Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

The proximal first exon architecture of the murine ghrelin gene is highly similar to its human orthologue

BACKGROUND: The murine ghrelin gene (Ghrl), originally sequenced from stomach tissue, contains five exons and a single transcription start site in a short, 19 bp first exon (exon 0). We recently isolated several novel first exons of the human ghrelin gene and found evidence of a complex transcriptional repertoire. In this report, we examined the 5' exons of the murine ghrelin orthologue in a range of tissues using 5' RACE. -----FINDINGS: 5' RACE revealed two transcription start sites (TSSs) in exon 0 and four TSSs in intron 0, which correspond to 5' extensions of exon 1. Using quantitative, real-time RT-PCR (qRT-PCR), we demonstrated that extended exon 1 containing Ghrl transcripts are largely confined to the spleen, adrenal gland, stomach, and skin. -----CONCLUSION: We demonstrate that multiple transcription start sites are present in exon 0 and an extended exon 1 of the murine ghrelin gene, similar to the proximal first exon organisation of its human orthologue. The identification of several transcription start sites in intron 0 of mouse ghrelin (resulting in an extension of exon 1) raises the possibility that developmental-, cell- and tissue-specific Ghrl mRNA species are created by employing alternative promoters and further studies of the murine ghrelin gene are warranted.

Systemic racism: the hidden barrier to educational success for Indigenous school students

Explanations for poor educational experiences and results for Australian Indigenous school students have, to a great extent, focused on intended or conscious acts or omissions. This paper adopts an analysis based on the legislation prohibiting indirect racial discrimination. Using the elements of the legislation and case law it argues that apparently benign and race-neutral policies and practices may unwittingly be having an adverse impact on Indigenous students' education. These practices or policies include the building blocks of learning, a Eurocentric school culture. Standard English as the language of assessment, legislation to limit schools' legal liability, and teachers' promotions.

Look before you leap : the epistemic violence that sometimes hides behind the word "inclusion"

This paper demonstrates how Indigenous Studies is controlled in some Australian universities in ways that continue the marginalisation, denigration and exploitation of Indigenous peoples. Moreover, it shows how the engagement of white notions of “inclusion” can result in the maintenance of racism, systemic marginalisation, white race privilege and radicalised subjectivity. A case study will be utilised which draws from the experience of two Indigenous scholars who were invited to be part of a panel to review one Australian university’s plan and courses in Indigenous studies. The case study offers the opportunity to destabilise the relationships between oppression and privilege and the epistemology that maintains them. The paper argues for the need to examine exactly what is being offered when universities provide opportunities for “inclusion”.

Critical Literacies in Schools : A Primer

Readers and writers use a variety of modes of inscription – print, oral and multimedia – to understand, analyze, critique and transform their social, cultural and political worlds. Beginning from Freire (1970), ‘critical literacy’ has become a theoretically diverse educational project, drawing from reader response theory, linguistic and grammatical analysis from critical linguistics, feminist, poststructuralist, postcolonial and critical race theory, and cultural and media studies. In the UK, Australia, Canada, South Africa, New Zealand and the US different approaches to critical literacy have been developed in curriculum and schools. These focus on social and cultural analysis and on how print and digital texts and discourses work, with a necessary and delicate tension between classroom emphasis on student and community cultural ‘voice’ and social analysis – and on explicit engagement with the technical features and social uses of written and multimodal texts.

How the whiteness embedded in health services impacts on the health and well-being of Aboriginal peoples

Aboriginal women are treated differently by non-indigenous health care providers based on perceptions of Aboriginality and skin colour and white race privilege within health care environments. The experiences shared below are from some of the Aboriginal woman respondents in a research project undertaken within Rockhampton, a regional area in Central Queensland (Fredericks, 2003). The experiences give an insight into how the Aboriginal women interviewed felt and their observations of how other Aboriginal women were treated within health care settings based on skin colour and perceptions of Aboriginality. A number of the women demonstrated a personal in-depth analysis of the issues surrounding place, skin colour and Aboriginality. For example, one of the women, who I named Kay, identified one particular health service organisation and stated that, ‘it is a totally white designed space. There is nothing that identifies me to that place. I just won’t go there as a client because I don’t feel they cater for me as a black woman’. Kay’s words give us an understanding of the reality experienced by Aboriginal women as they move in and out of places within health environments and broader society. Some of these experiences are examples of direct racism, whilst other examples are subtle and demonstrate how whiteness manifests and plays out within places. I offer acknowledgement and honour to the Aboriginal women who shared their stories and gave me a glimpse of their realities in the research project from which the findings presented in this chapter are taken. It is to this research project that is the subject of this chapter.

There is nothing that 'identifies me to that place' : Aboriginal Women's perceptions of health spaces and places

There is a growing body of literature within social and cultural geography that explores notions of place, space, culture, race and identity. The more recent works suggest that places are experienced and understood in multiple ways and are embedded within an array of politics. Memmott and Long, who have undertaken place-based research with Australian Indigenous people, present the theoretical position that ‘place is made and takes on meaning through an interaction process involving mutual accommodation between people and the environment’. They outline that places and their cultural meanings are generated through one or a combination of three types of people–environment interactions. These include: a place that is created by altering the physical characteristics of a piece of environment and which might encompass a feature or features which are natural or made; a place that is created totally through behaviour that is carried out within a specific area, therefore that specific behaviour becomes connected to that specific place; and a place created by people moving or being moved from one environment to another and establishing a new place where boundaries are created and activities carried out. All these ideas of places are challenged and confirmed by what Indigenous women have said about their particular use of, and relationship with, space within several health services in Rockhampton, Central Queensland. As my title suggests, Indigenous women do not see themselves as ‘neutral’ or ‘non-racialised’ citizens who enter and ‘use’ a supposedly neutral health service. Instead, Aboriginal women demonstrate they are active recognisers of places that would identify them within the particular health place. That is, they as Aboriginal women didn’t just ‘make’ place, the places and spaces ‘make’ them. The health services were identified as sites within which spatial relations could begin to grow with recognition of themselves as Aboriginal women in place, or instead create a sense of marginality in the failure of the spaces to identify them. The women’s voices within this paper are drawn from interviews undertaken with twenty Aboriginal women in Rockhampton, Central Queensland, Australia, who participated in a research project exploring ‘how the relationship between health services and Aboriginal women can be more empowering from the viewpoints of Aboriginal women’. The assumption underpinning this study was that empowering and re-empowering practices for Aboriginal women can lead to improved health outcomes. Throughout the interviews women shared some of their lived realities including some of their thoughts on identity, the body, employment in the health sector, service delivery and their notions of health service spaces and places. Their thoughts on health service spaces and places provide an understanding of the lived reality for Aboriginal women and are explored and incorporated within this paper.

Phylogenetic placement of the sugarcane orange rust pathogen Puccinia kuehnii in a historical and regional context

Sugarcane orange rust, caused by Puccinia kuehnii, was once considered a minor disease in the Australian sugar industry. However, in 2000 a new race of the pathogen devastated the high-performing sugarcane cultivar Q124, and caused the industry Aus$150–210 million in yield losses. At the time of the epidemic, very little was known about the genetic and pathogenic diversity of the fungus in Australia and neighbouring sugar industries. DNA sequence data from three rDNA regions were used to determine the genetic relationships between isolates within two P. kuehnii collections. The first collection comprised only recent Australian field isolates and limited sequence variation was detected within this population. In the second study, Australian isolates were compared with isolates from Papua New Guinea, Indonesia, China and historical herbarium collections. Greater sequence variation was detected in this collection and phylogenetic analyses grouped the isolates into three clades. All isolates from commercial cane fields clustered together including the recent Australianfield isolates and the Australian historical isolate from 1898.The other two clades included rust isolates from wild and garden canes in Indonesia and PNG. These rusts appeared morphologically similar to P. kuehnii and could potentially pose a quarantine threat to the Australian sugar industry. The results have revealed greater diversity in sugarcane rusts than previously thought.

Whiteness, Epistemology and Indigenous representation

With its focus on Australia, Whitening Race engages with relations between migration, Indigenous dispossession and whiteness. It creates a new intellectual space that investigates the nature of racialised conditions and their role in reproducing colonising relations in Australia.