DNA-directed enzyme synthesis

During the last few years, the method of DNA directed enzyme synthesis in vitro has become a powerful tool in the study of gene expression. 

For any DNA-directed enzyme synthesis in vitro, the conditions in the living cell should be more or less exactly copied. This is best approached in an extract prepared simply by disruption of the cells. Indeed such a crude extract supports the synthesis of enzymes (Schweiger et al., 1972). Unlike in intact cells, nucleases and proteases are free to attack nucleic acids and the products... 

These two cell-free preparations are the ones which have found the widest application for DNA-directed enzyme synthesis in vitro. Because they were developed for quite different purposes (phage enzyme synthesis—E. coli enzyme synthesis), a detailed comparison will be of interest. 

From the concentration dependence curve described above, an approximate measure of the activity of the ribosomes can be obtained. The number of rounds of translation by ribosomes in T7 DNA-directed enzyme synthesis was also estimated Crude ribosomes translate—on the average—10 times, preincubated ribosomes translate the messenger twice. The data must be taken with great reservation, because they depend on a large number of assumptions. 

Fig. 11. Dependence on template of DNA directed enzyme synthesis. SAMase s5nithesis was performed in the presence of varying amounts of X3 DNA per incubation mixture. Incubation mixture as in Table 2. Enzyme assay as described by Herrlich and Schweiger...

Magnesium can, to some extent, be substituted by spermidine or putrescine (Herrlich et al., 1971b). These polyamines have a differential influence on DNA directed enzyme synthesis in vitro. Spermidine for instance stimulates... 

DNA-Directed Enzyme Synthesis in vitro Table 7. DNA directed enzyme synthesis in vitro... 

Richter, D., Herrlich, P., Schweiger, M. Phage DNA directed enzyme synthesis in in vitro system from yeast mitochondria. Nature (Lond.) New Biol. 238, 74-76 (1972). 

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