The RNA polymerases encoded by bacteriophages T3 and T7 have similar structures, but exhibit nearly exclusive template specificities. We have determined the nucleotide sequence of the region of T3 DNA that encodes the T3 RNA polymerase (the gene 1.0 region), and have compared this sequence with the corresponding region of T7 DNA. The predicted amino acid sequence of the T3 RNA polymerase exhibits very few changes when compared to the T7 enzyme (82% of the residues are identical). Significant differences appear to cluster in three distinct regions in the amino-terminal half of the protein. Analysis of the data from both enzymes suggests features that may be important for polymerase function. In particular, a region that differs between the T3 and T7 enzymes exhibits significant homology to the bi-helical domain that is common to many sequence-specific DNA binding proteins. The region that flanks the structural gene contains a number of regulatory elements including: a promoter for the E. coli RNA polymerase, a potential processing site for RNase III and a promoter for the T3 polymerase. The promoter for the T3 RNA polymerase is located only 12 base pairs distal to the stop codon for the structural gene.

Wild-type Salmonella typhimurium can use carnosine (,8-alanyl-L-histidine) as a source of histidine, but carnosine utilization is blocked in particular mutants defective in the constitutive enzyme peptidase D, the product of the pepD gene. Biochemical evidence for assigning carnosinase activity to peptidase D (a broadspecificity dipeptidase) includes: (i) coelution of carnosinase and dipeptidase activity from diethylaminoethyl-cellulose and Bio-Gel P-300 columns; (ii) coelectrophoresis of carnosinase and dipeptidase on polyacrylamide gels; and (iii) inactivation of carnosinase and dipeptidase activities at identical rates at both 4 and 42°C. Genetic evidence indicates that mutations leading to loss of carnosinase activity map at pepD. Several independent pepD mutants have been isolated by different selection procedures, and the patterns of peptide utilization of strains carrying various pepD alleles have been studied. Many pepD mutations lead to the production of partially active peptidase D enzymes with substrate specificities that differ strikingly from those of the wild-type enzyme. The growth yields of carnosinase-deficient strains growing in Difco nutrient broth indicate that carnosine is the major utilizable source of histidine in this medium.purity. The Gly-Pro-Ala produced only one ninhydrinpositive spot after descending paper chromatography with 72% phenol and 28% ammonia (4). Analysis by Manuel Ricardo on a Beckman Instruments model 89B automated amino acid sequencer indicated the 361 on August 5, 2020 by guest http://jb.asm.org/ Downloaded from c"Parental culture treated with diethyl sulfate and small colony selected on minimal medium supplemented with excess Gly-Leu (0.2 mM) and a low concentration of L-leucine (0.01 mM).d Other JK strains used in this study and carrying supQ-pro deletions are described by Kemper (9).

The DNA of bacteriophage T3 was characterized by cleavage with seven restriction endonucleases. AvaI, XbaI, Bgm, and HindIII each cut T3 DNA at 1 site, KpnI cleaved it at 2 sites, MboI cleaved it at 9 sites, and HpaI cleaved it at 17 sites. The sizes of the fragments produced by digestion with these enzymes were determined by using restriction fragments of T7 DNA as molecular weight standards. As a result of this analysis, the size of T3 DNA was estimated to be 38.74 kilobases. The fragments were ordered with respect to each other and to the genetic map to produce a restriction map of T3 DNA. The location and occurrence of the restriction sites in T3 DNA are compared with those in the DNA of the closely related bacteriophage T7.T3 and T7 are closely related bacteriophages which share many features but differ in interesting and potentially significant ways (7). For example, both viruses elaborate phage-specified RNA polymerases during infection which, although they are similar, do not transcribe the heterologous DNA efficiently (3, 5). Although the two viruses have similar genetic organizations (1, 7), a detailed description of the physical structure of the genome (e.g., by restriction analysis) existed only for bacteriophage T7 (10, 12). Restriction maps of both viruses would be useful for locating and comparing regulatory signals in the DNAs and could serve as a basis for estimating the evolutionary divergence of the two viruses. In this paper, we describe the derivation of a restriction map for bacteriophage T3 DNA and compare it with the map of T7 DNA. MATERIALS AND METHODSBacterial and phage strains and preparation of DNA. Bacteriophage T3 Hausmann (15) and Escherichia coli B were from the laboratory of E. K. F. Bautz. Deletion mutants of T3 (16) were obtained from F. W. Studier.DNA was isolated from CsCl-purified virus particles as previously described (9). For [nP]DNA, lysates were prepared as previously described (9), and the phage particles were harvested by centrifugation at 35,000 x g for 80 min, suspended in buffer (10 mM Tris, pH 7.5,5 mM Mg9l2, 50 mM NaCI), and clarified by centrifugation at 12,000 x g before isolation of DNA.Cleavage of DNA and electrophoresis of fragments. AvaI, XbaI, KpnI, BglII, HindIII, MboI, and HpaI (11) were purchased from Bethesda Research Laboratories, Rockville, Md. MboI has the same specificity as DpnII, which was used previously to map T7 DNA (10, 11). The conditions of cleavage were those recommended by the supplier. For double digestions, the first enzyme was inactivated by the addition of 10 mM EDTA, followed by heating to 650C for 5 min.The DNA was precipitated with ethanol and sodium acetate, washed with 70% ethanol, dried in vacuo, and taken up in buffer suitable for cleavage by the second enzyme. DNA fragments were analyzed by electrophoresis through agarose or polyacrylamide gels, as previously described (10,12).Isolation ofindividual fragments from agarose gels. Individual fragments were resolved by electrophoresis through 0.6% agarose gels and visualized by sta...