The Schizosaccharomyces pombe cdc6 gene encodes the catalytic subunit of DNA polymerase δ

The cdc6 mutants of Schizosaccharomyces pombe have been classified as being defective in progression through the G2 phase of the cell cycle. We cloned an S. pombe gene that could complement the temperature-sensitive growth of the cdc6-23 mutant. Unexpectedly, the cloned gene was allelic to pol3, which encodes the catalytic subunit of DNA polymerase δ. Integration mapping confirmed that cdc6 and pol3 are identical. The cdc6-23 mutant carries one amino acid substitution in the conserved N3 region of Pol3. Received: 17 October 1996 / Accepted: 19 November 1996     

Refined genetic analysis of the region II che mutants in Salmonella typhimurium

Summary From a detailed complementation analysis of the region II che mutants of Salmonella typhimurium, we have located five che genes, cheA, cheW, cheR, cheB, and cheY. We have shown that corrections are required in the previous assignment of the mutations in four strains: both SL2514 and SL2515 which have been reported to be cheY mutants are cheR mutants, SL2539 is not a cheA but a cheW mutant, and ST171 which has been reported to be a cheZ mutant is a double mutant with defects in both cheA and cheB. Since ST171 is the only cheZ mutant so far isolated, the idea that the cheZ gene might play an essential role in chemotaxis in S. typhimurium as in Escherichia coli has lost its experimental basis. Furthermore, a number of deletion mutants in region II resulting from the excision of Tn10 have been isolated and analysed. From these experiments, we propose that the gene order in region II is flaK-flaE-motA-motB-cheA-cheW-cheR-cheB-cheY-flaM-flaC, which is identical with that in E. coli.     

Locations of hook-associated proteins in flagellar structures of Salmonella typhimurium.

Hooks of the flagella of Salmonella typhimurium were purified from an flaL mutant. Hook-associated proteins, namely HAP1, HAP2, and HAP3, were separated from them, and the antibody against each HAP was prepared. By immunoelectron microscopic observation, these three kinds of antiHAP antibodies were found to bind on the distal ends of hooks of filamentless mutants consistently with their composition of HAPs. The antiHAP2 antibody bound to the very tops of the claw-shaped ends of the hooks which contain all three HAPS. The antibodies against HAP1 and HAP3 bound to the basal areas and the middle areas, respectively, of the claw-shaped ends. The order of disassembly of the component proteins by heat treatment of the hook structure from the filamentless mutants was (HAP2, HAP3) greater than HAP1 greater than hook protein. These observations were consistent with our layered structure model: HAP1, HAP3, and HAP2 are assembled at the distal end of the hook in this sequence. All three HAPs were detected in the hook-filament complexes prepared from a flagellate strain. When the hook-filament structure was treated with antibody against HAP1 and with the anti-rabbit immunoglobulin G antibody, the antibody aggregate was observed in the region corresponding to the boundary between filament and hook. This observation strongly suggests that HAP1 is the protein connecting filament with hook. The locations of HAP2 and HAP3 in the hook-filament structure were not clarified with the same procedure.     

Genetic analysis of three additional fla genes in Salmonella typhimurium

In Salmonella typhimurium, 27 fla genes responsible for formation of flagella have been identified and assigned to three regions on the genetic map, termed fla regions I to III. By genetic analysis of 1984 non-flagellate mutants obtained from a phase-1 stable strain of S. typhimurium, SJW1103, three additional fla genes were identified; one, termed flaW, was assigned to fla region I and the other two, termed flaV and flaX, to fla region III. By intergeneric complementation tests, the flaW, flaV and flaX genes were shown to be functionally homologous with flaS, flbC and flaP of Escherichia coli, respectively. Electron microscopy showed that flaW and flaV mutants carried hook-basal body structures.     

Genetic analysis of H2, the structural gene for phase-2 flagellin in Salmonella

Non-flagellate H2 mutants were isolated from a phase-2 stable strain, SJW806 H1-gt- H2-enxon vh2-, a derivative of Salmonella typhimurium. By transductional crosses a deletion map and a recombination map of the H2 gene were made. There are three regions especially rich in nonflagellate mutational sites. By the use of the deletion map, mutational sites of 21 flagellar shape mutants were also determined. Most of them were located at two regions which coincide with two of the three regions rich in non-flagellate mutational sites. A gene, vh2, is closely linked to the promoter side of the H2 gene. Three-factor transductional crosses showed that the vh2 gene was on the left of the H2 gene in the present map. The H2 gene forms part of an operon with the distal gene rh1 which specifies the H1 repressor. Thus, a polarity effect of the H2 mutations on the expression of the rh1 gene was examined by observing whether a wild-type H1 allele introduced into the H2 mutants was expressed or not. Many of the H2 mutations were polar, and most of the strongly polar mutations were located in the left (promoter-proximal) half of the H2 gene, while most of the mutations in the right half of the gene were weakly polar or non-polar.     

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

A new class of site-specific endodeoxyribonucleases. Endo.Sce I isolated from a eukaryote, Saccharomyces cerevisiae

We had found that yeasts had intracellular endodeoxyribonucleases that cut phage DNA into a set of double-stranded fragments with discrete chain lengths. We purified one of them to apparent homogeneity from Saccharomyces cerevisiae and designated it Endo.Sce I. Sequence analysis around 5 cleavage sites in plasmid DNA and phage DNA revealed that Endo.Sce I cuts a defined phosphodiester bond in each strand of double helix at the cleavage sites and produces free cohesive ends consisting of 4 nucleotides protruding at 3'-termini. However, unlike in the case of prokaryotic type II-restriction endonucleases, (i) Endo.Sce I seems to consist of two nonidentical subunits, (ii) no common palindrome or consensus sequence including more than 5 base pairs is detected at or near these cleavage sites, and (iii) Endo.Sce I can cut the DNA isolated from the cells that produced Endo.Sce I. All of the 5 cleavage sites are included in inverted repeats, but these inverted repeats are variable in size, nucleotide sequence, and distance between repeating units. An inverted repeat itself is not a structure recognized by Endo.Sce I. This study shows that Endo.Sce I is the first example of eukaryotic site-specific endonuclease and has properties, as described above, which distinguish it from prokaryotic restriction endonucleases.     

Gravitropism of maize and rice coleoptiles: dependence on the stimulation angle

Gravitropism of maize and rice coleoptiles was investigated with respect to its dependence on the angle of displacement or the initial stimulation angle (ISA). Close examination of curvature kinetics and the response to a drop in stimulation angle (SA) indicated that the gravtropic response during an early but substantial part of the curvature development is directly related to the ISA, there being no effect of the reduction of SA resulting from the curvature response itself. On the basis of this finding, the relationship between the steady SA and the curvature rate was determined. In maize, the curvature rate increased linearly with the sines of SAs up to an SA of 90 degrees. Rice coleoptiles, however, showed a saturation curve in the same range of SAs. The saturation profile was nearly identical between coleoptiles grown in air and those submerged in water, although the latter elongated much faster. Rice coleoptiles appeared to be far more sensitive to gravity than maize coleoptiles. It is concluded that the sensitivity to gravity, assessed through dependence on ISA, is a property inherent to a given gravitropic organ. Long-term measurements of curvature indicated that the coleoptiles bend back past the vertical. This overshooting was marked in submerged rice coleoptiles.