Abstract
The location within the virion of the A, C, and D minor coat proteins of the filamentous bacteriophage fl has been analyzed. The A protein is present in approximately 5 copies/particle and is located at the tip of normal length phage, miniphage, and fl/pBR322 chimeric phage, a longer than normal length phage. The mole ratios of the A, C, and D proteins are the same for each type of particle, consistent with a model of phage organization in which the minor coat proteins are clustered near or at the ends of the phage. Normal length phage were fragmented by passing them through a French press, and those fragments that contained the A protein were separated from those that did not by treating the mixture with anti-A protein antibody. Analysis of the protein compositions of the two populations of fragments showed that the A and D proteins were found together in one population of fragments and that most, it not all, of the C protein was found in the other. These results show that the D protein is located near or at the A protein end of the phage and that the C protein is located in a region near or at the opposite end. Treatment of the virion with proteases which lowered the infectivity of the phage resulted in particles in which only the A protein was cleaved to any detectable extent. These particles remained resistant to the action of micrococcal nuclease.
Highlights
From the +Departmentof Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 a n d the CjDepartment of Molecular Biophysics a n d Biochemistry, Yale University, New Haven, Connecticut 06510
The mole mentous phage genome has led to the production of phage ratios of the A,C, and D proteins are the same for eachparticles which are longer than normal (For type of particle, consistent with a model of phage or- example see Refs. 11-14)
We show that theA andD minor coat proteins lations of fragments showed that the A andD proteins are located near or at one end of the normal length phage werefound together in one population of fragments particle and that the C protein is located nearor a t the and that most, it not all, of the C protein was found in opposite end
Summary
From the +Departmentof Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 a n d the CjDepartment of Molecular Biophysics a n d Biochemistry, Yale University, New Haven, Connecticut 06510. Fragmentation of Bacteriophage-Wild type (normal length) fl phage labeled with either [32P]orthophosphateor ["5S]cysteinewere diluted to a concentration of 5 X phage particles/ml in0.10 M NaCl, 0.001 M EDTA, 0.05 M Tris-HC1 (pH 7.8) containing 1 mg/ml of bovine serum albumin. The material that sedimented onto the metrizamide shelf (Peak X) or the material that remained in the middle of the gradient (Peak Y)was diluted 6-fold with 0.10 M NaCl, 0.001M EDTA, 0.01 M Tris-HC1(pH 7.6) containing 1 mg/ml of bovine serum albumin, and 0.1 mg of unlabeled phage was added as carrier. To determine the mole ratios of the A, C, and Dproteins, whole or fragmented phage particles labeled with ["Slcysteinewere solubilized, subjected to gel filtration on A-5magarose, and electrophoresed on SDS-polyacrylamide gels as described byLin et al(5).The procedures used for slicing gels, for extracting radioactivity from gel slices, and for the determination of the mole ratios of the minor coat proteins have been given previously (5). Particle lengths for normal length phage, miniphage, and maxiphage were determined by tracing micrographs that were enlarged 3 times
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