Effects of Inhibition and Restoration of Protein Synthesis on the Replication of the R Factor Rts1 in Proteus mirabilis

The effect of inhibition of protein synthesis on the replication of the R factor Rts1 in Proteus mirabilis was examined by using the technique of CsCl density gradient centrifugation. Only 12% of the copies of Rts1 were found to replicate during amino acid starvation, whereas there was a 30% increase in the amount of P. mirabilis chromosomal deoxyribonucleic acid (DNA) during the same period. Essentially the same amount of Rts1 and host chromosome replication was observed when chloramphenicol was used to inhibit protein synthesis. The replication of Rts1 DNA was also examined in experiments in which cultures were starved for amino acids in (14)N-labeled medium and then transferred to (15)N-labeled medium containing the required amino acids. These experiments showed that Rts1 replication took place throughout the first generation in (15)N-labeled medium and that each copy of Rts1 was replicated one time during the first generation of chromosomal DNA synthesis in (15)N-medium.     

Solution structures and DNA binding properties of the N-terminal SAP domains of SUMO E3 ligases from Saccharomyces cerevisiae and Oryza sativa

SUMO E3 ligase of the Siz/PIAS family that promotes sumoylation of target proteins contains SAP motif in its N-terminal region. The SAP motif with a consensus sequence of 35 residues was first proposed to be as a new DNA binding motif found in diverse nuclear proteins involved in chromosomal organization. We have determined solution structures of the SAP domains of SUMO ligases Siz1 from yeast and rice by NMR spectroscopy, showing that the structure of the SAP domain (residues 2-105) of rice Siz1 is a four-helix bundle with an up-down-extended loop-down-up topology, whereas the SAP domain (residues 1-111) of yeast Siz1 is comprised of five helices where the fifth helix alpha5 causes a significant change in the alignment of the four-helix bundle characteristic to the SAP domains of the Siz/PIAS family. We have also demonstrated that both SAP domains have binding ability to an A/T-rich DNA, but that binding affinity of yeast Siz1 SAP is at least by an order of magnitude higher than that of rice Siz1 SAP. Our NMR titration experiments clearly showed that yeast Siz1 SAP uses alpha2-helix for DNA binding more effectively than rice Siz1 SAP, which would result from the dislocation of this helix due to the existence of the extra helix alpha5. In addition, based on the structures of the SAP domains determined here and registered in Protein Data Bank, general features of structures of the SAP domains are discussed in conjunction with equivocal nature of their DNA binding.     

Fruit-body production of test cultures ofFlammulina velutipes preserved for seven years by freezing at three different temperatures

Two strains ofFlammulina velutipes were cultured on PDA plates, and mycelial disks punched out using a cork borer were used for preservation. Five disks of a strain were put into a vial containing one of three cryoprotectants, 10% glycerol, 5% DMSO or 10% polyethylene glycol. Vials were then stored for 7 yr at −20°C, −85°C or liquid nitrogen temperature. The mycelial growth on PDA plates of the cryopreserved mycelial disks, as well as the usual subcultures, were tested two times. After the second test, spawns were prepared for fruit-body production tests by bottle cultivation from selected plates of the second growth tests. The yields of fruit-bodies varied among the cultures derived from the mycelial disks of the same strain preserved under different conditions. Variation in yields was observed even among the mycelial disks preserved at liquid nitrogen temperature, although the range of yield variation was narrower. The yield variation was obvious for the cultures which showed large retardation in the growth test. Four mycelial disks out of the six preserved at −20°C showed higher yields than those preserved at other temperatures. Among the cultures derived from strain FMC224, the control cultures preserved by subculture showed the lowest yield.     

Functional diversification of kir7.1 in cichlids accelerated by gene duplication

Mutation in the inward rectifier potassium channel gene, kir7.1, was previously identified as being responsible for the broader stripe zebrafish skin pattern mutant, jaguar/obelix. An amino acid substitution in this channel causes a broader stripe pattern than that of wild type zebrafish. In this study we analyzed cichlid homologs of the zebrafish kir7.1 gene. We identified two kinds of homologous genes in cichlids and named them cikir7.1 and cikir7.2. Southern hybridization using cichlid genome revealed that cichlids from the African Great Lakes, South America and Madagascar have two copies of the gene. Cichlids from Sri Lanka, however, showed only one band in this experiment. Database analysis revealed that only one copy of the kir7.1 gene exists in the genomes of the teleosts zebrafish, tetraodon, takifugu, medaka and stickleback. The deduced amino acid sequence of cikir7.1 is highly conserved among African cichlids, whereas that of cikir7.2 has several amino acid substitutions even in conserved transmembrane domains. Gene expression analysis revealed that cikir7.1 is expressed specifically in brain and eye, and cikir7.2 in testis and ovary; zebrafish kir7.1, however, is expressed in brain, eye, skin, caudal fin, testis and ovary. These results suggest that gene duplication of the cichlid kir7.1 occurred in a common ancestor of the family Cichlidae, that the function of parental kir7.1 was then divided into two genes, cikir7.1 and cikir7.2, and that the evolutionary rate of cikir7.2 might have been accelerated, thereby effecting functional diversification in the cichlid lineage. Thus, the evolution of kir7.1 genes in cichlids provides a typical example of gene duplication--one gene is conserved while the other becomes specialized for a novel function.     

Occurrence of Chloramphenicol-acetylating Enzymes in Various Gram-negative Bacilli

The occurrence of a chloramphenicol-acetylating enzyme, similar to that found in Escherichia coli, carrying an R factor was investigated in various gram-negative bacilli. The acetylated products of chloramphenicol were identified by chromatography and quantitatively assayed after benzene extraction. The investigated strains were of the Salmonella-Arizona group, the Klebsiella-Aerobacter group, Serratia marcescens, the Proteus group, and Pseudomonas aeruginosa, most of which were isolated from 1947 to 1957. Both chloramphenicol-sensitive and -resistant strains were included, but none of them was able to transfer chloramphenicol resistance by conjugation. In the Proteus group, a significant level of a chloramphenicol-acetylating enzyme was found in most strains, whether they were sensitive or resistant to chloramphenicol; the resistant strains showed higher levels of the enzyme. Some chloramphenicol-sensitive strains lacked this enzyme. Only the sensitive strains containing the enzyme could easily produce chloramphenicol-resistant mutants with higher enzyme activity. Thus, the chloramphenicol resistance of this group can be reasonably explained on the basis of the chloramphenicol-acetylating enzyme. All of the Pseudomonas aeruginosa strains were resistant to chloramphenicol, and most strains showed low levels of the enzyme (which, however, did not appear sufficient to explain their resistance). All of the strains of the other groups (except one strain of Enterobacter cloacae) lacked the enzyme, although most strains of the Klebsiella-Aerobacter group and of S. marcescens were resistant to chloramphenicol. With respect to the origin of the resistance gene of the R factor, it is noteworthy that the strains of Proteus mirabilis isolated in 1947 possessed this enzyme before the discovery of chloramphenicol.     

Effects of reserpine on dopamine metabolite in the nucleus accumbens and locomotor activity in freely moving rats

The effect of reserpine (2 mg/kg i.p.) on both locomotor activity and the turnover of dopamine metabolite in the rat nucleus accumbens was estimated by using an activity monitor (Animex) and by in vivo brain microdialysis. Three to five hours after reserpine administration locomotor activity was reduced and there was a concomitant increase in the level of the dopamine metabolite, homovamillic acid. These findings suggest that depletion of dopamine from the nucleus accumbens may result in decreased locomotor activity. The data support the notion that dopamine in this tissue contributes to the control of locomotion.     

Mannose-Binding Lectin Inhibits the Motility of Pathogenic Salmonella by Affecting the Driving Forces of Motility and the Chemotactic Response

Mannose-binding lectin (MBL) is a key pattern recognition molecule in the lectin pathway of the complement system, an important component of innate immunity. MBL functions as an opsonin which enhances the sequential immune process such as phagocytosis. We here report an inhibitory effect of MBL on the motility of pathogenic bacteria, which occurs by affecting the energy source required for motility and the signaling pathway of chemotaxis. When Salmonella cells were treated with a physiological concentration of MBL, their motile fraction and free-swimming speed decreased. Rotation assays of a single flagellum showed that the flagellar rotation rate was significantly reduced by the addition of MBL. Measurements of the intracellular pH and membrane potential revealed that MBL affected a driving force for the Salmonella flagellum, the electrochemical potential difference of protons. We also found that MBL treatment increased the reversal frequency of Salmonella flagellar rotation, which interfered with the relative positive chemotaxis toward an attractive substrate. We thus propose that the motility inhibition effect of MBL may be secondarily involved in the attack against pathogens, potentially facilitating the primary role of MBL in the complement system.     

Localization of Rh1(D), 2(C), 3(E), 4(c), 5(e) and 25(LW) antigens of human Rh blood groups in fetal erythrocyte membranes

The fetal erythrocyte membranes were partially solubilized with Triton X-100 at the low concentration (0.5%). The localizations of Rh1(D), 2(C), 3(E), 4(c), 5(e) and 25(LW) were investigated. Using hemagglutination inhibition assay, Rh1(D) antigen activity was observed in the Triton-treated membrane (Triton shell) containing mainly band 1, 2 (spectrin), band 5 (actin), band 4.1 and a part of band 3, while Rh2(C), 3(E), 4(c), 5(e) and 25(LW) antigens were detected in the supernatant containing band 3, 6, 2.2, 2.3 and 4.2. It is suggested that: Rh1(D) antigen would associate with cytoskeleton matrix of fetal erythrocyte membranes; Rh1(D) and Rh25(LW) antigens might be integral membrane proteins, while Rh2(C), 3(E), 4(c) and 5(e) antigens would be surface membrane proteins which are easily released from membranes by EDTA, mercaptoethanol and alkaline treatments.