Incorporation of photolabile azido fatty acid probes inNeurospora crassa andSaccharomyces cerevisiae

Summary Photolabile azido fatty acids supported the growth of fatty acid auxotrophs, thecel mutant ofNeurospora crassa and the KD115 mutant ofSaccharomyces cerevisiae. These fatty acids were incorporated into the membrane lipids of thecel and KD115 mutants, but were not used by the wild type strain.This work was presented at the 26th International Congress of Pure and Applied Chemistry, Session 1, Tokyo, September 4–10, 1977, abstracts p. 241.Support was provided by the Govt. of India to B.C. and by NCERT, New Delhi India to D.N.C.To whom requests for reprints should be sent.     

Thiamine-binding activity ofSaccharomyces cerevisiae plasma membrane

Summary The specific binding activity to [¹⁴C]thiamine was found to be located in the plasma membrane ofSaccharomyces cerevisiae. The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant ofSaccharomyces cerevisiae. Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.     

The synthesis of stereoselectively labelled porphobilinogen and its incorporation into protoporphyrin-IX

Summary 11-(R)-²H porphobilinogen, stereospecifically labelled with deuterium in the aminomethylene group has been incorporated into protoporphyrin-IX by haemolysates of chicken erythrocytes. High field NMR spectroscopy confirms that the overall biochemical process is stereospecific, deuterium being retained at the -, -and -meso positions and lost from the -meso position.27 August 1986     

Comparison of wing margin structures ofvestigial and wild-typeDrosophila melanogaster grown at 31°C

Summary The development of the middle row of triple row wing bristles was examined in flies homozygous for thevestigial mutant, grown at 31°C. While bristle number, length and spacing all improved toward the wild-type condition, the mean values for both sexes were only about 10% of those of wild-type, although development in males was significantly more complete than in females. The results suggest an explanation for the apparent lack of regenerative ability in this tissue.     

thiD locus ofEscherichia coli

Summary A mutant strain ofEscherichia coli K-12 lacking phosphomethylpyrimidine kinase activity was produced from the polyauxotrophic female strain, JC1552. The locus of its lesion, for which we propose the designationthiD, was mapped at about 46 min on the chromosome.     

An increase in the influx of calcium ions into cilia induces thigmotaxis inParamecium caudatum

To understand the role of calcium ions in thigmotaxis inParamecium caudatum, the effects of caffeine, ruthenium red and lanthanum (LaCl₃) on thigmotaxis were examined. Thigmotaxis in the CNR mutant, which lacks voltage-dependent Ca²⁺-channels in the ciliary membrane, was also examined. Ruthenium red and LaCl₃ suppressed thigmotaxis inP. caudatum, while caffeine enhanced it. The CNR mutant showed hardly any thigmotaxis. It can be thought that an increase in Ca²⁺ influx and the intraciliary concentration of Ca²⁺ ions induces thigmotaxis inParamecium.     

A blocked mutant ofClaviceps purpurea accumulating chanoclavine-I-aldehyde

Summary An alkaloid-blocked mutant ofClaviceps purpurea was isolated from a strain which produces ergotoxine alkaloids. The mutant accumulates chanoclavine-I and the corresponding aldehyde. It lacks the ability to form tetracyclic ergolines.Dedicated to Prof. Dr Dr h.c. K. Mothes on the occasion of his eightieth birthday.     

β-Lactamases as models for protein-folding studies

This review traces some of the key features of the folding of β-lactamases and their relevance to the way proteins fold in general. Studies on the enzymes have highlighted the nature and role of equilibrium and transient condensed states. The kinetics of folding are multiphasic, and when monitored by acrylamide quenching of the tryptophan fluorescence, an early phase provides evidence for the transient accumulation of a nonnative intermediate involving burial of tryptophan in a nonpolar environment. Intermediate phases can be understood in terms of progressive folding of different parts of the molecule. The later, slow phases are associated with proline isomerization in the TEM-1 enzyme and, in its P167T mutant form, with isomerization from trans to cis of the E166 T167 peptide bond. Coupled with kinetic and X-ray crystallographic studies of the β-lactamase from Staphylococcus aureus and its D179Q mutant, it appears that the final stage of folding is that of collapse and packing of the Ω-loop on to the main body of the protein.     

Deletion mapping of a new gustatory mutant inDrosophila melanogaster

Summary A gustatory mutant ofDrosophila melanogaster insensitive to the taste of salt has been isolated. Genetic crosses and a deletion mapping analysis show that this mutation, designatedgust-M ₁ is located in the 93C_3–6-93D_6–7 region of the third chromosome.gust-M ₁ is also insensitive to the taste of quinine sulfate. The behavior of this mutant may be explained by assuming thatgust-M ₁ could be a mutation perturbing functions in the central nervous system affecting the responses to both compounds.     

Temperature-dependent responses to a developmental gradient in theDrosophila wing

Summary The mutant hairy (h) increases the number of sensillae on theDrosophila wing. This allows us to quantify a gradient that determines the type of sense organ that forms along the third long vein. Temperature significantly shifts the positional responses to this underlying gradient.This research was supported by National Science Foundation grant BSR-8300025. We thank Laura Karcher for the figure.     

A spontaneous benomyl-resistant mutant ofPodospora anserina exhibiting a diurnal growth rhythm

Summary A spontaneous mutant ofP. anserina isolated by screening for benomyl resistance exhibited a diurnal growth rhythm dependent on light-dark cycles. The rhythmic character, the benomyl resistance and a growth rate reduced to 50% of that of the wild type were inherited together over more than 10 generations. The locus was mapped on linkage group II, 0.35 map units distal to the locusz (=0.81 map units from the centromere).     

Ammonia assimilatory enzymes in a nif−III mutant ofAzotobacter chroococcum

Summary The levels of ammonia assimilatory enzymes have been studied in a nif^–III mutant and a wild strain ofAzotobacter chroococcum grown in media containing different nitrogen sources. It is suggested that the nif^–III mutant may be defective in transport or incorporation of molybdenum.     

Growth ofNostoc muscorum mutants in the presence of diuron (DCMU) and L-methionine-DL-sulfoximine

Summary Diuron (DCMU) is inhibitory to the photoautotrophic and photoheterotrophic growth of the N₂-fixing blue-green algaNostoc muscorum at concentrations of 1.0×10^–5 M and 2.0×10^–5 M, respectively. A mutant of this organism resistant to 5.0×10^–5 M DCMU under its photoheterotrophic growth conditions, with the ability to utilize DCMU as a carbon and nitrogen source for growth, and complete inability to grow photoautotrophically has been isolated. With the apparent defect in its photosynthetic ability, it is suggested that theDCMU ^r mutant lacks the step inhibited by 1.0×10^–5 M DCMU, and metabolizes DCMU by an existing enzyme system in the absence of such inhibition. That this enzyme may be glutamine synthetase (GS) is explained with the help of a L-methionine-DL-sulfoximine (MSO)-resistant mutant ofN. muscorum which is able to grow faster with 2.0×10^–5 DCMU and is known to contain an altered GS.Thanks are due to the Council of Scientific and Industrial Research, CSIR Complex, Govt. of India, New Delhi-110012, for appointing the author to the Scientists' Pool for undertaking researches on the physiological and genetic controls of nitrogen metabolism in blue-green algae, a part of which is presented in this literature.     

Loss of cytosolic serine hydroxymethyltransferase in a formate mutant ofNeurospora crassa

Summary In the wild type ofNeurospora crassa serine hydroxymethyltransferase occurs in the cytosol and mitochondria. The formate mutant (FGSC 9) lacks the cytosolic activity but has increased levels of isocitrate lyase and glyoxylate aminotransferase. Glycine is derived from glyoxylate in the mutant.     

Improving the thermal stability of lactate oxidase by directed evolution

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 °C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution. Received 15 September 2006; received after revision 21 October 2006; accepted 2 November 2006     

Mg2+-ATPase defective mutant ofEscherichia coli and thiamine transport

Summary Mg²⁺-ATPase deficient mutant ofEscherichia coli showed an evident dependency of thiamine uptake on the oxidative metabolism of glucose, whereas the parent strain did not. In both cells, this uptake was completely inhibited by H⁺ conductors.Acknowledgment. We are indebted to Miss M. Abe for her excellent technical assistance.     

Gel-electrophoretic description of European populations ofTerellia virens (Loew) (Diptera,Tephritidae); implications for its use as an agent for the biological control ofCentaurea spp. (Asteraceae) in North America

Allozyme frequencies of 15 enzyme loci, 14 of which were polymorphic, were used to characterize sevenTerellia virens populations originating from three allopatrically distributedCentaurea species. The two populations whose origins were geographically furthest apart, from Israel (onC. iberica) and from Switzerland (onC. vallesiaca), showed relatively high values of genetic distance from the 5 populations sampled in Austria and Hungary (onC. maculosa) (Nei's D>0.07). The latter five displayed a high degree of genetic similarity. No diagnostic (fixed) allelic differences were observed between these three groups ofT. virens populations, but they could be well characterized by significant differences in allelic frequencies at 9 enzyme loci. Independently of this study, the populations from Switzerland (C. vallesiaca) and eastern Austria (C. maculosa) were selected as potential source populations for future introductions into North America for the biological control of introducedC. maculosa andC. diffusa. Based on the observed genetic differences and results from field experiments on the host specificity of these two potential source populations, it is argued that host specificity screening tests should be conducted separately for local (host plant) populations, as such populations might accept a different set of hosts. Biotype mismatch and the risk of spill-overs to native species could thus possibly be reduced.     

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P_1B-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.     

The control of melanoblast differentiation in the periodic albino mutant ofXenopus

Summary Studies on the incidence of melanophores in older ventral trunk tissues and in isolated regions of periodic albino embryos ofXenopus suggest that melanin granule formation in mutant melanoblasts depends on an environmental contribution which arises at stage 43 in the endodermal tissues.     

A new type of interaction between spore colour mutants ofAspergillus niger

Summary A new white-spored mutant of the imperfect fungusA. niger is described. Behind the heterokaryotic heads formed with non-allelic spore colour mutants, it shows as a result of crossfeeding a uniform change of colour of the conidial heads in a band up to 4–5 mm deep. This mutation with pleiotropic effects on colour and crossfeeding occurs relatively frequently inA. niger, although it has not been reported previously.Acknowledgment. Thanks are due to Dr A. J. Clutterbuck (Department of Genetics, University of Glasgow, Glasgow) for helpful comments on this work.