Itaru Watanabe

Molecular Genetic Analysis of Myelin-Deficient Mice: Shiverer Mutant Mice Show Deletion in Gene(s) Coding for Myelin Basic Protein

Abstract: The gene expression of myelin basic proteins (MBPs) in shiverer mutant mice was investigated by the Northern and Southern hybridization techniques. In the control mice RNA molecules from the brains which were about 2,300 nucleotides in length were hybridized to cDNA of 1.8 kb encoding for a mouse MBP, but RNA from the brains of 3‐week‐old shiverer mutant mice contained no detectable amount of MBP transcripts hybridizing to this probe. Moreover the shiverer mutant mice lost several restriction fragments that hybridized to the same probe in the control mice when each of the five restriction enzymes, i.e., HindIII, PstI, PvuII, AccI, and StuI, was used. These data suggest that the shiverer mutation may correspond to the deletion of a large portion of MBP exon(s) in the gene, and this deletion causes inefficient transcription leading to the depletion of MBPs in the myelin and the dysmyelination observed in these mice.

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).

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.

Relationships among four groups of RNA coliphages based on the template specificity of GA replicase

Abstract In order to elucidate the intergroup relationships among four groups of RNA coliphages, we investigated the template activity of various phage RNAs with GA replicase in an in vitro complementary (minus strand) RNA synthesizing system. Phage RNAs of groups I and II showed almost equal template activities with GA replicase, those of group III revealed no template activity with the same replicase, while those of group IV showed lower but distinct template activity with the same replicase. On this basis, some affinity between group II and IV phage RNAs may exist.

SPECIFIC INHIBITOR FOR RNA REPLICASE

It was thought that multiplication of phage RNA i s independent of the nucleic acid synthesis of the host cell. If this is the case , there may be an inhibitor for phage RNA multiplication which does not interf e re with the host nucleic acid synthesis. The isolation in 1965 of the RNA replicase in bacteriophage Q P by I. Haruna and S. Spiegelmanl permit us to tes t the validity of this hypothesis by enzymatic methods. That is, a specific inhibitor for RNA replicase may be found which will protect the host cell f rom RNA phage infection without any ser ious damage. plant virus containing RNA. case.

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.

Suppression of phototaxis in silkworm larvae

Abstract A study of the olfactory and visual organs of the larvae of the silkworm ( Bombyx mori ), using electrophysiological and surgical techniques, indicates that olfactory stimuli from mulberry leaves, conducted through the antennae or the maxillary palps, cause continuous suppression of the phototactic response, and that the central nervous system plays an important role in this ‘control’ of phototaxis. Such phototactic suppression lasts for 30 h in fifth instar larvae, even after mulberry leaves have been removed.

Three complementation subgroups in group IV RNA phage SP

Abstract We isolated twelve suppressor sensitive ( sus ) mutants from the RNA phage SP which belongs to group IV and classified them into three cistrons (genes 1, 2, and 3) by complementation tests. Using representative mutants of each cistron, synthesis of infectious RNA, phage antigen (serum blocking power) and defective particles under nonpermissive conditions were examined. Gene 1 mutants had a defect in maturation protein synthesis, gene 2 mutants in coat protein synthesis, and gene 3 mutants in RNA synthesis, respectively. Thus, group IV RNA phage SP has (at least) three genes corresponding to RNA replicase, coat protein, and maturation protein, as already shown in group I (f2, R17) and group III (Qβ) RNA phages. From intergroup complementation tests between several sus mutants of groups I, III, and IV, Qβ(III) had a fairly close relationship to SP(IV), while f2(I) had no such close relationships to Qβ(III) or SP(IV). These are consistent with the results of grouping by several biological and physicochemical criteria and support the idea that groups I and II may be assembled into one group and groups III and IV into another.

Continuous Survey of the Distribution of RNA Coliphages in Japan

In order to demonstrate the stability and continuity of RNA coliphages (phages) in their natural habitats, we investigated the amount and group types of RNA phages in sewage samples collected continuously from domestic drainage in Japan proper and islands in the seas adjacent to Japan (abbreviated simply as islands, hereafter) over a 5‐yr period from 1973 to 1977. It was found that the frequencies of isolation of RNA phages were fairly high and constant. The group types of RNA phages isolated were also stable in the three cities, Choshi, Niigata, and Toyama in Japan proper. The average for the three cities was group II: III = 3: 1. The investigation in islands revealed that the frequencies of isolation of RNA phages were fairly high as in the case of the above three cities in Japan proper and the group types of RNA phages isolated were also stable. That is to say, group II phages were predominant on Rishiri Island, Rebun I., Iki I., and Tsushima I., which are located relatively near to mainland Japan, while group III phages were predominant on Amamiohshima I., mainland Okinawa, Ishigakijima I., and Iriomotejima I., which are located south of Kyushu. It can thus be said that the RNA phages in the domestic drainage of Japan proper and islands remained more or less stable over at least the 5‐yr period, and an apparent difference in the geographical distribution of RNA phages in Japan exists between Kyushu and Amamiohshima I.