Insulin increases transcription of rat gene 33 through cis-acting elements in 5'-flanking DNA.

Gene 33 is a multihormonally-regulated rat gene whose transcription is rapidly and markedly enhanced by insulin in liver and cultured hepatoma cells. To examine the mechanism by which insulin regulates transcription, we have constructed chimeric plasmids in which expression of the bacterial cat gene, encoding chloramphenicol acetyltransferase (CAT), is governed by gene 33 promoter elements and contiguous sequences in DNA flanking the transcription start point (tsp). When transfected into H4IIE hepatoma cells, these constructs gave rise to stably transformed cell lines producing the bacterial CAT enzyme. This expression was increased by insulin treatment in a fashion resembling the effect of this hormone on transcription of the native gene. In vitro transcription assays in nuclear extracts also revealed increased transcription of the chimeric plasmids when the extracts were prepared from insulin-treated rat hepatoma cells. The results demonstrate that induction by insulin is mediated by cis-acting nucleotide sequences located between bp -480 to +27 relative to the tsp.

Molecular cloning and analysis of full-length cDNAs cognate to a rat gene under multihormonal control.

Gene 33 is a multihormonally regulated rat gene whose transcription is rapidly and markedly enhanced by glucocorticoids, insulin, or cAMP. A cDNA clone (p216) containing a nearly full-length DNA complementary to the mRNA of this gene was isolated from a cDNA library and sequenced. The cDNA represents the entire mRNA transcript, except for three bases at the 5' terminus. The message is 2970 nucleotides long and can encode a protein of 459 amino acids with a molecular mass of 49,919 Da. The deduced amino acid sequence is rich in proline and serine but bears little homology to any known sequences. The 5' untranslated region, rich in G + C, is 270 nucleotides long and contains two apparently nonfunctional AUG initiator codons. The 3' untranslated sequence, rich in A + U, is 1323 nucleotides long and has a polyadenylation signal 13 bases upstream of the poly(A) tract. Two mRNA transcripts of the gene, which appear to be the consequence of alternative usage of splice sites, have been identified. The less abundant mRNA is truncated by 228 nucleotides in the protein coding region. The reading frame is maintained and this mRNA can code for a protein of 383 amino acids with a calculated molecular mass of 42,204 Da.

Structure of a multihormonally regulated rat gene.

Gene 33, a rat gene transcriptionally enhanced by glucocorticoids, insulin, or cyclic AMP, was isolated from a library of rat genomic DNA and characterized by sequence comparison to a full-length cDNA. The structural gene spans 13,500 bp encoding 2970 bp of exon sequences interrupted by three introns of about 9600, 101 and 811 bp, respectively. Exons (5' to 3') are 198, 194, 77 and 2501 bp in length; the first of these initiates at the transcriptional start point determined by S1 nuclease mapping. The 5'-flanking DNA contains several putative transcriptional control elements including TATA and CAAT boxes and a binding site for the Sp1 transcription factor in the usual locations proximal to the start point. Sequences resembling known glucocorticoid and cyclic AMP regulatory elements are also found upstream. A chimeric plasmid was constructed containing putative gene 33 regulatory elements fused to the Escherichia coli gene cat, encoding the enzyme chloramphenicol acetyltransferase, and transfected into cultured fibroblasts. Transient expression assays established that this gene 33 DNA is effective in promoting transcription.

Changes in hepatic differentiation following treatment of rat fetuses with 5-azacytidine.

Rat fetuses of 20 days gestational age were treated in utero with 5-azacytidine. Within 14 to 18 h after treatment several significant changes in the fetal livers were observed, including a dramatic maturation of hepatocyte morphology with little alteration in hematopoietic elements. Assessment of mRNA levels by hybridization to cloned cDNAs, together with other measures of gene expression, established that the change in hepatocyte morphology was associated with strong activation of expression of genes normally activated later in development, including those coding for the liver enzymes tyrosine aminotransferase and phosphoenolcarboxykinase and a gene of unknown specificity that is regulated in liver much like the aminotransferase. Rates of transcription of two of these genes, measured in isolated nuclei, were significantly increased after 5-azacytidine treatment. Expression of alpha-fetoprotein, normally declining during the perinatal period of development, was reactivated following treatment with the drug, while albumin expression was somewhat enhanced. For the most part the changes observed reflect temporal advancement of events normally programmed to occur later in differentiation of the liver. These changes appear to be the consequence of multiple effects of 5-azacytidine, including enhanced gene transcription and stabilization of gene products.

Gene-specific acquisition of hormonal responsiveness in rat liver during development.

Cloned cDNAs were used in hybridization analyses to assess hormonal responsiveness of two similarly regulated genes in livers of late-term fetal rats. Transcription of the tyrosine aminotransferase gene and of gene 33 (Lee et al.: J Biol Chem 260:16433-16438, 1985) is enhanced by glucocorticoids and by each of the usually antagonistic hormonal agents, insulin and cAMP, in adult liver, and that of both genes is developmentally activated at or just prior to birth. The mRNA of gene 33 was found to be significantly increased by each of the hormonal regulators in livers of fetuses treated in utero. Expression of the nearly silent aminotransferase gene in fetal liver was appreciably increased by cAMP but was refractory to control by either glucocorticoids or insulin; capacity of this gene to respond to insulin was not realized until several days postpartum. The data indicate specificity in the developmental acquisition of the capacity of individual genes to respond to hormonal regulators.

Different mechanisms control developmental activation of transcription of genes subject to identical hormonal regulation in adult liver.

Developmental changes in expression of two genes subject to identical hormonal controls in adult liver were examined in livers of fetal and newborn rats. Both mRNA concentrations and transcription of tyrosine aminotransferase were very low throughout gestation and increased sharply at birth. The mRNA of gene 33 (Lee et al., J. Biol. Chem. 260: 16433-16438, 1985) and its transcription were also low in fetal liver until a significant increase occurred just prior to birth, followed by a further increase at birth. In mutant mice carrying a deletion that prevents developmental activation of aminotransferase transcription, that of gene 33 was not affected. The data indicate that different mechanisms control developmental activation of these genes, in contrast to hormonal regulation of their expression.

Insulin enhances transcription of the tyrosine aminotransferase gene in rat liver.

The mechanism of insulin-mediated induction of tyrosine aminotransferase in rat liver was investigated using a cloned cDNA probe. The level of aminotransferase mRNA increases about fourfold following administration of the hormone. This induced mRNA accumulation does not require de novo protein synthesis. Nuclear runoff transcription assays in isolated liver nuclei demonstrate that insulin has a rapid and time-dependent stimulatory effect on aminotransferase gene transcription. The magnitude of enhanced transcription can fully account for the increase in the mRNA. We conclude that the induction of tyrosine aminotransferase in rat liver by insulin is primarily a consequence of a selective increase in the rate of transcription of the aminotransferase gene.

Molecular cloning of cDNAs cognate to genes sensitive to hormonal control in rat liver.

Poly(A)-RNAs were prepared from livers of rats treated with hydrocortisone and cycloheximide, then enriched for large mRNAs by successive sucrose gradients and gel electrophoresis. The size-selected RNAs were used as templates for synthesis of double-stranded cDNAs that were cloned in Escherichia coli using the pBR322 plasmid vector. Recombinant plasmids characterized as carrying inserts of potential interest were further analyzed by differential hybridization to mRNAs from untreated and hydrocortisone-treated rats. Seven of the cloned cDNAs were identified as complementary to mRNAs whose content in liver is sensitive to modulation by the steroid. Further screening for hormonal responsiveness revealed that two of the cloned cDNAs hybridize to a 3.4-kilobase mRNA that is rapidly induced in liver by treatment with insulin or cAMP as well as by hydrocortisone; restriction enzyme analysis demonstrated that the cDNAs from these two clones are derived from the same mRNA. In isolated nuclei, the rate of transcription of this mRNA is increased by each of the inducing hormones. This unusually regulated mRNA codes for a protein of 53 kDa on denaturing gels, undergoes rapid intracellular degradation that is prevented by cycloheximide, and appears to be the product of a single copy gene.

Activation of tyrosine aminotransferase expression in fetal liver by 5-azacytidine.

Rat fetuses of 20 days gestational age were treated in utero with the inhibitor of DNA methylation, 5-azacytidine. The liver enzyme tyrosine aminotransferase, normally expressed at very low levels until several hours after birth, was increased by the drug in the fetal livers after a lag period of about 9 hours, reaching a level 70-fold above control levels 18 hours after treatment. The high levels attained after 5-azacytidine treatment are comparable to those of glucocorticoid-treated adult livers, and were not further increased by administration of hydrocortisone to dams carrying treated fetuses. Cytidine and two other analogs, cytosine arabinoside and 6-azacytidine, were essentially without effect.

Development of tyrosine aminotransferase in perinatal rat liver: changes in functional messenger RNA and the role of inducing hormones.

Expression of the hepatic enzyme tyrosine aminotransferase was analyzed in the perinatal period of development in the rat, when this expression undergoes significant changes associated with hepatocyte differentiation. In late prenatal liver both enzyme and functional mRNA gene products are present at levels 10- to 15-fold below those in the fully differentiated adult liver. This low level of expression in fetal liver is refractory to induction by glucocorticoids, but both gene products are increased to a limited extent by cyclic AMP. This induction by cyclic AMP (cAMP) does not confer glucocorticoid-responsiveness on expression. By 3 hr after birth both functional mRNA and enzyme levels are significantly increased, an increase which continues until a peak is reached at 12 hr that is appreciably above the adult levels. Both gene products then decline until adult levels are reached by 24 hr. The postnatal shift in aminotransferase expression is accompanied by acquisition of the capacity to respond to glucocorticoids. Treatment of newborns with an antiglucocorticoid steroid or with glucose suppresses the postnatal overshoot of expression, but neither treatment affects the increase from fetal to adult levels of expression. The results indicate that prior to birth, expression of the aminotransferase gene is partially repressed, a repression that is lifted essentially immediately upon birth. The hormones capable of inducing aminotransferase synthesis have no apparent necessary role in this process.

Concanavalin A alters the turnover rate of tyrosine aminotransferase in cultured hepatoma cells.

Concanavalin A added to monolayer cultures of Reuber H-35 hepatoma cells caused a rapid inactivation of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, E.C. 2.6.1.5) and loss of reactivity with antibody against the native, dimeric enzyme. Analysis of treated cells with an antibody raised against carboxymethylated, denatured enzyme showed that the inactivated enzyme was reactive with this reagent, which does not react with the native enzyme. Subsequent addition of alpha-methyl-D-mannopyranoside to remove concanavalin A restored both enzyme activity and reactivity to antibody against native enzyme. After long-term treatment with concanavalin A, the restored enzyme levels were significantly higher than in controls treated with the sugar but not the lectin. Analysis of the turnover of the enzyme by two methods revealed that the rate of its degradation is reduced about 2-fold in concanavalin A-treated cells. Treatment with H-35 cells with concanavalin A thus effects an alteration in conformation of tyrosine aminotransferase, rendering it somewhat less sensitive to intracellular degradation.

Selective effect of the metallocarcinogen beryllium on hormonal regulation of gene expression in cultured cells.

Effects of the metallocarcinogen beryllium on regulation of gene expression were assessed by analysis of hormonal regulation of synthesis of tyrosine aminotransferase in beryllium-treated hepatoma cell cultures. Cell growth was not affected by exposure of the cells to 1 micro M BeSO4 throughout their 4- to 5-day growth cycle. In cells pretreated in this way, the induction by glucocorticoids was specifically impaired, the extent of induced enzyme synthesis being reduced about 50%. Inductions by insulin or cyclic adenosine 3':5'-monophosphate were not influenced by the metal. The results suggest that low concentrations of beryllium selectively interfere with regulatory mechanisms controlling transcriptional events in gene expression.

Effects of insulin on messenger RNA activities in rat liver.

Liver poly(A) RNA, isolated from adrenalectomized rats after insulin treatment, was translated in a nuclease-treated lysate of rabbit reticulocytes and quantitated for both total activity and the capacity to synthesize the insulin-inducible enzyme tyrosine aminotransferase. Analysis of the translated products from poly(A) RNA isolated 1 h after insulin treatment showed a 2.7-fold increase in activity of tyrosine aminotransferase mRNA. During the same interval, the capacity of poly(A) RNA to direct the synthesis of total protein in lysates also changed, showing a 30 to 40% increase in translational activity/unit of RNA. Increased translatability was apparent in all fractions of poly(A) RNA separated by centrifugation on sucrose gradients. Insulin thus appears to mediate a generalized change in mRNAs leading to increased capacity for translation; induction of tyrosine aminotransferase may reflect sensitivity to effect of the hormone.

Changes in hepatic levels of tyrosine aminotransferase messenger RNA during induction by hydrocortisone.

Messenger RNA specific for tyrosine aminotransferase was quantitated by microinjection into oocytes of Xenopus laevis. The heterologously translated enzyme was identified by specific immunoprecipitation and found to be identical with authentic aminotransferase by several criteria. The level of functional message present in rat liver increases during hydrocortisone induction, and this increase is directly proportional to the increased rate of synthesis of the enzyme. Kinetic analysis of the changes in tyrosine aminotransferase mRNA levels during induction and withdrawal indicates that the steroid does not affect the stability of the message, which has a half-life of approximately 1.2 h. Hydrocortisone, therefore, acts to increase the rate of synthesis of the specific messenger by stimulating either its transcription or processing to functional mRNA.

Role of coenzyme in aminotransferase turnover.

The role of coenzyme in determining intracellular contnet of pyridoxal enzymes was assessed by analyzing effects of pyridoxine deficiency on the rapidly degraded, readily dissociable tyrosine aminotransferase (EC 2.6.1.5) and the slowly degraded, nondissociable alanine aminotransferase (EC 2.6.1.2) of rat liver. Synthesis of the tyrosine enzyme was reduced, leading to a decreased amount of this enzyme, much of which was present as active apoenzyme. Synthesis of alanine aminotransferase was unchanged but much of this enzyme was present as an inactive apoenzyme which retained immunological reactivity. Degradation rates of both enzymes (t1/2 about 1.5 h, tyrosine aminotransferase; about 3 days, alanine aminotransferase) were not changed in pyridoxine deficiency. Hence, interaction with coenzyme is not a significant determinant in intracellular degradation of these aminotransferases. Coenzymes dissociation and intracellular stability probably reflect structural features of the proteins which determine both properties.

Separation of cellular and viral DNA polymerases by affinity chromatography on polynucleotide-Sepharose.

Polyguanylate- and poly(2'-O-methyl)uridylate-Sepharose have been prepared for affinity chromatography of DNA polymerases of viral origin (reverse transcriptase). Both cellular DNA polymerases and reverse transcriptase bind to polyguanylate-Sepharose. The cellular polymerases can be eluted from the column between 0.32 and 0.42 M NaCl while reverse transcriptase eluted between 0.56 and 0.78 M NaCl. However, only reverse transcriptase adheres to poly(2'-O-methyl)uridylate-Sepharose and can be eluted at approximately 0.35 M NaCl. The columns were used to partially purify RNA-dependent DNA polymerase from spleens of mice infected with Rauscher leukemia virus. The enzyme preparation is about 1300-fold purified and is inhibited by antiserum prepared against purified reverse transcriptase from Rauscher leukemia virus to the same extent as the virion enzyme.

Differential degradation of messenger RNAs in mammalian cells.

Through the use of an assay that measures cellular capacity for specific enzyme synthesis, mRNA of alanine aminotransferase (EC 2.6.1.2; L-alanine:2-oxoglutarate aminotransferase) was found to be degraded with a half-life of 12-14 hr in cultured Reuber H-35 cells; mRNA of tyrosine aminotransferase (EC 2.6.1.5; L-tyrosine:2-oxoglutarate aminotransferase) has a half-life of 2 hr in the same cells. Rates of degradation of the mRNAs are the same whether new mRNA accumulation is blocked by removal of the steroid inducer or by inhibition of mRNA synthesis (actinomycin). Cycloheximide inhibits the normally rapid turnover of tyrosine aminotransferase mRNA, but agents such as puromycin and sodium fluoride, which disrupt polysome structure, do not alter the turnover rate of the tyrosine and alanine aminotransferase mRNAs. The tyrosine and alanine aminotransferase mRNAs appear to be translated at equivalent rates. The data suggest that the degradation rate of these two mRNAs is determined by the polynucleotide structure of the mRNA molecules at or near the site for ribosome binding and initiation.