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Biological role and regulation of Pur-[alpha]: 'in vivo' and 'in vitro' studies
Dissertation

Biological role and regulation of Pur-[alpha]: 'in vivo' and 'in vitro' studies

Vandhana Muralidharan
Doctor of Philosophy (Ph.D.), Medical College of Pennsylvania and Hahnemann University
Feb 1999
DOI:
https://doi.org/10.17918/00007261
pdf
Muralidharan_Vandhana_19994.34 MB
PDF Access upon request, Email title, URL, or DOI to archives@drexel.edu

Abstract

Microbiology
Progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the human central nervous system (CNS) is caused by the replication of the human neurotropic virus, JCV. An early viral protein, JC large tumor (T)-antigen, mediates JC viral replication and the assembly of viral particles. JC T-antigen transgenic mice developed severe dysmyelination with reduced myelin basic protein (MBP) levels, suggesting the importance of T-antigen in JCV induced PML. Transcription factors that regulate MBP gene expression were investigated as one of the possible mechanisms through which JC T-antigen causes dysmyelination. Pur-[alpha] is a transcription factor that upregulates MBP (myelin basic protein) promoter expression in in vitro assays. Pur-[alpha] protein associates with JC T antigen in hamster glial cells as demonstrated by immunoprecipitation. This led to the hypothesis, 'the abrogation of the function of Pur-[alpha] as transcription factor for MBP gene expression by T antigen could lead to demyelination in PML'. To test this hypothesis, Pur[alpha]-/- mice were created and their myelin status was analyzed. Pur-[alpha] null mice survived the embryonic development. However, around postnatal day 14, they exhibited slack posture, kyphosis, resting tremors, growth retardation and eventually died around day 25. This demonstrates that Pur-[alpha] is not essential for the embryonic survival of the mice, but vital for the survival of adult animals. Compared to the wild type mice, Pur-[alpha]-/- mice did not show any significant difference in the (i) levels of MBP, PLP and MAG mRNA by Northern analysis; (ii) distribution and in the amount of MBP protein by MB immunohistochemistry; (iii) amount of MBP mRNAs from the sciatic nerve and spinal cord as demonstrated by quantitative RT-PCR. The above analysis demonstrated that Pur-[alpha] is not an essential transcription factor for MBP gene expression. Since Pur-[alpha] deficient mice exhibited normal myelination, it was concluded that inactivation of Pur-[alpha] alone by T antigen is not sufficient to cause demyelination in PML. Parallely, the transcriptional start site of Pur-[alpha] gene was identified. The promoter fragment of Pur-[alpha] gene was characterized and negative regulation of Pur-[alpha] on its own promoter was identified demonstrating the negative auto regulation of Pur-[alpha].

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