245, University of Florida, College of Medicine,. Gainesville,. FL 32610. suggest that elevated ..... narrowing the list of possible functions for the putative AS1.
THE JOURNAL OF BIOLOGICAL CHEMISTRY B 1990 by The American Society for Biochemistry
Molecular Cloning Starvation-induced)
Vol. 265, No. 29, Issue of October 15, pp. 17844-17648, 1990 Printed in U.S. A.
and Molecular Biology, Inc.
of an Amino Acid-regulated in Rat Hepatoma Cells*
mRNA
(Amino
Acid
(Receivedfor publication, April 26, 1990) Neil F. Shay, Harry From the Department Medicine, Gainesville,
S. Nick, and Michael S. Kilbergt
of Biochemistry
Florida
and Molecular
Biology,
J. Hillis
Miller
Health
Center,
University
of Florida,
College
of
32610
Using the combination of a subtracted library and differential hybridization, a 409-base pair cDNA was identified that corresponds to a mRNA that is induced 2-3-fold when rat Fao hepatoma cells are subjected to amino acid starvation for 12 h. While this mRNA species was induced during starvation, others such as flactin, Cu-Zn superoxide dismutase, glyceraldehyde-3P, and histone H4 were decreased in abundance to 2550% of their original levels. The induction of the amino acid starvation-induced (ASI) mRNA was repressed when starved cells were returned to a medium supplemented with amino acids. Tissue distribution analysis showed the AS1 mRNA, approximately 650 base pairs in length, to be present in every rat tissue tested. The cDNA clone has been sequenced and appears to correspond to the 3’-most end of the mRNA. The cDNA sequence includes the poly(A) tail, two potential polyadenylation signal sequences, and an open reading frame that we presume to be a portion of the coding sequence. The AS1 cDNA will be used to investigate the molecular mechanisms for amino acid-dependent regulation of protein expression by mammalian cells.
Metabolite control of gene expression has been well documented in both bacteria and yeast, particularly control exerted by carbohydrates and amino acids. In bacteria for example, the nutrient-dependent regulation of the lactose, histidine, and tryptophan operons by the respective substrates has been well characterized (1). Starvation of bacteria for certain amino acids will activate genes coding for enzymes involved in the biosynthesis of those same amino acids (1). In yeast, a more general control of amino acid biosynthesis also exists which involves both transcriptional and posttranscriptional elements (2). In mammalian cells, changes in amino acid availability initiate a response that is characterized by altered plasma membrane amino acid transport mediated by the System A carrier (3-5). Substrate starvation results in increased transport that can be blocked by inhibitors of transcription, translation, and glycoprotein biosynthesis (3-6). Furthermore, plasma membrane vesicles isolated from amino acid-starved cells retain elevated transport rates when compared to similarly prepared vesicles from control cells (7). These results * This research was supported by Grant DK-31580 from the Institute for Diabetes, Digestive, and Kidney Diseases, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, J. Hillis Miller Health Center, Box J245, University of Florida, College of Medicine, Gainesville, FL 32610.
suggest that elevated gene transcription and subsequent translation generate a membrane-bound glycoprotein, perhaps the transporter itself, that is responsible for the increased transport rates following amino acid starvation. Conversely, there is a repression of transport activity when cells are maintained in the presence of amino acid-containing medium (3-5, 8, 9). Although there are exceptions (10, ll), amino acids which are not System A substrates, such as the branched chain and aromatic amino acids, have no repressive effect on carrier protein expression. The amino acid-dependent repression of System A also occurs by a RNA- and protein synthesis-dependent mechanism (3, 8, 9). For liver tissue, alanine, asparagine, glycine, proline, serine, and threonine are the most effective repressor amino acids (9). It has been proposed that a molecular regulatory system functions to induce System A transporter gene expression when concentrations of intracellular amino acids become too low (12, 13). Conversely, when adequate substrate levels are restored, the excess functional plasma membrane transporters are inactivated or degraded and the elevated transcription rate is slowed. The laboratory of Englesberg (14-17) has employed genetic mutational analysis to obtain data showing that control of System A gene expression in Chinese hamster ovary cells is the result of at least two different regulatory genes. Translation of Chinese hamster ovary mRNA by microinjetted Xenopus oocytes has confirmed the increased levels of System A mRNA in the mutant cell line (18). Interestingly, the Chinese hamster ovary mutant cell lines which exhibit constitutively high levels of System A activity also contain increased amounts of NaK-ATPase mRNA and protein (19). To understand the molecular mechanisms responsible for amino acid-dependent regulation of gene expression in mammalian cells, it will be necessary to obtain both cDNA and genomic clones for an amino acid-regulated gene. To identify specific transcripts, other than that of System A, that are increased in abundance during amino acid starvation, we have used a combination of “subtracted” cDNA library construction and differential hybridization (20). We report here the cloning of a cDNA (amino acid starvation-induced, ASI),’ for which the corresponding mRNA concentration is elevated by amino acid starvation. This cDNA will be a useful tool for future studies on nutrient-dependent control of protein expression in mammalian cells. MATERIALS
AND
METHODS
Cell Culture and RNA Preparation-Rat Fao hepatoma cells were maintained in modified Eagle’s medium, pH 7.4, supplemented with 24 mM NaHC03, 2.5 mM g&amine, 100
