Abstract. The activity of 10 enzymes separated by acrylamide disc gel electrophoresis of leaf and stem extracts from Dianthus grown under summer and winter conditions was studied.While banding was constant and highly reproducible under each environment, differences between the 3 cultivars and between the tissues were evident. No significant differences in the isozyme patterns of glutamate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and catalase were observed between the 2 environments. Loss

A cell-free extract from cultured rat hepatoma cells is described that incorporates amino acids into chains of tyrosine aminotransferase that are identical, by several criteria, with tyrosine aminotransferase from rat liver. Extracts from steroid-induced cells are 6-to 10-times more active for tyrosine aminotransferase synthesis than extracts from uninduced cells, although the total incorporation rates are identical.Glucogenic adrenal steroids induce a 5-to 15-fold increase in the rate of synthesis of tyrosine aminotransferase (TAT) in liver (1) and in cultured rat hepatoma (HTC) cells (2). To clarify the mechanism of the induction, it would obviously be desirable to be able to observe all the steps of the process in a cell-free extract. The initial steps in the cell-steroid interaction, in which inducing hormones bind to a specific cytoplasmic receptor molecule, followed by association of the steroid-receptor complex with DNA-containing nuclear sites, have already been reproduced in cell-free extracts (3, 4). In the present communication, we describe a cell-free extract prepared from HTC cells in which polypeptide chainsidentified as TAT by their chromatographic, electrophoretic, and immunological properties-are labeled by added radioactive amino acids and released from polyribosomes. Furthermore, extracts prepared from induced and uninduced cells differ in their ability to label TAT in proportion to the difference in the rates of TAT synthesis measured in the intact cells from which the extracts are derived. These preparations should, therefore, be useful in determining which specific reactions in TAT synthesis are accelerated upon induction.Numerous reports have appeared describing the cell-free synthesis of mammalian proteins in homologous and heterologous systems. However, the relatively large amount of information available about the biology of TAT induction (5), and the report that TAT-containing polyribosomes can be concentrated (6), should make the system described here particularly valuable for studying the molecular basis of hormonal induction of a specific protein. MATERIALS AND METHODS6 Liters of a suspension of HTC cells were grown (7) in modified Swim's S-77 medium to a density of 4 X 105 cells per ml. 15 hr before collection of cells, the culture was divided; dexamethasone was added to 3 liters, to a final concentration of 1 gM.The remaining 3-liter culture was left as a control. Both batches of cells were collected by centrifugation at 800 X g for 10 min, the pellets were washed twice in 200 ml of isotonic saline and finally in 50 ml of solution A [2.5 mM Tris HCl (pH 7.2)-25 mM KCI-5 mM MgC12-0.25 M sucrose]. A 3-liter culture yields a pellet of about 6 ml of washed cells.The wash and subsequent operations were performed at 2-4'.A volume of solution A equal to the packed cell volume was added to the cell pellet, and the suspension was homogenized with 30 strokes in a Potter-Elvehjem Homogenizer (size C, Teflon pestle, clearance 0.15 mm). The homogenate was centrifuged for 10 min at 800 ...

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The effect of 14 combinations of photoperiod, soil and air temperature, and growth substance applications on the cold hardiness of Chrysanthemum morifolium 'Astrid' rhizomes was evaluated. Both triphenyl tetrazolium chloride and regrowth tests were used to determine the viability of the cold-stressed rhizome tissues. The rhizomes exhibited different degrees of cold hardiness under these environmental conditions. A combination of short photoperiod and low air and soil temperatures induced maximum cold hardiness. Low soil temperature accompanied by long photoperiods and warm aerial temperatures did not induce rhizome hardening, while some hardening in cool soils was evident under either short photoperiods or low aerial temperatures. Warm soils reduced rhizome hardening under the normally inductive short photoperiod-cool aerial conditions. Since the induction of rhizome hardening was dependent on the induction of the aerial organs, the involvement of translocatable hardiness promoters is indicated. Foliar applications of low levels of gibberellic acid (GAj) or abscisic acid only slightly influenced rhizome hardiness.

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