Spontaneous tandem sequence duplications reverse the thermal stability of carboxyl-terminal modified 3-isopropylmalate dehydrogenase.

A mutant strain of Thermus thermophilus which contains deletions in the 3'-terminal region of its leuB gene showed a temperature-sensitive growth phenotype in the absence of leucine. Three phenotypically thermostable mutants were isolated from the temperature-sensitive strain by spontaneous evolution. Each pseudorevertant carried a tandem sequence duplication in the 3' region of its leuB gene. The mutated 3-isopropylmalate dehydrogenases encoded by the leuB genes from the pseudorevertants were more thermostable than the enzyme from the temperature-sensitive strain. Structural analyses suggested that the decreased thermostability of the enzyme from the temperature-sensitive strain was caused by reducing hydrophobic and electrostatic interactions in the carboxyl-terminal region and that the recovered stability of the enzymes from the pseudorevertants was due to the restoration of the hydrophobic interaction. Our results indicate that tandem sequence duplications are the general genetic way to alter protein characteristics in evolution.     

Nuclear behavior during the formation of appressoria byAlternaria alternata

Nuclear behavior in the developmental process of appressoria inAlternaria alternata was investigated. In pregerminated conidia, approximately 94% of the conidial cells were uninucleate. The migration of a nucleus into an elongating germ tube from a germinating conidium was confirmed after 2h of incubation at 24±1°C in PDB. Peak frequencies of binucleate and trinucleate germ tubes were detected 1 and 2h after the peak frequency of uninucleate germ tubes, respectively. Four-and five-nucleate germ tubes did not show marked peak frrequencies. A marked peak frequency of the six-nucleate germ tubes occurred about 1 h after the peak frequency of the trinucleate germ tubes, suggesting that the nuclei in the trinucleate germ tubes each divided once within 1 h. The significance of early establishment of multinucleate appressorial cells in the colonization of host plants by pathogenicA. alternata was discussed.     

Two chlorophyll-binding subunits of the photosystem 2 reaction center complex isolated from the thermophilic cyanobacterium Synechococcus sp

The reaction center of photosystem 2 has been highly purified from digitonin-solubilized thylakoid membranes of the thermophilic cyanobacterium Synechococcus sp. by means of sucrose density gradient centrifugation and electrophoresis on polyacrylamide gels containing digitonin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated reaction center complex yielded four chlorophyll a proteins named CP2-a, CP2-b, CP2-c, and CP2-d. When reelectrophoresed, CP2-a was transformed to CP2-d, and CP2-b was converted to CP2-a and CP2-d. The reaction center complex consisted of two major polypeptides of 47,000 and 40,000 Da and several minor polypeptides. CP2-b contained a 47,000-Da polypeptide together with 66,000- and 31,000-Da polypeptides, while CP2-a and CP2-d had only a 47,000-Da polypeptide. The apoprotein of CP2-c was a 40,000-Da polypeptide. Absorption spectra of CP2-a, -b, and -d were similar to each other but distinctly different from those of CP2-c at liquid nitrogen temperature. The reaction center complex showed two fluorescence emission bands at 686 and 694 nm at 77 degrees K. CP2-a, -b, and -d emitted the band at 694 nm, whereas the fluorescence peak at 686 nm was associated with CP2-c. It is concluded that the photosystem 2 reaction center complex contains two chlorophyll-binding subunits, CP2-d (or CP2-a) which may be the site of the primary photochemistry of photosystem 2 and CP2-c which may function as the antenna of the reaction center of photosystem 2.     

Isolation of deuterated histones from yeast grown on media dissolved in H2O

We have succeeded in growing Saccharomyces cerevisiae (baker's yeast) on media containing ²H₂O and isolating the core histones highly deuterated in the non-exchangeable positions. The deuterated histones obtained here are of great value for their possible widespread use for structural studies of chromatin.     

Cloning and sequence determination of the Schizosaccharomyces pombe rpb1 gene encoding the largest subunit of RNA polymerase II.

The gene, rpb1, encoding the largest subunit of RNA polymerase II has been cloned from Schizosaccharomyces pombe using the corresponding gene, RPB1, of Saccharomyces cerevisiae as a cross-hybridization probe. We have determined the complete sequence of this gene, and parts of PCR-amplified rpb1 cDNA. The predicted coding sequence, interrupted by six introns, encodes a polypeptide of 1,752 amino acid residues in length with a molecular weight of 194 kilodaltons. This polypeptide contains eight conserved structural domains characteristic of the largest subunit of RNA polymerases from other eukaryotes and, in addition, 29 repetitions of the C-terminal heptapeptide found in all the eukaryotic RNA polymerase II largest subunits so far examined.     

Dormancy in Dioscorea: Rapid Germination of Detached Embryos from Dormant Seeds of D. tokoro

The intact dormant seeds of Dioscorea tokoro germinate slowlyif at all between 11-23°C; for full and rapid germinationthey require prior chilling treatment [Okagami and Kawai (1982)Bot. Mag. Tokyo 95: 155]. The germination abilities of zygoticembryos detached from dormant seeds of this species were studiedunder various nutritional and temperature regimes. For germinationof embryos, the minimum nutritional components in Murashigeand Skoog's (1962) medium that were required were sucrose andNO^–₃ or SO^2–₄. As the source of carbohydrate forgermination of detached embryos, sucrose, mannose and maltosewere effective; glucose and fructose were less effective; andrhamnose was entirely unable to support germination. Embryos detached from dormant seeds, incubated with the sucroseplus KNO₃, germinated more rapidly with increasing temperatureup to 35°C. However, application of sucrose and KNO₃ didnot induce germination of intact seeds above 26°C. Therefore,it is very possible that the endosperm exerts an inhibitoryfunction on germination at such high temperatures. When seeds were incubated after a cut was made over a smallpart of the edge of the endosperm in which the radicle of theembryo is encased, germination occurred rapidly but the increasein germination percentage was slight. This result suggests thatthe endosperm suppots part of the germination inhibition bymeans of a mechanical barrier or its impermeability to wateror gases. Physiological features of the endosperm alone or interactionsbetween the embryo and endosperm may contribute significantlyto the characteristics of dormancy of intact seeds of this species. (Received May 30, 1988; Accepted January 11, 1989)     

Heterogeneity of the calcium-dependent phosphatidylinositol phosphodiesterase in rat brain

1. The Ca²⁺-dependent phosphatidylinositol phosphodiesterase (phospholipase C-type) from the cytosolic supernatant of rat brain was active against exogenous [³²P]-phosphatidylinositol from pH5.0 to pH8.5. However, the activity in the range pH7.0–8.5 could not be recovered after precipitation with (NH₄)₂SO₄; most of the enzyme activity was recovered in the 30–50% fraction and showed a single sharp pH optimum at 5.5. 2. The cytosolic supernatant was analysed by isoelectric focusing on acrylamide gels, and assay at pH5.5. Four peaks of phosphodiesterase activity were found at pI ranges 7.4–7.2, 6.0–5.8, 4.8–4.4 and 4.2–3.8. 3. The cytosolic supernatant was also applied to a chromatofocusing column, and again assayed at pH5.5. Four peaks were eluted: minor, but consistent, activity at the beginning of the elution with a pI of near 7.2 or above; a second peak at pH6.0–5.85; a third broad peak with a wide range pH5.3–4.2; and a fourth peak, which was eluted by washing the column with 1m-NaCl, suggesting an isoenzyme with a pI below 4.0 (supported by the result of the isoelectric focusing). 4. If all the chromatofocusing fractions were assayed at pH7.0 or 8.0 (at 1mm-Ca²⁺), only a single sharp peak was detected, with a pI of 4.6–4.8. This peak disappeared on (NH₄)₂SO₄ fractionation (30–50%) of the cytosolic supernatant, whereas the four peaks with activity at pH5.5 were virtually unaffected. 5. The four activities (assayed at pH5.5) separated by chromatofocusing produced inositol 1:2-cyclic monophosphate, inositol 1-monophosphate and diacylglycerol as enzymic products. 6. We conclude that the Ca²⁺-dependent phosphatidylinositol phosphodiesterase exhibits considerable heterogeneity, both with respect to pH optima of activity, and its isoelectric properties.     

Transposition of the oxacillin-hydrolyzing penicillinase gene.

We have found that the oxacillin-hydrolyzing penicillinase gene (oxa) encoded by plasmid RGN238 transposes to various plasmids in a recA background. We call this transposable element Tn2603. Tn2603 encodes the genes for streptomycin, sulfonamide, and mercury resistance in addition to the oxa gene. Tn2603 has a molecular size of 19.6 kilobase pairs and appears to be flanked by small inverted repeat sequences of about 200 base pairs long.     

Mode of action of furosemide on the chloride-dependent short-circuit current across the ciliary body epithelium of toad eyes

Summary The effects of furosemide on the chloride-dependent short-circuit current across the toad ciliary epithelium were examined. Under control conditions, the short-circuit current obeyed Michaelis-Menten kinetics against medium chloride concentration, the Michaelis constant (K _m ) for chloride being 90mm and the maximal short-circuit current (V _max) 128 A/cm². Furosemide added to the aqueous side of the epithelium rapidly reduced the short-circuit current; the effect was reversible. The effect of furosemide addition to the stromal side was much smaller and slower than that from the aqueous side. The dose-dependent range of furosemide action was from 0.1 m to 1mm with 50% inhibition occurring at about 3 m. Line-weaver-Burk plot of the short-circuit current against the chloride concentration showed that furosemide decreased the value ofV _max and increased theK _m ; the inhibition being of mixed type. A Hill plot of the dose-response curve yielding a slope of unity suggested one furosemide molecule combines with one chloride transport site. Probenecid, a competitive inhibitor of organic acid transport, reduced the effects of furosemide significantly when added simultaneously. The involvement of organic acid transport system in the mechanism of furosemide action on chloride transport was suggested.Department of Ophthalmology.     

Organics in chimneys and water samples from deep-sea hydrothermal systems: implications for sub-vent biosphere.

Searching for life in extreme terrestrial environments can be a model of that for extraterrestrial life. Submarine hydrothermal system is one of promising sites for the frontier of life on the earth. Here seawater and vent chimnies were collected from deep-sea hydrothermal vents at Suiyo Seamount, Izu-bonin arc, Pacific Ocean as a part of Archaean Park Project. Pure seawater sample of 300 degrees C (purity>97%) could be collected. Dissolved and total hydrolyzable amino acids were determined by ion-exchange HPLC, and their enantiomeric ratio was measured by reversed-phase HPLC for the first time. Glycine and serine were two most abundant amino acids, followed by other proteinous amino acids such as alanine, glutamic acid and aspartic acid. Non-proteinous amino acids were detected as minor constituents. Most of the amino acids detected were of the L-form. Thus amino acids of abiotic origin were quite minor, and most of the amino acids detected were formed biologically. These results, together with analytical results of the vent chimney samples, suggest that there is active microbial activities near the hydrothermal systems.