Abstract
The product of gene C of bacteriophage phi X174 is required for the replication of phiX174 single-stranded DNA in Escherichia coli cells infected with phi X174. The protein has been purified to homogeneity using an in vitro complementation system. The protein exhibits a molecular weight of 5800 under denaturing conditions. The NH2-terminal amino acid sequence of the protein is (NH2)-Met-Arg-Lys, which is consistent with the sequence predicted from the nucleotide sequence of gene C. The protein has an affinity for single-stranded DNA but less for double-stranded DNA.
Highlights
Theproduct of gene C of bacteriophage 4x174 is required €or the replication of @X174 single-stranded DNA in Escherichia colicells infected with @X174.The protein has been purified to homogeneity using an in vitro complementation system
In order to study the function of gene C protein, as well as other phage-encoded proteins, we have developedan in vitro DNA-synthesizing system reconstituted from purified phage components and host protein fractionthat is capable of producing infectious phage(6).We report here the purification and properties of geneC protein
Purification of +X174 Gene C Protein fold from 170g of @X174N11-infected E. coli cells with a yield of 1.8%(Table I)
Summary
Bacterial and PhageStrains--E. coli HF4704 ( @ X , thy, at37 “C) is a laboratory stock. MM EDTA, 10% (v/v) glycerol, and 1 mM dithiothreitol;BufferC contained 50 mM Tris-HC1 (pH7.5), 15 mM EDTA, 0.1 M KCI, and 1 mg/ml of bovineplasmaalbumin.Lysissolutioncontained. H570 infected with +X174 am3och as described previously (9) and gene C protein to be assayed. Nine microliteorfs the reaction mixture were mixed with 0.2 rnl of stop solution (30 mM EDTA and 60 mM sodium pyrophosphate) at zero time to determine the background and the remaindewr as incubated at30 “C for 30 min and placed in an ice water bath. Nine microlitersof this reaction mixture werme ixed with 0.2 ml of stopsolutiontodeterminetheamount of DNaseresistantparticles.Trichloroaceticacid-insolubleradioactivitywas determined as described(5).One unit of gene C protein is defined as. 0.1 nmol of deoxyribonucleotidesincorporatedintotrichloroacetic acid-insoluble, DNase-resistant materials in min
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