Characterization of two cDNAs for novel drought-inducible genes in the highly drought-tolerant cowpea

Ten cDNAs for drought-inducible genes were isolated using differential screening of a cDNA library prepared from 10-hr dehydrated cowpea plants,Vigna unguiculata (S. Iuchi, K. Yamaguchi-Shinozaki, T. Urao, T. Terao, K. Shinozaki; Plant Cell Physiology, 1996 in press). Two of the cDNA clones, designated CPRD12 and CPRD46, were sequenced and characterized. The CPRD12 and CPRD46 cDNAs encode putative proteins related to nonmetallo-short-chain alcohol dehydrogenase (CPRD12) and chloroplastic lipoxygenase (CPRD46). Northern blot analysis revealed that these genes are induced by high-salinity stress and exogenous abscisic acid, but not by cold stress. The CPRD46 gene is also responsive to heat stress and methyl jasmonate and salicylic acid. Genomic Southern blot analysis suggested that CPRD12 constitutes a small gene family, but that CPRD46 is a single copy gene. We discuss the possible functions of these two CPRD gene products under drought stress.     

Analysis of the multi-stem clump structure ofLitsea japonica Juss. growing in a coastal dwarf forest

The multi-stem clump structure of a coastal dwarf forest dominated byLitsea japonica Juss. was investigated in order to clarify the sprouting characteristics and self-maintenance of clumps by stem alternation. The size and age distribution of multi-stem clumps were analyzed using cumulative relative frequency curves.L. japonica had a large number of stems and an even height distribution or young age-biased distribution of stems within a clump. These results indicated the sequential flushing of sprouts at high frequency. Height distribution within a clump ofL. japonica was relatively even compared to other species. This clump structure suggested the stable self-maintenance of individuals in all ranges of size and age without disturbances. It originated specific sprouting characteristics as a response to the severe stress of salty wind.Ardisia sieboldii Miq. had few stems within a clump. Although the stem height distribution of large individuals tended to be even, most clumps had a large size-biased distribution of stem height which indicated simultaneous sprouting. From this structure, sprouts of this species were thought to be of less significance in the stable self-maintenance of individuals thanL. Japonica.     

Automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography

An automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography (HPLC) with column switching is described. The system consists of a reversed-phase column, a cation-exchange column, a column switch, four sets of ultraviolet absorbance detectors, a microcomputer and other conventional equipment. As this system permits the simultaneous determination of urinary orotic acid, uracil, dihydrouracil, pseudouridine, xanthine, 2,8-dihydroxyadenine and succinyladenosine, it offers a useful method for the detection of orotic aciduria, dihydropyrimidine dehydrogenase deficiency, diphydropyrimidinuria, xanthinuria, adenine phosphoribosyltransferase deficiency and adenylosuccinase deficiency.     

Conformational changes of α-lactalbumin induced by the stepwise reduction of its disulfide bridges: The effect of the disulfide bridges on the structural stability of the protein in sodium dodecyl sulfate solution

Four disulfide bridges of bovineα-lactalbumin (α-lact) were selectively reduced to obtain its derivatives with three, two, and zero disulfide bridges (designated as 3SS, 2SS, and OSSα-lact, respectively). The original helicity was almost maintained in 3SSα-lact missing only the Cys6-Cysl20 bridge. Upon the reduction of both Cys28-Cys111 and Cys6-Cys120 bridges, various changes occurred in the protein. In particular, the maximum fluorescence of 1-anilinonaphthalene-8-sulfonic acid was observed in this stage. Upon the reduction of all disulfide bridges, the hydrophobic box of the protein, formed by Trp60, Ile95, Tyr103, and Trp104, was disrupted and an internal helical structure was destroyed. The conformation of each derivative was examined mainly in a solution of sodium dodecyl sulfate. In the surfactant solution, the helicity increased from 33% to 37% in 3SSα-lact, from 26% to 31% in 2SSα-lact, and from 18% to 37% in OSSα-lact, as against from 34% to 44% in intactα-lact. On the other hand, the tryptophan fluorescence of each derivative was affected in very low surfactant concentrations, suggesting that the tertiary structure considerably changed prior to the secondary structural change in the surfactant solution.     

Effects of estradiol and testosterone on the synthesis,expression and degradation of androgen receptor in human uterine endometrial fibroblasts

The mechanism of known receptor-mediated androgen effects on the endometrial stroma was studied in endometrial fibroblasts derived from human uterus. 17-Estradiol (E) induced the expressions of androgen receptor (AR) mRNA, and predominantly increased the level of testosterone-binding sites (TBS) in uterine endometrial fibroblasts. The effect on the level of dihydrotestosterone-binding sites (DHTBS) was similar but smaller. This result suggests that the AR mRNA expressed might encode TBS, but probably not DHTBS. The TBS level increased by estrogen was down-regulated by testosterone (T) + E, but the AR mRNA expression increased by E was not down-regulated by E + T in the fibroblasts. Although the synthesis rate of AR was slightly increased (p<0.05) by E alone or E + T, the degradation rate of AR was significantly accelerated (p<0.05) by E + T in the fibroblasts. This result suggests that T might stimulate the metabolic rate of TBS, but does not inhibit the synthesis rate of AR mRNA to TBS in endometrial fibroblasts.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene

Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC₅₀) of the enzyme activity was measured to be 400 M, whereas the IC₅₀ value was 15 M for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S. cerevisiae complemented the FAS2 pheno-type and showed cerulenin resistance. These data indicate that one amino acid substitution (Gly Ser) in the subunit of fatty acid synthase is responsible for the cerulenin resistance of the mutant KNCR-1.