Characterization of phenobarbital inducible rabbit cytochrome P450IIC4 and related genes

Chapter one of my thesis presents the work of cloning and characterization of the P450IIC4 related genes. The partial gene structures of cytochromes P450IIC4 and P450IIC5 were elucidated by sequence analysis of two isolated genomic clones. Clone 31 contains the last three of nine exons of P450IIC5 gene, whereas clone 32 contains the first five of nine exons of P450IIC4 gene. Nucleotide sequences of all exon/intron boundaries follow the GT/AG rule. The poly(A) attachment site for P450IIC5 was determined to be 355 base pairs downstream from the termination codon by comparison with the corresponding cDNA sequence. For the 5$\sp\prime$ flanking region of P450IIC4, the major transcription initiation site was assigned by mung bean nuclease protection mapping to an "A", 22 base pairs upstream from the translation initiation codon. A "TATA" sequence, TATAAA, was found 25 base pairs further upstream from the transcription initiation site. Sequences similar to those for proposed liver specific regulatory factors, HNF-1 and CCAAT, are found. The P450IIC4 gene exhibited high similarity to both P450IIC1 and P450IIC2 for about 150 nucleotides upstream from the RNA initiation site. Southern analysis using probes from the 5$\sp\prime$ region of the P450IIC4 gene suggests that there are at least three closely related members in the P450IIC4 subgroup in agreement with previous studies. After phenobarbital treatment, levels of P450IIC4 mRNA increased about 4-fold as measured by dot blot hybridization with a P450IIC4 specific oligonucleotide probe or by single-strand nuclease mapping of the 5$\sp\prime$ end of the mRNA. Together with the dot blot experiment using cDNA 3$\sp\prime$ untranslated region as probe, these three pieces of evidence have established that the P450IIC4 gene characterized in this study is responsive to phenobarbital.Chapter two of my thesis presents the work of our preliminary work in functional characterization of 5$\sp\prime$ flanking region of P450IIC4 gene. Using the gel mobility shift assay, we have defined a 100 bp region which interacts with a liver nuclear factor. The possibility that this factor is homologous to HNF-1 is discussed.U of I OnlyETDs are only available to UIUC Users without author permissio