Two transcripts encode rat cytochrome b5 reductase.
A cDNA expression library in lambda gt11 was screened with affinity-purified polyclonal anti-rat cytochrome b5 reductase antibodies. One positive clone out of 450,000 clones was isolated and found to be incomplete. This clone was used to rescreen the library, and a second, overlapping clone that contained the entire coding sequence was isolated. RNA gel blots showed that the two overlapping clones contained approximately 90% of the reductase mRNA sequence. Sequencing data showed (i) that rat reductase has a 93% sequence similarity with bovine and human reductase and (ii) that reductase is not synthesized as a high molecular weight precursor. Results of Southern blot analysis were consistent with the hypothesis that a single gene codes for the soluble and membrane-bound (microsomal and mitochondrial) forms of the reductase, present in erythrocytes and liver, respectively. The cloned cDNA was used to study reductase transcripts in liver and reticulocytes. Two antisense RNA probes that together covered the entire coding region and part of the noncoding region of reductase mRNA were used in RNase A protection experiments. These probes detected only one transcript in liver, suggesting that endoplasmic reticulum and mitochondrial reductase are translated from the same mRNA. In contrast, two transcripts were detected in reticulocytes, one of which mismatched the liver probe approximately 30 nucleotides downstream from the initiation codon. Since the soluble and membrane form of the reductase are known to differ at the N terminus, we suggest that this second transcript encodes soluble reductase.
Pietrini
G
G
Consiglio Nazionale delle Ricerche, Center for Cytopharmacology, University of Milan, Italy.
Carrera
P
P
Borgese
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003579:Cytochrome Reductases / Q000235:genetics*; D042966:Cytochrome-B(5) Reductase; D004247:DNA / Q000032:analysis; D015347:RNA Probes; D051381:Rats; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D014158:Transcription, Genetic
D015347:RNA Probes; D004247:DNA; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D012259:Ribonuclease, Pancreatic