The identification of prereplicative bacteriophage T4 proteins.

Abstract Amber and deletion mutants of bacteriophage T4 were used to identify prereplicative T4 proteins on sodium dodecyl sulfate acrylamide gels. The molecular weights of 25 identified T4 proteins are given.

The succeeding two papers describe the transcriptional and translational patterns which occur following bacteriophage T4 infection of Escherichia coli (1,2). Most of the analyses described are dependent on the assignment of prereplicative gene products to specific proteins detected by electrophoresis in sodium dodecyl sulfate-acrylamide gels. We present here the assignments we have made thus far by analyzing phage-specified proteins in cells infected with nonsense mutants or deletion mutants of T4. MATERIALS AND METHODS Preparation of Radioactive Proteins in Phage-infected Cells-Mutant stocks of coliphage T4 were prepared from single plaques by infection of E. coli B or CR63 in M9 media (3) plus tryptophan (10 pg per ml) at 30". All phage mutants, which are noted in Table I, were originally from the collections of Drs. W. Wood, C. Yegian, R. Epstein, S. Champe, L. Black, D. Parma, J. Cornett, and J. Weil. We thank each of them for their generous aid.
All infections were performed at a multiplicity of eight in E. coli B (cell density 3 X 10s cells per ml) in M9 media plus tryptophan (10 pg per ml) at 30". Racteria were irradiated with ultraviolet light prior to infection in order to diminish host transcription and translation (which normally masks T4 proteins synthesized at early times after infection (14)). The cells were irradiated for 13 min with an ultraviolet lamp (UVSL 25, Ultraviolet Products Incorporated); the lamp was placed 6 inches above the cells, which were gently stirred in a 150.mm diameter Petri dish. The cells were transferred to a flask and aerated for 10 min before infection. After irradiation in this manner, T4-infected cells synthesize proteins at four times the rate of uninfected cells. This rate of protein synthesis is 20 to 40% of the rate measured in T4-infected unirradiated cells.

Phage and mixed 14C-amino acids (Schwarz-Mann
No. 3122-09) were added simultaneously to the cells. The final concentration of radioactive amino acids was 0.375 PCi per ml. Cells labeled by this method incorporated about 25y0 of the input amino acids into protein.
The cells were lysed and prepared for electrophoresis by resuspension in 1 volume of water plus 4 volumes of SDS'-sample buffer (10). Complete lysis was ensured by heating the samples at 90" for 1 min.
Gel Electrophoresis and Autoradiography-Radioactive protein samples were run on the vertical slab apparatus described by F. W. Studier and J. Maizel.2 The SDS-gel and buffer system used is that of Laemmli and Maizel, described by Laemmli (10). A 12.5% separating gel was poured and always allowed to sit overnight before electrophoresis. A 4.5y0 stacking gel was poured the following morning and, following polymerization, the gel was used immediately for electrophoresis. Gels were run for 6 hours at constant current (10 ma per gel for a gel thickness of >$z inch).
After electrophoresis, the proteins were fixed in the gel with 50% trichloroacetic acid for 30 min, and then stained with 0.1 y0 Coomassie brilliant blue solution in 50% trichloroacetic acid for 30 min. The gels were destained overnight by repeated washing in 7oj, acetic acid. The gels were dried (10) and placed under film (Kodak No-Screen medical x-ray film) for various lengths of time. The gel patterns shown here were obtained in Boulder.
At Stanford % (as H&04) was used as the radioactive precursor (15); identical assignments were made for many of these genes.

AND DISCUSSION
The autoradiograms of SDS-gels in Fig. 1 show some of the T4 prereplicative proteins which are synthesized during the first 12 min of infection of E. coli B. Each column represents a separate infection using either a nonsense or deletion mutant of T4. The arrows indicate the gene product which is absent in each infection, when compared with that of a wild type T4Dinfected culture. '  Escherichia coli B was infected with various mutants of T4. The infected cells were labeled with l*C-amino acids for the first 12 min of infection.
The uvows indicate the missing band(s) in each infection. a, b, c, d, e refer to infections carried out on the same day and run on the same gel.
In Table I we have listed the genes whose protein products The sum of the molecular weights of the prereplicative proteins we have identified.
We have listed the molecular weights of (encoded by both mapped and unmapped genes) identified on these proteins and the mutants which were used in the assigngels is about 1,120,OOO. This total accounts for about 17% ments.
In addition, we list several unmapped deletion mutants of the coding capacity of T4 DNA. By the criterion of RNAwhich yield missing (presumably nonessential) prereplicative DNA saturation hybridization, prereplicative T4 RNAs are protein bands.
derived from about 50% of the total DNA (16). Thus, a com- The molecular weights were derived by comparing the electrophoretic migration of T4-specific proteins in SDS-polyacrylamide gels with the migration of the following standards : bovine serum albumin, D-lactoglobulin, catalase, carbonic anhydrase, pepsin, E. coli RNA polymerase, E. coli p factor, hemoglobin, myoglobin, ovalbumin, creatine phosphokinase, and trypsin. The references for published molecular weights are given in the c An additional band disappears. d These mutants of gene 44 appear to be polar on gene 62. e Deletion (39-56)l has been described previously (13). f The assignment for gene 55 was based on '%-amino acid pulses from 9 to 14 min after infection.
Those gels are not shown in Fig. 1. 0 Dr. B. Alberts, personal communication.
plete catalogue of prereplicative T4 proteins on gels is by no means complete.
There still remain 27 genetically defined prereplicative cistrons whose gene products cannot be identified by our present technique.
As more nonsense or deletion mutants become available to us this catalogue will be expanded.