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RNAs synthesized in Escherichia coli infected with virulent phages T4, T5, T7 and BF23 were labelled with 32PO4 3- after phage infection. [32P]RNAs of low molecular weight were separated by two-dimensional polyacrylamide gel electrophoresis, in which electrophoresis was carried out in two dimensions at different concentrations of acrylamide. The fractionated RNAs were characterized by RNA-fingerprint patterns made after T1 ribonuclease digestion. The two-dimensional gel of 10% yields 20% acrylamide was suitable for RNA of less than 200 nucleotides, while that of 5% yields 10% was preferred for RNAs of about 150--400 nucleotides. With T4 phage, 16 RNA species were separable on a single slab gel. Among those, 11 were identified as the known RNA species, including eight T4 tRNAs, one tRNA precursor and two non-tRNA molecules. In the case of T5 and BF23, more than 20 RNA species were separated on a slab gel; 15 or more RNAs were found in the 4-S RNA region, and several in 5-S and 6-S region. The RNA-fingerprint patterns of many BF23 RNAs were very similar to those of corresponding RNAs of T5. Pseudouridine and ribosylthymidine, minor nucleosides generally present in tRNA, were found in several BF23 4-S RNAs tested. Possibility of those BF23 4-S RNAs as tRNAs is discussed. With phage T7, three RNAs were detected, two of which were much smaller than tRNAs.

An efficient method was devised to isolate temperature sensitive mutants of E. coli defective in tRNA biosynthesis. Mutants were selected for their inability to express suppressor activity after su3(+)-transducing phage infection. In virtually all the mutants tested, temperature sensitive synthesis of tRNA(Tyr) was demonstrated. Electrophoretic fractionation of (32)P labeled RNA synthesized at high temperature showed in some mutants changes in mobility of the main tRNA band and the appearance of slow migrating new species of RNA. Temperature sensitive function of mutant cells was also evident in tRNA synthes: directed by virulent phage T4 and BF23. We conclude that although the mutants show individual differences, many are temperature sensitive in tRNA maturation functions. In spite of much information on the structure and function of transfer RNA (tRNA), our knowledge concerning the biosynthesis of tRNA is relatively poor. It is generally assumed that complete tRNA molecules are made via a series of processing steps from the original transcription products of tRNA genes which are presumably unmodified and longer than mature tRNA molecules. In the case of tyrosine suppressor tRNA of su3(+), an unmodified precursor RNA carrying additional residues at the 3' and 5' ends has been isolated (1,2), and an endonuclease cleaving at the 5' side of this precursor has been identified in E. coli (3). In the case of T4 encoded tRNA, a large precursor molecule for several tRNA's has been reported (4). Some enzymes that catalyze the modifications have also been described (5). However, the over-all picture and the precise mechanisms of tRNA maturation are as yet largely unkown. For study of tRNA biosynthesis in E. coli, a genetic approach may prove useful, as has been the case in other biosynthetic pathways. In order to obtain mutants blocked in any of the intermediary steps of tRNA synthesis, we have developed an efficient selection system that enriches these mutants. Since any mutational block in tRNA biosynthesis might well be lethal, we looked for conditional lethal mutants in which the defect in tRNA synthesis occurs only at high temperature. In this selection system, the su3 gene carried by a temperate phage was newly introduced into cells(su(-)) and those cells incapable of synthesizing su3(+) tRNA at high temperature were selected. Such mutants were easily enriched by using conditions in which cells expressing suppressor activity were killed by two virulent phages. In this communication, we report the method for isolation of mutants and some characterization of tRNA synthesis in these mutants. Recently, Schedl and Primakoff (6) have independently isolated thermosensitive mutants of E. coli defective in tRNA synthesis which may or may not be different types from ours.

About fifty suppressor sensitive (sus) mutants of phage phi80 were isolated after hydroxylamine treatment, and these were classified into fourteen cistrons. In vitro and in vivo complementation experiments revealed that at least five cistrons were concerned with head formation and that at least six cistrons were concerned with tail formation in phi80. Some presumed "early" mutants were also found. Defective lysogens were isolated from a phi80-lysogenic nonpermissive strain using the colicin plate method (Gratia, 1966). In these strains deletions which affected the colicin B receptor gene extended for varying distances into prophage phi80. Marker rescue experiments were carried out with these deletion lysogens by infecting various sus mutants, and the gene order in prophage phi80 was determined. Clustering of all head genes and also of tail genes, as in phage lambda, were demonstrated by the results of the prophage deletion mapping as well as the two-factor crosses performed among the sus mutants. Moreover, the gross gene arrangement of phi80 was also similar to that of phage lambda: namely, the cluster of head genes was found to be located at one end of the vegetative map of phi80, being followed by that of tail genes, and presumed "early" genes are located at the other end of the map.

Growth of phage BF23 was restricted in Escherichia coli K-12 strains carrying a colicin I factor (ColIb); most infected cells lysed early without producing progeny phages. Either addition of chloramphenicol before phage infection or ultraviolet irradiation of phage prevented early abortive lysis, an indication that certain phage functions are required for this phenomenon. Very little or no phage-induced lysozyme was synthesized in the infected ColI(+) cells. This result suggests that early abortive lysis was not due to the lysozyme action. A small fraction (0.05) of BF23-infected ColI(+) cells showed normal phage growth. This "escaped growth" may reflect the physiological state of the host bacteria rather than the heterogeneity of the infecting phage. Host-controlled modification was not observed. A phage mutant, BF23hI, able to grow on ColI(+) cells, was isolated and was characterized to be recessive to the wild-type BF23 in its ability to undergo early abortive lysis. Among the T series phages, T5 induced early abortive lysis, and growth of T5 was restricted upon infection to ColI(+) cells. These results and the other observations, including the occurrence of phenotypic mixing between BF23 and T5, suggest that these two phages are related to each other even though the receptor sites for BF23 and T5 are apparently different.