Isolation and identification of the Aspergillus nidulans gdhA gene encoding NADP-linked glutamate dehydrogenase

Summary The Neurospora crassa am gene was used as a heterologous probe to identify clones from two independently constructed Aspergillus nidulans gene libraries. These clones have a common HindIII 1.85 kb fragment. This A. nidulans nucleotide stretch hybridises to a N. crassa 2.7 kb BamHI fragment of wild type DNA but not to a co-migrating fragment from the DNA of the N. crassa am132 deletion mutant. One A. nidulans clone was shown to complement the N. crasse am132 deletion strain. The N. crassa transformants show low levels (5%) of heterologous glutamate dehydrogenase activity. The A. nidulans gdhA gene was found to locate in N. crassa at both the homologous (i.e. am) site as well as non-nomologous sites. Partial nucleotide analysis of the fragment has revealed the 5 end of the locus and considerable homology with the N. crassa am gene. We concluded that we have cloned the A. nidulans gdhA gene.Abbreviations bp base pairs - kb 1,000 bp - GDH NADP-Iinked glutamate dehydrogenase - NADP nicotinamide adenine dinuc leotide phosphate - SDS sodium dodecyl sulfate - phage lambda - DTT dithiothreitol - SSC 0.15 M NaCl, 0.015 Na₃ citrate pH 7.0     

Phenotypic and epistatic grouping of hypo- and hyper-rec mus mutants in Aspergillus

The mutants musK to musS of Aspergillus nidulans are sensitive to methyl-methanesulfonate (MMS) and several of them are meiotic-defective and alter mitotic recombination frequencies. All were found to be cross-sensitive to 4-nitro-quinoline-N-oxide (4-NQO) but unexpectedly none of them was hypersensitive to -rays and few to UV light. Double mus;uvs mutants were constructed to test for interactions with uvs mutations of the four epistatic groups of Aspergillus, UvsF, UvsC, UvsI, and UvsB. All meiotic-defective mus mutations caused some lethal interactions, usually with uvsF. None of them showed epistasis with UvsF or UvsB group mutants and one, musO, may represent a new group. Three mus mutations that affect recombination were assigned to the UvsC group, namely musN and K, and also musL which is recombination-defective and closely resembles uvsC. While uvsC mutants are mutators and lack UV-mutagenesis, most mus mutants had no effects on mutation. Only musR, which appeared epistatic with uvsI, showed reduced UV-reversion frequencies similar to uvsI. The recombination-proficient mus mutants appeared to be epistatic with more than one group, but in several cases sensitivities were slight and overlaps insufficient to obtain corroborating results with MMS and 4-NQO.     

Wild chromosomal variants inAspergillus nidulans

Pulsed-field gel electrophoresis and a chromosome-specific cosmid DNA library were used to determine the karyotypes of wild-typeAspergillus nidulans isolates from around the world. Overall, little structural variation was found, with a few major exceptions. One isolate possessed a non-essential B-chromosome of about 1.0 million base pairs (mb). Another isolate had undergone a non-reciprocal translocation of about 1.6 mb of chromosome VI onto chromosome VIII. Other than these chromosomal differences, these isolates appeared phenotypically normal. To analyze its effects on meiosis, the translocation isolate was outcrossed with another wild-type derivative that had a normal electrophoretic karyotype. This cross produced a range of phenotypes, including duplicated progeny that had a barren phenotype similar to that described forNeurospora partial disomics. The duplication was somewhat vegetatively unstable. This is the first association of sterility with chromosomal duplication inA. nidulans.     

Co-transformation with autonomously-replicating helper plasmids facilitates gene cloning from an Aspergillus nidulans gene library

Autonomously-replicating, marker-less helper plasmids were added to transformations of Aspergillus nidulans with plasmids which normally transform by chromosomal integration. This resulted in as much as a 200-fold increase in transformation efficiency. Recovery of autonomously-replicating plasmid co-integrates indicated that co-transformation involves recombination between integrating and helper plasmids, which occurs at a high frequency. Increasing DNA sequence-homology between pairs of plasmids used in simultaneous transformations enhanced co-transformation efficiency. Using helper plasmids and an A. nidulans gene library in a normally-integrating vector, the genes adC and adD were cloned as part of such a co-integrate. In effect, the addition of helper plasmid converts an integrating into an autonomously-replicating gene library in vivo.     

Aspergillus nidulans 5S rRNA genes and pseudogenes

Summary The sequence of four Aspergillus nidulans 5S rRNA genes and of two pseudogenes has been determined. A conserved sequence about 100 by upstream of the 5S rRNA coding sequences has been found in three genes and one pseudogene. The two pseudogenes correspond to the 5 half of the 5S rRNA coding sequence and their 3 flanking sequences which are not homologous to 5S rRNA are strongly conserved.     

Transformation of Penicillium nalgiovense with the amdS gene of Aspergillus nidulans

Summary Penicillium nalgiovense was transformed with the amdS gene from Aspergillus nidulans as a selectable marker. The vector apparently integrated at multiple sites into the chromosomes of the transformants, which were mitotically stable. A transformation efficiency of 12 transformants/g vector DNA was achieved when the expression phase was prolonged to 8 h.     

The cloning and sequencing of thealcB gene,coding for alcohol dehydrogenase II,inAspergillus nidulans

Alcohol dehydrogenase II (ADH II, structural genealcB) was purified from a strain H1035,biA1; alcE1; alc500 alcD1, which produces 100-times more ADH II activity than thealcAalcR deletion strain (alc500). Antibodies were raised against this ADH, and were used to screen a cDNA library in gt11. We have isolated the gene for an ADH which is over-expressed in H1035, and which we believe to be thealcB gene; cDNA and genomic clones were sequenced. The sequence contains three introns and encodes a protein of 367 amino acids. This protein shows a clear level of identity to a range of alcohol dehydrogenases, but is no more closely related to the ADH I and ADH III previously described inA. nidulans than to the ADHs ofS. pombe andS. cerevisiae. The significance of consensus sequences found in the 5 region of the gene is discussed in relation to the regulation of the gene.     

Chromosomal mapping and gene disruption of the ADHIII gene in Aspergillus nidulans

Summary There are at least three alcohol dehydrogenases in Aspergillus nidulans. ADHIII has no obvious physiological function. We describe here the cloning of the ADHIII gene (alcC), its mapping on linkage group VII by reverse genetics, and the properties of multicopy transformants tested for their ability to grow on a range of alcohols (butan-1-ol being the best substrate tested for growth). We were unable to detect any obvious alteration in phenotype of a strain carrying a disrupted copy of the ADHIII gene.     

An efficient cell-free translation system from Aspergillus nidulans and in vitro translocation of prepro-a-factor across Aspergillus microsomes

Summary We describe the preparation of an in vitro translation system from heat shock-treated Aspergillus nidulans, capable of supporting efficient and faithful synthesis of proteins from natural and in vitro transcribed eukaryotic messages. In vitro synthesized prepro--factor was translocated across Aspergillus nidulans microsomal membranes in either the homologous A. nidulans or a yeast cell-free system. The translocated prepro--factor was protected from digestion by protease and glycosylated to higher MW forms.     

ω-Grammotoxin blocks action-potential-induced Ca2+ influx and whole-cell Ca2+ current in rat dorsal-root ganglion neurons

Field-potential stimulation of rat dorsal-root ganglion (DRG) neurons evoked action-potential-mediated transient increases in intracellular free calcium concentration ([Ca²⁺]_i) as measured by indo-1-based microfluorimetry. Field-potential-evoked [Ca²⁺]_i transients were abolished by tetrodotoxin, and their dependence on stimulus intensity exhibited an abrupt threshold. -Conotoxin GVIA (-CgTx, 100 nM) inhibited action-potential-mediated Ca²⁺ influx by 79%, while nitrendipine (1 M) had little effect. -Grammotoxin SIA (-GsTx, 267 nM), a peptide toxin purified from the venom of the tarantula spider, Grammostola spatulata, blocked action-potential-mediated Ca²⁺ influx as effectively as did -CgTx, suggesting that -GsTx blocks N-type Ca²⁺ channels. In contrast to block by -CgTx, the block produced by -GsTx reversed upon washout of the peptide. -GsTx (270 nM) blocked 80%, and -CgTx (1 M) blocked 64%, of whole-cell Ca²⁺ current (I _Ca) elicited by step depolarization to 0 mV from a holding potential of –80 mV. -GsTx completely occluded inhibition of I _Ca by -CgTx. However, when applied after -CgTx, -GsTx produced an additional inhibition of 27%, indicating that -GsTx also blocked a non-N-type Ca²⁺ channel. BayK8644 (1 M) elicited an increase in I _Ca in the presence of maximally effective concentrations of -GsTx, suggesting that -GsTx does not block L-type channels. Thus, -GsTx displays a selectivity for Ca²⁺ channel subtypes which should prove useful for studying Ca²⁺ channels and Ca²⁺-channel-mediated processes.     

Nitrogen metabolite repression in Aspergillus nidulans: A farewell to tamA?

Summary Previous work has established that nitrogen metabolite repression in Aspergillus nidulans is mediated by the positive acting regulatory gene areA. Pateman and Kinghorn (1977) proposed that the gene tamA plays an equally important regulatory role in nitrogen metabolite repression as the result of work with tamA^r-50, an allele leading to inability to utilise nitrogen sources other than ammonium, and tamA^d-1, an allele leading to nitrogen metabolite derepression. Both tamA^r-50 and tamA^d-1 were subsequently lost. We have therefore attempted to reconstruct Pateman and Kinghorn's work with tamA. We propose that tamA^r-50 was in fact a pyroB^– tamA^– double mutation. pyroB^– mutations lead to a block in vitamin B₆ biosynthesis which can be supplemented by extremely high concentrations of ammonium. tamA^– mutations, possibly as the result of a membrane alteration, reduce the concentration of ammonium required to supplement the pyroB^– auxotrophy. There is, however, no evidence that pyroB^– or tamA- mutations, alone or in combination, affect the regulation of the levels of a number of enzymes subject to nitrogen metabolite repression. Reversion of pyroB^– strains constitutes a powerful positive selection technique for obtaining a wide variety of mutations in glnA, the probable structural gene for glutamine synthetase. We suggest that the nitrogen metabolite derepressed phenotype attributed to tamA^d-1 might have resulted from an extremely leaky glnA^– mutation.     

Chromosomal mapping of an alcC disruption with respect to amdA in Aspergillus nidulans

Summary We report the use of the riboB gene for a gene replacement in the alcC gene of Aspergillus nidulans, and show by reverse genetics that the alcC gene is very closely linked to the amdA gene.     

Einstellzeit der Pt-Elektrode bei Messungen nicht stationärer O2-Partialdrucke

Zusammenfassung Das Meßsignal bei sprunghaftenpO₂-Änderungen wird anhand des Diffusionsfeldes der Elektrode beschrieben. Es wird das zeitliche Verhalten des Meßsignals von blanken und membranbespannten Elektroden in gasförmigen und nicht gasförmigen Meßmedien betrachtet. Aus dem Verhalten des Meßsignals kann jeweils die Einstellzeit alssystematischer Meßfehler abgeleitet werden. In nicht gasförmigen Medien (z. B. biologisches Gewebe) übersteigt das Meßsignal nach einempO₂-Sprung zu höheren Werten das stationäre Endsignal. Daraus ergibt sich eine besondere Betrachtung der Einstellzeit in solchen Medien.Die Einstellzeit für Pt-Elektroden mit einfacher und doppelter Membran wird explizit angegeben. Schließlich wird für biologische Medien die Einstellzeit mit dem Diffusionsfehler [8] verglichen. Die Forderungen an eine Membran der Pt-Elektrode mit kleiner Einstellzeit und gleichzeitig kleinem Diffusionsfehler sind zusammengestellt.

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Erklärung der Symbole a Verhältnis der Diffusionskoeffizienten zweier Membranen - Bunsenscher Löslichkeitskoeffizient des Mediums - _m Bunsenscher Löslichkeitskoeffizient der Membran - b Verhältnis der Diffusionsleitfähigkeiten von Membran und Medium - C ₁,C ₂ Proportionalitätskonstanten zwischen Meßsignal und O₂-Partialdruck - D Diffusionskoeffizient des Mediums - D _m,D _m Diffusionskoeffizienten der Membranen - Diffusionskoeffizient der effektiven Membran - DF Diffusionsfehler - DGl Differentialgleichung - d _m,d _m Dicke der Membranen - Dicke der effektiven Membran - dimensionsloser Parameter des Diffusionsfehlers - erf Fehlerfunktion - exp Exponentialfunktion - F Faradaykonstante - grad Gradient - I stationäres Meßsignal vor dempO₂-Sprung - I stationäres Meßsignal nach dempO₂-Sprung - I(t), I(),I() instationäres Meßsignal als Funktion der Zeit bzw. zeitabhängiger dimensionsloser Parameter - K Diffusionsleitfähigkeit des Mediums - K _m Diffusionsleitfähigkeit der Membran - Diffusionsleitfähigkeit der effektiven Membran - dimensionsloser Parameter - n Summationsindex - pO₂ O₂-Partialdruck - pO₂ als Funktion von Ort und Zeit bzw. zeitabhängiger dimensionsloser Parameter; Diffusionsfeld der Elektrode - p _c konstanterpO₂ vor dempO₂-Sprung - p _c konstanterpO₂ nach dempO₂-Sprung - p(r ₀+d _m , ) pO₂ an der Grenze Membran/Medium in Abhängigkeit des Zeitparameters - p(r,o) Diffusionsfeld zum Zeitpunkt (t=0) despO₂-Sprunges - p(r₀+d_m, o) pO₂ an der Grenze Membran/Medium zum Zeitpunkt despO₂-Sprunges - R Radius der ebenen, kreisförmigen Elektrode - r ₀ Radius der Elektrode mit halbkugelförmiger Pt-Oberfläche - r Kugelkoordinate - ₁,₂ dimensionslose ortsabhängige Parameter - T ₉₀,T ₉₅ Zeit, bis 90% bzw. 95% des Signalunterschiedes nach dempO₂-Sprung ausgeglichen sind (Einstellzeit) - T ₉₀,T ₉₅ Einstellzeit der Elektrode mit Doppelmembran - T _90*,T _95* Zeit, bis sich das Signal nach Übersteigen des stationären Endwertes diesem auf 10% bzw. 5% angenähert hat - dimensionslose Parameter zu den vorangegangenen Einstellzeiten - t Zeitkoordinate - , dimensionslose zeitabhängige Parameter - t _max, _max Zeit maximaler Signalhöhe nachpO₂-Sprung und zugehöriger dimensionsloser Parameter - V(t) Diffusionsgesamtfluß zur Pt-Oberfläche - Stromdichtevektor der diffundierenden O₂-Moleküle - x, y, z Kartesische Koordinaten - Integrationsvariable - ² Laplace-Operator - partielle Ableitung nach der Zeit - Integral über eine Fläche - gerichtetes Flächenelement     

Identification and characterization of U1 small nuclear RNA genes from two crustacean isopod species

Four different units containing three variants of the U1 snRNA gene have been identified in the genome of Asellus aquaticus and only one unit has been identified in the genome of Proasellus coxalis. All four identified U1 snRNA genes can be folded according to the proper secondary structure and possess the functionally useful conserved sequences. Moreover, in the 3 flanking regions, all genes present both the 3 box, a conserved sequence required for 3 processing of mature snRNA, and a polyadenylation signal which is unusual for these genes. The PCR products were used as probes in fluorescent in-situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus and P. coxalis.     

Trennung der intestinalen β-Galactosidasen beim lactose-toleranten Erwachsenen durch Ultrazentrifugation im Dichtegradienten

Zusammenfassung Die intestinalen-Galactosidasen von 4 lactose-toleranten, erwachsenen Mitteleuropäern wurden im Saugbiopsie-Gewebe nach Solubilisierung mit Triton X-100 in einem linearen Mannitol-Gradienten (5–20%) auf der Ultrazentrifuge bei 4°C und 44000 U/min getrennt. Bei 12stündiger Zentrifugation fanden sich 3 Fraktionen, von denen die beiden schneller sedimenticrenden Lactose spalten. Alle 3 Fraktionen hydrolysieren p-Nitrophenyl--Galactosid. Die 3 isolierten-Galactosidasen entsprechen wahrscheinlich der neutralen Bürstensaum-Lactase, der sauren lysosomalen Lactase und einer cytoplasmatischen Hetero--Galactosidase.     

GM1-gangliosidosis: Chromosome 3 assignment of theβ-galactosidase-A gene (β GAL A )

The structural gene (GAL_A) coding for lysosomal -galactosidase- A (EC 3.2.1.23) has been assigned to human chromosome 3 using man-mouse somatic cell hybrids. Human -galactosidase-A was identified in cell hybrids with a species-specific antiserum to human liver -galactosidase-A. The antiserum precipitates -galactosidase-A from human tissues, cultured cells, and cell hybrids, and recognizes cross-reacting material from a patient with G_M1 gangliosidosis. We have analyzed 90 primary man-mouse hybrids derived from 12 separate fusion experiments utilizing cells from 9 individuals. Enzyme segregation analysis excluded all chromosomes for GAL_A assignment except chromosome 3. Concordant segregation of chromosomes and enzymes in 16 cell hybrids demonstrated assignment of GAL_A to chromosome 3; all other chromosomes were excluded. The evidence suggests that G_M1 gangliosidosis is a consequence of mutation at this GAL_A locus on chromosome 3.     

Proteinase — Antiproteinase imbalance in meningitis: Determination of α 1 proteinase inhibitor (α 1PI), elastase — α-1PI complex,and elastase inhibition capacity in cerebrospinal fluid

Summary Mortality and long-term neurologic sequelae are still frequent complications of meningitis despite effective antibiotic treatment. This suggests that pathogen-independent inflammatory mechanisms may play an important role in the course of this illness.Neutrophil granulocytes form the primary immune defense in meningitis. Once activated, these cells release elastase into the cerebrospinal fluid (CSF). Elastase may induce tissue damage if local antiproteinase capacity is low as under normal conditions.To define the relevance of this mechanism we studied 22 patients with meningitis. Concentrations of elastase in complex with the main antiproteinase 1-proteinase inhibitor (elastase- 1PI), 1-proteinase inhibitor ( 1PI), and elastase inhibition capacity (EIC) were measured in CSF of 9 patients with bacterial meningitis (BM), aged 1 month-214 years; 13 patients with non-bacterial meningitis (NBM), aged 1 month–15 years; and 20 patients in whom meningitis was excluded after spinal tap (control group), aged 6 months–15 years. The concentration of elastase- 1PI in the BM group (median 552 g/l) was significantly higher than in either the NBM group (median 30 g/l,p<0.01) or the control group (median 30 g/l,p<0.01). Similarly, the 1PI-concentration in the BM group was significantly higher (median 113 mg/l) than either the NBM group (median 13.7 mg/l,p<0.025) or the control group (median 6.3 mg/l,p<0.001). The concentration of elastase- 1PI shows a significant correlation with the duration of the infectious symptoms before admission to the hospital (r=0.51,p<0.02), but not with the number of neutrophil granulocytesr=0.23, p=0.21).Free elastolytic capacity in CSF could be demonstrated in 4 patients: 1 with BM, 2 with NBM, and 1 with pertussis pneumonia and enzephalitis.The measured insufficiency of the proteinase-antiproteinase system may indicate high-risk patients in need of additional anti-inflammatory therapy, e.g., with corticosteroids, during the initial phase of meningitis.Abbreviations 1PI 1-proteinase inhibitor, 1-antitrypsin - elastase- 1PI complex elastase- 1-proteinase inhibitor complex - EIC elastase inhibition capacity - BM group: bacterial meningitis - NBM group: non-bacterial meningitis - CSF cerebrospinal fluid     

Molecular cloning of the 3-phosphoglycerate kinase (PGK) gene from Aspergillus nidulans

Summary The Aspergillus nidulans 3-phosphoglycerate kinase gene (PGK) has been isolated from a phage genomic library, using the equivalent yeast gene as a hybridization probe. The location of the PGK gene within the cloned DNA has been physically mapped. The DNA sequence of a small region of the putative PGK has been determined and found to code for amino acids corresponding to the N-terminal end of the PGK protein. In contrast to the yeast PGK gene the Aspergillus gene contains a 57 base pair intron occurring between the coding sequences for amino acid 22 and 23.A DNA fragment encompassing the PGK gene was shown to hybridize a 1,700 base poly(A) mRNA, sufficient to encode the PGK polypeptide.     

Interactions of the RAD7 and RAD23 excision repair genes of Saccharomyces cerevisiae with DNA repair genes in different epistasis groups

Summary The RAD7 and RAD23 genes of S. cerevisiae affect the efficiency of excision repair of UV-damaged DNA. We have examined the UV survival of strains carrying the rad7 or rad23 deletion mutation in combination with deletion mutations in genes affecting different DNA repair pathways. As expected, the rad7 and rad23 mutations interact epistatically with the excision repair defective rad1 mutation, and synergistically with the rad6 and rad52 mutations that affect the postreplication repair and recombinational repair pathways, respectively. However, the rad7rad6 and the rad23rad6 mutants exhibit the same level of UV sensitivity as the radlrad6 mutant. This observation is of interest since, in contrast to the rad7 or the rad23 mutations, the rad1 mutant is very UV sensitive and highly excision defective. This observation suggests that RAD6 and RAD7 and RAD23 genes compete for the same substrate during DNA repair.     

Direction of DNA sequences within chromatids determined using strand-specific FISH

The 5 to 3 direction of DNA strands within chromatids of metaphase chromosomes can be determined by using simultaneous hybridization of a single strand of the telomere probe and a single strand of a repetitive sequence to slides pretreated for strand-specific hybridization. The telomere probe identifies the direction of the DNA helical strand remaining in each chromatid of the metaphase chromosomes. The direction of the repetitive sequence is then determined from the direction of the strand to which it hybridizes. This method was used to determine the 5 to 3 direction of three repetitive DNA sequences, each for a different human repeat family.