Molecular cloning of cDNA for rat L-type pyruvate kinase and aldolase B

Two double-stranded cDNA recombinant pBR322 plasmid libraries were constructed starting from high carbohydrate diet rat liver poly(A)+ mRNA, either fractionated by denaturing sucrose gradient centrifugation for the cloning of L-type pyruvate kinase cDNA, or nonfractionated for aldolase B. Both libraries were screened with single-stranded cDNA probes reverse transcribed from fasted or high carbohydrate diet rat liver mRNAs. mRNAs from fasted animals were also fractionated by sucrose gradient centrifugation and mRNAs from the fed animals were, in addition, further purified by high performance liquid gel filtration chromatography. Those clones hybridizing with the "positive" probe (from animals fed the high carbohydrate diet) and not with the "negative" one (from fasted animals) were preselected and their plasmid DNA was purified and analyzed by positive hybridization-selection. Thirty of 4500 bacteria colonies transformed by recombinant plasmids were preselected by differential screening for pyruvate kinase, and 8 of 864 colonies for aldolase B. Twenty-two recombinant plasmids for pyruvate kinase and two for aldolase B were shown to contain specific cDNA inserts by positive hybridization-selection. Plasmids DNAs of some pyruvate kinase and aldolase B clones (whose inserts ranged from 700 to 1050 bases in length) were labeled by nick translation and used as probes for Northern blot hybridization. The pyruvate kinase cDNA probes recognized mainly a 3400-base RNA species which was detected in high carbohydrate diet rat liver, but not in fasted rat liver and in tissues which do not synthesize L-type pyruvate kinase. In addition, some pyruvate kinase probes hybridized with minor RNA species of about 2000 bases in length, only observed after carbohydrate diet. For aldolase B, the recombinant plasmid DNA hybridized with a single RNA species of 1750 bases. This RNA, detected in kidney, small intestine and liver, was induced by a high carbohydrate diet and increased with liver development. The rat probe cross-hybridized with human aldolase B messenger RNA.     

Early modifications of gene expression induced in liver by azo-dye diet

The expression and regulation of the phosphoenolpyruvate carboxykinase gene were not grossly modified by feeding rats a 3'-methyl-4-(dimethylamino)azobenzene-containing diet despite maximum expression of the L-type pyruvate kinase gene being dramatically reduced as early as the 24th hour of the carcinogenic diet. Inhibition of aldolase B mRNA synthesis occurred more slowly, being maximum at the 3rd day. After stopping administration of the carcinogen, a very rapid, but transient increase of the L-type pyruvate kinase mRNA was observed at the 24th hour, whereas aldolase B mRNA increased only slowly. The amount of aldolase A mRNA fell quickly after termination of carcinogen administration, levels being normal at the 2nd-3rd day. At this time, the histological structure of the liver was indistinguishable from that of animals still receiving the azo-dye diet. It appears, therefore, that in the rat both administration and withdrawal of the azo-dye carcinogen induce rapid modifications of the expression of some genes, before any cellular modification is distinguishable.     

In vivo hormonal control of L-type pyruvate kinase gene expression. Effects of glucagon, cyclic AMP, insulin, cortisone, and thyroid hormones on the dietary induction of mRNAs in the liver

Using a cDNA probe complementary to rat L-type pyruvate kinase mRNAs, we studied the respective roles of glucocorticoids, thyroid hormones, glucagon, and insulin in the induction of specific mRNAs in the liver of animals refed either a maltose-rich or a fructose-rich diet. Neither adrenalectomized nor thyroidectomized nor diabetic animals could express L-type pyruvate kinase mRNAs in their liver when refed the carbohydrate-rich diets. When the animals were given the missing hormone, the level of hybridizable mRNAs returned to normal values but administration of the hormone alone failed to induce mRNA synthesis in fasted animals. Both glucagon and cyclic AMP abolished the induction of L-type pyruvate kinase mRNAs in refed animals. Exogenous insulin, whatever the dose, could not reverse the inhibitory action of glucagon. Insulin has usually been regarded as the main regulator of L-type pyruvate kinase gene expression. It appears now that glucagon, beside regulating the enzyme activity by phosphorylation mechanisms, may also modulate L-type pyruvate kinase synthesis at a pre-translational level. Consequently, our results show that three conditions are required for the synthesis of liver L-type pyruvate kinase mRNAs: (i) the presence of dietary carbohydrates, (ii) the cessation of glucagon release, and (iii) the presence of permissive hormones, including insulin.     

In vivo developmental modifications of the expression of genes encoding muscle-specific enzymes in rat

cDNA clones for rat muscle-type creatine kinase and glycogen phosphorylase and aldolase A were isolated from a rat muscle cDNA library. An additional clone recognizing an unidentified 2.7-kilobase pair mRNA species was also isolated. These cDNA clones were used as probes to investigate the expression of the corresponding mRNAs during muscle development. Two aldolase A mRNA species were detected, one of 1650 bases expressed in non-muscle tissues, fetal muscle, and adult slow-twitch muscle, the other of 1550 bases was highly specific of adult fast-twitch skeletal muscle differentiation. These aldolase A mRNAs were shown by primer extension to differ by their 5' ends. The accumulation of muscle-type phosphorylase and creatine kinase and muscle-specific aldolase A mRNA accumulation during muscle development seems to be a coordinate process occurring progressively from the 17th day of intrauterine life up to the 30th day after birth. In contrast, the 2.7-kilobase pair RNA species is maximally expressed at the 1st week after birth as is the neonatal form of myosin heavy chain mRNA.     

Dietary and hormonal regulation of L-type pyruvate kinase gene expression in rat small intestine

L-type pyruvate kinase is an enzyme of the glycolytic pathway whose activity and mRNA levels fluctuate in the small intestine according to dietary status. Both the enzyme activity and mRNA concentration decline during fasting and increase upon refeeding either a glucose-rich or a fructose-rich diet. Using a single-strand M 13 phage complementary to L-type pyruvate kinase mRNA as probe, we determined the level of the mRNA in the small intestine of normal, adrenalectomized, thyroidectomized, diabetic and glucagon-treated or cAMP-treated animals refed either a glucose-rich or a fructose-rich diet. The specific mRNA is present in the small intestine of normal fasted rats and increases twofold and threefold on refeeding glucose and fructose respectively. However, the hormonal control of the gene expression differs according to the dietary carbohydrate. The L-type pyruvate kinase mRNA increase, induced by glucose feeding, is hormone-dependent and requires the presence of thyroid hormones and insulin. In fructose-fed rats a certain level of mRNA increase occurs regardless of the hormonal status of the animals, but the full induction of the mRNA by fructose requires the presence of glucocorticoids, thyroid hormones and insulin. Thus, the hormonal regulation of L-type pyruvate kinase gene expression in the small intestine is largely similar to that described in normal rat liver but the basal mRNA level and the stimulation of the mRNA increase by fructose are higher in the small intestine.     

Regulation of the gene expression of glucokinase and L-type pyruvate kinase in primary cultures of rat hepatocytes by hormones and carbohydrates

The regulation of the gene expression of two important glycolytic enzymes, glucokinase and L-type pyruvate kinase, by hormones and carbohydrates was studied, in primary cultures of adult rat hepatocytes. Insulin caused time- and dose-dependent increases in the amounts of the mRNAs of the two enzymes in hepatocytes, although glucokinase responded to this hormone faster than L-type pyruvate kinase. The induction of glucokinase mRNA by insulin did not require the presence of glucose itself, but that of the L-type isozyme was dependent on the glucose concentration. For this effect, fructose and glycerol could partially substitute for glucose, but pyruvate and 2-deoxyglucose, a nonmetabolizable glucose analog, could not. The time course of insulin induction in the presence of fructose, but not of glycerol, was similar to that in the presence of glucose. In the presence of glycerol, the mRNA increased in a diphasic manner: the first increase, which probably reflected the effects of fructose and glycerol in normal liver, reached a maximum after 3 h, whereas the second increase corresponded to the increase in the presence of glucose. These results suggested that some metabolite of glucose was required for the insulin-induced increase in L-type pyruvate kinase mRNA. Cycloheximide inhibited the effects of insulin on the two mRNAs, suggesting that ongoing protein synthesis is required in both cases. The addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, also inhibited the effects of insulin. However, phorbol 12-myristate 13-acetate alone did not induce the two mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue-specific expression of rat aldolase A mRNAs. Three molecular species differing only in the 5'-terminal sequences

Three species of aldolase A mRNA (mRNAs I, II, and III) only differing in the structure of the 5'-terminal noncoding region were detected in rat tissues. The cDNA clones for mRNAs II and III were prepared from ascites hepatoma AH60C and sequenced. The mRNA II is 1393 nucleotides long excluding poly(A) tail, while the mRNA III is 1440 nucleotides long, some 50 nucleotides longer than the mRNA II. The mRNAs II and III differ in the sequence between -25 and the 5' termini from the previously reported skeletal muscle aldolase A mRNA (mRNA I, 1343 nucleotides long). By contrast, the residual 5' noncoding sequence (-24 to -1) and the coding and 3' noncoding sequences are common to all the mRNAs. By dot spot hybridization and S1 mapping the distribution of these mRNAs in the various tissues was determined. The mRNA I appears exclusively in a skeletal muscle and some in heart and hepatoma AH60C, whereas the mRNAs II and III appear more or less in all the tissues examined, implying that their appearances are under tissue-specific control. Furthermore, partial nucleotide sequence analysis of the fetal liver aldolase A mRNA supports that aldolase A mRNA that reappeared in hepatoma is really a resurgence of the gene product expressed in the fetus.     

A developmental biological study of aldolase gene expression in Xenopus laevis

We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source, aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism. We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that i