Yoshio Inokuchi

Analysis of the complete nucleotide sequence of the group IV RNA coliphage SP.

We report the nucleotide sequence of the Group IV RNA bacteriophage SP. The entire sequence is 4276 nucleotides long. Four cistrons have been identified by comparison with the related Group III phage Q beta. The maturation protein contains 449 amino acids, the coat protein contains 131 amino acids, the read-through protein contains 330 amino acids and the replicase beta-subunit contains 575 amino acids. SP is 59 nucleotides longer than Q beta. We have analyzed both sequence and structural conservation between SP and Q beta and shown that the sequences for the coat and central region of the replicase are strongly conserved between the two genomes. We also show that the S and M replicase binding sites of Q beta are strongly conserved in SP. Interestingly, the base composition of SP and Q beta differ significantly from one another, and most of the differences can be accounted for by a strong preponderance of U in the third position of each codon of Q beta relative to SP. We also compare conserved hairpins associated with potential coat protein and replicase binding sites.

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.

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).

Distribution of RNA Coliphages in Senegal, Ghana, and Madagascar

The distribution patterns of RNA coliphages (phages) in Senegal, Ghana, and Madagascar were investigated by collecting sewage samples from domestic drainage in November, 1980. In Senegal, among 65 sewage samples collected mainly from Dakar and its vicinity, 14 (22%) contained RNA phages (16 strains). By serological analysis, 13 of the 16 strains were found to belong to group III. This is consistent with the distribution pattern of RNA coliphages in tropical and subtropical regions of Asia. In Ghana, however, among 106 samples collected from Accra, Suhum, and their vicinities, only seven (7%) contained RNA phages (seven strains) (groups I, II, and III [1:3: 3]). In Madagascar, among 124 samples collected from Antananarivo, Moramanga, and their vicinities, seven (6%) contained RNA phages (seven strains) (groups I, II, III, and IV [1:1:1:4]). In spite of the low isolation frequency, it can be said that Madagascar appears to have a unique distribution pattern (abundance of group IV phages) which differs from that of any other countries we have examined. The generality of the distribution pattern of RNA phages in the tropical region (abundance of group III phages) was thus verified at least in Senegal.

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).

Cloning of TPA-inducible early (TIE) genes by differential hybridization using TPA-Nonresponsive variant of mouse 3T3-L1 cells

The tumor promoter 12-O -tetradecanoylphorbol-13-acetate (TPA) induces DNA synthesis in quiescent 3T3-L1 cells but not in its variant VT-1 cells. A λgt10 cDNA library was constructed using poly(A)+ RNA from 3T3-L1 cells that were stimulated by TPA for 20 min. Radioactive cDNA probes were prepared from mRNAs of TPA-treated 3 T3-L1 and VT-1 cells and used for screening of the 3T3-L1 cDNA library by differential hybridization. Nine of 6000 phage plaques hybridized only to the 3T3-L1 cDNA probe. Analysis of the nucleotide sequence of five of these clones indicated a high degree of homology with human or mouse type I and type III collagen genes. Three other independent clones showed no homology with any known DNA sequences. These isolated clones of TPA-inducible early (TIE) genes may be useful to study the signal transduction pathway of phorbol esters.