Vol. 45, 1959 BIOCHEMISTRY: KORNBERG ET AL. 773 



METHODS AND MATERIALS 



Preparation of Cell Extracts. — E. coli B was grown at 37° with vigorous aeration 

 in M-9 medium^* modified to contain per liter: KH2PO4, 3 gm, Na2HP04, 6 gm, 

 NH4CI, 1 gm, MgS04-7H20, 0.49 gm, glucose, 5.0 gm, FeS04-7H20, 0.5 mg, CaClz,' 

 55 mg. Growth at a logarithmic rate continued to 4-5 X 10^ cells per ml with a 

 generation time of about 50 min. 



Cell extracts were prepared in two ways : 



Method I: Cultures grown to 2 X 10* cells per ml were chilled, centrifuged, and 

 the cells resuspended at 4 X 10^ cells per ml in cold growth medium. Five T2r+'^ 

 or T5 per cell were added and after a 4-min adsorption period at 0°, the culture was 

 diluted twenty-fold into fresh growth medium at 37° and aeration continued. The 

 time of dilution was taken as "zero minutes." 50-ml aliquots were pipetted rapidly 

 onto crushed ice at intervals. The cells were sedimented by centrifuging for 5 min 

 at 10,000 X g, M'ere resuspended in 1 ml. of 0.5 M glycylglycine buffer, pH 7.0, 

 containing 0.001 M glutathione, and were stored for 1 to 3 days at -15°. The 

 cells were disrupted in a 10 kc Raytheon sonic oscillator. After removal of a small 

 amount of debris by centrifugation, the extracts contained about 2 mg of protein per 

 ml. 



Method II: Cultures were grown to 2 X 10» cells per ml, in the modified medium 

 without CaCl2 and four T2r+ per cell added ("zero minutes"). 50-ml aUquots 

 were pipetted rapidly onto crushed ice at intervals. The cells were sedimented and 

 resuspended in 4 ml of 0.05 M glycylglycine buffer, pH 7.0, containing 0.001 M 

 glutathione and disrupted as above. Extracts containing about 6 mg of protein 

 per ml were obtained after centrifugation. 



All results refer to extracts prepared by Method I unless otherwise stated. While 

 Method II was less effective for phage multiplication (see below), a description of 

 this method is included since it provided an alternative and efficient technique for 

 obtaining concentrated extracts in kinetic studies. The activities per mg protein 

 for the several enzymes studied were found to be at levels similar to those obtained 

 by Method I. 



Bacteriophage Determinations. — Bacteriophage was assayed by standard tech- 

 niques. '^ Intracellular phage was measured after "lysis from without" essentially 

 as described by Doermann.^^ The formation of infectious units in both T2r+ and 

 T5 infected cells (Fig. 1) was found to proceed in normal fashion in cells infected 

 as described in Method I. T2r+-infections produced by Method II yielded only 

 about 2 phage per original cell at 25 min when measured after "lysis from without," 

 although after clearing of the culture a yield of several hundred phage per original 

 cell was obtained. 



Enzyme Assays and Preparations. — Phosphorylation of the deoxynucleoside 

 monophosphates (kinase activities) was measured, as described before, ^ by using 

 a 5'-P^Mabeled mononucleotide as substrate and assaying the amount of label 

 which becomes resistant to the action of semen phosphatase.^* Formation of 

 dHMC-5-P from dC-5-P (hydroxymethylase) was assayed according to Flaks and 

 Cohen.7 The assay of DNA synthesis ("polymerase") was measured by the con- 

 version of a C^^-labeled deoxynucleoside triphosphate into an acid-insoluble product.^ 

 "Polymerase" fraction VII from uninfected E. coli was prepared as previously 

 described.^ 



126 



