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Title: An investigation of c-myc autoregulation
Author: Laufer, Edward Marc
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1990
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C-myc protein, when expressed at sufficient levels, can repress the initiation of transcription from its own promoter. The DNA sequences within the human c-myc promoter through which the c-myc protein acts, and the proteins which bind these sequences in vitro, were investigated. Chimaeric c-myc promoter:indicator constructs were stably transfected into Rat-1 fibroblasts, and the resultant colonies pooled. The cells were then infected with a retroviral vector which directs expression of myc protein, and polyclonal populations again pooled. The responsiveness of each construct to myc protein was assessed by quantitation of indicator mRNA. Deletion analysis revealed that sequences from -71 to +47 base pairs relative to the P2 promoter mRNA cap site are sufficient to impart myc sensitivity to P2 promoter-dependent transcription. Systematic mutation of this region suggests that it contains multiple, redundant response elements which are also involved in the control of basal transcription. Several protein complexes were found to specifically interact with these sequences in gel retardation assays. Two of these, MAC II and MAC I were investigated in detail. MAC II is recognised by two c-myc peptide-specific monoclonal antibodies, and may contain c-myc protein. MAC II recognises both sequence dependent and indepedent properties of a double stranded, plasmid-derived linear DNA fragment of the P2 promoter, maximally extending from positions -63 to +47. The sequence independent component appears to be stabilised by hydrogen bonding, and may involve the formation of a secondary structure. MAC I is recognised by one c-myc peptide-specific monoclonal antibody, and contains myc related protein which is probably distinct from c-myc. It recognises a single stranded DNA sequence on the antisense strand, which spans the P2 promoter mRNA cap site and extends from approximately positions -30 to +31. Possible models of the relationship between these complexes, c-myc autoregulation and myc protein function are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available