Regelung der Synthese von Dihydrofolsäure‐Reduktase bei synchronisierten Hefezellen
Authors: SCHOLZ K. , JAENICKE L. 
Published: March 2005

The activity of FH2 reductase, an enzyme intimately related to cell duplication, has been studied in synchronously growing yeast cells. The method of synchronisation consists of (a) a period of growth in a rich medium during which the temperature of the culture is changed at regular intervals between 30° and 38°, (b) aeration in a starvation medium, (c) repetition of (a), followed by fractionation of the yeast cells by low‐speed centrifugation.

After the appearance of buds, enzyme activity doubles rapidly and then remains constant, so that in each generation increase in activity follows a step‐wise pattern. Since periodic fluctuations in the specific activity and stability of the enzyme could not be detected, the periodicity of the increase in activity must be attributed to discontinuous de novo enzyme synthesis. This periodic enzyme synthesis is not subject to induction or repression. Thus FH2, the natural substrate of FH2 reductase, has no influence on enzyme formation. Moreover, periodic synthesis persists even in the presence of 10‐3 M amethopterin which has been shown to specifically inhibit FH2 reductase intracellularly. Quantitative removal of the inhibitor leads to complete reactivation of the enzyme.

Yeast cells are made permeable to actinomycin C for several hours by being grown in a medium containing 0.09% deoxycholic acid. Addition of actinomycin C, to a final concentration of 0.8×10‐4M, to a culture during a period of activity increase causes quite rapid and complete inhibition of the synthesis of FH2 reductase. This implies a direct connection between the synthesis of the enzyme and the fromation of RNA. The increase in activity occurring after the addition of actinomycin C is attributable to the translation of existing amounts of the messenger RNA which codes for FH2 reductase. From the duration of the increase in enzyme activity after the addition of inhibitor the half‐life of this mRNA is estimated to be about 5 minutes.

Enzyme is synthesised only once in a cell cycle when the cells are growing logarithmically. Retardation of DNA synthesis by addition of amethopterin to a concentration of 1.6×10‐2M — the intracellular concentration was determined experimentally to be < 10‐5M — leads to a slackening of the rate of enzyme synthesis but the step‐wise pattern is maintained. On the other hand, the synthesis of the total soluble cell protein proceeds as rapidly as in the controls. This finding points to a direct connection between DNA replication and the synthesis of FH2 reductase.

In contrast to logarithmically growing cells, the first generation in the lag phase shows two phases of synthesis, one occurring before, and the other after, the onset of DNA replication.

The model which has been developed to interpret these observations is based on the assumption that the structural gene for FH2 reductase exists in two different functional states, in only one of which can the short‐lived messenger be replicated. The change from one state to the other gives rise simultaneously to the step‐wise pattern of synthesis of FH2 reductase.