Two transcripts encode rat cytochrome b5 reductase

G. Pietrini, P. Carrera, N. Borgese

Research output: Contribution to journalArticlepeer-review

Abstract

A cDNA expression library in λgt11 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 ~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 ≃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.

Original languageEnglish
Pages (from-to)7246-7250
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number19
Publication statusPublished - 1988

ASJC Scopus subject areas

  • General
  • Genetics

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