The molecular basis for the pituitary‐specific expression of the human growth hormone (hGH) gene was investigated, by gene transfer and protein footprinting experiments. Plasmid constructs in which CAT or Neo transcription units are fused to a 0.5 kb fragment of the hGH 5′ sequences were efficiently expressed in GC and GH3 cells, derived from a pituitary tumor, but not in cell lines of other origins, indicating the presence of a tissue‐specific promoter. DNaseI footprinting experiments have identified at least three factors that specifically bind to the hGH 5′ region. While two of these factors were also detected in extracts of non‐expressing cells, the third factor, GHF‐1, was detected only in extracts of GH expressing pituitary tumor cells. Mutagenesis experiments suggest that binding of GHF‐1 and some of the other more ubiquitous factors is required for optimal hGH promoter activity in vivo. Tissue specificity of the hGH promoter therefore seems to be determined by the binding of at least one tissue‐specific trans‐acting factor, acting in concert with several other more ubiquitous, yet specific, DNA binding proteins.

Insulin-induced gene 1 (INSIG-1) is a key regulator in the processing of the sterol regulatory element-binding proteins (SREBPs). We demonstrated that

We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. AU showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four-to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA.In animal cells ornithine decarboxylase (ODC) is an essential enzyme, the activity of which is regulated by a wide variety of hormonal, developmental, and cell growth-related stimuli (35). It is the initial and rate-limiting enzyme in the synthesis of polyamines; its product, putrescine, is the precursor of the polyamines spermidine and spermine. In general, cells that are proliferating exhibit more activity than their nonproliferating counterparts. There is evidence for multiple forms of regulation of ODC activity, including mRNA steady-state level, polypeptide synthesis, and polypeptide turnover.In S49 mouse T-lymphoma cells cyclic AMP modulates cell growth and ODC activity (7,10,22). ODC activity is also changed by treatments that modulate cellular polyamine levels, such as exposure to putrescine, the direct product of ODC. We generated ODC-overproducing variants of S49 cells by selecting for cells resistant to the ODC inhibitor difluoromethylornithine (DFMO) (19,20). These variants allowed us to identify the ODC polypeptide and to clone the ODC cDNA (21) DFMO. Clone Z.12 was selected from an unmutagenized wild-type (clone 24.3.2) S49 cell population by serially increasing the concentration of DFMO as described previously (20). The D2 clones were selected from a mutagenized population in a single step as follows. Wild-type cells were mutagenized with 2.5 ,g of N-methyl-N'-nitro-N-nitrosoguanidine per ml for 4.5 h. This treatment resulted in 30% cell survival and induction of resistance to 6-thio...

(a) skeletal muscle PPARgamma1 expression does not differ between normal and diabetic subjects, and is not induced by short-term hyperinsulinemia; (b) skeletal muscle PPARgamma1 expression was higher in subjects whose percent body fat exceeded 25%, and this may be a compensatory phenomenon in an attempt to maintain normal insulin sensitivity.

We have transferred a mutant hamster gene coding for an altered dihydrofolate reductase to wild-type cultured mouse cells by using total genomic DNA from methotrexate-resistant Chinese hamster ovary A29 cells as donor. By demonstrating the presence of hamster gene sequences in transformants we have provided direct evidence for gene transfer. Transformants selected for increased resistance to methotrexate contain increased amounts of the newly transferred gene. We have used this mutant dhfr gene to introduce the Escherichia coli antibiotic resistance plasmid pBR322 into animal cells. Amplification of the dhfr sequences results in amplification of the pBR322 sequences as well. The use of this gene may allow the introduction and amplification of virtually any genetic element in various new cellular environments. The ability to transfer purified genes into cultured cells provides a unique opportunity to study the function and physical state of exogenous genes in new cellular environments. The development of systems for DNA transfer in animal cells originated with the lytic transfection of cells by using purified viral DNA (1, 2) and progressed to the stable transfer of viral transforming functions to appropriate recipient cells (3). Subsequently, viral genes from the herpesviruses coding for the biochemically selectable marker thymidine kinase (TK) (4-6) were transferred to enzyme-deficient mutant cells. Restriction fragments of herpes simplex virus type 1 encoding TK were isolated (6) and subsequently cloned into bacterial plasmids (7). Through the use of this selectable marker, virtually any gene can now be introduced into recipient cells (8, 9); however, these cells must be tk-mutants. Other potential selection systems are available, and several laboratories have recently demonstrated the DNA-mediated transfer of cellular genes coding for selectable markers such as TK (10), adenine phosphoribosyltransferase (11) and hypoxanthine phosphoribosyltransferase (12,13 Transformation and Selection. Ltk-aprF cells and NIH 3T3 cells were transformed with genomic DNA by the calcium phosphate coprecipitation method (2) as described (11). All DNAs were sterilized by ethanol precipitation and resuspended in 1 mM Tris-HCl/1 mM EDTA, pH 7.9. For tk+ transformation, cells were exposed to hypoxanthine/aminopterin/thymidine selective medium as described (10). Transformants resistant to Mtx were select~d in growth medium containing either 0.1 or 0.2 jqg of Mtx per ml with the same feeding schedule as for tk selection. Afte 2-3 weeks, colonies were isolated from individual dishes with cloning cylinders to ensure that each transformant arose from an independent event. In transformation with ligated DNAs, no more than 1 ,ug of pBR322 DNA was added to 106 cells per dish because higherThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.3567Abbreviations: DHFR, dihydrofolat...

No abstract

A novel nonthiazolidinedione dual peroxisome proliferator-activated receptor (PPAR)-␣/␥ agonist, LY465608, was designed to address the major metabolic disturbances of type 2 diabetes. LY465608 altered PPAR-responsive genes in liver and fat of db/db mice and dose-dependently lowered plasma glucose in hyperglycemic male Zucker diabetic fatty (ZDF) rats, with an ED 50 for glucose normalization of 3.8 mg ⅐ kg -1 ⅐ day -1 . Metabolic improvements were associated with enhanced insulin sensitivity, as demonstrated in female obese Zucker (fa/fa) rats using both oral glucose tolerance tests and hyperinsulinemic-euglycemic clamps. Further characterization of LY465608 revealed metabolic changes distinct from a selective PPAR-␥ agonist, which were presumably due to the concomitant PPAR-␣ agonism, lower respiratory quotient, and less fat accumulation, despite a similar impact on glycemia in male ZDF rats. In addition to these alterations in diabetic and insulin-resistant animals, LY465608 dose-dependently elevated HDL cholesterol and lowered plasma triglycerides in human apolipoprotein A-I transgenic mice, demonstrating that this compound significantly improves primary cardiovascular risk factors. Overall, these studies demonstrate that LY465608 beneficially impacts multiple facets of type 2 diabetes and associated cardiovacular risk, including those facets involved in the development of micro-and macrovascular complications, which are the major sources for morbidity and mortality in these patients.

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