Efficient disruption of a polyketide synthase gene ( pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis.

Glarea lozoyensis produces pneumocandin B(0), a potent inhibitor of fungal glucan synthesis. This industrially important filamentous fungus is slow-growing, is very darkly pigmented, and has not been easy to manipulate genetically. Using a PCR strategy to survey the G. lozoyensis genome for polyketide synthase (PKS) genes, we have identified pks1, a gene that consists of five exons interrupted by four introns of 56, 400, 50 and 341 bp. It encodes a 2124-amino acid protein with five catalytic modules: ketosynthase, acyltransferase, two acyl carrier sites, and thioesterase/Claisen cyclase. The transcriptional initiation and termination sites were found 43 bp upstream of the translational start codon and 295 bp downstream of the translational stop codon, respectively. Cluster analysis of 37 fungal ketosynthase modules grouped the Pks1p with PKSs involved in the biosynthesis of 1,8-dihydroxynaphthalene melanin. Disruption of pks1 yielded knockout mutants that displayed an albino phenotype, suggesting that pks1 encodes a tetrahydroxynaphthalene synthase. Gene replacement was achieved by Agrobacterium-mediated transformation, which proved to be simple and efficient. Loss of pigmentation occurred in more than half the transformants, and examination of six non-pigmented transformants showed that the functional genomic copy of the pks1 gene had been replaced by the disruption cassette in each case. A putative 1215-bp ORF (dsg) devoid of introns was present downstream from pks1. BLAST analysis of the 405-amino acid sequence of its predicted product showed a high degree of similarity with Zn(II)(2)Cys(6) binuclear cluster DNA-binding proteins, a class of fungal transcription factors involved in the regulation of polyketide production and other pathways.

Evolutionary relationship of the ligand-gated ion channels and the avermectin-sensitive, glutamate-gated chloride channels.

Two cDNAs, GluClalpha and GluClbeta, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707-711, 1994). Expression studies in Xenopus oocytes showed that GluClalpha and GluClbeta have pharmacological profiles distinct from the glutamate-gated cation channels as well as the gamma-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClalpha and GluClbeta genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClalpha and GluClbeta form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClalpha and GluClbeta, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClalpha and GluClbeta belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels.

Identification of a Drosophila melanogaster glutamate-gated chloride channel sensitive to the antiparasitic agent avermectin.

Glutamate-gated chloride channels, members of the ligand-gated ion channel superfamily, have been shown in nematodes and in insects to be a target of the antiparasitic agent avermectin. Two subunits of the Caenorhabditis elegans glutamate-gated chloride channel have been cloned: GluCl-alpha and GluCl-beta. We report the cloning of a Drosophila melanogaster glutamate-gated chloride channel, DrosGluCl-alpha, which shares 48% amino acid and 60% nucleotide identity with the C. elegans GluCl channels. Expression of DrosGluCl-alpha in Xenopus oocytes produces a homomeric chloride channel that is gated by both glutamate and avermectin. The DrosGluCl-alpha channel has several unique characteristics not observed in C. elegans GluCl: dual gating by avermectin and glutamate, a rapidly desensitizing glutamate response, and a lack of potentiation of the glutamate response by avermectin. The pharmacological data support the hypothesis that the DrosGluCl-alpha channel represents the arthropod H-receptor and an important target for the avermectin class of insecticides.

A receptor in pituitary and hypothalamus that functions in growth hormone release.

Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.

The mechanism of action of avermectins in Caenorhabditis elegans: correlation between activation of glutamate-sensitive chloride current, membrane binding, and biological activity.

Xenopus laevis oocytes were injected with mRNA isolated from the free-living nematode Caenorhabditis elegans and the activation and potentiation of a glutamate-sensitive chloride current by a series of avermectin analogs and milbemycin D were determined. There was a strong correlation between the EC50 value determined for current activation in oocytes, the LD95 value for nematocidal activity, and also for the Ki value determined in a [3H]ivermectin competition binding assay. Four of the analogs were tested for potentiation of glutamate-sensitive current and the rank order for potentiation correlated with the EC50 for direct activation of current. We conclude that avermectins and milbemycins mediate their nematocidal effects on C. elegans via an interaction with a common receptor molecule, glutamate-gated chloride channels.

Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans.

The avermectins are a family of macrocyclic lactones used in the control of nematode and arthropod parasites. Ivermectin (22,23-dihydroavermectin B1a) is widely used as an anthelmintic in veterinary medicine and is used to treat onchocerciasis or river blindness in humans. Abamectin (avermectin B1a) is a miticide and insecticide used in crop protection. Avermectins interact with vertebrate and invertebrate GABA receptors and invertebrate glutamate-gated chloride channels. The soil nematode Caenorhabditis elegans has served as a useful model to study the mechanism of action of avermectins. A C. elegans messenger RNA expressed in Xenopus oocytes encodes an avermectin-sensitive glutamate-gated chloride channel. To elucidate the structure and properties of this channel, we used Xenopus oocytes for expression cloning of two functional complementary DNAs encoding an avermectin-sensitive glutamate-gated chloride channel. We find that the electrophysiological and structural properties of these proteins indicate that they are new members of the ligand-gated ion channel superfamily.

Expression of a glutamate-activated chloride current in Xenopus oocytes injected with Caenorhabditis elegans RNA: evidence for modulation by avermectin.

Membrane currents were recorded from Xenopus laevis oocytes injected with C. elegans poly(A)+ RNA. In such oocytes glutamate activated an inward membrane current that desensitized in the continued presence of glutamate. Glutamate-receptor agonists quisqualate, kainate, and N-methyl-D-aspartate were inactive. The reversal potential of the glutamate-sensitive current was -22 mV, and exhibited a strong dependence on external chloride with a 48 mV change for a 10-fold change in chloride. The chloride channel blockers flufenamate and picrotoxin inhibited the glutamate-sensitive current. Ibotenate, a structural analog of glutamate, also activated a picrotoxin-sensitive chloride current. Ibotenate was inactive when current was partially desensitized with glutamate, and the responses to low concentrations of glutamate and ibotenate were additive. The anthelmintic/insecticide compound avermectin directly activated the glutamate-sensitive current. In addition, avermectin increased the response to submaximal concentrations of glutamate, shifted the glutamate concentration-response curve to lower concentrations, and slowed the desensitization of glutamate-sensitive current. We propose that the glutamate-sensitive chloride current and the avermectin-sensitive chloride current are mediated via the same channel.

Avermectin-sensitive chloride currents induced by Caenorhabditis elegans RNA in Xenopus oocytes.

Avermectins are a family of potent broad-spectrum anthelmintic compounds, which bind with high affinity to membranes isolated from the free-living nematode Caenorhabditis elegans. Binding of avermectins is thought to modulate chloride channel activity, but the exact mechanism for anthelmintic activity remains to be determined. In this report, the properties of an avermectin-sensitive membrane current were evaluated in Xenopus laevis oocytes that were injected with poly(A)+ RNA from C. elegans. In such oocytes, avermectins increased inward membrane current at a holding potential of -80 mV. An avermectin analog without anthelmintic activity had no effect. Half-maximal activation of current was observed with 90 nM avermectin. The reversal potential for avermectin-sensitive current was -19.3 +/- 1.9 mV, and it shifted with external chloride, as expected for a chloride current. Avermectin increased membrane current in C. elegans-injected oocytes that were also injected with the Ca2+ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The response to avermectin was greatest in the 1.0-2.5-kilobase class of size-fractionated C. elegans poly(A)+ RNA. Oocytes that responded to avermectin were insensitive to gamma-aminobutyric acid and the avermectin-induced current was blocked by picrotoxin.

Solubilization and characterization of a high affinity ivermectin binding site from Caenorhabditis elegans.

Ivermectin is a member of the avermectin family of compounds that are used to treat helminth and arthropod diseases in humans, domestic animals, and plants. A membrane-bound high affinity ivermectin binding site was extracted from Caenorhabditis elegans with the nonionic detergent 1-O-n-octyl-beta-D-glucopyranoside. The free-living nematode C. elegans is highly sensitive to the avermectins and was used as a model of parasitic nematodes. The membrane-bound and detergent-solubilized ivermectin binding sites are stable and exhibit high affinity binding, with dissociation constants of 0.11 nM and 0.20 nM, respectively. The maximum binding of [3H]ivermectin is 0.54 pmol/mg of membrane protein and 0.66 pmol/mg of detergent-soluble protein. Kinetic analysis of ivermectin binding shows that the ivermectin binding sites form a slowly reversible complex with ivermectin. The rates of dissociation of [3H]ivermectin with the solubilized and membrane-bound binding sites are 0.005 min-1 and 0.006 min-1, respectively. The association rate of the soluble binding site is 0.053 nM-1 min-1, slightly slower than that observed for the membrane-bound site, 0.074 nM-1 min-1. To characterize the ivermectin binding site, competition experiments were performed by inhibiting [3H]ivermectin binding with several avermectin derivatives and the neurotransmitter gamma-aminobutyric acid (GABA). The order of potency was 22,23-dihydroavermectin B1a monosaccharide greater than 22,23-dihydroavermectin B1a aglycone greater than 3,4,8,9,10,11,22,23-octahydro B1 avermectin for both the membrane-bound and NOG-soluble binding sites. GABA did not compete with ivermectin binding, although it has been suggested that ivermectin acts at the GABA-gated chloride channel in some invertebrate systems. Optimum ivermectin binding and assay conditions have been determined. The detergent-soluble ivermectin binding site appears to be negatively charged and has a pl of 4.0 and an apparent Mr in Triton X-100 micelles of 340,000. Detergent solubilization of a high affinity ivermectin binding site will enable the subsequent purification and characterization of a putative site of ivermectin action.

Glutamine synthetase of Streptomyces cattleya: purification and regulation of synthesis.

Glutamine synthetase (GS; EC 6.3.1.2) from Streptomyces cattleya was purified using a single affinity-gel chromatography step, and some of its properties were determined. Levels of GS in S. cattleya cells varied by a factor of 8 depending upon the source of nitrogen in the growth medium. Of 24 nitrogen sources examined only glutamine or NH4Cl utilization resulted in very low GS activity. Addition of NH4Cl to a culture with high GS levels appeared to stop further synthesis and resulted in a progressive decrease in the specific activity of the enzyme. The GS inhibitor methionine sulphoximine (MSX) inhibited GS activity but had no effect on exponentially growing cells. The presence of MSX either lengthened or shortened the period between spore inoculation and initiation of exponential growth, depending on the source of nitrogen. In glutamine minimal medium MSX produced earlier and more efficient spore germination while in glutamate or nitrate minimal medium germination was delayed by its presence.

Unstable variant of NADH methemoglobin reductase in Puerto Ricans with hereditary methemoglobinemia.

The electrophoretic mobility of erythrocyte NADH methemoglobin reductase in five hereditary methemoglobinemia patients from three Puerto Rican kindreds was 118% of normal at pH 8.6. The methemoglobin ferrocyanide reductase activity of the enzyme in erythrocyte hemolysates was 3.2-6.4% of normal. Electrophoresis of hemolysates prepared from the blood of patients from two different families at six pH values between 4.6 and 9.3 did not differentiate between the variant enzymes. Examination of the deficient enzymes extracted from the erythrocytes of one patient from each kindred revealed altered affinity for NADH and dichloroindophenol dye and decreased thermal stability. The quantitative similarity of the abnormal findings, together with the Puerto Rican origin of the kindreds, suggested that the cyanotic patients possessed the same abnormal enzyme and were thus homozygous for the same rare mutant gene. Consanguinity of the kindreds could not be established. The rates of decline of the normal and variant NADH methemoglobin reductase enzymes in vivo were measured in erythrocyte fractions of increasing cell age. The rate of decline of the variant enzyme was increased 20-fold by comparison with the normal enzyme. The methemoglobin percentage in erythrocyte fractions of increasing cell age correlated inversely with the activity of the variant. The variant enzyme averaged 37% of normal mean activity in young cells and 1% in old cells. The normal enzyme, on the other hand, lost only one-sixth of its activity as the cells aged, and the methemoglobin content in old normal cells did not rise. These observations support the hypothesis that the deficient activity and the heterogeneous pattern of methemoglobin accumulation in vivo arise principally from the accelerated inactivation of variant NADH methemoglobin reductase during the life-span of the red blood cell.