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R W Hanson

Publications and source records attributed to R W Hanson.

At least 91 records · Page 5Linked to original sources
PubMed1986

Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter-regulatory region. I. Multiple hormone regulatory elements and the effects of enhancers.

Transcription of the gene for cytosolic Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) from rat liver is increased by cAMP and glucocorticoids and decreased by insulin. A PEPCK-thymidine kinase (TK) chimeric gene was transfected into FTO-2B rat hepatoma cells, which were TK-deficient. Previous studies showed that a cAMP regulatory element is located at the 5' end of the PEPCK gene. In this report, we demonstrate that the 5' end of the gene also contains a glucocorticoid regulatory element, but not one for insulin. Regions of the PEPCK gene that contain these regulatory elements were attached to the Herpes simplex virus TK structural gene containing its own promoter. The hormone regulatory elements within the 5' flanking region of the PEPCK gene conferred cAMP and glucocorticoid responsiveness on the TK gene after transfection into FTO-2B cells. Like viral enhancer elements, these regulatory elements functioned properly when placed in either orientation at various positions 5' or 3' to TK. The presence of the SV40 enhancer element upstream from the PEPCK-TK gene had little effect on the basal level of expression or hormonal regulation of the chimeric gene.

Animals↗
PubMed1986

Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter-regulatory region. II. Identification of cAMP and glucocorticoid regulatory domains.

Hormonal regulatory elements within the phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) promoter region were mapped using a series of 5' deletions linked to the amino-3'-glycosyl phosphotransferase structural gene. These deletion mutants were stably transfected into the genome of FTO-2B hepatoma cells. A 47-base pair region of the PEPCK promoter was identified which was essential for stimulation by dibutyryl cAMP. A 12-base pair core sequence (CTTACGTCAGAG) within this region shows significant homology with sequences in four other cAMP-regulated genes. There are two glucocorticoid regulatory elements within the promoter, as well as an inhibitory element which depresses the level of basal gene transcription. The deletion of this inhibitory sequence prevents the induction of the chimeric gene by dexamethasone. The existence of the hormone regulatory domains within the PEPCK promoter was confirmed by attaching these elements upstream of the heterologous Herpes simplex virus thymidine kinase structural gene, containing its own promoter.

Animals↗
PubMed1986

Trans regulation of the phosphoenolpyruvate carboxykinase (GTP) gene, identified by deletions in chromosome 7 of the mouse.

Livers from newborn mice homozygous for either one of the lethal deletions c14CoS or c3H in chromosome 7 have drastically reduced levels of cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] activity when compared with normal littermates. The structural gene for the enzyme maps on chromosome 2 and appears intact and not grossly rearranged in deletion homozygotes. These mice also have negligible levels of hepatic mRNA encoding this enzyme. Studies of the transcription rate of the gene showed that it was reduced to 25-50% of normal in hepatic nuclei obtained from mice homozygous for either deletion. We suggest that, in addition to the reduction in the level of transcription, the deletions in chromosome 7 may also cause alterations in messenger stability, processing, or transport from the nucleus.

Animals↗
PubMed1986

Nucleotide sequence of the mRNA encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken.

We have determined the sequence of the mRNA encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from the chicken and have deduced the primary structure of the protein. The message for the enzyme is 2762 bases long and encodes a protein of 622 amino acids with a molecular mass of 69,522 daltons. The 5' untranslated region is 246 nucleotides long and contains two nonfunctional AUG initiator codons. The 3' untranslated sequence is 649 bases long and contains multiple polyadenylylation signals. There are regions of dyad symmetry and an A + U-rich region within the 3' translated and untranslated sequences of the message. Such regions are also present in the mRNA for the enzyme from the rat and may be of functional significance. Conserved regions of the enzyme, that may interact with substrates, were identified by comparing the amino acid sequence of phosphoenolpyruvate carboxykinase with that of other proteins that use guanine nucleotides and phosphoenolpyruvate as substrates.

Amino Acid Sequence↗
PubMed1986

Neurologic outcome and blood glucose levels during out-of-hospital cardiopulmonary resuscitation.

We examined the interrelations of outcome, time elapsed during cardiopulmonary resuscitation (CPR), and blood glucose levels drawn from 83 patients with out-of-hospital cardiac arrest. Levels rose significantly during CPR. Although slope and intercept of regression lines differed for those dying in the field and those admitted, regression lines were similar for those who awoke and never awoke after admission. These results suggest that the previously reported association between poor neurologic recovery and high blood glucose level on admission after cardiac arrest is best explained by prolonged CPR, leading to both higher rise of blood glucose and worse neurologic outcome.

Aged↗
PubMed1985

Thyroid hormone regulates transcription of the gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) in rat liver.

Using an in vitro assay with isolated rat nuclei, we have determined that thyroid hormone causes a 4-6-fold increase in the synthesis of mRNA coding for phosphoenolpyruvate carboxykinase. Proportional changes were seen in the steady-state cytosolic mRNA levels for phosphoenolpyruvate carboxykinase. Dibutyryladenosine cyclic 3',5'-monophosphate, which stimulates transcription of the phosphoenolpyruvate carboxykinase gene in normal rats, remained effective in hypo- or hyperthyroid animals. The effect of epinephrine on transcription of the gene for phosphoenolpyruvate carboxykinase appears to be modulated by thyroid hormone.

Animals↗
PubMed1985

Effect of hormones on transcription of the gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) in rat kidney.

The effect of hormones on the transcription rate of cytosolic phosphoenolpyruvate carboxykinase and level of mRNA for this enzyme in the rat kidney has been investigated. In renal nuclei isolated from rats given dibutyryladenosine cyclic 3',5'-phosphate (Bt2cAMP) or 8-bromoadenosine cyclic 3',5'-phosphate (8-Br-cAMP), [32P]UMP incorporation into hybridizable phosphoenolpyruvate carboxykinase mRNA increased severalfold within 1 h. Changes in the concentration of cytosolic phosphoenolpyruvate carboxykinase mRNA, measured by hybridization of [32P]cDNA to poly(A)+ mRNA, paralleled alterations in the transcription rate. Dexamethasone treatment of adrenalectomized rats increased the transcription rate and the level of phosphoenolpyruvate carboxykinase mRNA 3-4-fold after 4 h. Both parameters then declined to control values by 8 h. When dexamethasone (5 mg/kg) and Bt2cAMP (25 mg/kg) were given together, the rate of phosphoenolpyruvate carboxykinase RNA synthesis and the level of cytosolic mRNA were not increased more than those with either drug alone. Transcription of the gene for renal phosphoenolpyruvate carboxykinase was not affected by diabetes or glucose refeeding but was increased 2-fold after 24 h of starvation and reduced by bicarbonate feeding after 2 h. We conclude that glucocorticoids and cAMP change the rate of transcription of the phosphoenolpyruvate carboxykinase gene in rat kidney, leading to changes of similar magnitude in mRNA level and, hence, enzyme activity. The results presented here and in previous work [Lamers, W., Hanson, R. W., & Meisner, H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5137] indicate that the transcription rate of the gene for phosphoenolpyruvate carboxykinase in liver and kidney responds to hormones in a tissue-specific manner.

Animals↗
PubMed1985

Processing of phosphoenolpyruvate carboxykinase (GTP) RNA in vivo.

Nuclear RNA precursors to the mRNA for rat cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and nuclear RNA species smaller in size than the mature enzyme mRNA have been identified by using hybridization probes specific for introns and exons from the gene. A number of processing intermediates, including an intact intron, were noted. Based on the size and distribution of the precursors observed, we propose a pathway for phosphoenolpyruvate carboxykinase RNA processing that involves splicing of introns starting at the 5' end of the RNA transcript and proceeding toward the 3' end.

Animals↗
PubMed1984

Induction by cAMP of the mRNA encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken. Identification and characterization of a cDNA clone for the enzyme.

Previous work from our laboratory (Hod, Y., Utter, M. F., and Hanson, R. W. (1982) J. Biol. Chem. 257, 13787-13794) has demonstrated that chicken kidney contains both mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and that the two forms are distinct proteins. Using poly(A+) RNA from chicken kidney, a double-stranded cDNA library was constructed. DNA clones containing sequences complementary to the mRNA for the cytosolic form of phosphoenolpyruvate carboxykinase were initially identified by colony hybridization with 32P-labeled cDNA transcribed from an RNA fraction enriched for the enzyme mRNA. The identity of plasmids containing phosphoenolpyruvate carboxykinase cDNA was confirmed by hybrid-selected translation. Mature mRNA for cytosolic phosphoenolpyruvate carboxykinase of the chicken is 2.8 kilobases in length, similar to that previously noted for mRNA coding for the same enzyme in the rat. The cDNA for the chicken enzyme hybridizes with several restriction fragments of the corresponding cDNA for the rat cytosolic phosphoenolpyruvate carboxykinase, indicating conservation of nucleotide sequences during evolution. Wide spread conservation of sequence homology is also demonstrated by the hybridization of the cDNA for the rat phosphoenolpyruvate carboxykinase with a 2.8-kilobase RNA from the livers of a variety of vertebrates including amphibian, avian, and primate species. Specific mRNA coding for the cytosolic form of phosphoenolpyruvate carboxykinase was present in chicken kidney but absent from the liver, even in animals starved for 48 h. However, the administration of cAMP to normal fed chickens caused a rapid induction of phosphoenolpyruvate carboxykinase mRNA. These findings suggest that the gene for the cytosolic enzyme in chicken liver can be expressed if the proper hormonal stimuli are present.

Animals↗
PubMed1984

The gene encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken.

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) from the chicken was isolated from a recombinant library containing the chicken genome in phage lambda Charon 4A. The isolated clone, lambda PCK1cc, contains the complete gene for the enzyme as well as both 5' and 3' flanking sequences. The gene is approximately 8 kilobases in length divided into 8 exons, as demonstrated by restriction endonuclease mapping and DNA-RNA heteroduplex analysis. Southern blotting of chicken chromosomal DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of lambda PCK1cc. The phosphoenolpyruvate carboxykinase gene is present as a single copy in the haploid chicken genome. The 5' region of the gene was defined by S1 nuclease mapping and by sequencing. Two mRNA species with discrete 5' ends were observed using S1 nuclease mapping. The ratio between the amounts of these multiple forms of mRNA is the same in chicken kidney and liver and is not affected by induction of the enzyme mRNA by cAMP. Examination of sequence homologies with the gene for rat cytosolic phosphoenolpyruvate carboxykinase indicates a putative control region contained in flanking sequences at the 5' end of the gene.

Animals↗
PubMed1984

Identification of a cAMP regulatory region in the gene for rat cytosolic phosphoenolpyruvate carboxykinase (GTP). Use of chimeric genes transfected into hepatoma cells.

cAMP stimulates the transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) in rat liver. We have investigated the nucleotide sequences required for regulation of PEPCK gene expression by cAMP. A chimeric gene was constructed in which a 620-base pair fragment of the 5'-end of the PEPCK gene (including 547 base pairs of 5'-flanking sequence) was ligated to the herpes simplex virus thymidine kinase (TK) structural gene. The PEPCK promoter fragment was introduced either in the proper orientation for transcription of the TK gene or in the opposite orientation. These fusion genes and the parent vector, pOPF, which contains the intact TK gene, were transfected individually into TK-deficient FTO-2B rat hepatoma cells. FTO-2B cells contain an active endogenous PEPCK gene which is stimulated by cAMP. Cells were selected in HAT medium and grown either as mass cell cultures or as individual clones. Dibutyryl cyclic AMP (Bt2cAMP) plus theophylline (16 h) stimulated TK activity 1.6-6.1-fold in cell lines transfected with the PEPCK-TK fusion gene containing the PEPCK promoter fragment in the correct orientation. However, the intact TK gene was not induced by Bt2cAMP after transfection, nor was there any expression of the PEPCK-TK fusion gene in cells which contained the PEPCK promoter fragment in the wrong transcriptional orientation. Bt2cAMP also increased the levels of TK mRNA in cells transfected with the PEPCK-TK fusion gene, but not in cells transfected with the intact TK gene. The chimeric PEPCK-TK mRNA initiated at the PEPCK start site, as determined by S1 nuclease mapping. There was no relationship between the number of copies of the PEPCK-TK gene integrated in the various cell lines and either the basal level of TK activity or its inducibility of Bt2cAMP.

Animals↗
PubMed1983

Decrease of renal phosphoenolpyruvate carboxykinase RNA and poly(A)+ RNA level by ochratoxin A.

Ochratoxin A, a nephrotoxin produced by Aspergillus ochraceus, decreases the activity of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) in the cytosol of rat kidneys, as well as inhibits renal gluconeogenesis (Meisner, H., and Meisner, P. (1981) Arch. Biochem. Biophys. 208, 146-153). Ochratoxin A greatly reduces the level of translatable mRNA for PEPCK in kidneys of rats fed the toxin for 2 days, while the efficiency of translation of poly(A)+ RNA is not affected. A species of poly(A)+ RNA coding for a 72,000 Mr protein is increased in relative amount. Although similar in molecular weight to PEPCK, this protein is not precipitated by an antibody to PEPCK, and has a different peptide map. The sequence abundance of PEPCK mRNA, as measured by either Northern blotting or the dot blot technique, is reduced, while the hepatic level of PEPCK mRNA is not changed. The total poly(A)+ RNA level is reduced 50% in kidneys, but not livers, of rats fed a standard dose of ochratoxin A for 3-5 days. In nuclei isolated from toxin-fed rats, the rate of transcription of total RNA or PEPCK mRNA, as measured by incorporation of [32P]UTP, is not reduced by the toxin. Ochratoxin A therefore lowers total renal mRNA concentration, and certain species, notably PEPCK, are reduced to a greater extent than the bulk of the RNA pool.

Cell Nucleus↗
PubMed1983

Isolation and characterization of the gene coding for cytosolic phosphoenolpyruvate carboxykinase (GTP) from the rat.

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from the rat was isolated from a recombinant library containing the rat genome in phage lambda Charon 4A. The isolated clone, lambda PCK1, contains the complete gene for phosphoenolpyruvate carboxykinase and approximately equal to 7 kilobases (kb) of flanking sequence at the 5' end and 1 kb at the 3' terminus. Restriction endonuclease mapping, R-loop mapping, and partial DNA sequence assay indicate that the gene is approximately equal to 6.0 kb in length (coding for a mRNA of 2.8 kb) and contains eight introns. Southern blotting of rat DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of lambda PCK1. A control region at the 5' end of the gene contained in a 1.2-kb restriction fragment was isolated and subcloned into pBR322. This segment of the gene contains the usual transcription start sequences and a 24-base sequence virtually identical to the sequence found in the 5'-flanking region of the human proopiomelonocortin gene, which is known to be regulated by glucocorticoids. The 1.2-kb fragment of the phosphoenolpyruvate carboxykinase gene can be transcribed into a unique RNA fragment of predicted size by an in vitro transcription assay.

Animals↗
PubMed1982

The mitochondrial and cytosolic forms of avian phosphoenolpyruvate carboxykinase (GTP) are encoded by different messenger RNAs.

Previous work from our laboratory (Watford, M., Hod, Y., Chiao, Y. B., Utter M. F., and Hanson R. W. (1981) J. Biol. Chem. 256, 10023-10027) indicated that in the chicken, hepatic phosphoenolpyruvate carboxykinase is in the mitochondria, whereas kidney contains both a mitochondrial and cytosolic form of the enzyme. In the present study the two forms of phosphoenolpyruvate carboxykinase were purified and shown to be distinct proteins which differ in size, charge, and immunochemical properties. Using a cell-free protein synthesis system, we demonstrate that the cytosolic isozyme is encoded only by kidney mRNA and that its translation form has similar properties to that of the mature protein. On the other hand, the mitochondrial enzyme is encoded by liver and kidney mRNA and synthesized as a protein about 2000 daltons larger than the mature form. The putative precursor of the mitochondrial phosphoenolpyruvate carboxykinase is processed to a mature size by isolated, respiring mitochondria. We further show that the mRNA species for the cytosolic and mitochondrial forms are separable and are about 3 and 4 kilobases, respectively. It is concluded that the two forms of phosphoenolpyruvate carboxykinase of the chicken are encoded by distinct mRNA species.

Animals↗