The role of ACTGCT motif in the expression of the minimal constitutive enhancer of the human papillomavirus type 18 in C33A cervical carcinoma cells

Hasnadka, Raghuram V. (1995) The role of ACTGCT motif in the expression of the minimal constitutive enhancer of the human papillomavirus type 18 in C33A cervical carcinoma cells. Masters thesis, Memorial University of Newfoundland.

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Abstract

The activity of the human papillomavirus type 18 minimal constitutive enhancer (nucleotides 7508-7738) was studied in C33A cervical carcinoma cells . Unidirectional deletion mutations were performed to delineate the minimal constitutive enhancer (MCE) region that is important for the human papillomavirus type 18 expression. Maximum reduction in the activity was seen when region V (nucleotides 7673-7738) was deleted. The deletion mutant enhancer pBK8315 (nucleotides 7508-7664) expressed only 6.5 percent of the full length MCE activity. Though the expression was the least with this mutant, all the regions appeared to be a requirement for the full activity of the MCE. Gel retardation assays with the full length MCE using C33A nuclear extract showed two complexes. Gel retardation assays were performed with deletion mutants having regions I-IV (nucleotides 7508-7664) and region V using C33A nuclear extract. The slower migrating complex associated with regions I-IV, while the faster migrating complex was bound to region V. To identify the important motif of region V and thereby the factor responsible for the expression, site directed mutagenesis was performed. The ACTGCT motif at nucleotides 7679-7684 was mutated. The in vivo activity of this site-directed mutant plasmid pBBl55 was reduced to the extent similar to pBK8315, signifying the importance of this motif in the context of the full length MCE. When gel retardation assay was performed with the site-directed mutant using C33A nuclear extract, the faster migrating complex was abolished, while the slower migrating complex was present. The mutant MCE competed with the slower migrating complex, while the faster migrating complex was still present. The in vitro binding assays correlated with the in vivo activity in C33A cells. To further investigate the binding property of the mutant motif, UV crosslinking was performed using C33A nuclear extracts with MCE sequences from nucleotides 7667-7693. A band present with the wild type sequence was absent when the mutant sequence was used in the reaction. These experiments demonstrate the importance of the ACTGCT motif at nucleotides 7679-7684 and suggest the involvement of an ACTGCT binding protein (ABP) in the regulation of HPV 18 MCE activity.

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