c-Myc activates multiple metabolic networks to generate substrates for cell-cycle entry.

Cell proliferation requires the coordinated activity of cytosolic and mitochondrial metabolic pathways to provide ATP and building blocks for DNA, RNA and protein synthesis. Many metabolic pathway genes are targets of the c-myc oncogene and cell-cycle regulator. However, the contribution of c-Myc to the activation of cytosolic and mitochondrial metabolic networks during cell-cycle entry is unknown. Here, we report the metabolic fates of [U-(13)C] glucose in serum-stimulated myc(-/-) and myc(+/+) fibroblasts by (13)C isotopomer NMR analysis. We demonstrate that endogenous c-myc increased (13)C labeling of ribose sugars, purines and amino acids, indicating partitioning of glucose carbons into C1/folate and pentose phosphate pathways, and increased tricarboxylic acid cycle turnover at the expense of anaplerotic flux. Myc expression also increased global O-linked N-acetylglucosamine protein modification, and inhibition of hexosamine biosynthesis selectively reduced growth of Myc-expressing cells, suggesting its importance in Myc-induced proliferation. These data reveal a central organizing function for the Myc oncogene in the metabolism of cycling cells. The pervasive deregulation of this oncogene in human cancers may be explained by its function in directing metabolic networks required for cell proliferation.

Analysis of gamma-globin expression cassettes in retrovirus vectors.

With the goal of optimizing retrovirus vectors for human gamma-globin, we studied the effect of several globin gene expression elements on vector titer, stability, and expression. We found that all combinations tested were genetically stable, but that vectors with therapeutic titers (0.5 to 2 x 10(6) colony-forming units/ml) could be achieved only by either partially or fully deleting the second intron of the Agamma-globin gene. Efficient transfer and high-level expression was achieved only when an optimized beta-globin promoter was linked to an Agamma-globin cassette containing an intact intron 1 and a 714-bp internal deletion of intron 2. When flanked by two copies of the HS-40 enhancer core from the alpha-globin locus, this cassette expressed gamma-globin mRNA at 46 +/- 19% per copy of mouse alpha-globin in the murine erythroleukemia cell line MEL585. Complete deletion of the first or second intron diminished expression to < or = 2.0%, and deletion of the HS-40 enhancer diminished expression to 7 +/- 8%. High-level, uniform expression of gamma-globin protein was confirmed in MEL585 clones (n = 12) transduced with the optimized vector. Efficient but variable expression of the optimized vector was also observed in erythroid progenitor colonies (n = 6) grown from transduced mouse bone marrow. Taken together, these studies demonstrate the role of intronic, promoter, and enhancer sequences on retrovirus vectors for human gamma-globin, and the development of an optimized vector capable of efficient expression in a murine erythroid cell line and primary cultures.

Inheritance and expression of chimeric genes in the progeny of transgenic maize plants.

We obtained transgenic maize plants by using high-velocity microprojectiles to transfer genes into embryongenic cells. Two selectable genes were used to confer resistance to either chlorsulfuron or phosphinothricin, and genes encoding either E. coli beta-glucuronidase or firefly luciferase were used as markers to provide convenient assays for transformation. When regenerated without selection, only two of the eight transformed embryogenic calli obtained produced transgenic maize plants. With selection, transgenic plants were obtained from three of the other eight calli. One of the two initial lines produced 15 fertile transgenic plants. The progeny of these plants contained and expressed the foreign genes. Luciferase expression could be visualized, in the presence of added luciferin, by overlaying leaf sections with color film.

The expression and perpetuation of inherent somatic variation in regenerants from embryogenic cultures of Pennisetum glaucum (L.) R. Br. (pearl millet).

Genetic analysis was conducted on the qualitative and quantitative traits of sexual progeny derived from embryogenic cultures of two inbred lines of Pennisetum glaucum (L.) R. Br. (pearl millet). These lines included a genetically stable inbred of Tift 23 BE and a genetic marker line, derived from Tift 23BE, which bore qualitative genetic markers for a dominant purple plant trait (P) and two recessive traits, early flowering (e1) and yellow stripe (ys). Tissue culture regenerant populations (R0) and progeny populations (R1) produced from these plants by selfing showed no qualitative genetic variation when derived from the genetically stable inbred Tift 23BE. In contrast, stably inherited qualitative variation for a number of genetic markers was observed in R0, R1, and R2 progeny of the genetic marker line. In a population of 1,911 plants regenerated over a 12-month period, 0.02% of the population lost or showed reduced expression of the purple plant trait and 92% of plants were chlorophyll deficient. Plants showing reduction or loss of anthocyanin synthesis also flowered later. None of the purple plants showed any significant variation in flowering time. The incidence of chlorophyll deficiency increased with time in culture, 51 % of the progeny regenerated after 1 month were chlorophyll deficient, while 100% of the plants regnerated after 12 months were chlorophyll deficient. Qualitative variation was also observed in control populations of the genetic marker line where 1 plant in a total of 1,010 lacked purple pigmentation and a total of 6% showed chlorophyll variation in the first generation (S0). The presence of qualitative variation in controls suggests that the inherent variation present in the original explant was expressed and perpetuated in vitro. Quantitative variation was observed for a number of traits in the first sexual cycle (R1) of the marker line but did not occur in a subsequent generation, suggesting that this variation was epigenetic.

DNA Methylation and Embryogenic Competence in Leaves and Callus of Napiergrass (Pennisetum purpureum Schum.).

Quantitative and qualitative levels of DNA methylation were evaluated in leaves and callus of Pennisetum purpureum Schum. The level of methylation did not change during leaf differentiation or aging and similar levels of methylation were found in embryogenic and nonembryogenic callus.

Comparative analysis of free DNA delivery and expression into protoplasts of Panicum maximum Jacq. (Guinea grass) by electroporation and polyethylene glycol.

Relative levels of gene expression were studied in protoplasts isolated from two cell lines of Panicum maximum following DNA delivery by electroporation and polyethylene glycol (PEG). Gene expression was evaluated by assaying for chloramphenicol acetyltransferase (CAT) activity expressed by the CaMV 35S promoter with a nopaline synthase 3' polyadenylation signal, approximately 48 hours after DNA delivery. The expression of the CAT gene was slightly higher in electroporated protoplasts in comparison to PEG mediated delivery. However, PEG treated protoplasts showed higher plating efficiency. The effect of different salts and the molecular weight of PEG used on gene expression was also studied.