THE STRUCTURE, ORGANIZATION AND DIFFERENTIAL EXPRESSION OF THE RAT GENE ENCODING BILIVERDIN REDUCTASE

Screening of phage lambda libraries and genomic polymerase chain reactions were employed to generate clones of the rat gene encoding biliverdin reductase (BVR), the penultimate enzyme in the heme metabolic pathway. This enzyme, which converts biliverdin to bilirubin, is unique among enzymes characterized to date in that it exhibits two pH optima, 6.75 and 8.7, and utilizes a different cofactor, NADH and NADPH, respectively, at each optimum. The gene, which is 12270 bp in length, consists of five exons and four introns; two introns are greater than or equal to 4 kb. Only two of the four splice sites conform to consensus donor/acceptor sequences. Primer extension indicates the presence of two distinct transcription start points (tsp) in kidney and brain, as well as an additional tsp present in kidney, but not in brain RNA. The gene lacks a conventional TATA-box; however, an overlapping pair of TATA-like sequences is found 80 nt upstream from the kidney-specific tsp. The promoter region contains binding sites for several known regulatory factors, including AP-1, HNF-5 and INF-1, as well as two partial (7/8) matches to the heat-shock (HS) transcription factor-binding site. However, the time-course of the increase in message level, as determined by Northern blot analysis, indicates that BVR is not an early HS protein in that the relative abundance of mRNA is increased 6 h after hyperthermia and not at 1 h after HS. The approx. 1.6-kb BVR message is abundantly expressed in kidney, spleen, liver and brain, and at lower levels in the thymus, with minimal levels being detected in testis. Northern hybridization further suggests the presence of two other messages in kidney that share significant homology with the BVR-coding region.