Cloning of cDNA sequences for murine ATP-citrate lyase. Construction of recombinant plasmids using an immunopurified mRNA template and evidence for the nutritional regulation of ATP-citrate lyase mRNA content in mouse liver

H. S. Sul, L. S. Wise, M. L. Brown, C. S. Rubin

Research output: Contribution to journalArticle

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Abstract

Mouse liver mRNA that was enriched in sequences coding for ATP-citrate lyase by polysome immunoadsorption was used as a template for cDNA synthesis. Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Escherichia coli RR1. Twenty-seven plasmids containing putative cDNA sequences for ATP-citrate lyase were identified by differential hybridization with single-stranded 32P-cDNAs synthesized from immunopurified mRNA, sucrose gradient-purified ATP-citrate lyase mRNA, and mRNA isolated from the livers of mice that were nutritionally induced or de-induced for ATP-citrate lyase biosynthesis. A subgroup of five recombinant plasmids was characterized further in hybridization-selection experiments. Each of these plasmids positively selected ATP-citrate lyase mRNA as determined by in vitro translation and specific immunoprecipitation. The length of ATP-citrate lyase mRNA was estimated to be 4900 bases in a Northern blot analysis. A 32P-cDNA probe derived from a 1500-base pair insert was used to investigate the basis for the 20-30-fold induction of ATP-citrate lyase that occurs when starved animals are fed a high carbohydrate/low fat diet. Dot-blot hybridization analysis disclosed that the relative content of liver ATP-citrate lyase mRNA increased 25-fold after 15 h of refeeding, indicating that the synthesis of the lipogenic enzyme is controlled at a pretranslational level in the nutritional paradigm.

Original languageEnglish (US)
Pages (from-to)1201-1205
Number of pages5
JournalJournal of Biological Chemistry
Volume259
Issue number2
Publication statusPublished - Jan 1 1984

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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