Polyamines in Paul's Scarlet rose suspension cultures

Polyamine concentrations have been determined at intervals in suspension cultures of Paul's Scarlet rose cells during a culture period of 2 weeks. The mean concentrations of the putrescine, spermidine and spermine in the cells of the inocula were respectively 73, 70 and 13 nmol/g fresh weight. Putrescine at fitst increased with a peak (160 nmol/g) after 6 h, declined to a minimum (14 nmol/g) after 2–3 days, increased to a second peak (180 nmol/g) after 5–6 days, and then declined slowly to the concentration of the inoculum (taken on day 14). Spermidine rose slowly (×2.6) to a broad peak over 3–6 days (180 nmol/g), then declined slowly to the concentration in the inoculum. Spermine showed a rapid increase to a peak (130 nmol/g) after 2–3 days, and then declined rapidly, reaching the inoculum concentration by day 6. In one experiment the three amines showed a minor peak at day 11. Changes in spermine and RNA contents appeared to be correlated. DNA content reached a peak after that of the RNA (day 3) and did not appear to be correlated with the content of putrescine or the polyamines.     

Methylation of ribosomal RNA in growing and resting cultures of Paul's Scarlet Rose

The role of methylation of rRNA on the differential rate of ribosome accumulation was studied in exponentially growing and resting cultures of rose cells. The cells were labelled with [³H] uridine and pre-rRNA and rRNA were prepared from the nuclei and ribosomes. The results demonstrate that in resting cultures, in which the ribosome accumultaion was reduced, the pre-rRNA and rRNA were 50% and 75% less methylated respectively, than those of the growing cells.     

Ammonium influence on nitrogen assimilating enzymes and protein accumulation in suspension cultures of Paul's Scarlet rose

The influence of NH₄⁺ on protein accumulation was examined by growing suspension cultures of Rosa cv. Paul's Scarlet on two defined media. Both contained 1920 μmol of NO₃^? but only one contained 72.8 μmol of NH₄⁺. At the conclusion of a 14-day growth period, cultures grown with NH₄⁺ possessed twice as much protein as cultures grown without NH₄⁺. The influence of NH₄⁺ did not appear to be a substrate effect, since the amount of NH₄⁺ provided accounted for only 10% of the nitrogen recovered in protein. The provision of NH₄⁺ in the starting medium increased the activity (μmol substrate. h^?1· g^?1 fr wt) of glutamate dehydrogenase and glutamate synthase, and reduced the activity of glutamine synthetase. A comparison of the total activity per culture for each of these enzymes with the rate of nitrogen incorporation into protein showed that the enzymatic potential of glutamine synthetase and glutamate dehydrogenase greatly exceeded the actual in vivo rate of nitrogen assimilation through the respective pathways. Thus it was concluded that the availability of either of these enzymes does not limit nitrogen assimilation in rose cells and the fluctuations in their level brought about by NH₄⁺ was of no physiological importance. The activity of glutamate synthase per culture approximated the rate of nitrogen incorporation into protein during early stages of growth, and for that reason may have limited nitrogen assimilation or caused a diversion of nitrogen through the alternative pathway to glutamate catalyzed by glutamate dehydrogenase.     

Nitrate reductase activity and growth in Paul's Scarlet rose suspension cultures in relation to nitrogen source and molybdenum

Growth and nitrate reductase activity were measured in Paul's Scarlet rose cell suspensions, cultured in media purified from molybdenum and containing nitrate or urea as sole nitrogen source with or without added Mo. Urea could replace nitrate to yield 80% of the fresh weight in nitrate medium. Nitrate reductase activities were compared by in vivo and in vitro assays. The latter varied due to inactivation during extraction. Compared with activities in cells in complete NO₃ ^- medium, activity in NO₃ ^--Mo cells was reduced to 30% and, in urea-grown cells, to trace amounts. Increases in nitrate reductase activity were found when NO₃ ^- alone was added to NO₃ ^- or urea+Mo cultures. In NO₃ ^--Mo cultures, Mo alone or with NO₃ ^- caused a similar increase in activity, whereas urea-Mo cultures required both NO₃ ^- and Mo for enzyme induction.Abbreviations FAD flavin adenine dinucleotide - Mo molybdenum - NADH reduced nicotinamide adenine dinucleotide - NO₃ ^-+Mo standard MX₁ culture medium - NO₃ ^--Mo MX₁ medium purified of Mo and used for continuous subculture with nitrate - NR nitrate reductase - PSR Paul's Scarlet rose - PVP polyvinylpyrrolidone - U urea - U+Mo MX₁ medium containing urea instead of nitrate - U-Mo MX₁ medium containing urea instead of nitrate and also purified of Mo     

Changes in protein and protease activity during the growth and senescence of Paul's Scarlet rose

A study was performed to determine the relationship between the protein content and protease activity in suspension cultures of rose (Rosa cv Paul's Scarlet) grown over a 30 day period. Protein levels and protease activity were calculated on a per culture and per cell basis. Older nongrowing 14 day-old cultures possessed the largest total protease activity, but the highest concentration of protease activity per cell was in young 4 day-old rapidly dividing cells.     

Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state

Thymidylate synthase (TS) activity is very low in resting mouse 3T6 fibroblasts but increases sharply in growth-stimulated cells at about the same time the cells enter S phase. To study the mechanism responsible for the increase in TS level, we isolated a 5-fluorodeoxyuridine (5-FdUrd)-resistant cell line (LU3-7) that overproduces TS and its mRNA about 50-100-fold. In this paper we show that the LU3-7 cells were able to rest in the G0 state of the cell cycle when maintained in medium containing 0.5% serum. When the serum concentration was increased to 10%, the resting cells reentered the cell cycle and began DNA replication about 12 hr later. TS activity remained at the resting level until DNA replication began, then increased at later times. The increase was not affected when the cells were stimulated in the presence of DNA synthesis inhibitors. The rate of synthesis of TS (as determined in a pulse-labeling experiment) remained at the resting level for the first 10 hr following stimulation, then increased 8-9-fold by 25 hr following serum stimulation. The half-life of TS in growing LU3-7 cells was measured in a pulse-chase experiment and found to be greater than 24 hr. Therefore the increase in TS activity was primarily due to an increase in the rate of synthesis of the enzyme. Since TS gene expression appears to be regulated in a similar manner in LU3-7 cells and in the parental 3T6 cells, the LU3-7 cells should be a good model system for detailed analysis of the mechanism for regulating TS gene expression in mammalian cells.     

Stability of the cytokinin requirement in Paul's scarlet rose cells in culture

Summary Cells ofRosa sp. cv Paul's scarlet have been reported to require cytokinin for growth in suspension culture. This report was verified in the present study. However, a rose cell line that was maintained for 2 yr in suspension culture by routine subculturing developed the capacity to grow without exogenous cytokinin. The stability of the cytokinin requirement and the basis for this altered response to cytokinin was investigated. The parental cell line, which has been maintained independently on agar-solidified medium, was subcloned and the cytokinin dependence of the subclones was determined. The subclones were found to exhibit a continuous spectrum of responses, ranging from a high degree of cytokinin dependence for growth to rapid growth upon the initial transfer to cytokinin-deficient medium. The average growth constant (K=1n W/Wo; Wo=initial fresh weight,W=fresh weight after growth for the stated time interval) of 30 subclones grown on medium containing 0.5 μM zeatin was 3.1, with a range of 1.1 to 4.0. The average growth constant of the same subclones when grown on medium lacking a cytokinin was 1.5, with a range of 0 to 3.9. By comparison, the parental cell line exhibited growth constants of 3.5 in the presence of 0.5 μM zeatin and 1.6 in the absence of exogenous cytokinin. Although the growth of some of the subclones after transfer to cytokinin-deficient medium suggested that they were cytokinin autotrophs, this was not the case because none of them grew after a second transfer to medium lacking cytokinin. Culture in medium containing cytokinin conferred upon the cells the capacity for a limited amount of growth after subsequent transfer to medium lacking cytokinin. The extent of this cytokinin-induced growth potential varied from subclone to subclone. Efforts to determine the frequency with which cytokinin autotrophs appeared in a subclone that required cytokinin suggested that it is a rare event and that the cytokinin requirement is a fairly stable phenotypic characteristic of these cells. This research was supported by Grant PCM 7722398 from the National Science Foundation.     

Nitrate reductase activity in Paul's scarlet rose suspension cultures and the differential role of nitrate and molybdenum in induction

Induction of nitrate reductase (EC 1.6.6.1) activity was measured in Paul's Scarlet rose cell suspensions cultured in media containing nitrate (NO ₃ ^- ) or urea (U) as nitrogen source, and with (+Mo) or without molybdenum (-Mo). There was a lag of 30 min during induction by NO ₃ ^- in +Mo cultures but no lag occurred during induction after adding Mo to NO ₃ ^- -Mo or to U-Mo cultures preincubated with NO ₃ ^- . Actinomycin D, cycloheximide, and puromycin completely blocked induction by NO ₃ ^- , but had no effect on the initial rate of induction by Mo. Cycloheximide and puromycin blocked induction by NO ₃ ^- more quickly than actinomycin D. Induction by NO ₃ ^- appeared to involve mRNA-dependent synthesis of apoprotein followed by rapid activation with molybdenum in intact cells independently of protein synthesis. Nitrate-induced apoprotein appeared less stable than the holoenzyme. When induced by NO ₃ ^- in the absence of Mo, apoprotein concentration was about half the amount of maximally induced nitrate reductase. Cycloheximide stabilised preformed nitrate reductase which disappeared steadily in the presence of puromycin. Apoprotein was not stabilised by either antimetabolite.Abbreviations Mo molybdenum - NO ₃ ^- +Mo standard, MX₁ culture medium - NO ₃ ^- -Mo MX₁ medium purified of Mo - NR nitrate reductase - PSR Paul's Scarlet rose - U urea - U+Mo MX₁ medium with NO ₃ ^- replaced by urea - U-Mo MX₁ medium with NO ₃ ^- replaced by urea and also purified of Mo     

Turbidostat culture of yeast during transition from a resting state to active growth

The age components of a Saccharomyces cerevisiae 14 culture and the kinetics of its growth were studied afer the quiescent state at the onset active growth. The following factors induced the quiescent state: the cessation of a chemostat flow for 24 h, growth inhibition with 2,4-dinitrophenol (DNP) for 24 h, the storage of a culture growing on agar in a refrigerator during 24 h. The process of transition from the point of growth activation to the maximum rate of growth was then studied in turbidostat. This process took the shortest time in a refrigerated culture as well as in a culture that had been limited with a phosphorus source and in a culture limited with a nitrogen source and grown in chemostat at D = 0.26 h-1. The process was longer in cultures that had been either limited with glucose or inhibited with DNP and longest in a chemostat culture limited with a nitrogen source at D = 0.15 and 0.05 h-1. The rate of initial mitosis phases in the yeast is presumed to exert the greatest effect on the duration of this transition process.     

Contribution of Nonautotrophic Carbon Dioxide Fixation to Protein Synthesis in Suspension Cultures of Paul's Scarlet Rose

Bicarbonate-¹⁴C was provided to 5- and 11-day-old suspension cultures of Paul's Scarlet rose, and the incorporation of ¹⁴C into lipid, protein, amino acids, and organic acids was determined. The rate of bicarbonate uptake was approximately the same by 5- and 11-day-old cells, but the distribution of ¹⁴C among cell constituents was markedly different. In 5-day-old cells a larger proportion of the ¹⁴C entered protein, whereas in 11-day-old cells there was a greater tendency for ¹⁴C to accumulate in malate.     

Exogenous nucleosides stimulate RNA synthesis in resting cells of a culture of scarlet rose

RNA synthesis in response to exogenous nucleoside precursors was studied in a suspension culture of rose cells. Exponentially growing and resting cells were prelabeled with [3H] uridine, an excess of unlabeled uridine added, and subsequent isotopic incorporation into nuclear and ribosomal fractions measured. The data were compared to control values in cells continuously labeled in the absence of unlabeled uridine. Addition of uridine to the growing culture reduced the further uptake, and incorporation of [3H] uridine into RNA. In contrast, in resting cells, the addition of uridine (or, purine nucleosides) enhanced the apparent utilization of [3H] uridine in RNA synthesis by 2- to 4-fold.     

Induction of anthocyanin accumulation in rose suspension-cultured cells by conditioned medium of strawberry suspension cultures

A conditioned medium (CM) prepared from suspended cultures of strawberry, Fragaria ananassa, which stimulated anthocyanin accumulation in cultured strawberry cells, was applied to the suspension-cultured cells of rose, Rosa hybrida sp which did not normally produce anthocyanin. When the rose cells were transferred into the CM, it induced anthocyanin formation and accumulation in the rose cells. It is suggested that the CM may be effective for inducing anthocyanin accumulation in cultured cells of other species. This revised version was published online in June 2006 with corrections to the Cover Date.