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.
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.
D001275:ATP Citrate (pro-S)-Lyase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D005502:Food; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism*; D013217:Starvation / Q000378:metabolism; D013698:Templates, Genetic
D012333:RNA, Messenger; D004247:DNA; D001275:ATP Citrate (pro-S)-Lyase