Genetics - Theses
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ItemCellular characterisation of the insulin-regulated aminopeptidase (IRAP) in the brain(University of Melbourne, 2006)Central infusions of AT4 ligands, including Angiotensin IV and LW-Hemorphin 7, enhance memory acquisition, retention and retrieval in fear-conditioned memory tasks and spatial learning. A single acute dose of an AT4 ligand is also sufficient to reverse memory deficits caused by scopolamine treatment, perforant pathway lesions and other forms of experimentally induced amnesia models. The robust effects of these ligands on learning and memory processes are thought to be mediated by binding to the catalytic site of the insulin regulated aminopeptidase (IRAP). I RAP is also found in muscle and adipose cells, where the enzyme is found colocalised with GLUT4, the insulin regulated glucose transporter, in specialised vesicles. In response to insulin stimulation, IRAP traffics with GLUT4 from an intracellular location to the plasma membrane, from where GLUT4 mediates insulin regulated glucose uptake. The N-terminal domain of IRAP is believed to interact with multiple proteins to regulate the intracellular tethering, trafficking and possibly recycling of these vesicles. It is unknown how AT4 ligands, via binding to the catalytic site of IRAP, promote such robust memory enhancing effects. One hypothesis is based on the role of IRAP in regulating the trafficking of GLUT4 containing vesicles in muscle and adipose, suggesting that the enzyme may be present in the brain in an analogous system. In this way, modulation of IRAP activity by centrally administered AT4 ligands may result in enhanced glucose uptake into neurones. This hypothesis is supported by the findings that exogenous glucose administration promotes an equally wide range of cognition enhancing effects as demonstrated for AT4 ligands. Therefore to investigate whether IRAP location and function in the brain is analogous to that characterised in muscle and adipose cells, the studies described in this thesis aimed to: 1) Map and characterise the cellular expression of IRAP in the rodent brain 2) Determine the subcellular localisation of IRAP in neurones 3) Determine if IRAP is associated with the facilitative glucose transporter in neurones as it is in muscle and adipose 4) Investigate if IRAP may be involved in neuronal glucose uptake Using a highly specific in-house antibody raised against the unique intracellular tail of IRAP, the studies in this thesis utilised an immunohistochemical approach to characterise IRAP in the brain. Firstly, IRAP immunoreactivity was visualised in specific nuclei throughout the brain. In particular, IRAP was highly expressed in cognitive associated areas, such as the medial septum, cerebral cortex and hippocampus. IRAP immunoreactivity was also abundant in many motor and motor associated nuclei. Dual label immunohistochemistry demonstrated IRAP was exclusively expressed in neurones and was partially associated with cholinergic neurones and their projection, supporting a role for IRAP in cognitive processes. The pattern of IRAP immunoreactivity within neurones was punctate and vesicular, throughout the cell soma and extending into proximal dendrites, indicating a predominantly post-synaptic localisation. At a subcellular level, IRAP was localised to vesicles containing VAMP2, but not small synaptic vesicles at nerve terminals. A clear overlap between IRAP and markers of the Trans Golgi network and endosomal membranes was evident, although IRAP localisation was not limited to these membranes. These findings were confirmed by electron microscopy studies, that visualised IRAP specific electron-dense precipitate associated with neurosecretory vesicles and cisterns of the rough endoplasmic reticulum and golgi apparatus. In summary, these findings demonstrated the subcellular location of I RAP in neurones is analogous to that characterised in muscle and adipose. The association between IRAP expression and the different facilitative glucose transporters present in the brain was examined. IRAP immunoreactivity was found to coincide specifically with GLUT4, such that in some brain regions the subcellular localisation of I RAP and GLUT4 appeared to overlap completely. Three patterns of co-localisation were mapped and quantitated with a high degree of co-localisation noted in cognitive associated nuclei, moderate co-localisation in motor associated nuclei while low co-localisation was noted in the hypothalamus and cerebellum. In areas of high co-localisation I RAP and GLUT4 appear to be localised to the same intracellular vesicles. This subcellular co-localisation is suggestive of a role for I RAP in regulating GLUT4 activity in cognitive associated regions such as the hippocampus. Finally the role of IRAP in neuronal glucose uptake was investigated by testing the effect of AT4 ligands on hippocampal glucose uptake. Both Ang IV and LVV-H7 significantly enhanced stimulated glucose uptake specifically in areas where I RAP and GLUT4 are co-localized. Furthermore, glucose uptake was visualised to occur in the pyramidal neurones that contain colocalised I RAP and GLUT4. These results strongly suggest a role for I RAP in neuronal glucose uptake. Taken together, these studies have demonstrated that IRAP is present in the brain in localisation analogous to that found in muscle and adipose. The presence of IRAP in vesicles with the regulated glucose transporter, GLUT4, in neurones involved in cognitive processing suggests I RAP and GLUT4 may mediate inducible glucose uptake in neurones, possibly in response to heightened energy demand in neurones. Therefore the hypothesis investigated in this thesis is confirmed. Additionally these results suggest a potential molecular mechanism that may underlie the cognition enhancing effects of AT4 ligands.
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ItemCellular characterisation of the insulin-regulated aminopeptidase (IRAP) in the brain(University of Melbourne, 2006)Central infusions of AT4 ligands, including Angiotensin IV and LW-Hemorphin 7, enhance memory acquisition, retention and retrieval in fear-conditioned memory tasks and spatial learning. A single acute dose of an AT4 ligand is also sufficient to reverse memory deficits caused by scopolamine treatment, perforant pathway lesions and other forms of experimentally induced amnesia models. The robust effects of these ligands on learning and memory processes are thought to be mediated by binding to the catalytic site of the insulin regulated aminopeptidase (IRAP). I RAP is also found in muscle and adipose cells, where the enzyme is found colocalised with GLUT4, the insulin regulated glucose transporter, in specialised vesicles. In response to insulin stimulation, IRAP traffics with GLUT4 from an intracellular location to the plasma membrane, from where GLUT4 mediates insulin regulated glucose uptake. The N-terminal domain of IRAP is believed to interact with multiple proteins to regulate the intracellular tethering, trafficking and possibly recycling of these vesicles. It is unknown how AT4 ligands, via binding to the catalytic site of IRAP, promote such robust memory enhancing effects. One hypothesis is based on the role of IRAP in regulating the trafficking of GLUT4 containing vesicles in muscle and adipose, suggesting that the enzyme may be present in the brain in an analogous system. In this way, modulation of IRAP activity by centrally administered AT4 ligands may result in enhanced glucose uptake into neurones. This hypothesis is supported by the findings that exogenous glucose administration promotes an equally wide range of cognition enhancing effects as demonstrated for AT4 ligands. Therefore to investigate whether IRAP location and function in the brain is analogous to that characterised in muscle and adipose cells, the studies described in this thesis aimed to: 1) Map and characterise the cellular expression of IRAP in the rodent brain 2) Determine the subcellular localisation of IRAP in neurones 3) Determine if IRAP is associated with the facilitative glucose transporter in neurones as it is in muscle and adipose 4) Investigate if IRAP may be involved in neuronal glucose uptake Using a highly specific in-house antibody raised against the unique intracellular tail of IRAP, the studies in this thesis utilised an immunohistochemical approach to characterise IRAP in the brain. Firstly, IRAP immunoreactivity was visualised in specific nuclei throughout the brain. In particular, IRAP was highly expressed in cognitive associated areas, such as the medial septum, cerebral cortex and hippocampus. IRAP immunoreactivity was also abundant in many motor and motor associated nuclei. Dual label immunohistochemistry demonstrated IRAP was exclusively expressed in neurones and was partially associated with cholinergic neurones and their projection, supporting a role for IRAP in cognitive processes. The pattern of IRAP immunoreactivity within neurones was punctate and vesicular, throughout the cell soma and extending into proximal dendrites, indicating a predominantly post-synaptic localisation. At a subcellular level, IRAP was localised to vesicles containing VAMP2, but not small synaptic vesicles at nerve terminals. A clear overlap between IRAP and markers of the Trans Golgi network and endosomal membranes was evident, although IRAP localisation was not limited to these membranes. These findings were confirmed by electron microscopy studies, that visualised IRAP specific electron-dense precipitate associated with neurosecretory vesicles and cisterns of the rough endoplasmic reticulum and golgi apparatus. In summary, these findings demonstrated the subcellular location of I RAP in neurones is analogous to that characterised in muscle and adipose. The association between IRAP expression and the different facilitative glucose transporters present in the brain was examined. IRAP immunoreactivity was found to coincide specifically with GLUT4, such that in some brain regions the subcellular localisation of I RAP and GLUT4 appeared to overlap completely. Three patterns of co-localisation were mapped and quantitated with a high degree of co-localisation noted in cognitive associated nuclei, moderate co-localisation in motor associated nuclei while low co-localisation was noted in the hypothalamus and cerebellum. In areas of high co-localisation I RAP and GLUT4 appear to be localised to the same intracellular vesicles. This subcellular co-localisation is suggestive of a role for I RAP in regulating GLUT4 activity in cognitive associated regions such as the hippocampus. Finally the role of IRAP in neuronal glucose uptake was investigated by testing the effect of AT4 ligands on hippocampal glucose uptake. Both Ang IV and LVV-H7 significantly enhanced stimulated glucose uptake specifically in areas where I RAP and GLUT4 are co-localized. Furthermore, glucose uptake was visualised to occur in the pyramidal neurones that contain colocalised I RAP and GLUT4. These results strongly suggest a role for I RAP in neuronal glucose uptake. Taken together, these studies have demonstrated that IRAP is present in the brain in localisation analogous to that found in muscle and adipose. The presence of IRAP in vesicles with the regulated glucose transporter, GLUT4, in neurones involved in cognitive processing suggests I RAP and GLUT4 may mediate inducible glucose uptake in neurones, possibly in response to heightened energy demand in neurones. Therefore the hypothesis investigated in this thesis is confirmed. Additionally these results suggest a potential molecular mechanism that may underlie the cognition enhancing effects of AT4 ligands.
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ItemSexual segregation in kangaroos : testing alternative evolutionary hypotheses(University of Melbourne, 2006)Males and females of many animal species live apart for most of the year, coming together only for mating. The causes of this sexual segregation are still debated, with most research being focused on ungulates. However, if we are to develop robust and inclusive explanations for this phenomenon we need better coverage of other vertebrate taxa. In this thesis, I investigate the occurrence of sexual segregation in Australian marsupials, and describe and quantify this phenomenon in western grey kangaroos, Macropus fuliginosus, and red kangaroos, M rufus, at Hattah-Kulkyne National Park in north-western Victoria, Australia. Exploiting some of the unique life-history characteristics of kangaroos, which are ecological analogues of ungulates, I test a number of hypotheses that have been proposed to account for the evolution of sexual segregation in ungulates. A literature review revealed that sexual segregation occurs in some Australian marsupials, as it does in some eutherians, but is most pronounced among the larger macropods, as it amongst their eutherian counterparts, the ungulates. Sexual segregation appears to be associated with body size dimorphism and birth synchrony in marsupials, as it is in ungulates: all species that exhibited sexual segregation were either dimorphic in body size, highly synchronous in breeding, or both. Species that were monomorphic, and gave birth year-round or only moderately synchronously, did not exhibit segregation. Sexual segregation occurs at the social, spatial and habitat level in western grey and red kangaroos, and the degree of social and spatial segregation is greater than that of habitat segregation in both species. The magnitude and timing of sexual segregation in these species is influenced by the synchrony and timing of their mating activity. In the synchronously breeding western grey kangaroo, segregation peaks during autumn, when females are in lactational anoestrus. In red kangaroos, which mate year round, segregation is weaker but occurs throughout the year. Intraspecific variation in body size in kangaroos, particularly amongst males, allows predictions of the four main hypotheses for sexual segregation (predation risk, forage selection, activity budget and social factors) to be tested independently of each other. I found that, in accordance with the predation risk hypothesis, female western grey kangaroos made greater use of secure habitats, large males made greater use of nutrient-rich, open habitat, while small males were intermediate between females and large males in their habitat use. Consistent with the social factors hypothesis, same-sex groups composed of females only, and males of both small and large body size, predominated. The activity budget hypothesis is an unlikely explanation for social segregation in western grey kangaroos since two assumptions of this hypothesis were not supported by my results. First, differences in body size did not result in activity budget differences, and consequently activity asynchrony: individuals of different body size did not have different transition times between resting and feeding, sex- size classes were not least synchronized when in mixed-body size groups, and synchrony was not lower in mixed-body size than same-body size groups. Second, activity asynchrony did not cause mixed-body size groups to be less cohesive than same-body size groups. Although mixed-body size groups were fused for less time than same-body size groups, synchrony was not correlated with group stability. An examination of the fission/fusion dynamics of groups of western grey kangaroos confirmed that the social factors hypothesis, and more specifically, social affinity among males, best explains social segregation in western grey kangaroos. As predicted by the male social affinity hypothesis, large males joined groups composed of other males more frequently than expected. This study contributes significantly to our understanding of the behavioural ecology of kangaroos, and more generally, identifies key factors involved in the evolution of sexual segregation in vertebrates.
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ItemSexual segregation in kangaroos : testing alternative evolutionary hypotheses(University of Melbourne, 2006)Males and females of many animal species live apart for most of the year, coming together only for mating. The causes of this sexual segregation are still debated, with most research being focused on ungulates. However, if we are to develop robust and inclusive explanations for this phenomenon we need better coverage of other vertebrate taxa. In this thesis, I investigate the occurrence of sexual segregation in Australian marsupials, and describe and quantify this phenomenon in western grey kangaroos, Macropus fuliginosus, and red kangaroos, M rufus, at Hattah-Kulkyne National Park in north-western Victoria, Australia. Exploiting some of the unique life-history characteristics of kangaroos, which are ecological analogues of ungulates, I test a number of hypotheses that have been proposed to account for the evolution of sexual segregation in ungulates. A literature review revealed that sexual segregation occurs in some Australian marsupials, as it does in some eutherians, but is most pronounced among the larger macropods, as it amongst their eutherian counterparts, the ungulates. Sexual segregation appears to be associated with body size dimorphism and birth synchrony in marsupials, as it is in ungulates: all species that exhibited sexual segregation were either dimorphic in body size, highly synchronous in breeding, or both. Species that were monomorphic, and gave birth year-round or only moderately synchronously, did not exhibit segregation. Sexual segregation occurs at the social, spatial and habitat level in western grey and red kangaroos, and the degree of social and spatial segregation is greater than that of habitat segregation in both species. The magnitude and timing of sexual segregation in these species is influenced by the synchrony and timing of their mating activity. In the synchronously breeding western grey kangaroo, segregation peaks during autumn, when females are in lactational anoestrus. In red kangaroos, which mate year round, segregation is weaker but occurs throughout the year. Intraspecific variation in body size in kangaroos, particularly amongst males, allows predictions of the four main hypotheses for sexual segregation (predation risk, forage selection, activity budget and social factors) to be tested independently of each other. I found that, in accordance with the predation risk hypothesis, female western grey kangaroos made greater use of secure habitats, large males made greater use of nutrient-rich, open habitat, while small males were intermediate between females and large males in their habitat use. Consistent with the social factors hypothesis, same-sex groups composed of females only, and males of both small and large body size, predominated. The activity budget hypothesis is an unlikely explanation for social segregation in western grey kangaroos since two assumptions of this hypothesis were not supported by my results. First, differences in body size did not result in activity budget differences, and consequently activity asynchrony: individuals of different body size did not have different transition times between resting and feeding, sex- size classes were not least synchronized when in mixed-body size groups, and synchrony was not lower in mixed-body size than same-body size groups. Second, activity asynchrony did not cause mixed-body size groups to be less cohesive than same-body size groups. Although mixed-body size groups were fused for less time than same-body size groups, synchrony was not correlated with group stability. An examination of the fission/fusion dynamics of groups of western grey kangaroos confirmed that the social factors hypothesis, and more specifically, social affinity among males, best explains social segregation in western grey kangaroos. As predicted by the male social affinity hypothesis, large males joined groups composed of other males more frequently than expected. This study contributes significantly to our understanding of the behavioural ecology of kangaroos, and more generally, identifies key factors involved in the evolution of sexual segregation in vertebrates.
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ItemNitrogen utilization and its regulation in Aspergillus nidulans(University of Melbourne, 2006)The filamentous fungus Aspergillus nidulans is able to utilize a wide range of compounds as sources of nitrogen. The presence of preferred nitrogen sources (ammonium and glutamine) signals nitrogen sufficiency, and genes required for the utilization of alternative sources are not expressed. In the absence of preferred nitrogen sources regulatory proteins activate expression of these genes. This constitutes nitrogen metabolite repression, and ensures the most efficient use of available nitrogen. In A.nidulans nitrogen metabolite repression is mediated by AreA, a positively acting GATA transcription factor. This thesis describes the investigation of two genes whose expression is subject to nitrogen metabolite repression and controlled by AreA, and the characterization of a fourth AMT/MEP gene in A. nidulans. areA102 is a specific mutation of the areA gene which results in a protein with altered promoter binding specificity, and areA102 mutants grow more strongly on a range of amino acids as nitrogen sources. Mutations at the sarA locus were first isolated as suppressors of the strong growth of an areA102 strain on histidine. In this study the sarA gene was characterized and confirmed to encode an L-amino acid oxidase (LAO) with broad substrate specificity. A sarA gene inactivation abolished LAO activity and suppressed the areA102 phenotype on histidine. An areA102 mutant was found to have increased utilization and stronger growth on amino acids which are LAO substrates. Investigation of sarA dependent and independent amino acid catabolism further defined the substrate specificity of LAO, and the contribution of other catabolic pathways was assessed. In A.nidulans the LAO was found to be the sole pathway for the catabolism of some amino acids, while for others this enzyme represents only a minor pathway. A.nidulans is known to possess four ammonium permeases differentially regulated by AreA. meaA encodes a low affinity ammonium transporter responsible for the majority of ammonium uptake. mepA has been shown to encode a high affinity permease which scavenges low concentrations of ammonium during nitrogen limitation, and mepB encodes a second high affinity permease only expressed during nitrogen starvation. To confirm the role of the fourth permease in ammonium acquisition, the mepC gene was cloned and its DNA and protein sequences analysed. The MepC ammonium transporter motifs differed somewhat from the consensus, and topology predictions indicated that mepC was more structurally divergent than the other A.nidulans ammonium transporters. The mepC gene was inactivated and the deletion strain was found to be indistinguishable from wildtype and from meaA, mepA, and mepB single, double and triple deletion backgrounds. However, strains over-expressing mepC from the highly inducible xylP promoter were able to partially complement the poor growth of an meaA?; mepA?; mepB? strain, indicating that MepC is capable of transporting ammonium. In other fungal systems certain ammonium permeases act as sensors of cellular nitrogen status. MeaA, MepA, and MepB do not act as nitrogen sensors. To determine whether MepC played a role in nitrogen sensing, amdS
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ItemNitrogen utilization and its regulation in Aspergillus nidulans(University of Melbourne, 2006)The filamentous fungus Aspergillus nidulans is able to utilize a wide range of compounds as sources of nitrogen. The presence of preferred nitrogen sources (ammonium and glutamine) signals nitrogen sufficiency, and genes required for the utilization of alternative sources are not expressed. In the absence of preferred nitrogen sources regulatory proteins activate expression of these genes. This constitutes nitrogen metabolite repression, and ensures the most efficient use of available nitrogen. In A.nidulans nitrogen metabolite repression is mediated by AreA, a positively acting GATA transcription factor. This thesis describes the investigation of two genes whose expression is subject to nitrogen metabolite repression and controlled by AreA, and the characterization of a fourth AMT/MEP gene in A. nidulans. areA102 is a specific mutation of the areA gene which results in a protein with altered promoter binding specificity, and areA102 mutants grow more strongly on a range of amino acids as nitrogen sources. Mutations at the sarA locus were first isolated as suppressors of the strong growth of an areA102 strain on histidine. In this study the sarA gene was characterized and confirmed to encode an L-amino acid oxidase (LAO) with broad substrate specificity. A sarA gene inactivation abolished LAO activity and suppressed the areA102 phenotype on histidine. An areA102 mutant was found to have increased utilization and stronger growth on amino acids which are LAO substrates. Investigation of sarA dependent and independent amino acid catabolism further defined the substrate specificity of LAO, and the contribution of other catabolic pathways was assessed. In A.nidulans the LAO was found to be the sole pathway for the catabolism of some amino acids, while for others this enzyme represents only a minor pathway. A.nidulans is known to possess four ammonium permeases differentially regulated by AreA. meaA encodes a low affinity ammonium transporter responsible for the majority of ammonium uptake. mepA has been shown to encode a high affinity permease which scavenges low concentrations of ammonium during nitrogen limitation, and mepB encodes a second high affinity permease only expressed during nitrogen starvation. To confirm the role of the fourth permease in ammonium acquisition, the mepC gene was cloned and its DNA and protein sequences analysed. The MepC ammonium transporter motifs differed somewhat from the consensus, and topology predictions indicated that mepC was more structurally divergent than the other A.nidulans ammonium transporters. The mepC gene was inactivated and the deletion strain was found to be indistinguishable from wildtype and from meaA, mepA, and mepB single, double and triple deletion backgrounds. However, strains over-expressing mepC from the highly inducible xylP promoter were able to partially complement the poor growth of an meaA?; mepA?; mepB? strain, indicating that MepC is capable of transporting ammonium. In other fungal systems certain ammonium permeases act as sensors of cellular nitrogen status. MeaA, MepA, and MepB do not act as nitrogen sensors. To determine whether MepC played a role in nitrogen sensing, amdS
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ItemCharacterisation of lipase genes in Helicoverpa armigera( 2006)Helicoverpa armigera (cotton bollworm) is a major agricultural pest in Australia, Asia and Africa. Analysis of a midgut cDNA library revealed a surprising diversity of genes encoding lipases, enzymes that metabolise lipids. Prior to and during this project, 13 neural lipases and three acidic ones were discovered. These were classified and compared to other insect lipases using sequence alignments and phylogenetic analysis. Novel non-catalytic subfamilies were discovered, including one containing an H. armigera lipase. At least three other subfamilies were found to contain H. armigera lipases. Lipidopteran lipases were found to be almost entirely distinct from Dipteran ones.
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ItemDNA methylation at the neocentromere( 2006-01)The Centromere is a vital chromosomal structure that ensures faithful segregation of replicated chromosomes to their respective daughter cells. With such an important structure, one would expect the underlying centromeric DNA sequence would be highly conserved across all species. It turns out that the underlying centromeric DNA sequences between species ranging from the yeast, fly, mouse to humans are in fact highly diverged suggesting a DNA sequence independent or an epigenetic mechanism of centromere formation. Neocentromeres are centromeres that form de-novo at genomic locations that are devoid of highly repetitive a-satellite DNA sequences of which normal centromeres are usually comprised from. To date, the 10q25 neocentromere is the most well-characterised, fully functional human centromere that has been used previously to characterise the extent of a number of centromeric protein binding domains and characterise the properties of the underlying DNA sequence. Along with other factors, the existence of neocentromeres has given rise to a hypothesis where centromeres are defined by epigenetic or DNA sequence independent mechanisms. The putative 10q25 neocentromere domain was recently redefined by high resolution mapping of Centromeric protein A (CENP-A) binding through a chromatin immunoprecipitation and array (CIA) analysis. The underlying DNA sequence was investigated to determine and confirm that the formation of the 10q25 neocentromere was through an epigenetic mechanism. Through a high-density restriction fragment length polymorphism (RFLP) analysis using overlapping PCR amplified DNA derived from genomic DNA representing the 10q25 region before and after neocentromere activation. No sequence polymorphisms, large insertions or deletions were detected and confirmed the epigenetic hypothesis of centromere formation. DNA methylation is one of many epigenetic factors that are important for cellular differentiation, gene regulation and genomic imprinting. As the mechanisms and functions of DNA methylation have been well characterised, its role at the 10q25 neocentromere was investigated to try and identify the candidate epigenetic mechanism involved in the formation of centromeres. DNA methylation across the neocentromere was assessed using sodium bisulfite PCR and sequencing of selected CpG islands located across the 10q25 neocentromere. Overall, the methylation level of the selected CpG islands demonstrated no difference in DNA methylation before and after neocentromere activation. However, significant hypomethylation upon neocentromere formation was detected close to the protein-binding domain boundaries mapped previously suggesting that this may have a role in demarcating protein binding domains at the neocentromere. Further analysis of DNA methylation investigated non-CpG island methylation at sites defined as CpG islets and CpG orphans. Interestingly, the DNA methylation level measured at selected CpG islets and CpG orphans across the 10q25 neocentromere were not completely hypermethylated as previously thought, but demonstrated variable methylation that became fully hypermethylated upon neocentromere activation in most sites investigated. These results suggested that a role for DNA methylation existed at the 10q25 neocentromere and that it occurred at sites devoid of CpG islands. This study has found that DNA methylation at non-CpG island sites was variable contrary to popular belief and, was linked with neocentromere formation through the observation of increased DNA methylation at the 10q25 neocentromere. Inhibition of DNA methylation demonstrated increased neocentromere instability and a decrease in methylation of these CpG islets and CpG orphans confirming the importance of DNA methylation at neocentromeres. This study has characterised a new class of sequences that are involved in the maintenance of chromatin structure through DNA methylation at the 10q25 neocentromere.