STUDIES ON THE RELATIONSHIP BETWEEN VIRUS AND HOST CELL: THE PREPARATION OF T2r⁺BACTERIOPHAGE LABELLED WITH RADIOACTIVE PHOSPHORUS

Abstract

T2r+ bacteriophage grown in its host, Escherichia coli B, in broth medium in the presence of radioactive inorganic phosphorus was labelled with the isotope. Purified suspensions of this virus had specific activities up to 50,000 c.p.m. per μgm. P. There was little or no exchange of P32 between virus and inorganic phosphate. Chemical analysis showed that at least 98% of the virus phosphorus was contained in nucleic acid; of the nucleic acid phosphorus 95.5% was associated with desoxypentose nucleic acid and 4.5% with pentose nucleic acid. More than 99% of the radioactivity of the labelled bacteriophage was contained in the nucleic acid fraction. Preparations of bacteriophage were obtained with sufficiently high specific activity to enable metabolism experiments to be carried out on the growth of the labelled virus in the host cell.

Preparation, Purification, and Properties of E. Coli Virus T2

“…Usually a maximum was reached in 2 days but sometimes 5 days were required. This observation is qualitatively similar to those of Lesley et al (25) and Luria et al (30).…”

“…The nucleic acid content as determined by the diphenylamine test (13), the cysteine color method (14), or the phosphorus analysis was over 50 per cent of the whole virus. Carrying along of non-constituent DNA is unlikely in the present instance since DNAse has been used in the purification procedure and The figure for the micrograms of phosphorus per infective particle which can be calculated from the data in Table III is in very good agreement with the average value of 3.0 × 10 xx reported by Lesley, French, and Graham (25).…”

“…The yield appeared to be inversely related to the quantity of dark gray colloidal material present in some preparations which was difficult to separate without a considerable sacrifice in the yield. Lesley et al (25) have satisfactorily solved this problem of the gray impurity. As might be expected from the results shown in Text- fig.…”

“…If this centrifugation is continued longer, i.e, 2 hours, a dark gray film often, but not always, collects over the pellet and the ratio of titer to turbidity ~s proportionally lower indicating that this gray material is an impurity. Lesley et al (25) who also encountered a similar material suggest a simple procedure for its removal.…”

“…Titers of the lysates varied from 2 to 5 X 1011 plaque-forming units per ml. In test tube quantities lysate titers have run as high as 8 X 10n/ml, Other workers (25,5,6) have also reported high titer lysates. Trial runs using the coli viruses T2, and T4 also yielded lysates titerlng 2 X 10n/ml.…”