with these two species. Hybrid survival in the seed bank, germination and seedling establishment in the field are important components of fitness. Seed banks established in the soil after the field trials described above germinated in subsequent spring seasons. The predominantly brassica weed populations were screened for herbicide resistance and the numbers of interspecific hybrids germinating compared to the original frequency in the field trial results. Frequency of interspecific hybrids was reduced in the populations compared to the original seed deposit. Seed with a known frequency of interspecific hybrid seed was sown in a separate trial, and the frequency of interspecific hybrids compared at 0, 4, 6, and 8 weeks after sowing. Poor germination resulted limited competition between seedlings, however the frequency of interspecific hybrids declined over time indicating low plant fitness. There were no significant population effects on any parameters tested. Interspecific hybrids grown in a glasshouse were backcrossed to the parental species and selfed within the plant and within populations. Pollen from the interspecific hybrids was found to have markedly reduced fertility. Interspecific hybrid plants had low female fertility, with the majority (88%) of the pollinated flowers aborting the siliques. Of the remaining siliques, most (98%) had only one to three seeds per silique. Inheritance of the herbicide resistance gene was regular in backcrosses but highly skewed following self pollination with an excess of herbicide-sensitive progeny. Production of a stochastic predictive model integrated the information acquired over the practical work phase of this thesis and utilised the capabilities of @risk, a new application of a risk analysis tool. The three outputs examined were the number of flowering plants resulting from backcrosses to rape and wild turnip and self pollination of the interspecific hybrid progeny. Five scenarios were modelled and all demonstrated the high likelihood of introgression failure in this system. In all scenarios, >75% of simulations resulted in no interspecific hybrid progeny surviving to flowering in the third generation. In all scenarios, and for all three outputs, the seed set on the interspecific hybrids of the second generation was the major factor that limited the number interspecific hybrid progeny surviving to flowering in the third generation.
16
McPhee, Scott William John. "Phenotypic characterisation of the tremor mutant and AAV mediated aspartoacylase gene transfer in the rat model of Canavan disease." Thesis, University of Auckland, 2004. http://wwwlib.umi.com/dissertations/fullcit/3136372.
Full textAbstract:
The doctoral studies described in this thesis involve the phenotypic characterization of the tremor rat, an animal model of Canavan disease, and a proof of principle gene transfer study in this model. The phenotype of the tremor rat is examined at the genetic, molecular, cellular, neurochemical, physical and behavioural levels, and tremor mutants are described within the context of Canavan disease. Tremor mutants appear to share many phenotypes with both human patients and to the knock-out mouse model. The deletion of aspartoacylase results in a total loss of the capacity to metabolize N-acetyl-aspartate to acetate and aspartate in brain, leading to elevations in brain N-acetyl-aspartate levels, changes in cell and tissue morphology, and physical and behavioural deficits including mild akinesia and loss of normal motor coordination and balance. Parallel to this work was the development of a gene transfer approach to treat Canavan disease, involving Adeno-associated virus mediated delivery of aspartoacylase to the mammalian central nervous system. Gene transfer was undertaken in tremor rat mutants, and analysis was made of gene expression and function as well as the effect of aspartoacylase expression on improving the phenotypic deficits observed in mutant animals. Gene expression was observed at the RNA and protein level, with recombinant protein observed in cell soma and processes. Although not significant the data suggested a trend of decreased NAA levels after aspartoacylase transfer in comparison to animals injected with a vector encoding green fluorescent protein. Improvement was noted in the rotorod phenotype with mutant animals receiving aspartoacylase gene transfer performing better at tests of balance and coordinated locomotion than animals receiving a control vector. The study provided evidence that Adeno-associated virus mediated aspartoacylase gene transfer to the brain improves some of the deficits in tremor mutants, and supports the rationale of human gene transfer for Canavan disease.Subscription resource available via Digital Dissertations only.
17
Gudex, B. W. "Sireline variation in neonatal lamb cold tolerance." Lincoln University, 2001. http://hdl.handle.net/10182/1055.
Full textAbstract:
The cost of lamb mortality caused by cold exposure has been estimated at approximately 40 million dollars per year. This value is probably conservative as it does not include the cost due to the reduction in productivity in hypothermic lambs that manage to survive or the cost of reduced selection potential incurred by fewer lambs surviving until selection. The objectives of this research was to investigate whether sire-line variation exists in neonatal lamb cold tolerance and whether polymorphism in the β₃ adrenergic receptor gene can be used as a genetic marker for lamb cold tolerance and lean muscle growth. The influence of the climate, birthweight, age of dam at lambing, gender and birth rank on neonatal lamb cold tolerance was also analysed. Neonatal lamb mortality due to cold exposure was analysed in four field trials that used neonatal lamb morality from cold exposure as a predictor of neonatal lamb cold tolerance. Sire-line variation in neonatal lamb morality was observed in all trials, though it appeared that this effect was largely mediated through sire-line variation in lamb birth weight. Variation in lamb birth weight between birth rank classed was also found to be responsible for the influence of birth rank on neonatal lamb mortality due to cold exposure. The age of dam at lambing and the lamb gender was not observed to influence neonatal lamb mortality due to cold exposure. The sires from the cold tolerance study and the progeny of the lean muscle growth study were genotyped for the β₃ adrenergic receptor locus. Other studies have found evidence that a major gene exists in the catecholamine stimulation of brown adipose thermogenesis and evidence that the β₃-AR gene is a likely candidate. However, this hypothesis and the hypothesis that polymorphism in the β₃-AR gene is also linked to lean muscle growth in lambs was not confirmed in this study. So while it appears that the results were confounded by experimental design, there is evidence that influence of polymorphism in the ovine β₃ AR gene on neonatal lamb mortality and/or lean muscle growth is not sufficient to be considered a major gene effect. The implications of this experiment on the sheep industry and sheep farmers in general are huge. While completely eliminating lamb deaths due to inadequate cold tolerance is impossible, this study shows that large gains in lamb survival could be possible through selective breeding.
18
Spiers, Andrew J. (Andrew Julian). "Molecular and genetic analysis of RepA from the P307 RepFIB replicon." Thesis, University of Auckland, 1992. http://hdl.handle.net/2292/2044.
Full textAbstract:
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.
19
Othman, Rashidi. "Biochemistry and genetics of carotenoid composition in potato tubers." Diss., Lincoln University, 2009. http://hdl.handle.net/10182/1336.
Full textAbstract:
Potato cultivars exhibit a wide variation in skin and flesh colour due to the presence of pigments. This study established that potato cultivars differ greatly with respect to types and concentrations of carotenoids in tubers. A total of 46 cultivars were evaluated for quantitative and qualitative carotenoid composition in different growing seasons, locations, storage conditions and disease symptoms. Factors controlling carotenoid accumulation were also tested by developing an in vitro minituber system as a new high-throughput model system for carotenogenesis in potato tubers. Tuber flesh colour was found to correlate with total carotenoid content in potato cultivars grown in both New Zealand and Netherlands. The main carotenoids identified in 32 potato cultivars in New Zealand were lutein, neoxanthin, violaxanthin and β-carotene. The ratio of these carotenoids varies between cultivars. Neoxanthin was detected in only 13 cultivars (10.59 to 69.21µg/g DW); violaxanthin was found only in 1 cultivar (32.76 µg/g DW). Whereas lutein and β-carotene were found in most of the cultivars but the concentration varied from (0.00 to 160.63 µg/g DW) and (0.00 to 13.62 µg/g DW) respectively. The main carotenoids identified in 12 cultivars grown in the Netherlands were neoxanthin, violaxanthin and lutein, whereas zeaxanthin was not found in any of the cultivars analysed. Marked differences were observed between the same potato cultivars grown in New Zealand and the Netherlands. Therefore cultivars were analysed over a second growing season to assess stability in carotenoids composition. The carotenoid profiles of the potato tubers grown for two different seasons showed highly significant differences between the cultivars, the seasons, the carotenoid pigments, and all combinations of interactions, indicating the complex nature of factors influencing carotenoid composition. Reflectance colorimeter measurement of yellow hue component in this study confirmed that the higher the total carotenoid content, the greater the yellow intensity colour. Eight cultivars were grown at three locations in New Zealand and Agria and Desiree were grown at eight locations in the Netherlands to further investigate the stability of carotenoid composition. Highly significant differences were observed between the cultivars, the locations, the carotenoid pigments, and all combinations of interactions, which emphasises that changes in carotenoid composition are complex and the responses are not consistent across cultivars. Reflectance colorimeter measurement of yellow hue component confirmed the relationship between the yellow colour intensity of tuber flesh, as well as confirming the interaction between colour and locations. Disease and post harvest storage conditions markedly influenced the levels of total carotenoid, neoxanthin, violaxanthin, zeaxanthin, lutein and β-carotene in potatoes. The magnitude of these effects depends on the cultivar, time of storage, and the intensity of powdery scab symptoms. Results showed that long term storage resulted in the accumulation of neoxanthin, violaxanthin and zeaxanthin with a concomitant decreased of lutein, β-carotene and total carotenoid content. Genotypes infected with disease (lower and higher scab score) resulted in accumulation of violaxanthin, β-carotene and total carotenoid with a concomitant decreased in neoxanthin and lutein. A high-throughput model system for investigating carotenoid biogenesis in potato tubers was developed. This involved in vitro potato minitubers and was validated by assessing the effects of environmental variables, such as drought stress, light intensity and nutrient availability on carotenoid accumulation. Light influenced the presence of zeaxanthin, whereas water stress and nutrient strength influenced the accumulation of neoxanthin and violaxanthin. Although these factors had an effect on the carotenoid content and profile, the most influential factor appeared to be cultivar selection.
20
Grievink, Hilbert. "Malignant hyperthermia: allele specific expression and mutation screening of the ryanodine receptor 1 : a dissertation presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry." Massey University, 2009. http://hdl.handle.net/10179/1051.
Full textAbstract:
Malignant hyperthermia (MH) is a dominant skeletal muscle disorder caused by mutations in the ryanodine receptor skeletal muscle calcium release channel (RyR1). Allele-specific differences in RyR1 expression levels might provide insight into the observed incomplete penetrance and variations in MH phenotypes between individuals. Firstly, an H4833Y allele-specific PCR (AS-PCR) assay was designed that allowed for the relative quantification of the two RYR1 mRNA alleles in heterozygous samples. In four MHS skeletal muscle samples and two lymphoblastoid cell lines (LCLs), the wild type allele was found to be expressed at higher levels than the mutant RyR1 allele. These differences were not caused by variations in RYR1 mRNA stabilities. Secondly, high-throughput amplicon sequencing was employed for the quantification of both the T4826I and H4833Y causative MH mutations in heterozygous MHS samples. With the exception of one, all detected H4833Y and T4826I mutation frequencies were about 50%. This included a control, which was constructed and proven to have a 3:1 ratio of the wild type (H4833) versus the mutant (Y4833) RYR1 allele. This suggested that that the high-throughput amplicon sequencing approach as used here, was not suitable for accurate quantification of the two RyR1 alleles in heterozygous H4833Y MHS samples. To detect possible variations in RyR1 alleles at the protein level, the RyR1 was to be isolated from microsomes prepared from a H4833Y MHS frozen skeletal muscle tissue. Microsomes isolated from MHS skeletal muscle tissues lacked the immunoreactive band that was believed to be the full length RyR1. Poor muscle quality, due to long term storage was believed to be the main cause of RyR1 depletion. Faster and less expensive screening methodologies are required for the identification of genetic variants in MH research. Thus, in an additional project inexpensive and high-throughput high-resolution melting (HRM) assays were developed to allow screening of the RYR1 gene, for mutations associated with MH and/or central core disease (CCD).
21
Chng, Soon Fang. "Microbial factors associated with the natural suppression of take-all in wheat in New Zealand : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Lincoln University, Canterbury, New Zealand /." Diss., Lincoln University, 2009. http://hdl.handle.net/10182/863.
Full textAbstract:
Take-all, caused by the soilborne fungus, Gaeumannomyces graminis var. tritici (Ggt), is an important root disease of wheat that can be reduced by take-all decline (TAD) in successive wheat crops, due to general and/or specific suppression. A study of 112 New Zealand wheat soils in 2003 had shown that Ggt DNA concentrations (analysed using real-time PCR) increased with successive years of wheat crops (1-3 y) and generally reflected take-all severity in subsequent crops. However, some wheat soils with high Ggt DNA concentrations had low take-all, suggesting presence of TAD. This study investigated 26 such soils for presence of TAD and possible suppressive mechanisms, and characterised the microorganisms from wheat roots and rhizosphere using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A preliminary pot trial of 29 soils (including three from ryegrass fields) amended with 12.5% w/w Ggt inoculum, screened their suppressiveness against take-all in a growth chamber. Results indicated that the inoculum level was too high to detect the differences between soils and that the environmental conditions used were unsuitable. Comparison between the Ggt DNA concentrations of the same soils collected in 2003 and in 2004 (collected for the pot trial), showed that most soils cropped with 2, 3 and 4 y of successive wheat had reduced Ggt DNA concentrations (by 195-2911 pg g-1 soil), and their disease incidences revealed 11 of the 29 test soils with potential take-all suppressiveness. Further pot trials improved the protocols, such that they were able to differentiate the magnitudes of suppressiveness among the soils. The first of the subsequent trials, using 4% w/w Ggt inoculum level, controlled conditions at 16°C, 80% RH with alternate 12 h light/dark conditions, and watering the plants twice weekly to field capacity (FC), screened 13 soils for their suppressiveness against take-all. The 13 soils consisted of 11 from the preliminary trial, one wheat soil that had been cropped with 9 y of wheat (considered likely to be suppressive), and a conducive ryegrass soil. The results revealed that 10 of these soils were suppressive to take-all. However, in only four of them were the effects related to high levels of microbial/biological involvement in the suppression, which were assessed in an experiment that first sterilised the soils. In a repeat trial using five of the soils H1, H3, M2, P7 (previously cropped with 3, 3, 4 and 9 y successive wheat, respectively) and H15 (previously cropped with 5 y of ryegrass), three of them (H1, H3 and M2) had reduced Ggt DNA concentrations (>1000 pg g-1 soil reductions), and were confirmed to be suppressive to take-all. A pot trial, in which 1% of each soil was transferred into a γ-irradiated base soil amended with 0.1% Ggt inoculum, indicated that soils H1 and H3 (3 y wheat) were specific in their suppressiveness, and M2 (4 y wheat) was general in its suppressiveness. The microbial communities within the rhizosphere and roots of plants grown in the soils, which demonstrated conduciveness, specific or general suppressiveness to take-all, were characterised using PCR-DGGE, and identities of the distinguishing microorganisms (which differentiated the soils) identified by sequence analysis. Results showed similar clusters of microorganisms associated with conducive and suppressive soils, both for specific and general suppression. Further excision, re-amplification, cloning and sequencing of the distinguishing bands showed that some actinomycetes (Streptomyces bingchengensis, Terrabacter sp. and Nocardioides sp.), ascomycetes (Fusarium lateritium and Microdochium bolleyi) and an unidentified fungus, were associated with the suppressive soils (specific and general). Others, such as the proteobacteria (Pseudomonas putida and P. fluorescens), an actinomycete (Nocardioides oleivorans), ascomycete (Gibberella zeae), and basidiomycete (Penicillium allii), were unique in the specific suppressiveness. This indicated commonality of some microorganisms in the take-all suppressive soils, with a selected distinguishing group responsible for specific suppressiveness. General suppressiveness was considered to be due to no specific microorganisms, as seen in soil M2. An attempt to induce TAD by growing successive wheat crops in pots of Ggt-infested soils was unsuccessful with no TAD effects shown, possibly due to variable Ggt DNA concentrations in the soils and addition of nutrients during the experiment. Increasing numbers of Pseudomonas fluorescens CFU in the rhizosphere of plants, during successive wheat crops was independent of the Ggt DNA concentrations and disease incidence, suggesting that increases in P. fluorescens numbers were associated with wheat monoculture. This study has demonstrated that TAD in New Zealand was due to both specific and general suppressiveness, and has identified the distinguishing microorganisms associated with the suppression. Since most of these distinguishing microorganisms are known to show antagonistic activities against Ggt or other soilborne pathogens, they are likely to act as antagonists of Ggt in the field. Future work should focus on validating their effects either individually, or interactively, on Ggt in plate and pot assays and under field conditions.
22
Wragg, Graham. "The comparative biology of Fluttering shearwater and Hutton's shearwater and their relationship to other shearwater species." Lincoln College, University of Canterbury, 1985. http://hdl.handle.net/10182/1635.
Full textAbstract:
The discovery and taxonomic history of fluttering shearwater (Puffinus gavia (Forster) and Hutton's shearwater (Puffinus huttoni Mathews) are reviewed. Taxonomic theory, where appropriate to this thesis, is discussed. The external morphology of P. gavia and P. huttoni is compared. No single external measurement or plumage character separates more than 60% of birds examined. The best system of identification is to compare the ratio of different body parts within an individual bird. The distribution of P. gavia and P. huttoni is compared. Hutton's shearwater feeds further out to sea and it is believed to be a migrant species wintering in north west Australian waters. The fluttering shearwater is believed to be a semi-migrant species with only the juveniles spending time in south east Australia. The red cell enzymes of P. gavia, P. huttoni and P. griseus are compared. There are differences in two esterase loci between gavia and huttoni, while P. griseus is more distantly related. Nei's genetic identity values are calculated. The systematic value of electrophoretic data is discussed. The relationship of an undescribed subfossil shearwater to P. gavia and P. huttoni is discussed. An outgroup analysis to other shearwater species is carried out according to phylogenetic (cladistic) theory. The subfossil shearwater is most closely related to the fluttering shearwater, and these two form a sister group to Hutton's shearwater. These three species are a sister group of P. opisthomelas. The relationship between the many P. assimilis subspecies, the black-backed Manx shearwaters, and the gavia, huttoni and opisthomelas group was not resolved. Puffinus nativitatis is more closely related to the Manx and the little shearwaters than to the P. griseus, P. tenuirostris group.
23
Xie, Zhi. "Modelling genetic regulatory networks: a new model for circadian rhythms in Drosophila and investigation of genetic noise in a viral infection process." Phd thesis, Lincoln University. Agriculture and Life Sciences Division, 2007. http://theses.lincoln.ac.nz/public/adt-NZLIU20070712.144258/.
Full textAbstract:
In spite of remarkable progress in molecular biology, our understanding of the dynamics and functions of intra- and inter-cellular biological networks has been hampered by their complexity. Kinetics modelling, an important type of mathematical modelling, provides a rigorous and reliable way to reveal the complexity of biological networks. In this thesis, two genetic regulatory networks have been investigated via kinetic models.
In the first part of the study, a model is developed to represent the transcriptional regulatory network essential for the circadian rhythms in Drosophila. The model incorporates the transcriptional feedback loops revealed so far in the network of the circadian clock (PER/TIM and VRI/PDP1 loops). Conventional Hill functions are not used to describe the regulation of genes, instead the explicit reactions of binding and unbinding processes of transcription factors to promoters are modelled. The model is described by a set of ordinary differential equations and the parameters are estimated from the in vitro experimental data of the clocks components. The simulation results show that the model reproduces sustained circadian oscillations in mRNA and protein concentrations that are in agreement with experimental observations. It also simulates the entrainment by light-dark cycles, the disappearance of the rhythmicity in constant light and the shape of phase response curves resembling that of experimental results. The model is robust over a wide range of parameter variations. In addition, the simulated E-box mutation, perS and perL mutants are similar to that observed in the experiments. The deficiency between the simulated mRNA levels and experimental observations in per01, tim01 and clkJrk mutants suggests some differences in the model from reality. Finally, a possible function of VRI/PDP1 loops is proposed to increase the robustness of the clock.
In the second part of the study, the sources of intrinsic noise and the influence of extrinsic noise are investigated on an intracellular viral infection system. The contribution of the intrinsic noise from each reaction is measured by means of a special form of stochastic differential equation, the chemical Langevin equation. The intrinsic noise of the system is the linear sum of the noise in each of the reactions. The intrinsic noise arises mainly from the degradation of mRNA and the transcription processes. Then, the effects of extrinsic noise are studied by means of a general form of stochastic differential equation. It is found that the noise of the viral components grows logarithmically with increasing noise intensities. The system is most susceptible to noise in the virus assembly process. A high level of noise in this process can even inhibit the replication of the viruses.
In summary, the success of this thesis demonstrates the usefulness of models for interpreting experimental data, developing hypotheses, as well as for understanding the design principles of genetic regulatory networks.
24
Nguyen, Lan K. "Dynamical modelling of feedback gene regulatory networks." Diss., Lincoln University, 2009. http://hdl.handle.net/10182/1340.
Full textAbstract:
Living cells are made up of networks of interacting genes, proteins and other bio-molecules. Simple interactions between network components in forms of feedback regulations can lead to complex collective dynamics. A key task in cell biology is to gain a thorough understanding of the dynamics of intracellular systems and processes. In this thesis, a combined approach of mathematical modelling, computational simulation and analytical techniques, has been used to obtain a deeper insight into the dynamical aspects of a variety of feedback systems commonly encountered in cells. These systems range from model system with detailed available molecular knowledge to general regulatory motifs with varying network structures. Deterministic as well as stochastic modelling techniques have been employed, depending primarily on the specific questions asked. The first part of the thesis focuses on dissecting the principles behind the regulatory design of the Tryptophan Operon system in Escherichia coli. It has evolved three negative feedback loops, namely repression, attenuation and enzyme inhibition, as core regulator mechanisms to control the intracellular level of tryptophan amino acid, which is taken up for protein synthesis. Despite extensive experimental knowledge, the roles of these seemingly redundant loops remain unclear from a dynamical point of view. We aim to understand why three loops, rather than one, have evolved. Using a large-scale perturbation/response analysis through modelling and simulations and novel metrics for transient dynamics quantification, it has been revealed that the multiple negative feedback loops employed by the tryptophan operon are not redundant. In fact, they have evolved to concertedly give rise to a much more efficient, adaptive and stable system, than any single mechanism would provide. Since even the full topology of feedback interactions within a network is insufficient to determine its behavioural dynamics, other factors underlying feedback loops must be characterised to better predict system dynamics. In the second part of the thesis, we aim to derive these factors and explore how they shape system dynamics. We develop an analytical approach for stability and bifurcation analysis and apply it to class of feedback systems commonly encountered in cells. Our analysis showed that the strength and the Hill coefficient of a feedback loop play key role in determining the dynamics of the system carrying the loop. Not only that, the position of the loop was also found to be crucial in this decision. The analytical method we developed also facilitates parameter sensitivity analysis in which we investigate how the production and degradation rates affect system dynamics. We find that these rates are quite different in the way they shape up system behaviour, with the degradation rates exhibiting a more intricate manner. We demonstrated that coupled-loop systems display greater complexity and a richer repertoire of behaviours in comparison with single-loop ones. Different combinations of the feedback strengths of individual loops give rise to different dynamical regimes. The final part of the thesis aims to understand the effects of molecular noise on dynamics of specific systems, in this case the Tryptophan Operon. We developed two stochastic models for the system and compared their predictions to those given by the deterministic model. By means of simulations, we have shown that noise can induce oscillatory behaviour. On the other hand, incorporating noise in an oscillatory system can alter the characteristics of oscillation by shifting the bifurcation point of certain parameters by a substantial amount. Measurement of fluctuations reveals that that noise at the transcript level is most significant while noise at the enzyme level is smallest. This study highlights that noise should not be neglected if we want to obtain a complete understanding of the dynamic behaviour of cells.
25
Peacock, Lora. "Eco-climatic assessment of the potential establishment of exotic insects in New Zealand." Lincoln University, 2005. http://hdl.handle.net/10182/1530.
Full textAbstract:
To refine our knowledge and to adequately test hypotheses concerning theoretical and applied aspects of invasion biology, successful and unsuccessful invaders should be compared. This study investigated insect establishment patterns by comparing the climatic preferences and biological attributes of two groups of polyphagous insect species that are constantly intercepted at New Zealand's border. One group of species is established in New Zealand (n = 15), the other group comprised species that are not established (n = 21). In the present study the two groups were considered to represent successful and unsuccessful invaders. To provide background for interpretation of results of the comparative analysis, global areas that are climatically analogous to sites in New Zealand were identified by an eco-climatic assessment model, CLIMEX, to determine possible sources of insect pest invasion. It was found that south east Australia is one of the regions that are climatically very similar to New Zealand. Furthermore, New Zealand shares 90% of its insect pest species with that region. South east Australia has close trade and tourism links with New Zealand and because of its proximity a new incursion in that analogous climate should alert biosecurity authorities in New Zealand. Other regions in western Europe and the east coast of the United States are also climatically similar and share a high proportion of pest species with New Zealand. Principal component analysis was used to investigate patterns in insect global distributions of the two groups of species in relation to climate. Climate variables were reduced to temperature and moisture based principal components defining four climate regions, that were identified in the present study as, warm/dry, warm/wet, cool/dry and cool/moist. Most of the insect species established in New Zealand had a wide distribution in all four climate regions defined by the principal components and their global distributions overlapped into the cool/moist, temperate climate where all the New Zealand sites belong. The insect species that have not established in New Zealand had narrow distributions within the warm/wet, tropical climates. Discriminant analysis was then used to identify which climate variables best discriminate between species presence/absence at a site in relation to climate. The discriminant analysis classified the presence and absence of most insect species significantly better than chance. Late spring and early summer temperatures correctly classified a high proportion of sites where many insect species were present. Soil moisture and winter rainfall were less effective discriminating the presence of the insect species studied here. Biological attributes were compared between the two groups of species. It was found that the species established in New Zealand had a significantly wider host plant range than species that have not established. The lower developmental threshold temperature was on average, 4°C lower for established species compared with non-established species. These data suggest that species that establish well in New Zealand have a wide host range and can tolerate lower temperatures compared with those that have not established. No firm conclusions could be drawn about the importance of propagule pressure, body size, fecundity or phylogeny for successful establishment because data availability constrained sample sizes and the data were highly variable. The predictive capacity of a new tool that has potential for eco-climatic assessment, the artificial neural network (ANN), was compared with other well used models. Using climate variables as predictors, artificial neural network predictions were compared with binary logistic regression and CLIMEX. Using bootstrapping, artificial neural networks predicted insect presence and absence significantly better than the binary logistic regression model. When model prediction success was assessed by the kappa statistic there were also significant differences in prediction performance between the two groups of study insects. For established species, the models were able to provide predictions that were in moderate agreement with the observed data. For non-established species, model predictions were on average only slightly better than chance. The predictions of CLIMEX and artificial neural networks when given novel data, were difficult to compare because both models have different theoretical bases and different climate databases. However, it is clear that both models have potential to give insights into invasive species distributions. Finally the results of the studies in this thesis were drawn together to provide a framework for a prototype pest risk assessment decision support system. Future research is needed to refine the analyses and models that are the components of this system.
26
Chong, Belinda Yuen Yee. "Molecular genetics of idiopathic hyperphosphatasia." 2004. http://hdl.handle.net/2292/1223.
Full textAbstract:
The subject of this thesis is the molecular genetic study of idiopathic hyperphosphatasia. The work in this thesis describes linkage analysis, mutation screening of candidate genes and the functional analysis of mutant proteins expressed in patients with a clinical diagnosis of idiopathic hyperphosphatasia. Idiopathic hyperphosphatasia is an autosomal recessive bone disease characterized by excessive bone resorption and bone formation. Affected children are normal at birth but develop deformities of long bones, kyphosis and acetabular protrusion with increasing severity as they pass through adolescence. There is considerable variability in phenotype, with some cases diagnosed in infancy and others in later childhood. A genome-wide search of a New Zealand family affected by idiopathic hyperphosphatasia suggested linkage to a locus on the long arm of chromosome 8 (8q24). The gene TNFRSF11B encoding osteoprotegerin (OPG), which lies within 8q24, was an obvious candidate gene given the critical role of OPG in regulating osteoclast development. Mutation screening of this gene indicated an apparent disease-causing mutation in exon 3 in affected individuals of the New Zealand family. Subsequently eight families, recruited by the members of the Idiopathic Hyperphosphatasia Collaborative Group in Turkey, Germany, Argentina and the United kingdom were also screened for mutations in the TNFRSF11B gene. Recombinant wild-type and mutant OPG CDNAs were expressed in human epithelial kidney cells, and secreted OPG was collected form the conditioned medium. In vitro measurements of osteoclastic bone resorption showed that wild type OPG suppressed bone resorption, whereas the mutant forms did not, confirming them to be inactivating mutations.
27
Lambie, Suzanne Claire. "Reverse genetic analyses of TERMINAL EAR-like RNA-binding protein genes in Arabidopsis thaliana (L.) Heynh. : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand." 2008. http://hdl.handle.net/10179/768.
Full textAbstract:
In maize, a loss-of-function mutation in a MEI2-like gene, terminal ear1 (te1), leads to morphological defects able to be traced back to the shoot apical meristem. One MEI2-like gene has been identified in maize, while six have been identified in rice and nine in Arabidopsis thaliana. In this thesis, a programme of reverse genetic analysis has been designed to investigate if Arabidopsis genes most closely aligned in parsimony trees with TE1, TERMINAL EAR-LIKE 1 (TEL1), TERMINAL EAR-LIKE 2 (TEL2), perform the same function as TE1. The expression pattern of TEL1 and TEL2 genes is restricted to the Shoot Apical Meristem (SAM) and the Root Apical Meristem (RAM) suggesting these genes are important in meristem maintenance or function. Results of the molecular genetic analysis of TEL genes in Arabidopsis support models in which these genes help maintain cells in a pluripotent state. For the first part of the thesis, analysis of lines carrying single knockouts of TEL1 and TEL2 and double knockout lines reveals a slightly accelerated rate of organogenesis, consistent with these genes normally acting to inhibit terminal differentiation pathways. Plants grown on medium containing gibberellic acid and sucrose, at higher than normal concentrations, present a further accelerated rate of organogenesis. As the second part of the thesis, in situ and promoter/reporter GUS fusion analyses indicate TEL1 is preferentially expressed in both the root and shoot apical meristems. Deletion analysis using GFP reporter constructs show that 5' sequences are sufficient to drive quiescent centre (QC) expression in the root while additional sequences are required for central zone (CZ) expression in the SAM. Physiological studies indicate expression of TEL1 in the root is sensitive to the hormones, auxin, gibberellic acid and zeatin, when added at physiological concentrations. To confirm the auxin effect, GFP expression is no longer visible after 12 hours of exposure to auxin transport inhibitors in plants containing GFP under the control of the TEL1 promoter, suggesting, in common with other QC markers, that TEL expression is sensitive to auxin levels. Analysis of mutant plants with altered root patterning suggests QC specific expression of TEL1 requires early acting genes, such as PLETHORA 1 and 2, but does not depend on later acting genes such as SCARECROW or SHORTROOT.
28
Sartie, Alieu Mortuwah. "Phenotypic assessment and quantitative trait locus (QTL) analysis of herbage and seed production traits in perennial ryegrass (Lolium perenne L.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph. D.) in Plant Science, Institute of Natural Resources, College of Sciences, Massey University, Palmerston North, New Zealand." 2006. http://hdl.handle.net/10179/1474.
Full textAbstract:
The aims of this study were to develop a genetic linkage map of perennial ryegrass, identify quantitative trait loci (QTL) for herbage and seed production traits, and to identify DNA markers associated with QTL for use in marker-assisted selection (MAS). Major traits identified for herbage production were leaf elongation rate (LER), leaf lamina length (LL), tiller number (TN) and tiller weight (TW), and for increased seed production were seed yield per head (SdYH), reproductive tiller number (RT), reproductive tillers with matured heads (TMH), florets per head (FH), spikelets per head (SH), florets per spikelet (FS), floret site utilization (FSU) and seed weight (TSW). A genetic linkage map spanning 582 centimorgans (cM) was constructed with EST-SSR (simple sequence repeat markers derived from expressed sequence tags) and used to identify QTL for herbage dry weight (DW) and seed yield per plant (SdYP), and their key component traits. Significant genotype by environment effects were encountered for herbage yield, with fewer QTL identified in spring than in autumn. For some traits, ranking of genotypes differed greatly between seasons and different QTL were identified. QTL for DW were identified on linkage groups (Lg) 1 and 6. The QTL on Lg 6 co-located with QTL for TN, while that on Lg 1 co-located with LER and LL. Markers at Lg 1 QTL (qDW-03-1.1) may be more useful for increasing herbage production by MAS because selection for high LER and long LL has been suggested to increase herbage production in perennial ryegrass. QTL for SdYP were identified on Lg 2 and Lg 6. The QTL on Lg 6 co-located with QTL for SdYH, FSU and TSW, while that on Lg 2 co-located with FH, SH and FS. For seed production, markers at Lg 6 QTL (qSdYP-03-6) may be very useful because this QTL co-located with QTL for SdYH, FSU and TSW, and SdYH has been identified previously as a key selection criterion for increasing seed yield. Marker-trait validation confirmed markers pps0495 and pps0698 identified by QTL analysis to be potentially useful for selecting for fast leaf appearance and long LL, respectively, in perennial ryegrass.
29
Song, Jiancheng. "Genetic diversity and flowering in Clianthus and New Zealand Sophora (Fabaceae) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Molecular Biology at Massey University, Palmerston North, New Zealand." 2005. http://hdl.handle.net/10179/1607.
Full textAbstract:
Clianthus and New Zealand Sophora species are woody legumes endemic to New Zealand, with high ornamental value and biodiversity significance. Research was conducted to address the fact that little is known about the details of their developmental characteristics, genetic structure and relatedness of the wild populations, and their molecular mechanism of flowering. Genetic diversity and relatedness of all remaining wild populations of Clianthus and samples of all New Zealand Sophora species were investigated using ISSR and AFLP markers. Genetic relationships were established for Sophora species, Clianthus wild populations and cultivars, and most individuals in each of the wild Clianthus populations. The molecular evidence did not support the recent separation on morphological grounds of the two Clianthus species, C. maximus and C. puniceus. Postharvest treatments were tested to extend vase life of the short-lived cut Clianthus maximus and Sophora tetraptera flowers. Appropriately treated Clianthus cut flowers lasted 10-12 days in the vase, with over 80% of flowers opening. Similar postharvest treatments did not improve the vase performance of cut Sophora flowers. Detailed calendars of vegetative and reproductive growth, and of floral ontogeny were developed for Clianthus and Sophora. Contrasting behaviours of both vegetative and reproductive growth were observed between these two legumes. A long period of summer-autumn dormancy of vegetative and reproductive growth in Sophora, and mass abortion of initiated Clianthus inflorescences during most of the year were observed. Unusual floral ontogeny processes, with precocious carpel initiation and delayed petal development, were observed in both species. An efficient two-step quantitative real-time RT-PCR protocol for detailed gene expression analysis of large numbers of samples was developed using SYBR Green DNA dye and a LightCycler instrument. The consistency of this protocol was optimised with regards to sample and template preparation, primer design, and determination of appropriate internal controls for gene expression quantification. Differences of gene expression in the range of 5-7 orders were effectively detected. Putative partial homologues of LEAFY, APETALAI, PISTILLATA, and AGAMOUS were isolated from both Clianthus and Sophora. Detailed temporal and spatial expression of each floral identity gene was investigated using quantitative real-time RT-PCR. The expression patterns, together with the sequence similarity, showed that these new isolated gene fragments were most probably LEAFY, APETALAI, PISTILLATA, and AGAMOUS homologues in Clianthus and Sophora, and that the ABC model of floral development is generally applicable to both species. However, there were important variations in temporal expression patterns compared to those of herbaceous species. A bimodal expression pattern of LEAFY and APETALAI homologues was observed in Sophora, but not in Clianthus, coincident with their contrasting patterns of floral initiation and development.
30
Atkinson, Kelly Rene LeFevre. "Proteomic biomarker discovery for preeclampsia." 2008. http://hdl.handle.net/2292/2565.
Full textAbstract:
Preeclampsia is a serious multisystem complication of late pregnancy with adverse effects for mothers and babies. Currently this disorder is diagnosed from clinical observations occurring late in the disease process. Unknown factors in the maternal circulation, possibly released by the preeclamptic placenta, have been linked to the pathophysiological changes characteristic of the disorder. The research in this thesis used proteomic techniques to identify putative preeclampsia biomarkers from two sources: secreted from a placental cell line undergoing differentiation, and directly sampled from the serum and plasma of women with late-onset preeclampsia. The first part of this research examined the secreted proteome of a placental choriocarcinoma cell line (BeWo) undergoing forskolin-mediated differentiation. Development of serum-free culture techniques enabled analysis of these secreted proteins by two-dimensional gel electrophoresis (2DE). Statistical testing revealed the significant involvement of seven spots during this differentiation model, with VE-cadherin and matrix metalloproteinase 2 among the proteins identified. In the second part of this research, maternal serum and plasma proteins were compared from women with preeclampsia and healthy pregnant women. Serum samples were analyzed using 2DE, and plasma was subjected to difference gel electrophoresis (DIGE). Bioinformatic analysis of both datasets identified multiple spot clusters able to classify samples according to disease state. Five of these serum proteins were differentially regulated in preeclampsia, including two isoforms of apolipoprotein E whose isoform-specific expression was confirmed using western blots. Analysis of plasma from preeclamptic women identified six proteins, again including apolipoprotein E. Proteins from both studies are linked to preeclampsia pathophysiology through lipid transport, complement, and retinol transport systems. The culture methods and secreted proteomic techniques developed in this work have uncovered proteins in a placental cell line and maternal serum and plasma that are associated with preeclampsia. These methods can be extended to any system where secreted proteins are of interest. The differentially regulated proteins found in this study provide an important first step towards developing effective biomarkers for diagnosing and/or predicting preeclampsia.
31
Clarke, Andrew Christopher. "Origins and dispersal of the sweet potato and bottle gourd in Oceania : implications for prehistoric human mobility : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand." 2009. http://hdl.handle.net/10179/1727.
Full textAbstract:
Origins and dispersal of the sweet potato and bottle gourd in Oceania :|bimplications for prehistoric human mobility : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
32
Collett, Michael Anthony. "Cloning, characterisation and evolutionary relationships of two pyr4 genes from an Acremonium endophyte of perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand." 1994. http://hdl.handle.net/10179/1300.
Full textAbstract:
A fragment of the Claviceps purpurea pyr4 gene, encoding the enzyme orotidine-5'-monophosphate decarboxylase (OMPdecarboxylase) was used to screen a genomic library to an isolate (designated Lp1) of an Acremonium sp. which grows as an endophyte in perennial ryegrass (Lolium perenne). Four positive clones, λMC11, λMC12, λMC14 and λMC20 were isolated. Three of these clones, λMC12, λMC14 and λMC20 were overlapping clones from the same locus, while λMC11 was from a different locus. Fragments of these clones which hybridised with C. purpurea pyr4 were sequenced and found to have similarity with pyr4 from other fungi of the Pyrenomycetes and related Deuteromycetes, suggesting that Lp1 has evolved from a sexual Pyrenomycetes species. The pyr4 from λMC12, λMC14 and λMC20 was designated pyr4-1 and that from λMC11 was designated pyr4-2. The predicted ORFs of the two genes were highly conserved and the 5' non-coding nucleotide sequences were the least conserved regions. RT-PCR and northern analysis of total RNA from Lp1 demonstrated that transcripts approximately 1.4 kb in length were produced from the two genes and present at similar levels. Genomic fragments containing pyr4-1 or pyr4-2 were transformed into a strain of Aspergillus nidulans which has a mutation in the pyrG gene (encoding OMPdecarboxylase). Both of the Lp1 pyr4 complemented a pyrG mutation in Aspergillus nidulans, confirming that both pyr4-1 and pyr4-2 encode functional OMPdecarboxylases. Comparisons of pyr4 restriction fragment length polymorphisms (RFLPs) from Lp1 and isolates of Epichloë typhina, E. festucae, A. lolii, A. uncinatum, and three endophyte taxonomic groupings from Festuca arundinacea: FaTG-1 (=A. coenophialum), FaTG-2 and FaTG-3 suggested that pyr4-1 originated from E. typhina, the ryegrass choke pathogen, and pyr4-2 originated from A. lolii, another endophyte from perennial ryegrass. This suggested that Lp1 is an interspecific hybrid, between E. typhina and A. lolii. Comparisons of the variable 5' non-coding nucleotide sequences from pyr4 of Lp1 and other isolates demonstrated that E. typhina, and A. lolii or E. festucae were the most likely ancestors of the two pyr4 found in Lp1. The A. lolii and E. festucae sequences were very similar, suggesting they are closely related. A. lolii has most probably evolved from an E. festucae, and in the process lost the sexual cycle. Analysis of single spore purified isolates of Lp1 revealed that Lp1 was a homokaryon for pyr4. A Southern blot of a CHEF gel of Lp1 and these single spored isolates was hybridised to a pyr4 probe and demonstrated that pyr4-1 and pyr4-2 were present on either two chromosomes of similar size, or one chromosome. The hybridisation that gave rise to Lp1 was concluded to have been a relatively recent event, given the similarity of pyr4-1 and pyr4-2 nucleotide sequences to those of their probable ancestors, and the fact that both genes are expressed and functional. Interspecific hybridisation is probably widespread in the asexual endophytes, and may be an important event in their evolution, and the evolution of other fungal species.
33
Binnie, Jan E. "Characterization of ACC oxidase from the leaves of Malus domestica Borkh. (apple) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biochemistry and Molecular Biology, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand." 2007. http://hdl.handle.net/10179/1445.
Full textAbstract:
The expression, accumulation and kinetic properties of 1 -aminocyclopropane- 1 -carboxylic acid (ACC) oxidase (ACO), the enzyme which catalyses the final step in the ACC-dependent pathway of ethylene biosynthesis in plants, is examined. The investigation is divided into three sections: (i) identification of two ACO genes in apple leaf tissue, designated MD-ACO2 and MD-ACO3, (ii) kinetic analyses of each of the three isoforms of ACO in apple (MD-ACO1, MD-ACO2 and MD-ACO3) expressed in E. coli, and (iii) temporal and developmental expression in vivo of each of the ACO genes and accumulation of the corresponding gene products in leaf and fruit tissue. The coding regions of putative ACO gene transcripts were generated from leaf tissue using RT-PCR. Sequence alignments and interrogation of the expressed sequence tags (ESTs) database indicated that the entire open reading frame (ORF) sequences were encoded by two distinct genes, and these are designated MD-ACO2 and MD-ACO3. A third ACO gene had been identified in apple by other research workers previously, and designated MD-ACO1. Differences are obvious in the number of base-pairs (bp) constituting the entire ORF of MD-ACO1 (942 bp), MD-ACO2 (990 bp) and MD-ACO3 (966 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9 % in the ORF but diverge in the 3' untranslated regions (3' -UTR) (69.5 %). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5 %) and MD-ACO2 (77.8 %) in the ORF, and in the 3'-UTR (MD-ACO1, 68.4 %; MD-ACO2, 71 %). A comparison of the gene structures show that the endonuclease restriction sites are unique to each individual MD-ACO sequence. Genomic Southern analysis, using probes spanning the 3'-UTR and the 3'-end of the coding region confirmed that MD-ACO3 is encoded by a distinct gene. However, while the distinction between MD-ACO1 and MD-ACO2 is not as definitive, different gene expression patterns adds credence to their distinctiveness. Each of the three deduced amino acid sequences contain all of the residues hitherto reported to be necessary for maximal ACO activity. Expression of MD-ACO1, MD-ACO2 and MD-ACO3 as fusion proteins in E. coli was induced using isopropy1-β-D-thiogalactopyranoside (IPTG), the recombinant proteins purified by nickel-nitrilotriacetic acid (NiNTA) affinity chromatography and the products had predicted masses determined from the nucleotide sequences, including the His-tag of 38.53 kDa (MD-ACO1), 40.39 kDa (MD-ACO2) and 39.3 kDa (MD-ACO3). Polyclonal antibodies were raised against the MD-ACO3 fusion in rabbit for western blot analysis. Antibodies had been raised previously against recombinant MD-ACO1, and while it was considered likely the MD-ACO2 would be recognized by the MD-ACO1 antibodies, MD-ACO2 does not appear to be recognized in vivo by the antibody. Analyses of the kinetic properties of the three apple ACOs was undertaken. Apparent Michaelis constants (Km) of 89.39 μM (MD-ACO1), 401.03 μM (MD-ACO2) and 244.5 μM (MD-ACO3) have been determined which suggests differences in the affinity of each enzyme for the substrate ACC. Maximum velocity (Vmax) was determined for MD-ACO1 (15.15 nmol), MD-ACO2 (12.94 nmol) and MD-ACO3 (18.94 nmol). The catalytic constant (Kcat) was determined for MD-ACO1 (6.6 x 10-2), MD-ACO2 (3.44 x 10-2) and for MD-ACO3 (9.14 x 10-2), with kcat/Km(μM s-1) values of 7.38 x 10-4 μM s-1 (MD-ACO1), 0.86 x 10-4 M s-1 (MD-ACO2) and 3.8 x 10-4 μM s-1 (MD-ACO3). The optimal pH for MD-ACO1 was 7.0 - 7.5, MD-ACO2 7.5 - 8.0 and MD-ACO3 7.0 - 8.0. All three isoforms exhibited absolute requirements for the co-substrate ascorbate in vitro with optimal activity at 30 mM. Similarly, ferrous iron (FeSO4.7H20; of 15 - 25 μM) and sodium bicarbonate (NaHCO3; of 30 mM) were required for optimal activity, and were the same for all isoforms. No significant difference in thermostability was found in this study between the MD-ACO isoforms at the P = 0.05 level. However, the activities of the enzyme differed significantly between temperatures over time. In vivo expression of each of the ACO genes in leaf tissue determined using RT-PCR and cDNA Southern analysis reveal that both MD-ACO2 and MD-ACO3 are expressed in young leaf tissue and in mature leaf tissue. While MD-ACO3 is expressed predominantly in young leaf tissue, MD-ACO2 (in common with MD-ACO1) is expressed predominantly in mature fruit tissue. None of the MD-ACOs were observed to be senescence associated genes (SAG). MD-ACO3 protein accumulated predominantly in young leaf tissue and less intensely in both mature leaf tissue and young fruit tissue, while MD-ACO1 accumulated only in mature fruit tissue. For the developmental studies, samples were collected at approximately 11 am in this study. MD-ACO2 and MD-ACO3 were also expressed in leaf tissue collected over a 24 h period in the spring and also in the autumn. For both genes transcripts accumulated in the presence of fruit but tended to disappear in the absence of fruit. These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed, and that MD-ACO3 is encoded by a gene distinct from MD-ACO1 and MD-ACO2. MD-ACO1 and MD-ACO2 are either allelic forms of the same gene or closely clustered. Although there is some variation in kinetic properties which may reflect different physiological environments, they do not vary greatly.
34
Thomas, Ludivine A. "Regulation of sulfur assimilation in onion (Allium cepa L.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Physiology at Massey University, Palmerston North, New Zealand." 2008. http://hdl.handle.net/10179/1396.
Full textAbstract:
Onion (Allium cepa L.) is an example of a species that accumulates very high levels of reduced sulfur (S)-containing compounds, particularly in the bulb as alk(en)yl-L-cysteine-sulfoxides (ACSOs) and it is these compounds, or their derivatives, that confers the distinct odour and pungent flavour. In common with higher plants, the S assimilation pathway in onion begins with the activation of uptaken sulfate (SO4 2-) to 5'-adenylylsulfate (APS), a reaction catalysed by ATP sulfurylase (ATPS; EC 2.7.7.4). Then, APS is reduced to sulfide (S2-) in a two-step process catalysed by the enzymes APS reductase (APSR; EC 1.8.4.9) and sulfite reductase (SiR; EC 1.8.7.1). To complete the reductive assimilation pathway, S2- is incorporated into the amino acid skeleton of O-acetylserine (OAS) to form cysteine, and this reaction is catalyzed by OAS (thiol)-lyase (OAS-TL; EC 4.2.99.8). While the regulation of the pathway is quite well defined in the plant model Arabidopsis, much less is known about its regulation in S accumulating species such as onion. The primary aim of this thesis, therefore, was to characterise the enzymes of the S assimilation pathway in onion, with a particular emphasis on ATPS. As part of this charaterisation two genotypes of onion were compared. These comprised a mild genotype, 'Texas Grano 438' (TG) with a lower level of S-containing compounds in the bulb tissues, and 'W202A' (W), a cultivar with a higher level of S containing compounds in the bulb tissues. As well, comparisons were made between seedlings (typically harvested at 7 weeks) and plants at a designated mature stage (at bulbing; typically after 4 months growth), and for plants grown in S-sufficient (S+) media or S-deficicnt (S-) media, as appropriate. In terms of plant growth, S-deprivation generally had a negative influence for both genotypes, with significant reductions in total biomass (measured as fresh weight) for TG at both the seedlings and mature stages. ATPS activity and accumulation were shown to be present in all tissues examined (leaf, root, bulb) as well as the chloroplasts, with highest activity measured in the roots, particularly in seedlings. ATPS activity and accumulation were also compared between the two genotypes (TG and W) with ATPS activity and accumulation higher in W, particularly at the seedling stage. In terms of the influence of S supply, in general higher ATPS activity was measured in chloroplast, leaf and root extracts from plants of both genotypes grown in the S- media, at the seedling stage. In roots of mature plants of both genotypes, a significant increase in activity was measured in response to S-deprivation, while in chloroplasts isolated from mature plants of both genotypes, highest activity was measure in those grown in the S+ media. Finally diurnal variations were observed in chloroplast, leaf and root extracts of both genotypes with a general trend of an increase in ATPS activity and accumulation a few hours after illumination and upon the onset of the dark period. Although a single gene coding for ATPS is presumed to be present in onion, the enzyme was characterized as two electrophoretic forms using 1D-PAGE during western analyses following fractionation of chloroplasts by anion exchange chromatography and also as an alignment of spots using 2D-PAGE. As protease inhibitors were routinely included in the extraction buffers, these forms suggest the occurrence of ATPS isoforms that may arise as a consequence of post-translational modifications. The regulation of ATPS by one mechanism of post-translational modification, phosphorylation, was therefore investigated using several techniques including the detection of a shift in molecular mass, a change in enzyme activity or pI (as determined by 2D-PAGE) and the capability to bind to 14-3-3 proteins using affinity chromatography. Following treatments of chloroplast extracts to promote either the phosphorylation (P+) or the dephosphorylation (P-) of proteins, no molecular mass change or change in activity was observed. However, after fractionation by 2D-PAGE, differences in the spot alignment of ATPS were visualized, suggesting that ATPS is a phosphoprotein. The enzyme was detected in pull-downs after affinity chromatography, suggesting that ATPS may also interact with 14-3-3 proteins (although this needs to be confirmed unequivocally). A model is advanced, therefore, in which upon phosphorylation, no variation in ATPS activity occurs but a change in the surface charged and possibly a change in conformation of the protein does occur to make the enzyme competent to interact with 14-3-3 proteins.
35
Chen, Chih-Ming. "Expression studies of the ACC oxidase gene family of white clover (Trifolium repens L.) : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Plant Biotechnology at Massey University, Palmerston North, New Zealand." 2005. http://hdl.handle.net/10179/1564.
Full textAbstract:
Four ACO promoters and four ACO genomic sequences have been isolated and cloned from Trifolium repens L. The promoter sequences were cloned using Gene WalkerTM technology, and are defined as the 5' flanking sequences upstream of the ATG translation start codon, and designated pTR-ACO1 (1006 bp), pTR-ACO2 (1510 bp), pTR-ACO3 (1350 bp), and pTR-ACO4 (1250 bp). To confirm that each 5' flanking sequences represents distinct genes, Southern analysis was undertaken with each of the 5' flanking sequences used as probes. For TR-ACO1 and TR-ACO2, Southern analysis indicated that the genome of white clover contains two copies of each gene, while single copies of TR-ACO3 and TR-ACO4 are evident. However, the pattern of recognition of pTR-ACO3 differs from pTR-ACO4 confirming TR-ACO4 as a newly identified member of the ACO gene family of white clover. The four genomic sequences isolated cover sequences downstream of the ATG codon to the stop codon, and each comprises 4 exons interspersed by 3 introns. In terms of sequence identity, for exon 1, identities over the four genes ranges from 69% to 94%, with 94% identity between exon 1 of TR-ACO3 and TR-ACO4, while for exon 2, identities range from 60% to 99%, with 99% identity between TR-ACO3 and TR-ACO4. For exon 3, sequence identities ranged from 71% to 89%, with 89% identity between TR-ACO3 and TR-ACO4, while for exon 4, identities range from 62% to 100%, with 100% sequence identity between TR-ACO3 and TR-ACO4. For the intron sequences, significantly lower identities are observed, with again, highest identities were observed for TR-ACO3 and TR-ACO4. For intron 1, identities ranged from 40% to 81% with the highest identity of 81% observed between TR-ACO3 and TR-ACO4. For intron 2, an identity range of 32% to 72% was observed with 72% identity between TR-ACO3 and TR-ACO4, while identity values of 13% to 79%, with 79% between TR-ACO3 and TR-ACO4. Analysis, in silico, of the 5' flanking sequences was undertaken to identify putative transcriptional binding domains using the PLACE and Mat-Inspector programmes. The TR-ACO1 5' flanking sequence contains a higher proportion of domains that are associated with young developing tissues, while the TR-ACO2 5' flanking sequence contains domains that are associated with environmental/hormonal cues. In contrast, the TR-ACO3 and TR-ACO4 5' flanking sequences contain a higher proportion of ethylene-response and wound associated domains. The expression pattern, in vivo, directed by all four 5' flanking sequences during leaf development has been examined using GUS fusions and transformation into both tobacco and white clover. In tobacco, the pTR-ACO1 directed expression in the terminal bud and in axillary buds of younger leaves, with expression declining in the older tissues. The pTR-ACO2 directed expression in the petioles and mature-green and senescent leaves, while the TR-ACO3 and TR-ACO4 promoters directed expression in the axillary buds, petioles and leaves of mature-green tissues and those in the early stages of senescence. In white clover, the TR-ACO1 5' flanking sequence directed highest expression in the apical tissues, axillary buds, and leaf petiolules in younger tissues and then declines in the ageing tissues, while the pTR-ACO2 directed expression in the axillary buds and leaf petiolules in mature-green tissues. The TR-ACO3 and TR-ACO4 5' flanking sequences direct more expression in the ontological older tissues, including the axillary buds and leaf petiolules. However, in association with this ontological pattern, all of the 5' flanking sequences directed expression in most cell types examined during leaf ontogeny. In younger tissues, the TR-ACO1 5' flanking sequence directed expression in the ground meristem and newly emerged leaf tissue at the apical bud of the stolon, the ground meristem tissue of axillary buds, vascular tissue, pith and cortex of the internode and node, and the cortex and vascular tissue of the leaf petiolule. In ontological older tissue, the TR-ACO3 and TR-ACO4 5' flanking sequences directed expression in the ground meristem of the axillary buds, the vascular tissue of the stolon and petiolule. However, staining could be observed in the pith and cortex of the stolon, and the cortex of the leaf petiolule, but at a reduced intensity. These expression studies suggest that in leaf development of white clover, the primary cues for the transcriptional regulation of the ACO gene family are ontological in nature.
36
Hills, Simon Francis Kahu. "Evolution in a marine gastropod : rocks, clocks, DNA and diversity : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Evolutionary Biology." 2010. http://hdl.handle.net/10179/1628.
Full textAbstract:
Comprehensive integration of paleontological and molecular data remains a sought-after goal of evolutionary research. This thesis presents a dataset unlike any previously studied to document changes over time in the evolutionary history of the New Zealand marine mollusc genus Alcithoe. In order to study evolutionary relationships in the Alcithoe, DNA sequence of approximately 8Kb of mitochondrial DNA was generated using universal and newly developed PCR primers. The gene composition of the resulting sequences has been thoroughly analysed, using a novel splits-based approach, to gain a clear understanding of the underlying phylogenetic signals in the data. Refinement of the phylogeny was achieved by considering subsets of both the taxa and genes. Taking these analyses into account the combined a robust phylogeny for the Alcithoe is presented for use in subsequent analyses. The Alcithoe genus includes species that are exemplars of the problem of correctly identifying species by morphological traits, in both the living and extinct taxa. Taxonomic assignments were explored in a population level analysis of the highly morphologically variable species A. wilsonae. Analyses revealed that the various recognised forms of A. wilsonae are genetically indistinguishable and that the previously recognised species A. knoxi is a synonym of A. wilsonae. This result has significant implications for the interpretation of the paleontological data, as A. knoxi specimens are known from the Tongaporutuan stage (10.92 – 6.5 Ma) of the New Zealand geological timescale. Therefore, this finding also has implications on the assignment of calibration data in molecular clock analysis. To ensure accurate estimation of divergence times and rates of molecular evolution, extensive explorations of parameter space in molecular-clock analyses were carried out. These analyses identified the most appropriate models and calibration settings for Alcithoe the dataset. The fossil data used to calibrate this analysis is amongst the most robust applied to molecular clock analyses to date. Statistical sampling uncertainty derived from the paleontological data was included in the calculation of prior distributions. Divergence dates inferred for the extant Alcithoe are largely consistent with the fossil record. However, the root of the tree was consistently inferred to be younger then expected. Rates of evolution in the species of Alcithoe included in this analysis are broadly consistent. However, some small rate differences are observed in some branches, for example, Alcithoe fusus appears to have a faster rate then the rest of the genus. This rate increase is the likely cause of topological inconsistencies observed for four closely related taxa, including A. fusus, and indicates that slight rate differences can cause phylogenetic instability when small genetic distances are involved. Direct comparison of diversification rates between the molecular and paleontological data for the Alcithoe illustrated that modern Alcithoe species have origins that are around 13 millions years younger then the oldest known Alcithoe fossils. The suggestion that A. fusus is descended from a series of fossil Leporemax species is directly contradicted by the molecular tree. In light of the molecular evidence this result highlights the problem of morphological convergence in the interpretation of fossil Alcithoe species. Comparison of the molecular and paleontological datasets was difficult for absolute speciation and extinction rates, as errors inherent to each dataset led to disparate estimates. For example, the fossil record clearly fails to record most recent speciation events observed in the molecular phylogeny, but the molecular data cannot sufficiently account for the amount of extinction evident in the fossil record. It is clear that the assumption of a constant and equal probability of speciation and extinction for all lineages is violated in the Alcithoe. However, the general long-term trends estimated for both datasets are concordant, and demonstrate an increase in both speciation and extinction rates over the Cenozoic era. The research described in this thesis represents significant progress toward the goal of more thorough integration of molecular and paleontological in the study of evolution. I have shown that reconciliation of molecular and paleontological data is not only possible, but can substantially improve the resulting interpretation of evolution. This study is the broadest analysis of the evolution in a single genus using combined molecular and paleontological data that the author is currently aware of. It illustrates the advantage of having quality paleontological data to compare to emerging molecular data, and how the molecular data can further inform the paleontological data. Furthermore, it adds support to the shift in perspective from an adversarial to a complementary approach to the consideration of molecular and paleontological data. This thesis is a comprehensive first step in the synthesis of molecular and paleontological data in the study of evolution of the New Zealand mollusc fauna, and alludes to many promising avenues for future study.
37
Shepherd, Lara Dawn. "Ancient DNA studies of the New Zealand kiwi and wattlebirds : evolution, conservation and culture : a thesis presented in fulfilment of the requirements of Doctor of Philosophy in Molecular BioSciences at Massey University, Albany, New Zealand." 2006. http://hdl.handle.net/10179/1499.
Full textAbstract:
Following content removed due to copyright restrictions: Lambert, D. M., King, T., Shepherd, L. D., Livingston, A., Anderson, S. & Craig, J. L. (2005). Serial population bottlenecks and genetic variation: translocated populations of the New Zealand saddleback (Philesturnus carunculatus rufusater). Conservation Genetics 6: 1–14. Perrie, L. R., Shepherd, L. D.& Brownsey, P. J. (2005). Asplenium ·lucrosum nothosp. nov.: a sterile hybrid widely and erroneously cultivated as ‘‘Asplenium bulbiferum’’ Plant systematics and evolution 250: 243–257Ancient DNA was used to provide a temporal perspective for examining a number of evolutionary, conservation and cultural questions involving members of the New Zealand avifauna. Ancient mitochondrial DNA (mtDNA) sequences were used to examine the past levels and patterns of genetic diversity in the five species of New Zealand kiwi (Apterygidae). Brown kiwi, particularly in the South Island, exhibited high levels of genetic structuring with nearly every population exhibiting private mitochondrial haplotypes. The extinction of a large number of brown kiwi populations has, therefore, led to the loss of a large amount of genetic variation in these species. The past ranges of great spotted kiwi and the three brown kiwi species, whose bones are morphologically indistinguishable, were determined. This information can aid conservation programmes aiming to re-introduce kiwi to regions where they are now extinct. In contrast to the high level of genetic structuring in South Island brown kiwi, the majority of little spotted kiwi samples from the South Island shared a common haplotype. The difference in phylogeography between brown kiwi and little spotted kiwi is hypothesised to relate to differences in their dispersal behaviour and/or their population histories. The addition of ancient samples of little spotted kiwi from the North Island indicated a complex relationship with great spotted kiwi. Nuclear microsatellite DNA markers were isolated from North Island brown kiwi and tested for cross amplification in the other kiwi species. Five loci were polymorphic in all kiwi species. Preliminary analyses of genotyping results indicated that the kiwi species were distinguished by assignment tests and that subdivision may occur within several of the species. An extensive reference database of modern and ancient mtDNA sequences was used to determine species and provenance of a number of unlabelled museum subfossil bones and skins. This method was also used to examine provenance of brown kiwi feathers from Maori artefacts (cloaks and baskets). Ancient DNA methodology was also used in a molecular examination of the relationships of a second endemic avian family, the New Zealand wattlebirds (Callaeatidae). Analyses of nuclear gene sequences, c-mos and RAG-1, revealed kokako, saddleback and huia comprised a strongly supported monophyletic group. A divergence time estimate for the New Zealand wattlebirds indicated that they are more likely to have arrived by transoceanic dispersal than have a Gondwanan origin. Sequences from three mtDNA genes, 12S, ND2 and cytochrome b, were also analysed but could not resolve the relationships between the three genera. Microsatellite DNA from the extinct New Zealand huia exhibited considerable genetic variation, exceeding that found in extant North Island saddleback, from which the loci were isolated. Assignment tests indicated no genetic structuring within huia, although interpretation was complicated by a lack of provenance details for many of the skins. The results presented here suggest that ancient DNA can not only provide information about the relationships of extinct taxa but also demonstrates the importance of placing the present day genetic diversity found in endangered taxa within the context of past patterns and levels of genetic variation.
38
Weake, Vikki Marie. "The x-linked LSP1α gene of Drosophila Melanogster is not acetylated by MOF, but is sex-specifically regulated by individual components of the MSL complex : a thesis presented in partial fulfilment of the requirements for the degree Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand." 2005. http://hdl.handle.net/10179/1626.
Full textAbstract:
Male Drosophila melanogaster double the transcription of most of the genes on their single X chromosome, to equal that from the two female X chromosomes, in a process termed dosage compensation. This process is mediated by the MSL complex, consisting of both protein and non-coding RNA components. This complex is only active in males due to the presence of MSL2, which is not translated in females. The X-linked Lsp1α gene of Drosophila melanogaster appears to escape dosage compensation, and exhibits two-fold higher levels of expression in females compared to males. The apparent lack of dosage compensation of Lsp1α could be due to the promoter being more active in females than in males, or to a lack of regulation by the MSL complex. In this study, the mechanism by which this happens has been addressed. Lsp1α is expressed exclusively in the fat body tissue of third instar larvae, and forms part of a multi-protein complex that acts as a nutrient reservoir during pupariation. In this study it has been shown that transgenes, in which the reporter gene, lacZ, is under the control of the Lsp1α promoter, exhibit variable levels of increased activity in female compared to male third instar larvae. At high levels of transgene expression, activity of the transgene is equal in female and male larvae. When the expression of the transgene is low, the activity of the transgene is much higher in female compared to male larvae. This increased sensitivity of the Lsp1α promoter to position effects in females appears to be mediated by one or more components of the MSL complex. Females ectopically expressing MSL2 exhibit decreased levels of transgene activity. Furthermore, overexpression of MSL1 causes an increase in the activity of transgenes subject to strong position effects. Despite these findings, the sex-specific regulation of the Lsp1α promoter does not account for the non-dosage compensated appearance of Lsp1α. Instead, unlike control dosage compensated X-linked genes, Lsp1α is not enriched for a histone modification, acetylation of lysine 16 of histone H4 that is essential for dosage compensation by the MSL complex. The developmental stage at which the four genes flanking Lsp1α are expressed has been determined using northern RNA hybridization. Expression of the gene immediately 3' of Lsp1α could not be detected at any developmental stage using northern RNA hybridization or in adults by RT-PCR. However, the two genes flanking Lsp1α are expressed in equal levels in male and female Drosophila as determined by quantitative RNase protection analysis. Furthermore, the regions between Lsp1α and these flanking dosage compensated genes do not prevent dosage compensation of an X-linked armlacZ reporter gene. Bioinformatic analysis shows that Lsp1α is present in three species closely related to D. melanogaster but is absent in more distantly related species. It is probable that because of its recent evolutionary origin, the Lsp1α gene lacks the DNA sequences that are required to attract the MSL complex. More generally, a model is proposed in which dosage compensation involves binding of the MSL complex to DNA sequences in actively transcribed regions with possible limited spreading to closely associated active genes.
39
Lane, Michael. "Biochemical and molecular characterisation of FliI and FliH from Helicobacter pylori : a thesis presented in partial fulfilment of Doctor of Philosophy in Microbiology at the Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand." 2006. http://hdl.handle.net/10179/1579.
Full textAbstract:
The bacterium Helicobacter pylori is a human pathogen that infects a large proportion of the world's population and is associated with serious diseases such as gastric ulcers and adenocarcinoma. The motility of this organism, by virtue of sheathed polar flagella is essential to colonisation and persistence in the human host. The sequencing of the H. pylori genome in 1996 identified homologues of the majority of the flagellar genes found in S. enterica serovai typhimurium. These included genes encoding the flagellum ATPase, FliI and FliH a presumptive inhibitor, the primary focus of this study. Sequencing did not originally identify an H. pylori homologue of the flagellar chaperone FliJ, and this is also considered in this study. Bioinformatic analysis and modeling suggests a structural and functional relationship between FliI and homologues such as F1-ATPase α- and β-subunit. In particular, residues 2-91 of FliI resemble the N-terminal domain of the F1-ATPase α- and β-subunits. Biochemical analyses reported in this thesis showed that a truncated FliI-(2- 91) protein was folded, although the N-terminal 18 residues were likely unstructured. Furthermore, deletion mutagenesis showed that this disordered segment of the protein mediates interaction with FliH and very likely forms an amphipathic α-helix upon forming of the FliI-FliH complex. The scanning mutagenesis of this interaction segment of FliI identified a cluster of conserved hydrophobic residues that was critical for the interaction with FliH. Thus, the interaction between FliI and FliH has similarities to the interaction between the N-terminal α-helix of the α-subunit and the globular domain of the δ-subunit of the F1-ATPase. This similarity suggests that FliH, by analogy with the δ-subunit of the F1-ATPase, may function as a molecular stator of the flagellum. The findings presented above have been published (96). The function of a putative H. pylori FliJ homologue, HP0256, was also investigated by knock-out mutagenesis. Disruption of this gene does not abolish flagellar assembly, however further research continued beyond this thesis showed that the knock-out mutant results in impaired motility.
40
Trainor, Vernon. "Functional analyses of the Terminal Ear 1-like RNA binding proteins of Arabidopsis thaliana : a thesis presented in partial fulfillment of the requirements of the degree of Doctorate of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand." 2007. http://hdl.handle.net/10179/1453.
Full textAbstract:
In the Shoot Apical Meristem (SAM) the position at which leaf primordia arise on the periphery, and their subsequent differentiation, have been shown to be (at least in part) to be directed by genetic programs of development. A candidate gene associated with this regulation is TERMINAL EAR 1 (TE 1) a maize gene identified by the irregular phyllotaxy of its mutant lines. Unlike most other genes associated with meristem function, TE 1 is a novel RNA binding gene of the RRM type. It has been shown to have orthologues in a variety of plants including Arabidopsis thaliana as well as unicellular eukaryotes including MEI2, a gene whose product is associated with the regulation of meiosis in Schizosaccharomyces pombe. In order to more fully understand TE1's role, a functional characterisation of two of the so-called Mei2-like genes was undertaken in the model plant A. thaliana. These genes are called Terminal Ear-Like 1 and 2 (TEL1 and TEL2). Constitutive overexpression of the cDNA of TEL2 using the Cauliflower Mosaic Virus 35S promoter (CaMV35S) revealed a phenotype involving an apparently prolonged vegetative phase. However this was only observed in a limited number of lines of the total screened, and the next generation did not reiterate this phenotype. These difficulties were overcome using the LhGpOP construct system for ectopic misexpression in specific domains as well as inducible ubiquitous expression. Ectopic expression of either TEL cDNA is shown to lead to a pleiotrophic spectrum of phenotypes, which in general, were associated with reduced determinant development outside the apical meristems and as well as a delayed overall developmental progression. This provided some evidence that the normal function of TEL genes within the apical meristems is the repression of differentiation associated with the regulation of plant growth and architecture.
41
McDonald, Michael Joseph. "The genetics of Pseudomonas fluorescens SBW25 : adaptation to a spatially structured environment : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Auckland Campus." 2009. http://hdl.handle.net/10179/1086.
Full textAbstract:
Experimental microbial populations provide powerful models for testing the most challenging problems in evolutionary biology. In the midst of the genome sequencing revolution microbial evolutionary genetics has flourished; promising high-resolution explanations for the underlying causes of evolutionary phenomena. This thesis describes four investigations into the adaptation of Pseudomonas fluorescesns SBW25 to a spatially structured environment. The first builds upon a large body of experimental work characterising the genetic and phenotypic causes of the ability of divergent Wrinkley Spreader (WS) types to colonise the air-liquid interface in spatially structured microcosms. The mws and aws genetic loci are described, which together with the previously described wsp locus, account for the location of the causal mutation for all known WS genotypes. It was found that if these loci were deleted from the P. fluorescens genome, it could still evolve the WS phenotype via a previously undiscovered locus (sws). This study provides the first explicit evidence that genetic biases can influence the outcome of evolution. The second study used a novel method to sample WS genotypes without the biasing effects of natural selection; the distribution of the fitness effects of these genotypes was measured and analysed from a unique perspective. The distribution of fitness effects of new mutations is found to best fit the normal distribution, facilitating the extension of the mutational landscape model of adaptation to include all possible adaptive walks. The third study investigates the underlying causes of genetic biases on evolution; many WS genotypes are obtained at different time points during colonisation of the air-liquid interface (including WS obtained without selection) and the causal mutations of many of these mutants determined. Together these results allowed the elucidation of the relative effects of natural selection, genetic architecture and mutation rate on evolutionary outcomes. The final study considers the WS mat as the product of cooperative interactions, and uses a group selection experiment to investigate the potential of WS mats to evolve group level adaptations. A novel strategy is developed to overcome cheating types, considered the main barrier to the evolution of group level complexity. Furthermore, WS groups evolved specialised cell types, the first example of a de novo evolution of a division of labour, a hallmark of complexity.
42
Chen, Xiaowei. "Non-protein-coding-RNA processing in the deep-branching protozoan parasite Giardia intestinalis : a thesis presented in partial fulfilment of the requirements for the degree of PhD in Molecular Genetics at Massey University, Palmerston North, New Zealand." 2008. http://hdl.handle.net/10179/777.
Full text
43
Johnston, Robyn Maree. "Characterisation of the maize leaf patterning mutants Wavy auricle in blade1-R and milkweed pod1-R : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand." 2007. http://hdl.handle.net/10179/1452.
Full textAbstract:
The maize leaf has three main axes of growth, with an asymmetric distribution of tissue types along each axis. This study focuses on three mutants, Wavy auricle in blade1-R (Wab 1-R), liguleless1-R (lg1-R) and milkweed pod1-R (mwp1-R) that disrupt axial patterning of maize leaves. Dominant Wab1 mutations disrupt both medial-lateral and proximal-distal patterning. Wab1 leaf blades are narrow and ectopic auricle and sheath-like tissues extend into the leaf blade. Previous analyses have shown that Lg1 acts cell-autonomously to specify ligule and auricle tissues. The current study reveals additional roles in defining leaf shape. The recessive lg1-R mutation exacerbates the Wab1-R phenotype; in the double mutants, most of the proximal blade is deleted and sheath tissue extends along the residual blade. A mosaic analysis of Wab1-R was conducted in Lg1 and lg1-R backgrounds to determine if Wab1-R affects leaf development in a cell-autonomous manner. Normal tissue identity was restored in all wab1/- sectors in a lg1-R mutant background, and in three quarters of sectors in a Lg1 background. These results suggest that Lg1 can influence the autonomy of Wab1-R. In both genotypes, leaf-halves with wab1/- sectors were significantly wider than non-sectored leaf-halves, suggesting that Wab1-R acts cell-autonomously to affect lateral growth. mwp1-R is a recessive mutation that specifically affects patterning of sheath tissue. Characterisation of the mwp1-R phenotype revealed that mwp1-R husk leaves and the sheaths of vegetative leaves develop pairs of outgrowths on the abaxial surface associated with regions of adaxialised tissue. In situ hybridisation confirmed that disruptions to adaxial-abaxial patterning are correlated with misexpression of leaf polarity genes. Leaf margins and fused organs such as the prophyll are most severely affected by mwp1-R. The first two husk leaves normally fuse along adjacent margins to form the bi-keeled prophyll. In the most severe cases the mwp1-R prophyll is reduced to an unfused, two-pronged structure and keel outgrowth is significantly reduced. We speculate that the adaxial-abaxial patterning system has been co-opted during evolution to promote outgrowth of the keels in normal prophyll development. The results of this study place Mwp1, wab1 and Lg1 in a network of genes that regulate leaf polarity and axial patterning.
44
Young, Carolyn Anne. "The indole-diterpene gene cluster from the ryegrass endophyte, Neotyphodium lolii, is required for the biosynthesis of lolitrem B, a bioprotective alkaloid : this thesis is presented as a partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph. D.) in Molecular Biology at Massey University, Palmerston North, New Zealand." 2005. http://hdl.handle.net/10179/1670.
Full textAbstract:
Content removed due to copyright: Young, C. A., Bryant, M. K., Christensen, M. J., Tapper, B. A., Bryan, G. T., & Scott, B. (2005). Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass. Molecular Genetics and Genomics, 274(1), 13-29.Lolitrems are indole-diterpene alkaloids produced by Epichloë and Neotyphodium endophytes in association with their host grass Lolium perenne. Some indole-diterpene (ID) alkaloids are proposed to have insecticidal properties, but lolitrem B is known as the causative agent of the animal syndrome ryegrass staggers. Lolitrems are preferentially synthesised in planta. which suggests that the genes required for lolitrem biosynthesis are symbiotically expressed. The lolitrem biosynthesis pathway has been proposed as a metabolic grid based on the identification of likely intermediates from endophyte-infected ryegrass. Closely related ID compounds are expected to serve as substrates for the same enzyme, but until recently these steps had not been validated. The identification and characterisation of a Petticillium paxilli gene cluster required for the synthesis of the ID paxilline has identified key enzymes required for the production of the ID backbone. Based on the similarity of lolitrem B to paxilline it was proposed that these two biosynthesis pathways would share orthologous early steps but later steps to convert paxilline to the more complex lolitrem B would require additional enzymes. The lolitrem biosynthesis genes (ltm) were isolated using degenerate PCR and from candidate genes identified as ESTs in cDNA libraries. Ten ltm genes were identified that had functions consistent with those required for lolitrem B biosynthesis. The 10 ltm genes were contained on three gene clusters that are separated by repetitive AT-rich sequences that contain remnants of retrotransposons. The ltm clusters 1 and 2 contain eight genes, seven of which are orthologues of the characterised P. paxilli paxilline biosynthesis gene cluster (pax). Functional characterisation of ltmM an FAD-dependent monooxygenase and ltmC a prenyl transferase confirmed these two genes were required for ID biosynthesis and were orthologues of paxM and paxC, respectively. All 10 ltm genes have similar expression profiles and were highly expressed in planta where the production of lolitrem B is most prevalent. The taxonomic distribution of the ltm genes has established which endophyte strains are likely to produce ID compounds. This work provides the basis for elucidation of the lolitrem biochemical pathway and opens the way for determining how the plant regulates the synthesis of this important group of bioprotective molecules.
45
Laverty, Corey. "The importance of the promoter in Drosophila dosage compensation : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand." 2009. http://hdl.handle.net/10179/1289.
Full textAbstract:
Dosage compensation is the equalisation of gene expression from unequal doses of genes. Drosophila males up-regulate transcription from their single X chromosome to equal that from the two female X chromosomes. Five malespecific lethal (msl) genes are required in males, and encode the main agents of the up-regulation. At least these proteins, together with either or both of two noncoding RNAs, form the MSL chromatin-modifying complex. Female-specific translational repression of a key component, msl2, limits the complex to males. The MSL complex binds to the X chromosome at hundreds of distinct loci, acetylates nucleosomes, and de-condenses the chromatin. Together with possibly many co-factors, the transcriptional up-regulation caused by MSL complex appears to counteract repressive factors to achieve an average effect of transcriptional doubling. Here, I have studied the initiation of MSL regulation on the X chromosome with a variety of approaches. In order to study early events, dosage compensation was induced in females with ectopic expression of msl2 from the tetracycline system. However, low background expression without activation prohibited further studies. To identify novel factors that affect dosage compensation, a reporter gene system based on variable eye size was evaluated. The system provided a dose-dependent phenotype, but could not report additional up-regulation by the MSL complex, and was thus unsuitable for the proposed mutational screen. The quantifiable lacZ gene was measured in a strict comparison of expression from an eye-specfic (GMR) or a constitutive (armadillo) promoter. At defined locations on the X chromsome, armadillo-lacZ acquired local compensation, but GMR-lacZ did not. Further modifications upstream of GMR-lacZ increased the response, and confirmed the importance of the promoter in attraction of dosage compensation. To corroborate this with the established importance of genic sequences in MSL attraction, a combinatorial model of attraction is proposed. The relative importance of early or constitutive expression was also tested, by providing GMR-lacZ with extra expression through the tetracycline system. A burst of embryonic expression, and constitutive expression, were both insufficient to increase dosage compensation of the transgene. Finally, the compensation of GMRmediated transgenes was confounded by ‘transvection’ effects of chromosome pairing. This effect may have wider implications on the study of compensation at individual genes.
46
Balasubramanain, Diana. "Loss of heterozygosity of the H4833Y mutation on RYR1 gene causing malignant hyperthermia : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North." 2010. http://hdl.handle.net/10179/1660.
Full textAbstract:
Malignant hyperthermia is a potentially fatal pharmacological disorder and is triggered by volatile anaesthetics in predisposed individuals. Mutations in the RYR1 gene, encoding the skeletal muscle calcium receptor channel have been linked to MH susceptibility. Over 200 point mutations have been have been found to date in the RYR1 gene linked to MHS worldwide. EBV-immortalization is regularly used worldwide as an effective procedure for inducing long-term growth of human B lymphocytes. In the current study, it was observed that immortalized lymphocytes from MHS patients heterozygous for the missense mutation H4833Y when initially cultured expressed both wild type and mutant allele but after a few weeks of culture they seemed to lose the mutant allele. High resolution melting assays and hybridization probe assays showed the loss of heterozygosity and this was confirmed using DNA sequencing. Genotyping and haplotype analysis using three intragenic RFLPs and two (CA)n repeat microsatellite markers tightly linked to the RYR1 gene showed a definite change in the haplotype, suggesting more widespread changes in the genome upon short-term culture of EBV-immortalized B-lymphocytes
47
Zhang, Xiuwen. "Functional analysis of a thiamine biosynthetic gene in the interaction of Epichloë typhina with perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand." 2004. http://hdl.handle.net/10179/1717.
Full textAbstract:
Appendix content (raw data and statistics; sequences) unavailable online, but available with print copyEpichloë/Neotyphodium endophytes are a group of clavicipitaceous fungi that form symbiotic associations with temperate grasses. The asexual N. lolii form asymptomatic mutualistic associations with ryegrass whereas the sexual E. typhina behaves similar to a mutualist during the vegetative phase of plant growth but switches to epiphytic growth and formation of an external stroma upon development of the floral inflorescence. The aim of this project was to study the metabolic interaction between these endophytes and their perennial ryegrass host. The role of endophyte thiamine biosynthesis in host colonisation and stroma development was chosen, because of the key role this coenzyme plays in primary cellular metabolism and because thiamine biosynthetic genes are induced in several fungal-plant interactions. The orthologue (thil) of Saccharomyces cerevisiae THI4 was isolated from N. lolii and E. typhina by PCR using degenerate primers designed to conserved regions of known thiazole biosynthetic genes. This gene is expressed in planta and in culture, and is alternatively spliced, with distinct patterns of the isoforms expressed under different nutritional conditions. Mutant with a deletion in the E. typhina thil gene was constructed and shown to have reduced hyphal density and branching compared to the wild-type on defined media lacking thiamine. Both thiamine and thiazole complemented this defect. Artificial inoculation of the mutants into plants showed that the thil mutant retained the ability to colonise the perennial ryegrass host and form stromata. However, the mutant had some differences in host colonisation and growth, including reduced hyphal branching and reduced detrimental effects on the host. In addition, glycogen-like deposits, which were abundant in the wild-type hyphae, were not evident in the mutants. Unexpectedly, both the thil mutant and wild-type strains formed some stromata on vegetative tissue. Electron microscopic examination revealed that the cells of epiphytic hyphae found on the vegetative tillers typically were enlarged, lacking in cytoplasm and highly vacuolated, an ultrastructure similar to that found for hyphae growing in reproductive tillers. The mutants retained the ability to form conidia on the outer layer of the stromata. Extensive vascular colonisation and hyphal ramification in the mesophyll were common characteristics of stromata bearing regions. Although the morphology and ultrastructure of stromata formed on vegetative tillers is very similar to those on reproductive tillers, one significant difference was the presence of abundant glycogen-like deposits in hyphae of vegetative tillers. Furthermore, there were dramatic differences in the levels of glycogen-like deposits in hyphae in different regions of the vegetative tillers, indicating that the energy demand changes during stroma development. This is the first report of E. typhina forming stromata on non-inflorescence tillers.
48
Jaya, Elizabeth S. K. D. "Morphological, physiological, and molecular studies on the effect of shoot architecture on phase change and floral transition in Eucalyptus occidentalis and Metrosideros excelsa : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand." 2007. http://hdl.handle.net/10179/1454.
Full textAbstract:
Shoot morphogenesis in Eucalyptus occidentalis and Metrosideros excelsa was analysed at the morphological, physiological and molecular levels to understand the regulation of phase change and the floral transition. Study of the regulation of these developmental plant processes is limited in woody species due to their long juvenile phase. Six ecotypes of E. occidentalis were grown to two predetermined architectures (free branching or single stem). Free branching plants of ecotype 13648 displayed adult shoot phenology (lanceolate leaves) earlier than single stem counterparts. In addition, changes in leaf morphology in free branching plants were accompanied with changes in leaf anatomy and gas exchange signifying that in E. occidentalis complexity of shoot architecture had a significant effect on rate of phase change. Flowering was observed in all but one ecotype irrespective of architecture demonstrating that vegetative phase change and floral transition are temporally uncoupled in this species. To understand the floral transition at the molecular level in E. occidentalis, partial homologues of the inflorescence meristem identity gene TERMINAL FLOWER1 and floral meristem identity genes LEAFY and APETALA1 were isolated. The expression patterns of these meristem identity genes during development of free branching and single stem plants were analysed by quantitative real-time PCR. Increased levels of expression of EOLFY and EOAP 1 (relative to α -TUBULIN) were displayed at more proximal nodes in free branching plants than in single stem plants. Elevated floral meristem identity gene expression levels correlated with flower initiation. Further, effects of architecture and environment on gene expression were monitored in E. occidentalis. The overriding effect of shoot architecture on the floral transition was observed under warm long day and ambient environments. Elevated levels of EOLFY and EOAP 1 were correlated with floral bud score and EOAP1 was found to be a reliable marker of floral transition in E. occidentalis. Low levels of EOTFLI expression were detected in buds irrespective of their position on the plant leading to the suggestion that this might have contributed to the precocious flowering observed in this species. In contrast to E. occidentalis, M excelsa attained adult shoot phenology (pubescent leaves) faster when grown as single stem plants than as free branching plants. It appears that growth as height is required for vegetative phase change in this species. However, floral transition occurred only once single stem plants were allowed to branch. Vegetative phase change and the transition to flowering seem to be coordinated in this species with the former being a pre-requisite for the latter.
49
Bryant, Michelle Kay. "Functional analysis of genes encoding hydrolytic enzymes in the interaction of Epichloë festucae with perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand." 2005. http://hdl.handle.net/10179/1563.
Full textAbstract:
Hydrolytic enzymes degrade macromolecules into smaller components. These enzymes are important in fungal nutrition and have been implicated in the pathogenicity and virulence of pathogenic fungi towards their hosts. However, it is unknown if hydrolytic enzymes play important roles in mutualistic symbioses. In this study, the function of two different classes of hydrolytic enzymes was examined in the mutualistic symbiosis between the fungal endophyte Epichloë festucae and perennial ryegrass (Lolium perenne cv. Nui). Nine members of a gene family encoding subtilisin-like proteases were identified in E. festucae. The prt2, prt3 and prt5 genes encode putative extracellular proteins belonging to the proteinase K subfamily 1, and prt1 and prt6 encode putative extracellular proteins belonging proteinase K subfamily 2. The prt7 and prt8 genes encoded pyrolysin-like enzymes from subfamilies 1 and 2. The prt4 gene encodes a putative vacuolar protease, while the kex2 gene encodes a putative proprotein convertase. Expression analysis showed that the prt1, prt3, prt5, prt4 and kex2 genes, but not the prt2 gene, were expressed in culture. The prt1 and prt3 genes appeared to be up-regulated in planta compared to culture. The function of prt1 and prt2 in the symbiotum between E. festucae and perennial ryegrass was characterised by expressing these genes under the control of the Aspergillus nidulans gpdA or the E. festucae F11 ltmM promoters. No major differences in hyphal or plant morphology were observed between symbioses containing wild type E. festucae or endophyte strains containing the prt1 or prt2 transgenes. The gcnl gene, which encodes a β-1,6-glucanase, was identified immediately downstream of the prt2 gene. The function of the gcnl gene was characterised by gene replacement and testing the phenotype during growth in culture and in planta. E. festucae ∆gcnl strains grew normally on glucose-containing media. On media containing the β-1,6-glucan pustulan, ∆gcnl strains did not form aerial hyphae or hydrolyse pustulan, which the wild type strain did. This phenotype was partially complemented by growth of the ∆gcnl mutant in close proximity to wild type strains, and fully complemented by insertion of the gcnl gene. This suggests that the gcnl gene encodes the major β-1,6-glucanase activity of E. festucae.
50
Shang, Yongjin. "How the pigment stripes form in snapdragon (Antirrhinum majus) flowers : a study of the molecular mechanism of venation pigmentation patterning in flowers : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Molecular Biology at Massey University, Palmerston North, New Zealand." 2006. http://hdl.handle.net/10179/1569.
Full textAbstract:
Floral stripes are a common pigmentation pattern in plants. Defining the molecular mechanisms of the striped pattern formation will aid understanding of how a gene can be differentially regulated across a population of similar cells. In the venation phenotype of Antirrhinum majus, the anthocyanin pigment is typically confined to the adaxial epidermal cells overlaying the petal veins. To explore how this pattern forms this study focused on the expression and regulation of Venosa, a Myb regulator of anthocyanin biosynthesis. Pigment complementation experiments demonstrated that the lack of a MYB factor caused the lack of pigment in the cells outside the venation pigmentation domain. An allele of Venosa was isolated and identified. It was a mutant version of functional Venosa due to the central part being replaced by a transposon. Phenotype / genotype analysis indicated that the venation pigmentation patterning was due to the functional Venosa. In situ mRNA hybridisation showed that Venosa was expressed from the xylem to the adaxial epidermis, and was controlled spatially and quantitatively by a signal associated with the petal veins. Venosa expression provided the longitudinal axis for venation pigmentation stripes, and determined the location and intensity of the pigmented cells. Because another factor required for pigmentation, a bHLH factor, is specifically expressed in epidermal cells and it provides the transverse axis. The pigmented stripes are the cross expression domain of these two kinds of factors. The transcriptional controlling property of a 2.4 kb (relative to the ATG) promoter region of the Venosa gene was analysed. The -900 bp fragment was characterised in detail using 5'-end deletion mutagenesis. A heterologous host, tobacco, was used for analysis in stable transgenics. The homologous host, Antirrhinum, was used for transient assays. The efficacy and efficiency of different reporter genes (intron-containing GUS, GFP, Venosa cDNA and genomic Venosa) and enhancement systems (transcriptional enhancer, translational enhancer, inhibitor of post transcriptional gene silencing and a two-step signaling amplification system) for the detection of low-level reporter gene expression were also tested. The strength of expression correlated to the length of the promoter fragment, and expression was detected using deletions down to -500 bp, although only weak expression was found. This expression was flower specific but not vein related in both plant hosts. No expression was detected in petals of either host with fragments shorter than -500 bp. The results suggest that the fragment from -380 bp to -900 bp positively affected Venosa expression at the transcriptional level, but might not be sufficient to define venation. A possibility is that the venation controlling property is negatively controlled at the epigenetic level, such as DNA methylation status and / or chromatin structure. The role of gibberellin and sugar in the pigment and venation patterning formation of Antirrhinum was studied. The results suggest that gibberellin is not required for pigmentation or venation patterning. Convincing evidence on the role of sugar signaling could not be obtained from the experiments, due to the difficulty in separating the impact on pigmentation from other functions of sugars in petal development. In addition, the in situ analysis detected the expression of a gene probably related to aurone biosynthesis that may be a regulatory gene of this biosynthetic pathway.