Isolation and identification of (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid from anAmanita of the sectionRoanokenses (Amanitaceae)

A chlorine-containing non-protein amino acid which was recently discovered from the fruit bodies ofAmanita gymnopus (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid, was isolated and crystallized for the first time from the fruit bodies of an unknown member ofAmanita belonging to the sectionRoanokenses, subsectionSolitariae. The results of elementary analyses, determination of optical rotations,¹H- and¹³C-NMR-spectra, and some chemical reactions supported an earlier proposed structure.Part 24 in the series Biochemical studies of nitrogen compounds in fungi. for Part 23, see Hatanaka, S. I. et al. 1994. this journal35: 391–394.     

A simplified and efficient method for in situ hybridization to whole Drosophila embryos, using electrophoresis for removing non-hybridized probes

We report a simplified and reliable method for non-radioactive in situ hybridization to whole Drosophila embryos. In the previous method (Tautz and Pfeifle, 1989) the post-hybridization wash, or the procedure for washing non-hybridized probe away from embryos depends simply on diffusion. We modified the method with application of electrophoresis to the wash. After hybridized with RNA probe, embryos were transferred to a small well where an electric charge was given to drive non-hybridized probe away from the embryos. This procedure enables us to acquire a much higher signal-to-noise ratio than that obtained from a conventional method. Furthermore, this is a time-saving method. We propose a term "electro-wash" for this procedure.     

Genetic analyses of signalling in flower development using Arabidopsis

Flower development can be divided into four major steps: phase transition from vegetative to reproductive growth, formation of inflorescence meristem, formation and identity determination of floral organs, and growth and maturation of floral organs. Intercellular and intracellular signalling mechanisms must have important roles in each step of flower development, because it requires cell division, cell growth, and cell differentiation in a concerted fashion. Molecular genetic analysis of the process has started by isolation of a series of mutants with unusual flowering time, with aberrant structure in inflorescence and in flowers, and with no self-fertilization. At present more than 60 genes are identified from Arabidopsis thaliana and some of them have cloned. Although the information is still limited, several types of signalling systems are revealed. In this review, we summarize the present genetic aspects of the signalling network underlying the processes of flower development.     

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.     

Isolation of an Internal Deletion Mutant of the Arabidopsis thaliana ABI3 Gene

An Arabidopsis thaliana mutant that produces green seeds thatare highly insensitive to exogenous ABA, non-dormant and severelydesiccation intolerant was isolated from a population of fastneutron-irradiated seeds. Molecular and genetic analysis ofthis mutant shows that these phenotypes are caused by an internaldeletion of approximately one third of the ABI3 gene. Thereforeabi3 mutants with the above phenotypes are representative ofnull alleles at this locus. (Received December 3, 1993; Accepted January 22, 1994)     

A Mutant of Arabidopsis thaliana That Defines a New Locus for Glycine Decarboxylation

A novel photorespiratory mutant of Arabidopsis thaliana, designatedgld2, was isolated based on a growth requirement for abnormallyhigh levels of atmospheric CO₂. Photosynthetic CO₂ fixationwas inhibited in the mutant following illumination in air butnot in atmosphere containing 2% O₂. Photosynthetic assimilationof ¹⁴CO₂ in an atmosphere containing 50% O₂ resulted in accumulationof 48% of the soluble label in glycine in the mutant comparedto 9% in the wild type. The rate of glycine decarboxylationby isolated mitochondria from the mutant was reduced to 6% ofthe wild type rate. In genetic crosses, the mutant complementedtwo previously described photorespiratory mutants of A. thalianathat accumulate glycine during photosynthesis in air due todefects in glycine decarboxylase (glyD, now designated gld1)and serine transhydroxymethylase (stm). Because glycine decarboxylaseis a complex of four enzymes, these results are consistent witha mutation in a glycine decarboxylase subunit other than thataffected in the gld1 mutant. The two gld loci were mapped tochromosomes 2 and 5, respectively. ³Present address: Department of Crop and Soil Sciences, MichiganState University, East Lansing, MI 48824, U.S.A. ⁴Present address: Department of Applied Bioscience, Facultyof Agriculture, Hokkaido University, Kita-Ku, Sapporo, 060 Japan ⁵Present address: Department of Biology, Carnegie Institutionof Washington, 290 Panama Street, Standford, CA 94305, U.S.A.     

Spatial distribution of root activity and nitrogen fixation in sorghum/pigeonpea intercropping on an Indian Alfisol

A medium-duration pigeonpea cultivar (ICP 1–6) and a hybrid sorghum (CSH 5) were grown on a shallow Alfisol in monocropping and intercropping systems. Using a monolith method, spatial distribution of nodulation, acetylene reduction activity (ARA) and root respiration were measured.The number, mass and ARA of nodules decreased exponentially with distance from the plant base except at the late reproductive stage. Nodulation and ARA tended to be higher in the intercrop than in the monocrop.Respiration rate of roots increased with distance from the plant base and reached a maximum value at about 20–30 cm. The rate was higher in pigeonpea than in sorghum and also higher in intercrop than in monocrop.This study suggests that pigeonpea roots are physiologically more active than sorghum roots, implying that pigeonpea may become a strong competitor for nutrients in the soil when intercropped. The nitrogen-fixing ability of pigeonpea may be enhanced by intercropping because the sorghum rapidly absorbed inorganic N which would otherwise inhibit N₂ fixation.     

Hyperactive TNF-α derivatives with combinational mutations in the amino and carboxyl terminal regions

Summary We prepared various TNF- derivatives by protein engineering techniques. Mutant 471, in which 7 N-terminal amino acids were deleted and Pro⁸Ser⁹Asp¹⁰ was replaced by ArgLysArg, had a 8-fold higher antitumor activity against mouse L929 cells than wild-type TNF-. The additional substitution of Ala¹⁵⁶ or Leu¹⁵⁷ by more hydrophobic amino acids enhanced the activity of mutant 471. These results suggested that the combinational mutations in the N- and C-terminal regions of TNF- are effective for the improvement of antitumor activity.