Delores J. Grant, Ph.D.

Dissertation research performed under the guidence of Dr. Nobuyo N. Maeda

ABSTRACT
    The haptoglobin gene complex (Hp) has served as a model to study the effect that base substitutions and a retrovirus-like element have on the transcriptional activity of the gene promoters. Using a tissue culture based system, I demonstrated that an A to C base substitution at nucleotide position -61 in the promoter region of the Hp² allele, which has been shown to be strongly associated with the haptoglobin 2-1 modified (Hp2-1mod) phenotype, decreases the transcriptional activity of the Hp² gene promoter. In HepG2 cells, which normally express their endogenous haptoglobin genes, chloramphenicol acetyl transferase (CAT) plasmid constructs with the -61C base change in the promoter had about 10-fold lower transcriptional activity after transfection than the Hp control construct. The -61C substitution also rendered the construct unresponsive to treatment with interleukin-6 after transfection into Hep3B2 cells, which normally do not express haptoglobin but do so in response to stimulation by acute phase reactants. These results support the hypothesis that the Hp2-1mod phenotype results from the -61C mutation in the promoter region of the Hp² allele. The Hpr gene, which has normal gene structure, may be inactivated by a retrovirus-like element (RTVL-Ia) present in the first intron. Three fragments containing different regions of a retrovirus-like insertion, RTVL-Ia, were inserted in both orientations into a 'mini intron' within a parental HprCAT construct. This construct consists of sequences derived from the promoter region and the first intron, including the 5' and 3' donor and acceptor splice sites of the Hpr gene. The resulting constructs were transiently transfected into HepG2 cells and promoter transcriptional activity was measured. The results show that a fragment which consists of putative gag and pol gene sequences and then another which consists of pol, env gene sequences and the 3'LTR of the RTVL-Ia element significantly decrease transcriptional activity when inserted in the same transcriptional orientation as the Hpr gene promoter. These results suggest that regions of the RTVL-Ia element may negatively affect promoter function.