Report of Progress, lay 1, 1957 Growth and Inheritance in Bacteriophage Grant C2158 Ae D. Hershey, Department of Genetics, Carnegie Institution of Washington, Cold Spring Harbor, New York Period from 1950 to 1957 A. Summary. Our genetic experiments with phage T2, 1945 to 1951, established that inheritance in this phage is regulated by a linear linkage system functionally equivalent to the visible chromosomes of other organisms. A phage particle is now believed to contain only one such “chromosome.” During 1948-1952 Doermann, combining our materials and techniques with his, demon- strated that phage particles multiply in a noninfective form, called vegetative phage, inside bacterial celis. Beginning about 1950, our interest shifted to chemical problems relating to growth and inheritance as exemplified by the transition from resting to veretative phage and back again during the infectious cycle. We found that only the phage DNA passes from phage particle into bacterium at the time of in- fection, that such chromosomal DNA can replicate autonomously in the cell, and that its incorporation into new phare particles occurs only as a terminal phase of growth during which protein synthesis is the dominant process. Needless to say» these statements skip over many agonizing questions. Be Statement of progress. The purely genetic phass of our work, ending in 1951, will not be summarized in detail. Its general nature is indicated by the titles of papers in the bibliography given below. Genetic experiments with phase are being continued in several laboratories in this country and abroad, including that of Streisinger at Carnegie Institution. The main fact pertinent to this report is that a phage particle contains a single linear "chromosome" comparable in function to the visible chromosomes of higher organisms which, however, are ca. 1000-fold greater in mass. In 1948 to 1950, Hershey, Kanens Kennedy, and Gest showed that phage particles containing assimilated radio-phosphorus are subject to "suicide" as a& consequence of nuclear reactions occurring in their DNA. This formed the starting point of the lines of research outlined below, Wie did nots, however, follow up our suicide experiments, chiefly because of the difficulty of obtain- ing suitable radioisotopes at the time. The method is being fully exploited at present in several laboratories, notably by Stent at University of California. By 1950, the pioneer work of Doermann and Luria and (further afield) Lwoff and Bertani was beginning to focus attention on new aspects of the multiplica-~ tion of phage; namely, qualitative transformations as opposed to simple numerical increase. The new point of view was surmarized in the paper by Hershey (1952), written in 1950, which introduced the term "vegetative phage" to distinguish multiplying from resting viral structures. We quote from this paper as follows: ",..deoxypentose nucleic acid synthesis changes from a minor to a major activity of the cell after infection...one of the big questions of biology is whether this is a qualitative as well as a quantitative change....Luria has suggested that Pa the bulk of the nucleic acid may be synthesized during the conversion of vegeta- tive into resting phage, rather than during the period of multiplication proper. (If so) genetic specificity of the phage is independent of its major DNA con- tent. ..-.perhaps the initial question (about vegetative phage) could be formulated as follavs. Does or does not the replication of phage-specific sub- stances occur within a phage-specific membrane?" At that time chemical experimentation with phage was developing in inter- esting directions under the leadership of Cohen, Putnan, and Kozloff, but had not yet furnished any clues to the nature of vegetative phage. It was knom that phage particles are composed of about equal parts of protein and nucleic acid, that the infected bacterium devotes its major energy to the synthesis of these mterialss and that some of the atomic constituents of the infecting phage particle reappear among the offspring. The first step in the identification of vegetative phage was reported by Ilershey and Chase (1952) who shaved that viral DNA but not viral protein entered the cell at the time of infection. Consistent with this fact, only the atoms of the parental viral DNA could be found among the offspring particles. The second step consisted in showing that all the DNA synthesized after infection is viral precursor, that such precursor DNA is synthesized in advance of the phage particles in which it is to appear, and that the DNA in this precursor pool is sampled at random to make phage particles, which are not therefore manufactured on the assembly line principle (Iershey et als, 1953; Iershey 1953). In the early stages of this work we discovered and made use of the unique base 5ehydroxynethyl cytosine in the DNA of T2 but did not identify it. ‘lyatt and Cohen independently discovered and identified it. The third step in the identification of vegetative phage consisted in shoving, by the use of chloramphenicol and isotopic labeling, that phage parti-~ cles cen be prepared containing DNA synthesized almost entirely in the presence of the antibiotic, and protein synthesized almost entirely during o subsequent period after chloramphenicol is removed (Hershey and lielechen, 1957). The fourth and final step, proof that phage preoursor DNA synthesized in the presence of chloramphenicol includes chromosomal DNA, is still incomplete. The folloving discovery by Tomizawa in our laboratory indicates one of two independent lines of attack that we are pursuing. If phage precursor DNA formed in the presence of chloramphenicol is irradiated with ultraviolet light while still in the bacteria, and these are afterward transferred out of chioram- phenicol so that protein synthesis can begin, phage particles form that are alnost all noninfective. The dead particles have properties similar to those produced by irradiating phage particles themselves. Owing to the remrkable mature of these properties, the dead particles are subject to genetic analysis, as shovrm by Deermann for particles irradiated outside the bacterium. In this history we have necessarily neglected the work of many peoples including much of our ome 3 Our conclusion, subjee+ to possible upsets by work in progress, is that the phage chromosame is a single DNA molecule that multiplies in infected bacteria independently of all those processes involving protein synthesis by which the typical, finished phage particle is formed, C. Future plans. (1) To complete the line of thought summarized above, (2) To attempt to identify chromosomal and nonchramosomal DNA among fractions that oan be separated by chromatography on columns of basic protein. (3) To study further the genetic significance of the transfer of nucleic acid from parental to offspring phage. D. Bibliography. (Asterisks denote review papers that do not acknowl- edge USPHS support). 1. Hershey, A. D. and Raquel Rotman. 1948 Linkage among genes controlling inhibition of lysis in a bacterial virus. Proc, Nat. Acad. Sci, vol. 34, No. 3, pp. 89-96. 2. Hershey, A. D. and Raquel Rotman. 1949 Genetic recombination between nost- range and plaque-type mutants of bacteriophage in single bacterial cells. Genetics vol. 34, pp. 44-71. 3. Hershey, A. D. and Harriet Davidson. 1951 Allelic and non-allelic genes controlling host specificity in a bacteriophage. Genetics, vol. 56, No. 6 pp. 667-675, 4, Hershey, A. D. and Martha Chase, 1951 Genetic recombination and hetero~ zygosis in bacteriophage. Cold Spring Harbor Symp. Quant. Biol. vol. i6, pp. 471-479, 5. Hershey, A. D, M. D. Kamen, J. W. Kemedy, and H. Gest. 1951 The mortality of bacteriophage containing assimilated radioactive phosphorus. J. Gen. Physiol, vol. 34, No. 3, pp. 305-519. 6. Hershey, A. D. 1952 Reproduction of bacteriophage. Intern. Rev. Cyto. vol. 1, pp. 119-154. *7, Hershey, A. D. 1953 Inheritance in bacteriophage. Advances in Genet. vol. 5, pp. 89-106. *8, Hershey, A. D. 1953 Intracellular phases in the reproductive cycle of bacteriophage T2. Ann. inst. Pasteur vol. 84, pp. 99-112. *9. Hershey, A. D. 195% Functional differentiation within particles of bacteriophage T2. Cold Spring Harbor Symp. Quant. Biol. vol 18, pp. 135-139, L965 10. Hershey, A. D., dune Dixon, and Martha Chase. / ilucleio acid economy in bacteria infected with bacteriophage T2. I. Purine and pyrimidine composition. d. Gen. Physiol. vol. 36, No. 6, pp. 777-789. ll. Hershey, A. D. 1953 Nucleic acid eConomy in bacteria infected with bacteriophage T2, II. Phage precursor nucleic acid. J. Gen. Physiol. vol. 37, No. 1, pp. 1-23. 12. Hershey, A. D., Alan Garen, Dorothy K. Fraser, and June Dixon Hudis. 1954 Growth and inheritance in bacteriophage. Carnegie Inst. Wash. Year Book Wo. 53, pp. 210-225. 13. Hershey, A. D, 1954 Conservation of nucleic acids during bacterial growth, J. Gen. Physiol. vol 38, No. 2, pp. 145~148. 14. Garen, Alan and Norton D, Zinder 1955 Radiological evidence for partial genetic homology between bacteriophage and host bacteria, Virology, vol. 1, pp. 347-876, 15. 21. 22. 23. Hershey, A. D. 1955 An upper limit to the protein content of the germinal substance of bacteriophage T2. Virology vol. 1, No. 1, pp. 108-127, Hershey, A. D. 1956 The organization of genetic material in bacterio- phacpe T2. Mutation: No. 8, pp. 6-16. (Brookhaven Syup. Biol.) Hershey, A. D. 1956 Chemistry and viral growth. In Currents in Bio» chemical Research, ed. David E. Green, pp. 1-28. interscience Publishers, Inc., New York. Hershey, A. D. 1957 Bacteriophage T2: Parasite or organelle? In The Harvey Lectures, series 51, New York, Academic Press. pp. 229-239. Hershey, A. D. 1956 Genetic structure and function in bacteriophage T2. In Enzymes: Unites of Biological Structure and Function. pp. .109-117. New York, Academic Press. Hershey, A. D., and Burgi, E. 1956 Genetic significance of the transfer of nucleic acid from parental to offspring phage. Cold Spring Harbor Symp. Quant. Biol. vol. 21, pp. 91-101, Hershey, A. D. and Norman &, Helechen 1957 Synthesis of phage-precursor nucleic acid in the presence of chloramphenicol, Virology vol. 3, pp. 207~236. Hershey, A. D. 1957 Experimental problems concerning role of deoxyribo- nucleic acid in growth of phage. Special Publ. 5, New York Acad, Soi., in press, Hershey, A. D. 1957 Bacteriophages as genetic and biochemical systems. In Advances in Virus Research. vol. 4, pp. 25-61. New York, Academic Press.