Akikazu Hirashima

Grouping of RNA coliphages based on analysis of the sizes of their RNAs and proteins.

Abstract In order to elucidate the intergroup relationships among four groups of RNA coliphages (RNA phages), we studied the sizes of their RNAs by measuring the sedimentation velocity of the RNA in a sucrose density gradient and the electrophoretic mobility of the RNA and those of proteins in polyacrylamide gel. The RNAs of group I, II, III, and IV phages (including serological intermediates) were found to have sedimentation coefficients of 24, 23, 27, and 28 S by sucrose density gradient centrifugation analysis and to have average molecular weights of 1.21, 1.20, 1.39, and 1.42 × 106 daltons by gel electrophoretic analysis, respectively. In the virions of group I and II phages, there were two kinds of protein (maturation protein and coat protein). In those of group III and IV phages, an additional protein, read-through (IIb or Al) protein (average molecular weights: 3.85 × 104 for group III, and 3.90 × 104 for group IV phages) was detected. The average molecular weights of coat protein from groups I, II, III, and IV were 1.40, 1.29, 1.69, and 1.73 × 104, respectively. Those of maturation protein were 4.48, 4.45, 4.50, and 4.8 × 104, respectively. Read-through protein was synthesized not only in cells infected with group III and IV phages, but also in a cell-free protein synthesizing system directed by groups III and IV phage RNAs. These results indicate that a distinct difference (about 20%) in molecular size of RNA exists between groups I and II and groups III and IV, which reflects the presence of readthrough protein in groups III and IV. The above results suggest that the molecular sizes of RNAs and virion proteins may offer a useful means for grouping RNA phages, because the present results were in good agreement with those of grouping of RNA phages based on serological property. In this respect, the serologically intermediate phages, JP34 and MX1, were classified into groups II and IV, respectively.

Nucleotide sequence from the ssRNA bacteriophage JP34 resolves the discrepancy between serological and biophysical classification

The nucleotide sequence of the coat and lysis genes of the single-stranded RNA bacteriophage JP34 is presented. Serological inactivation studies classified this phage as an intermediate between groups I and II. We show that the nucleotide similarity with group I is less than 45% but more than 95% for group II, classifying JP34 as a member of group II. The altered serotype of JP34 is most likely due to the change of three critical amino acids of the coat protein to residues present in group I phage MS2 at the homologous positions. Serological characterization of RNA bacteriophages is thus not unambiguous. Phylogenetic sequence comparison between JP34, GA, and MS2 confirms the existence of a conserved helix in the coat gene of group I and group II phages. We also show that the JP34 coat and lysis genes can be expressed in cDNA clones and that the translation of the lysis gene is coupled to coat gene translation analogous to the regulation found in the group I phages.

Comparison of the nucleotide sequences at the 3′-terminal region of RNAs from RNA coliphages

Abstract In order to study the genealogical relationships among four groups (I to IV) of RNA coliphages, we sequenced 200 to 260 nucleotides from the 3′ termini of 14 phage RNAs according to the method of Sanger et al. (1977), and compared the results. It was found that the sequences of phage RNAs in the same group were extremely homologous (about 90%). On the other hand, when the sequences were compared with those from other groups, they were seen to be only about 50 to 60% homologous between group I and group II, and about 50% homologous between group III and group IV. In other combinations, such as groups I (or II) and III, and groups I (or II) and IV, however, the extent of homology was small. Furthermore, the sequences up to 30 residues from the 3′ end were found to be about 90% homologous between groups I and II, and between groups III and IV. These results confirm our previous findings, that the sequences located in the proximity of the 3′ end of phage RNA in the same group were well-conserved (Inokuchi et al., 1979), and that close relationships exist between groups I and II, and between groups III and IV (Furuse et al., 1979).

Enhancing effect of magnesium ion on cell-free synthesis of read-through protein of bacteriophage Qβ

Abstract This report describes the enhancing effect of magnesium ion on the synthesis of read-through protein of bacteriophage Qβ in a cell-free protein synthesizing system from E. coli . At 6 mM of magnesium acetate, the major product was coat protein. At 12 mM of magnesium, it was replaced by read-through protein. This enhanced synthesis was substituted by the addition of 0.25 mM of spermine or 1 mM of spermidine to 6 mM of magnesium. These results suggest that magnesium or combination of magnesium and polyamines causes leaky termination at the end of the coat protein cistron of Qβ-RNA.

Characterization of an Intergroup Serological Mutant from Group II RNA Phage GA

Starting from the group II RNA phage GA which has an amber mutation in the maturation protein cistron, a spontaneous mutant of group II phage GA, whose serological and electrophoretic properties became similar to those of group I phage MS2, was isolated and analyzed. The mutant has now become sensitive to anti‐MS2 serum and resistant to anti‐GA serum. Analysis of the nucleotide sequence of the coat protein gene revealed that G↔A transition was the main change. The deduced amino acid sequence showed that five amino acids were substituted in the mutant, and three of the five became identical to MS2, resulting in increased molecular weight of the coat protein. However, it did not complement MS2. These results suggested that the serological change from group II phage GA type to group I phage MS2 type is induced spontaneously at high frequency by minor nucleotide changes in coat protein gene, and confirmed the previous results at the RNA level that MS2 and GA were related although the closeness between them seems somewhat remoter than that of groups III and IV (18, Inokuchi et al, unpublished data for the nucleotide sequence of group IV phage SP).

Electrophoretic properties of RNA coliphages.

Miyake et al (9, 10, 12) previously reported that RNA coliphages showed group specific electrophoretic profiles on cellulose acetate membranes. Based on this characteristic, we extensively studied the interand intra-group relationships of RNA coliphages classified into four groups (I to IV) according to serological and certain physicochemical properties (5, 9, 13). All RNA phages used here, except for MS2 (1), f2 (7), and R17 (11), were originally isolated in our laboratory and were classified into four groups (2-5,9, 12, 13). Growth of the phages was carried out as described previously (8). Electrophoresis of the phages on cellulose acetate membranes (Separax, jookoo Sangyo Co., japan) was performed as described by Miyake et al (10) with slight modifications (see legend to Fig. 1). As shown in Fig. 1, among the four groups, the electrophoretic profile of group I phage was more group specific than that of the other groups. For example, all the group I phages tested here migrated to the anode (+) side (Fig. 1(A)). Among these phages, MS2 and FRI migrated to the position of fraction 7. Phages f2, R17, ZR, and BOI moved slightly faster than MS2, and jP50l to the furthest (+) side. These results suggest that jP50l is apparently distinct from other group I phages [subgroup I(a)] in this character and should be assigned to a subgroup of I(b). This is supported by the fact that jP50l is serologically different from other group I phages (Furuse et al, unpublished data). Most of the group II phages such as GA, SD, THI, BZ13, and KUl migrated to the position of fraction 3 in a direction opposite to the group I phages (Fig. 1 (B)). It should be noticed in this panel that phages jP34 and jP500, which are serologically close to the group I phage MS2 (2) but classified into group II on the basis of other characters (5,6), moved to a position adjacent to that of MS2. On this basis, group II phages can be divided into two subgroups: (a) GA, SD, THl, BZ13, and KUl, and (b) jP34 and jP500. The electrophoretic profiles of group III phages were slightly complicated. As shown in Fig. 1(C), phages Qf3 and VK migrated to the cathode (-) side, whereas TW18 and ST migrated to the (+) side. Although the mobility of Qf3 was almost the same as that of VK, the mobility of ST was distinctly different from that of TW18. Group III phages can thus be separated into three subgroups on the basis of this character as follows: (a) Qf3 and VK, (b) TW18, and (c) ST.

The Host-Dependent Restriction of Growth of an RNA Coliphage FI

Phage FIC is a spontaneous host‐dependent mutant of phage FI which is classified into the fourth group of RNA Escherichia coli phages (RNA coliphages). The mutant phage (FIC) grows normally in E. coli strain Q13 (permissive host), but poorly in strain A/λ (non‐permissive host) (9). Attempts to elucidate the regulatory mechanism of growth of the mutant phage in the non‐permissive host revealed the following: (a) growth of the mutant phage was specifically restricted in E. coli strains that have certain suppressor genes for amber mutation; (b) the mutant phage RNA (FIC‐RNA) could not produce progeny in the spheroplasts of the non‐permissive host; (c) adsorption of the mutant phage to, and penetration of the mutant phage RNA into, the non‐permissive host were normal; and (d) biosynthesis of the phage‐specific late protein and RNA did not occur in the non‐permissive host. Based on these results we conclude that phage FIC is a spontaneous azure‐type mutant of the fourth group of RNA coliphage FI.

Analysis of Coat Proteins from Group IV RNA Coliphages

At present, RNA coliphages can be divided into four groups (I to IV) on the basis of serological and several physicochemical properties of the phage particles (1, 11, 13). Some of the coat proteins from group I and III phages have been sequenced (14), and the amino acid composition of the coat proteins in group II phages is known (12). However, analysis of the coat proteins of group IV phages has not been reported. In this paper, we report the amino acid composition and peptide map analysis of the coat proteins of group IV phages SP, FI, and ID2. Phages SP, FI, and ID2 were grown separately in Escherichia coli A/). or Ql3 in peptone-glucose medium supplemented with 0.25% yeast extract and 10 mM CaCh, and purified as described previously (7). Purification of the coat protein was performed as described by Hofstetter et al (8). The N-terminal amino acids of the coat proteins were determined according to the method of Gray (4) and the Ctermini were determined with the use of carboxypeptidase A and Y (6). The amino acid composition of the coat proteins was determined with an amino acid analyzer after the proteins were hydrolized with 6 N HCl. For the peptide map analysis, a mixture of radioactive and nonradioactive proteins was digested with TPCK-trypsin. Then the tryptic peptides were separated by the two-dimensional peptide mapping method. First, we examined the N(Fig. 1) and C(Fig. 2) termini of the coat proteins of these three phages. Since the N-termini of the coat proteins of groups I to III phages are known to be alanine (12), we expected them to be alanine in group IV phages also. As seen in Fig. lA, when the mixture of the hydrolysates of dansylated (DNS-) coat proteins ofID2 and QfJ (a group III phage) was developed by two-dimensional ascending chromatography, a major spot appeared at the DNSalanine position. To confirm this, commercial DNS-alanine and a sample of ID2 were spotted. The result again indicated alanine (Fig. IB). We therefore concluded that the N-terminus of the ID2 coat protein is alanine. Similarly, the Ntermini of SP and FI coat proteins were found to be alanine (Fig. 1, C and D). Figure 2 shows the release of amino acids from the ID2 coat protein digested with carboxypeptidase Y to identify the amino acid sequence of the C-terminal side of the coat protein. Tyrosine was first released in the early stage of incubation, fol-