Biomedical subjects
Publications and source records attributed to A Wynshaw-Boris.
The important advances that have occurred in molecular and cell biology have great potential for metabolic and nutritional research. This review will focus on the application of genetic manipulation to metabolism and nutrition, with special emphasis on the tissue-specific expression and regulation of the newly introduced genes.
Hepatoma cells were infected with replication-incompetent murine retroviruses containing the selectable gene for amino-3'-glycosyl phosphotransferase (neo) and/or the nonselectable gene for bovine growth hormone (bGH). Expression of these genes was controlled by the promoter regulatory region of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) from the rat, which contains hormone and tissue-specific regulatory elements. Expression of the transduced PEPCK-neo gene was stimulated by Bt2cAMP and glucocorticoids and inhibited by insulin. The amount of RNA which initiated within the retroviral 5' long terminal repeat (5' LTR) was inhibited when internal promoters were present in the retroviral vector. When no internal promoter was present, expression from the 5' LTR was higher and stimulated by glucocorticoids, due to the presence of a glucocorticoid regulatory element in the 5' LTR. Infection of cells with retroviruses altered the basal expression and hormonal regulation of the endogenous PEPCK gene, but had no effect on the expression of the tyrosine aminotransferase gene, which is regulated in a similar manner by cAMP and glucocorticoids. A segment of the PEPCK promoter acted as a hormonally regulated enhancer, bringing the SV40 early promoter under the control of Bt2cAMP. A second, nonselectable gene (PEPCK-bGH), contained in the retroviral vector together with PEPCK-neo, was expressed and regulated appropriately when introduced into hepatoma cells. The proviruses were initially integrated randomly into the host cell genome, but after prolonged selection for expression of the transduced PEPCK-neo gene, cells were selected which contain a predominant site(s) of integration. Among populations of cells, however, the predominant site(s) of proviral integration was different. The selection of cells with a specific site of integration from a population was accelerated by the presence of PEPCK promoter sequences in the provirus. Despite the need to better characterize their effects on the host cell, retroviruses appear to be versatile tools for the specific introduction of regulated genes into cells.
A series of transgenic mice was produced by microinjection of a segment of DNA, containing 460 base pairs of the phosphoenolpyruvate (P-enolpyruvate) carboxykinase promoter-regulatory region ligated to the bovine growth hormone structural gene, into the male pronucleus of fertilized mouse eggs. Founder animals which contained the gene were selected for further analysis and for breeding. The concentration of bovine growth hormone in the serum of animals which were shown to contain the gene ranged from a low of 5 ng/ml serum to approximately 2300 ng/ml serum. Mice with high levels of bovine growth hormone had growth rates double that of their litter mates which did not contain the transgene. The transgene was expressed only in the liver and kidney of the animals studied, and the level of specific mRNA for bovine growth hormone in these tissues could be regulated by diet in a manner similar to the endogenous P-enolpyruvate carboxykinase gene. Feeding the animals a diet high in carbohydrate for 1 week caused a 90% decrease in the concentration of bovine growth hormone in the blood, suggesting that the expression of the chimeric P-enolpyruvate carboxykinase/bovine growth hormone gene is sensitive to insulin. When the same animals were then refed a diet high in protein, but devoid of carbohydrate, the concentration of bovine growth hormone in their blood was induced 30-fold within a week. The administration of dibutyryl cyclic AMP to the transgenic mice caused a 2-fold induction in the level of bovine growth hormone in the serum within 90 min. Thus the region between -460/+73 in the P-enolpyruvate carboxykinase promoter-regulatory region contains sequences which can direct the tissue-specific expression, as well as hormonal and dietary responsiveness, of a linked structural gene.
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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.
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.
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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.
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.
Heterozygous mutation or deletion of Pafab1b1 (LIS1) in humans is associated with syndromes with type 1 lissencephaly, a severe brain developmental disorder resulting from abnormal neuronal migration. We have created Lis1 heterozygous mutant mice by gene targeting. Heterozygous mutant mice are viable and fertile, but display global organizational brain defects as a result of impaired neuronal migration. To assess the functional impact of the mutation, Lis1 heterozygous mice and their wild-type littermates were evaluated on a wide variety of behavioral tests. Lis1 mutant mice displayed abnormal hindpaw clutching responses and were impaired on a rotarod test. Lis1 heterozygous mice were also impaired in the spatial learning version of the Morris water task. Impaired motor behavior and spatial learning and memory in Lis1 mutant mice indicates that impaired neuronal migration can have functional effects on complex behavioral responses. The behavioral findings also support the use of the Lis1 mutant mice as a model from human type 1 lissencephaly.