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Title: The control of retroviral gene expression in pluripotential embryonic stem cells
Author: Prince, Victoria Emma
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1991
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The Moloney murine leukaemia virus (Mo-MuLV) and its close relative the Moloney murine sarcoma virus (Mo-MSV) are retroviruses incapable of productively infecting early embryonic cells. The viral long terminal repeat (LTR) sequences are unable to direct transcription in embryonic cells but are highly active in their differentiated derivatives; hence these viruses provide a useful probe for investigating transcriptional control in stem cells. Protein extracts of F9 embryonal carcinoma stem cells and their differentiated derivatives were used in gel retardation experiments with Mo-MSV LTR sequences. The interactions were compared to those with sequences from the LTRs of mutant derivatives of Mo-MuLV which are transcribed in early embryonic cells. Two mutant viruses, the myeloproliferative sarcoma virus (MPSV) and its derivative PCMV, share a point mutation at -166 base-pairs (bp) with respect to the transcriptional start, which generates a consensus binding site for the well characterized transcription factor Sp1. The PCMV sequence around -166 interacted with a protein of equivalent immuno- reactivity, DNA-binding specificity and electrophoretic mobility to purified human Sp1 protein. The wild-type sequence had 5-fold lower affinity for this protein. Consistent with this observation, transient transfection experiments showed that the point mutation at -166 increased transcription 6-fold. Gel retardation experiments showed that the wild-type sequence around -345 bp interacted with an EC cell-specific protein. The protein had 10-fold lower affinity for the equivalent PCMV sequence, which has a point mutation at -345. As PCMV can be transcribed in EC cells the protein is an excellent candidate for a repressor of transcription. However, the mutation did not increase transcription in transient transfection experiments. The gene products of the adenovirus early gene, E1A, down-regulate papovavirus transcription. Co-transfection experiments showed that E1A also down-regulates transcription of Mo-MSV in an enhancer dependent fashion.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available