DNA of a new parvo-like virus isolated from the silkworm,Bombyx mori

Parvo-like virus, which was designated as “Ina-flacherie virus (Ina-FV),” was isolated from the silkworm, Bombyx mori, and the properties of its DNA were characterized. Purified Ina-FV had a diameter of 22 ± 0.5 nm and a sedimentation coefficient of 102 S. On density gradient separation in CsCl, particles were found at densities of 1.40 and 1.45 g/ml. The DNA content of Ina-FV was 28 ± 2%. The DNA in low-salt buffer possessed properties typical of a single-stranded (ss) molecule. Double-stranded (ds) DNA was extracted under conditions of appropriate high salt and elevated temperature. Electron microscopical examination revealed that the ds DNA was composed of linear molecules with an average length of 1.7 μm and other less well-defined structures. The linear ds molecule had a molecular weight of about 3.4 × 10⁶ determined by electron microscopy (EM) and agarose gel electrophoresis. When the ds DNA was alkali-denatured and examined in an EM, linear ss molecules with approximate length of 1.7 μm were observed, indicating that the linear ds molecule was formed from the annealing of the linear ss molecules of unit length. These data suggest that Ina-FV is closely related to members of the densovirus subgroup.     

Identification of regions in which positive selection may operate in S-RNase of Rosaceae: Implication for S-allele-specific recognition sites in S-RNase

A stylar S-RNase is associated with gametophytic self-incompatibility in the Rosaceae, Solanaceae, and Scrophulariaceae. This S-RNase is responsible for S-allele-specific recognition in the self-incompatible reaction, but how it functions in specific discrimination is not clear. Window analysis of the numbers of synonymous (d_S) and non-synonymous (d_N) substitutions in rosaceous S-RNases detected four regions with an excess of d_N over d_S in which positive selection may operate (PS regions). The topology of the secondary structure of the S-RNases predicted by the PHD method is very similar to that of fungal RNase Rh whose tertiary structure is known. When the sequences of S-RNases are aligned with the sequence of RNase Rh based on the predicted secondary structures, the four PS regions correspond to two surface sites on the tertiary structure of RNase Rh. These findings suggest that in S-RNases the PS regions also form two sites and are candidates for the recognition sites for S-allele-specific discrimination.     

Studies on the polyhedrosis virus forming polyhedra in the midgut-cell nucleus of the silkworm,Bombyx mori: I. Purification procedure and form of the virion

A procedure for the isolation and purification of a new polyhedrosis virus that forms polyhedra in the midgut-cell nucleus is described. The method is a modification of that used by Hayashi and Bird (1970) for the isolation of free cytoplasmic-polyhedrosis virus (CPV). The purified virions were spherical in shape with several projections that measured 62 nm in diameter. The sedimentation coefficient of this virion was 430 S. These features are very similar to those of CPV. No significant difference was observed between this virus and CPV in the sedimentation profiles of preparations. Electron microscope observations indicated that the purified virus preparations contained many more empty particles than those of CPV.From these results, it is concluded that this virus is quite similar to CPV.     

Virus-specific RNA synthesis in the midgut of silkworm, Bombyx mori, infected with cytoplasmic-polyhedrosis virus

Virus-specific RNA synthesis in the midgut of silkworm infected with cytoplasmic-polyhedrosis virus was investigated under the condition inhibiting host RNA synthesis by actinomycin D injection. Two species of virus-induced RNA were formed; one was sensitive to ribonuclease (RNase) but the other was resistant. The resistant RNA had a sedimentation coefficient of 15 S and was considered as viral progeny with doublestranded RNA. The sensitive RNA, presumably single-stranded RNA, consisted of two classes with 15 S and 22 S sedimentation coefficients. Annealing the single-stranded RNA with heat-denatured CPV-RNA indicated that the single-stranded RNA was transcribed from viral genome RNA. The function of 22 S and 15 S single-stranded RNAs was discussed from the viewpoint of virus multiplication.     

Activation of the prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mamestra brassicae

The effects of JHA (ZR-515) application or brain implantation on metamorphosis and adult development were examined in the last instar larvae and pupae of Mamestra brassicae. When JHA was applied to neck-ligated 4- or 5-day-old larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands taken from 5-day-old larvae, the insects pupated. Dauer pupae and diapausing pupae treated with JHA showed adult development. By contrast, pupation could not be induced by the application of JHA to 2- or 3-day-old neck-ligated larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands from 0-day-old larvae. Implantation of a brain into neck-ligated 3- or 5-day-old larvae (at the beginning of gut emptying and wandering) caused pupation of the host. A similar result was obtained when both a brain and the prothoracic glands from 0- or 5-day-old larvae were implanted into the isolated abdomens of 5-day-old larvae. These results indicate that activation of the prothoracic glands by application of JHA is temporally restricted to the last part of the last larval instar and to the pupal stage, while the activation by prothoracicotropic hormone (PTTH) can occur throughout the last larval instar and the pupal stage. In addition, the implantation of brains or application of JHA to neck-ligated 5-day-old larvae 25 days after ligation seldom induced pupation of the hosts, a result which suggests that larval prothoracic glands maintained under juvenile hormone (JH) or PTTH-free conditions for long periods of time may become insensitive to reactivation by both hormones.     

Comprehensive Structural Analysis of the Genome of Red Clover (Trifolium pratense L.)

With the aim of establishing the basic knowledge and resourcesneeded for applied genetics, we investigated the genome structureof red clover Trifolium pratense L. by a combination of cytological,genomic and genetic approaches. The deduced genome size was440 Mb, as estimated by measuring the nuclear DNA content byflow cytometry. Seven chromosomes could be distinguished bymicroscopic observation of DAPI stained prometaphase chromosomesand fluorescence in situ hybridization using 28S and 5S rDNAprobes and bacterial artificial chromosome probes containingmicrosatellite markers with known positions on a genetic linkagemap. The average GC content of the genomes of chloroplast, mitochondrionand nucleus were shown to be 33.8, 42.9 and 34.2%, respectively,by the analysis of 1.4 Mb of random genomic sequences. A totalof 26 356 expressed sequence tags (ESTs) that were grouped into9339 non-redundant sequences were collected, and 78% of theESTs showed sequence similarity to registered genes, mainlyof Arabidopsis thaliana and rice. To facilitate basic and appliedgenetics in red clover, we generated a high-density geneticlinkage map with gene-associated microsatellite markers. A totalof 7159 primer pairs were designed to amplify simple sequencerepeats (SSRs) identified in four different types of libraries.Based on sequence similarity, 82% of the SSRs were likely tobe associated with genes. Polymorphism was examined using twoparent plants, HR and R130, and 10 F₁ progeny by agarose gelelectrophoresis, followed by genotyping for the primer pairsshowing polymorphisms using 188 F₁ plants from the mapping population.The selected 1305 microsatellite markers as well as the previouslydeveloped 167 restriction fragment length polymorphism markerswere subjected to linkage analysis. A total of 1434 loci detectedby 1399 markers were successfully mapped onto seven linkagegroups totaling 868.7 cM in length; 405 loci (28%) were bi-parental,611 (43%) were specific to HR and 418 (29%) were specific toR130. Each genetic linkage group was linked to a correspondingchromosome by FISH analysis using seven microsatellite markersspecific to each of the linkage groups as probes. Transferabilityof the developed microsatellite markers to other germplasmswas confirmed by testing 268 selected markers on 88 red clovergermplasms. Macrosynteny at the segmental level was observedbetween the genomes of red clover and two model legumes, Lotusjaponicus and Medicago truncatula, strongly suggesting thatthe genome information for the model legumes is transferableto red clover for genetic investigations and experimental breeding.     

Cardiolipin biosynthesis and mitochondrial respiratory chain function are interdependent

Cardiolipin (CL) is an acidic phospholipid present almost exclusively in membranes harboring respiratory chain complexes. We have previously shown that, in Saccharomyces cerevisiae, CL provides stability to respiratory chain supercomplexes and CL synthase enzyme activity is reduced in several respiratory complex assembly mutants. In the current study, we investigated the interdependence of the mitochondrial respiratory chain and CL biosynthesis. Pulse-labeling experiments showed that in vivo CL biosynthesis was reduced in respiratory complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) and oxidative phosphorylation complex V (ATP synthase) assembly mutants. CL synthesis was decreased in the presence of CCCP, an inhibitor of oxidative phosphorylation that reduces the pH gradient but not by valinomycin or oligomycin, both of which reduce the membrane potential and inhibit ATP synthase, respectively. The inhibitors had no effect on phosphatidylglycerol biosynthesis or CRD1 gene expression. These results are consistent with the hypothesis that in vivo CL biosynthesis is regulated at the level of CL synthase activity by the DeltapH component of the proton-motive force generated by the functional electron transport chain. This is the first report of regulation of phospholipid biosynthesis by alteration of subcellular compartment pH.