Modeling and optimization of alpha-amylase production in a recombinant yeast fed-batch culture taking account of the cell cycle population distribution.

A simple mathematical model describing the cell cycle dependency of rice alpha-amylase production by a recombinant yeast was constructed to investigate the efficiency of cell cycle population control. First, the effects of the glucose concentration and cultivation temperature on the specific growth rate, the specific production rate of rice alpha-amylase, and the distribution of the cell cycle population were studied under balanced growth conditions. On the basis of the results, parameter values for the mathematical model were then estimated. The proposed model was shown to be applicable for unbalanced as well as balanced growth phases. The optimal control strategy in respect of temperature and glucose concentration for maximum rice alpha-amylase production, taking into account the cell cycle population, was determined and the result was compared with that obtained by a simple mathematical model in which cell cycle distribution was not considered. Finally, the effect of the initial population of each cell cycle phase on the final amount of the product under optimal operational conditions was investigated. The simulation and experimental data coincided well with each other, and the model was used to optimize the control strategy for maximum alpha-amylase production.     

Yeast L double-stranded ribonucleic acid is synthesized during the G1 phase but not the S phase of the cell cycle.

The cytoplasm of Saccharomyces cerevisiae contains two major classes of protein-encapsulated double-stranded ribonucleic acids (dsRNA's), L and M. Replication of L and M dsRNA's was examined in cells arrested in the G1 phase by either alpha-factor, a yeast mating pheromone, or the restrictive temperature for a cell cycle mutant (cdc7). [3H]uracil was added during the arrest periods to cells prelabeled with [14C]uracil, and replication was monitored by determining the ratio of 3H/14C for purified dsRNA's. Like mitochondrial deoxyribonucleic acid, both L and M dsRNA's were synthesized in the G1 arrested cells. The replication of L dsRNA was also examined during the S phase, using cells synchronized in two different ways. Cells containing the cdc7 mutation, treated sequentially with alpha-factor and then the restrictive temperature, enter a synchronous S phase when transferred to permissive temperature. When cells entered the S phase, synthesis of L dsRNA ceased, and little or no synthesis was detected throughout the S phase. Synthesis of L dsRNA was also observed in G1 phase cells isolated from asynchronous cultures by velocity centrifugation. Again, synthesis ceased when cells entered the S phase. These results indicate that L dsRNA replication is under cell cycle control. The control differs from that of mitochondrial deoxyribonucleic acid, which replicates in all phases of the cell cycle, and from that of 2-micron DNA, a multiple-copy plasmid whose replication is confined to the S phase.     

Cyclin B targets p34cdc2 for tyrosine phosphorylation.

A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.     

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.     

Oocyte activation and passage through the metaphase/anaphase transition of the meiotic cell cycle is blocked in clams by inhibitors of HMG-CoA reductase activity

Cell cycle progression for postembryonic cells requires the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R), the enzyme which catalyzes the production of the isoprenoid precursor, mevalonate. In this study, we examine the requirements of HMG-R activity for cell cycle progression during the meiotic and early mitotic divisions using oocytes and dividing embryos from the surf clam, Spisula solidissima. Using two different inhibitors of HMG-R, we find that the activity of this enzyme appears to be required at three distinct points of the cell cycle during meiosis. Depending on the stage at which these inhibitors are added to synchronous clam cultures, a reversible cell cycle block is triggered at the time of activation or at metaphase of either meiosis I or II, whereas there is not block to the mitotic cell cycle. Inhibition of HMG-R activity in activated oocytes does not affect the transient activation of p42MAPK but results in a block at metaphase of meiosis I that is accompanied by the stabilization of cyclins A and B and p34cdc2 kinase activity. Our results suggest that metabolites from the mevalonate biosynthetic pathway can act to influence the process of activation, as well as the events later in the cell cycle that lead to cyclin proteolysis and the exit from M phase during clam meiosis.     

Induction of a mitosis delay and cell lysis by high-level secretion of mouse alpha-amylase from Saccharomyces cerevisiae

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse alpha-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in alpha-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of alpha-amylase. Our data also suggest that high levels of alpha-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous alpha-amylase in S. cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis.     

Relocation and distinct subcellular localization of p34cdc2-cyclin B complex at meiosis reinitiation in starfish oocytes.

M phase promoting factor (MPF) is a major element controlling entry into the M phase of the eukaryotic cell cycle. MPF is composed of two subunits, p34cdc2 and cyclin B. Using indirect immunofluorescence staining with specific antibody against starfish cyclin B, we monitored the dynamics of the subcellular distribution of MPF during meiosis reinitiation in starfish oocytes. We found that all of the cyclin B is already associated with p34cdc2 in immature oocytes arrested at the G2/M border and that this inactive complex is present exclusively in the cytoplasm. After its activation, part of the p34cdc2-cyclin B complex moves into the germinal vesicle before nuclear envelope breakdown, independently of either microtubules or actin filaments. Thereafter, some part of the complex accumulates in the nucleolus and condensed chromosomes. Another portion of the complex accumulates on meiotic asters and spindles, while the rest is still present throughout the cytoplasm. As these patterns of localization are detected in the detergent-extracted oocytes, we propose at least four distinct subcellular states of the p34cdc2-cyclin B complex: freely soluble, microtubule-associated, detergent-resistant cytoskeleton-associated and chromosome-associated. Thus, in addition to the intramolecular modification of p34cdc2-cyclin B complex, its intracellular relocation plays a key role in promoting the M phase.     

Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture

The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. (c) 1994 John Wiley & Sons, Inc.     

Interaction of cAMP with the CDC25-mediated step in the cell cycle of budding yeast

Addition of exogenous cAMP to cultures of the start mutant cdc25-1 of Saccharomyces cerevisiae shifted to restrictive temperature causes a partial reversion of the mutated phenotype, with a marked increase of the percentage of budded cells. This effect is coupled to a progression in the cell cycle, as demonstrated by DNA histograms obtained by flow cytometry. Moreover cdc25 cells have a high intracellular cAMP content also at restrictive temperature, and no change in the cAMP content was seen during a transition from restrictive to permissive temperature. These data suggest that CDC25 gene product allows cell proliferation by interacting with a cAMP-mediated mechanism.     

Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes.

The mammalian homologue of the cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a p34cdc2 cyclin-dependent kinase that regulates the cell cycle of a wide variety of cell types. Resting murine T lymphocytes contained no detectable p34cdc2 protein, histone kinase activity, or specific mRNA for the cdc2 gene. Activation of the T cells by immobilized anti-CD3 resulted in the expression of specific mRNA late in the G1 phase of the cell cycle, and p34cdc2 protein was detectable at or near G1/S. At this point in the cell cycle, the protein was phosphorylated at tyrosine and displayed no H1 histone kinase activity. As the cells progressed through the cycle, the amount of specific mRNA and p34cdc2 increased, and H1 histone kinase activity was detectable when the cells were blocked at G2/M by nocodazole. The activation of T cells by phorbol dibutyrate induced the expression of IL-2R but failed to induce the synthesis of IL-2 or the expression of cdc2-specific mRNA. Under these conditions, the activated cells failed to enter the S phase of the cell cycle. Because the presence of IL-2 added exogenously during activation by phorbol dibutyrate resulted in the expression of cdc2-specific mRNA and progression through the cell cycle, either IL-2 or the interaction with IL-2R may be involved in the expression of cdc2 and regulation of the G1/S transition.     

Identification of MSA1, a cell cycle-regulated,dosage suppressor of drc1/sld2 and dpb11 mutants

The Dpb11 and Drc1/Sld2 proteins form a complex that is critical for the initiation of DNA replication. In this study we identify MSA1 as a high copy suppressor of a drc1-1 mutant. MSA1 overproduction can also suppress the temperature sensitivity of dpb11-1 and pol2-12 mutants. Reciprocally, msa1 deletion exacerbates the mutant phenotypes of both drc1/sld2 and dpb11 mutants and msa1 deletion alone results in a delay in S phase entry of synchronous cells indicating a positive role for MSA1 in DNA replication. Paradoxically, MSA1 overproduction is deleterious to cdc6-1, cdc7-1, cdc28-1N and cdc14-1 mutants indicating a complex relationship with DNA replication and cell cycle regulatory genes. The Msa1 protein is tightly cell cycle regulated. Msa1 and its paralog, Msa2, both accumulate in highly modified forms just as cells commit to enter S phase and then are rapidly destroyed. MSA1 represents a new cell cycle regulated gene important for S phase entry.     

Reovirus-Induced ς1s-Dependent G2/M Phase Cell Cycle Arrest Is Associated with Inhibition of p34cdc2

Serotype 3 reoviruses inhibit cellular proliferation by inducing a G(2)/M phase cell cycle arrest. Reovirus-induced G(2)/M phase arrest requires the viral S1 gene-encoded sigma1s nonstructural protein. The G(2)-to-M transition represents a cell cycle checkpoint that is regulated by the kinase p34(cdc2). We now report that infection with serotype 3 reovirus strain Abney, but not serotype 1 reovirus strain Lang, is associated with inhibition and hyperphosphorylation of p34(cdc2). The sigma1s protein is necessary and sufficient for inhibitory phosphorylation of p34(cdc2), since a viral mutant lacking sigma1s fails to hyperphosphorylate p34(cdc2) and inducible expression of sigma1s is sufficient for p34(cdc2) hyperphosphorylation. These studies establish a mechanism by which reovirus can perturb cell cycle regulation.     

Relationship between hybridoma growth and monoclonal antibody production.

Factors affecting cell growth and antibody production in a mouse hybridoma were investigated. Antibody was produced during the growth and decline phases of a batch culture with an increase in the specific rate of antibody production during the decline phase. The specific rate of antibody production was also increased in cells arrested by 2 mM thymidine, suggesting that cell proliferation and antibody production can be uncoupled. Reduced serum concentrations resulted in lower cell growth rates but increased antibody production rates. However, this trend was reversed in hybridomas which had been arrested by thymidine, since the highest antibody production rate was associated with high serum concentrations. Likewise, in proliferating cells, the optimum pH for antibody production (pH 6.8) was lower than the optimum pH for cell growth (pH 7.2), whereas in thymidine-blocked cells, the highest antibody production rate was at pH 7.2. High antibody production rates and product yields were also associated with low growth rates in continuous cultures. The possibility that antibody was under cell cycle control was investigated in synchronized hybridoma cultures. Antibody production occurred during G1 and G2 with a decline in the M phase and evidence of a further decline in the S phase. Thus antibody production was not restricted to the G1 and S phase in this hybridoma.     

Specific monoclonal antibody productivity and the cell cycle-comparisons of batch,continuous and perfusion cultures

A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G₁ phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of G₁ phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.