Sequence and gene expression of rabbit cytochrome P450 IIC16: comparison to highly related family members.

Cytochrome P450s are heme containing proteins which evolved from an ancestral gene(s) to form a large superfamily of enzymes. We have isolated a unique cDNA from the rabbit P450 IIC subfamily, IIC16, which is 2028 bp in length. Nucleotide sequence determination indicated an ATG start codon 66 bp from the 5' end of the molecule, and an open reading frame coding for a protein of 487 amino acids. P450 IIC16 protein is greater than or equal to 90% identical in sequence to rabbit P450 IIC4, IIC5, and to the partial sequence available for IIC15. Northern and slot blot experiments demonstrated that the P450 IIC16 gene is expressed constitutively in liver, lung, testes, and kidney, and is inducible by phenobarbital in each tissue with the exception of the kidney, where mRNA levels are repressed. Alignment analysis of eight rabbit P450 IIC proteins revealed conserved and variable regions common to all IIC enzymes, and specific areas are suggested which may be important with respect to structure and function.     

Differences in deoxyribonuclease I hypersensitive sites in phenobarbital-inducible and constitutive rabbit P450IIC genes.

DNase I hypersensitivity of nuclear chromatin near the rabbit cytochrome P450IIC genes was investigated by indirect end-labeling genomic Southern analysis. Major DNase I hypersensitive sites were observed in proximal (-200 base pairs) and distal (-2000 to -2200 base pairs) regions of the genes. The presence of the proximal site correlated well with the expression states of the individual genes. In contrast, the distal site was present in DNA from both liver and kidney nuclei and in untreated and phenobarbital-treated animals irrespective of the expression state of the genes. However, the distal site was present only in genes that respond to phenobarbital (cytochromes P450IIC1 and P450IIC2) and was not detected in the constitutive cytochrome P450IIC3 gene. The nucleotide sequences of 500 base pairs in the distal site regions of cytochromes P450IIC1 and P450IIC2 were 67% similar, and hepatocyte nuclear factor 1 like motifs were conserved in these two sequences. These results are consistent with the hypothesis that distal regions cooperate with the proximal promoter regions in the regulation of cytochrome P450IIC gene expression.     

The actin gene in yeast Saccharomyces cerevisiae: 5'' and 3'' end mapping, flanking and putative regulatory sequences

The 5' and 3' flanking regions of the yeast actin gene have been sequenced and the ends of the actin mRNA were determined by the single-strand nuclease mapping procedure. The mRNA starts with a pyrimidine residue 141 (or 140) nucleotides upstream from the initiation codon. The actin gene lacks a typical "TATA" box 30 base pairs upstream from the mRNA start site but it contains a region homologous to the canonical sequence 5'-GGCTCAATCT-3' which is found in several eukaryotic genes 70 to 80 bp upstream from the mRNA cap site. Judging from the S1 nuclease mapping, there are two populations of actin mRNA terminating 98 and 107 nucleotides downstream from the stop codon. The 3' termini are preceded by three AATAAA sequences found in most eukaryotic polyadenylated mRNAs.