The presence of intervening sequences in the alpha-fetoprotein gene of the mouse. (1979)

‘lkli. ~JOURNAL OF BIOI.OCICAL Vol. 254. No. 15. Issue of August Prmfed m U.S.A The Presence Mouse* CHEMISTRY 10, pp. 7393-7399, of Intervening Sequences in the a-Fetoprotein (Received Shirley Ingram From M. Tilghman,$ the Fels Research Dimitris Institute, Kioussis, Temple Michael University School is a major constituent of fetal species which have been examined in the mouse, AFP’ is synthesized sac and liver and secreted After birth, the amount serum in all (l-4). During by the yolk into the blood and amniotic of AFP in serum decreases of u-fetoprotein synthesis in the adult Garcia Philadelphia, Ruiz, Pennsylvania March and Robert S. under a select number of pathological conditions: following liver injury (6, 7), in hepatocarcinomas (1, 2), and in testicular teratocarcinomas (8, 9). In addition, a number of congenital birth defects, including congenital nephrosis, severe Rh-he- molytic disease, and several open neural tube defects are associated with abnormally elevated concentrations of AFP in the maternal and fetal circulation and amniotic fluid (10-12). These striking and reproducible increases in the production of AFP in fetal abnormalities, in liver regenerative diseases, and in carcinomas of the liver and germ cells in humans has generated interest in the mechanism of control of the expres- sion of AFP. Recent studies by Tamaoki and his co-workers (13,14) have used cell-free translation assays to demonstrate that the rate of synthesis of a-fetoprotein in fetal and neonatal mouse liver is related to the concentration of translatable AFP mRNA. Taking this analysis one step further, Innis and Miller (15) reported the isolation of rat AFP mRNA. Complementary DNA to the AFP mRNA was used in hybridization kinetic experiments to show that the actual amount of AFP mRNA in adult liver mRNA was less than 1% of that in hepatoma cells. These studies imply that the control of AFP synthesis occurs at the level of mRNA transcription or processing, or both. Distinguishing between these possibilities will require large amounts of specific hybridization probes to the a-fetoprotein gene, as well as intimate knowledge about its structural or- ganization in the genome. Toward this end, we have con- structed chimeric plasmids containing portions of the mouse AFP mRNA sequence, and used them to identify three EcoRI fragments of mouse genomic DNA which encode in a discon- tinuous manner the single copy AFP gene. fluid. EXPERIMENTAL markedly in most strains of mice to a basal level which represents less than 0.01% of that in fetal serum (3, 5). This decline has recently been shown by Olsson et al. (5) to be inherited as an autosomal recessive trait, which they termed regulation of a- fetoprotein (ran. Reinitiation of Medicine, J. Predes for publication, occurs * This work is supported by Grants CA 23572 to S.M.T. from the National Institutes of Health and CA 12227 to the Fels Research Institute. 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 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of an American Cancer Society Junior Faculty Award. 0 Trainee of the Medical Scientist Training Program, National Institutes of Health Grant GM 07170. ’ Abbreviations used are AFP, a-fetoprotein; dsDNA, double- stranded DNA; SSC, 0.15 M NaCl, 0.015 M sodium citrate; bp, base pairs; kb, kilobase pairs; Hepes, N-(2-hydroxyethyl)-l-piperazine-eth- anesulfonic acid, SDS, sodium dodecyl sulfate. PROCEDURES Purification of a-Fetoprotein-Amniotic fluid, pooled from 16- to Id-day mouse fetuses, was subjected to 35 to 70% ammonium sulfate precipitation. The 70% pellet was dialyzed against 50 mu Tris.HCl (pH 7.5) and electrophoresed through a nondenaturing 7% polyacryl- amide gel (16). The AFP band was identified by a comparison to fetal and adult serum protein markers, extracted and concentrated by ethanol precipitation (16). This material was used to raise antisera to AFP by bimonthly injections into a rabbit. The rabbit antisera were regularly tested for anti-AFP activity by Ouchterlony analysis (17) against purified AFP, amniotic fluid, and adult mouse serum. Preparation of RNA-Yolk sac and livers, obtained from 16- to 18-day-old Swiss mouse fetuses were frozen in liquid Na. Poly(A) RNA was prepared by phenol extraction of total cellular RNA (18) followed by oligo(dT)-cellulose chromatography (19). The RNA was fractionated on an 8 to 25% sucrose gradient in 2 mM EDTA, 100 mM NaCl, 10 mM Hepes (pH 7.6), which was centrifuged at 29,000 rpm for 22 h in a Beckman SW40 rotor. Individual fractions were precipitated by the addition of 2 volumes of ethanol, resuspended in Hi0 and tested for AFP mRNA activity using a cell-free synthesizing system derived from wheat germ (20, 21). After treatment with 200 pg/ml of Downloaded from http://www.jbc.org/ at UCLA-Louise Darling Biomed. Lib. on April 25, 2017 Messenger RNA for the oncofetal protein cu-fetopro- tein was isolated from yolk sacs of 16- to H-day-old mouse embryos. This RNA was used as a template to synthesize full-length double-stranded DNA containing predominantly a-fetoprotein coding sequences. Chi- merit plasmids containing the double-stranded DNA were constructed in two ways. First, molecular linkers containing the recognition site for the restriction en- donuclease HindI were ligated to the blunted ends of the DNA, and following cleavage with HindIII, the re- combinant molecules were ligated to the single Hind111 site in the plasmid vehicle pBR322. Second, internal Pst I fragments of the double-stranded DNA were ligated to the single Pst I site of pBR322. Following transfor- mation of the host Escherichiu coli x1776, colonies con- taining those chimeric plasmids which had incorpo- rated n-fetoprotein sequences were identified by selec- tive hybridization and arrest translation procedures. Two cloned sequences derived from the 5’ and 3’ regions of the mRNA were used as hybridization probes to detect the presence of the a-fetoprotein gene within a two-dimensional fingerprint of mouse genomic DNA cleaved with EcoRI, an enzyme which does not cleave within the messenger RNA sequence. The detection of 3 EcoRI fragments demonstrates that the single cu-fe- toprotein gene must be represented discontinuously in the mouse genome, with at least two intervening se- quences. a-fetoprotein mammalian development B. Gorin,@ Gene of the