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.     

Na+/H+ exchanger and reperfusion-induced ventricular arrhythmias in isolated perfused heart: possible role of amiloride

The roles of the Na⁺/H⁺ exchange system in the development and cessation of reperfusion induced ventricular arrhythmias were studied in the isolated perfused rat heart. The hearts were perfused in the working heart mode with modified Krebs Henseleit bicarbonate (KHB) buffer and whole heart ischemia was induced by a one-way ball valve with 330 beat/min pacing. Ischemia was continued for 15 min followed by 20 min of aerobic reperfusion (control). Amiloride (1.0mM), an inhibitor of the Na⁺/H⁺ exchange system, was added to the KHB buffer only during reperfusion (group B) or only during ischemic periods (group C). Electrocardiographic and hemodynamic parameters were monitored throughout the perfusion. Coronary effluent was collected through pulmonary artery cannulation and PO₂, PCO₂, HCO ₃ ^– and pH were measured by blood-gas analyzer.The incidence of reperfusion induced ventricular arrhythmias was 100%, 100% and 0% in control, group B and group C, respectively. The mean onset time of termination of reperfusion arrhythmias was significantly shorter in group B than in control. PCO₂ increased from 39.0±0.9 to 89.3±6.0 mmHg at the end of ischemia in control and from 40.6±0.4 to 60.5±5.8 in group C, the difference between groups was statistically significant. HCO ₃ ^– level decreased from 21.8±0.1 to 18.3±0.5 mmol/l in control, however, this decrease was significantly inhibited in group C (from 22.0±0.5 to 20.3±0.2). The increase in PCO₂ and the decrease in HCO ₃ ^– in group B were similar over time to those observed in control. The decrease in pH produced by ischemia was marked in control (from 7.35±0.01 to 6.92±0.04) and group B (from 7.34±0.01 to 6.94±0.02), whereas a decrease in pH was significantly prevented in group C (from 7.34±0.01 to 7.15±0.04). There were no significant differences in PCO₂, HCO ₃ ^– or pH among the three groups during reperfusion.These experiments provide evidence that amiloride significantly prevented the incidence of reperfusion arrhythmias when added only during ischemia and significantly terminated reperfusion arrhythmias when added only during reperfusion. Amiloride may prevent a decrease in pH, due to alterations in PCO₂ and/or HCO ₃ ^– . These changes in PCO₂ and HCO ₃ ^– might be indirectly influenced by inhibition of the Na⁺/H⁺ exchange system via Cl^–/HCO ₃ ^– exchange. The mechanism by which amiloride terminates reperfusion arrhythmias seems to involve electrophysiological effects which were not directly addressed in this experiment.