Induction of mitotic crossing-over in diploid strains of Aspergillus nidulansusing low-dose X-rays

As a contribution towards detecting the genetic effects of low doses of genotoxic physical agents, this paper deals with the consequences of low-dose X-rays in the Aspergillus nidulans genome. The irradiation doses studied were those commonly used in dental clinics (1-5 cGy). Even very low doses promoted increased mitotic crossing-over frequencies in diploid strains heterozygous for several genetic markers including the ones involved in DNA repair and recombination mechanisms. Genetic markers of several heterozygous strains were individually analyzed disclosing that some markers were especially sensitive to the treatments. These markers should be chosen as bio-indicators in the homozygotization index assay to better detect the recombinogenic/carcinogenic genomic effects of low-dose X-rays.

Asexual Recombination in a uvsH Mutant of Aspergillus nidulans

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.

Parasexuality in asexual development mutants of Aspergillus nidulans

The parasexual cycle with parameiosis has been characterized previously by the occurrence of genetic recombination and haploidization inside heterokaryotic hyphae prior to conidial formation. The aim of current research was to characterize, through genetic and cytological analyses, an asexual development mutant strain of A. nidulans and to use it to obtain parameiotic segregants. Analyses showed the medusa phenotype of the B84 strain, whose mutant allele was mapped in the chromosome I. The heterokaryons B84(med)//G422(med+) and B84(med)//G839(brl) were formed in liquid MM+2% CM and inoculated in the appropriate selective media. Two mitotic segregant groups were obtained: aneuploids and haploid stable recombinants. Mitotic segregants, wild-types, and developmental mutants, which did not produce new visible mitotic sectors in the presence of Benomyl and which showed normal meiotic behavior during the sexual cycle, were classified as parameiotics.

Genotoxic evaluation of sodium nitroprusside in Aspergillus nidulans

The exogenous nitric oxide donor, sodium nitroprusside, evaluated the recombinogenic potential of nitric oxide. Drug inhibited mycelial growth and conidiation in A757 Aspergillus nidulans master strain. Two heterozygous diploid strains, one wild (uvsH+//uvsH+) and the other defective to DNA repair (uvsH//uvsH) were used for recombinagenesis tests. Sodium nitroprusside recombinogenic effect was evaluated by the induction of homozygosis of recessive genes, originally present in heterozygous condition. Results show that sodium nitroprusside (40 uM, 80 uM and 160 uM) is effective in inducing mitotic crossing-over in diploid cells of A. nidulans

Benznidazole-induced genotoxicity in diploid cells of Aspergillus nidulans

Genotoxic effects of benznidazole were studied by the induction of homozygosis of genes previously present in heterozygous. UT448//A757 diploid strain was used in the benznidazole's recombinagenesis test. Although toxic effects on growth of colonies were not observed, 75 and 100 æM benznidazole induced an increasing of mitotic recombination events in diploid strain. Results were related to the induction of chromosomal breaks by the antiparasitic drug.

Molecular characterization of ABC transporter-encoding genes in Aspergillus nidulans

As a preliminary step towards characterizing genes encoding ATP-binding cassette (ABC) transporters that confer pleiotropic drug resistance in Aspergillus, we used a PCR-based approach to isolate four DNA fragments corresponding to different ABC type transporter genes. DNA sequencing and Southern blot analysis confirmed that they were distinct genes, which were designated abcA-D. One of these genes, abcD, was cloned and characterized. It was found to have a predicted 1,452-amino acid translation product with a calculated molecular mass of 147,467 kDa. The abcD gene specifies a single transcript of approximately 5.0 kb; there was a two- to six-fold enhancement of mRNA levels following exposure to miconazole, camptothecin, methotrexate, and ethidium bromide

Estudio toxicogenético de un extracto fluido de Ocimun basilicum L (albahaca blanca) / Toxicogenetic study of a fluid extract of Ocimun basilicum L (white sweet basil)

Se presentan los resultados obtenidos al evaluar el potencial toxicogenético de un extracto fluido con un menstruo etanólico al 70 porciento de Ocimun basilicum L. en 3 sistemas de ensayos a corto plazo, se emplean 2 modelos in vitro el sistema Salmonella/microsoma (Ames) y el de segregación mitótica con el hongo diploide Aspergillus nidulans D-30 y otro in vivo que utiliza el ensayo de inducción de micronúcleos en médula ósea de ratón. En el ensayo de Ames se observó una respuesta positiva con las cepas de Salmonella typhimurium TA-98 con activación metabólica en el rango de concentraciones de 1 500 y 5 000 m g/placa y TA-1535 sin activación metabólica en el rango de concentraciones de 50, 150, 500 y 1 500 m g/placa. En el ensayo de segregación mitótica, se evaluaron concentraciones desde 0,005 a 1,00 mg de sólidos totales/mL, sin detectar aumentos significativos concentración-dependiente en la Frecuencia de Sectores Segregantes por Colonias (FSC). En el ensayo de inducción de micronúcleos se ensayaron dosis de 500, 1 000 y 2 000 mg/kg de peso corporal (pc). Los resultados obtenidos demuestran que el extracto de Ocimun basilicum L. fue mutagénico en el sistema Salmonella/microsoma, moderadamente citotóxico en el ensayo de segregación mitótica sin mostrar daño genético en esta prueba. En el ensayo in vivo no se observó respuesta genotóxica en las dosis estudiadas

Electrophoretic characterization of Aspergillus nidulans strains with chromosomal duplications

Linhagens de Aspergillus nidulans que apresentam duplicaçäo cromossômica Dp(I-II) foram caracterizadas por eletroforese em campo pulsado. Foram analisados variantes morfologicamente deteriorados e melhorados. O cariótipo eletroforético demonstrou que em ambas as linhagens duplicadas (A e B) a banda de 4,2 Mb, que corresponde ao cromossomo II, näo estava presente e foi encontrada uma nova banda. Foram feitas hibridizaçöes usando os genes uapA (cromossomo I) e wA (cromossomo II), que demonstraram que a nova banda corresponde ao cromossomo II mais o segmento duplicado do cromossomo I. O tamanho da duplicaçäo foi determinado como aproximadamente 1,0 Mb. A análise das bandas cromossômicas da linhagem morfologicamente melhorada mostrou que o segmento duplicado do cromossomo I foi completamente perdido. Os variantes morfologicamente deteriorados V9 e V17 mostraram o mesmo cariótipo eletroforético apresentado pelas linhagens duplicadas. Contudo, o variante deteriorado V5 perdeu parte do cromossomo I e apresentou um rearranjo envolvendo o cromossomo V. Esse rearranjo pode ter resultado do tratamento mutagênico usado para a obtençäo dos marcadores genéticos. Os resultados obtidos nesse trabalho demonstram que a técnica de eletroforese em campo pulsado é uma ferramenta excelente para a localizaçäo de rearranjos cromossômicos.

Characterization and mapping of an informational suppressor in Aspergillus nidulans

The present work was undertaken to characterize a suppressor gene present in a mutant strain of A. nidulans obtained with NTG (N-Methyl-N'-Nitro-N-Nitrosoguanidine). Analyses of this mutant have shown that this suppressor, designated suO1, induces phenotypic co-reversion of several auxotrophic mutations and makes the strain sensitive to aminoglycoside antibiotics and lower temperatures. suO1 has shown to be on linkage group VIII. The vegetative growth of the mutant strain is very unstable because the suppressor gene induces the production of prototrophic mitotic sectors. The strains bearing the suO1 gene produce cleistothecia containing a reduced number of viable ascospores during the sexual cycle. The segregation of the genetic markers has also been observed in the mutant strain self crossed. From the above results it may be concluded that suO1 is an informational suppressor.

Phenylalanine transport in Aspergillus nidulans: demonstration of role of phenylalanine binding proteins.

Crude shock proteins extracted by two stage osmotic shock were further purified by affinity chromatography to obtain ligand (phenylalanine) specific binding protein (phebip) a component of phenylalanine (phe) transport system from wild type and a phe transport mutant fpaD11 of Aspergillus nidulans. A new eluent 0.1 M Tris-HCl containing 1.5 N NaCl and 0.5 N Na2CO3, pH 8 was used during the investigation. The elution profile of mutant phebip exhibited one simple and two compound peaks instead of three simple ones as exhibited by the wild type phebip. SDS-PAGE profile of mutant phebip showed faster electrophoretic mobility than that of wild type one. It is therefore evident that the mutant phebip has reduced molecular mass (M(r)) due to deletion of a segment that somehow has bearing on the binding capacity of the active site of phebip. The resultant erosion in the binding capacity of the mutant phebip is in turn responsible for its incapability to stimulate transport of ligand across the plasma membrane.

Does gene palB regulate the transcription or the post-translational modification of Pi-repressible phosphatases of Aspergillus nidulans?

When grown on low-Pi medium, the chaA1 pabaA1 palB7 mutant of Aspergillus nidulans excretes an acid phosphatase with steady-state kinetic properties, temperature sensitivity and electrophoretic mobility different from those of the enzyme excreted by the pabaA1 strain. The enzyme excreted by the pabaA1 strain at pH 6.5 showed PNP-P activity with negative cooperativity (K0.5 = 0.87 + or - 0.06 mM, n = 0.68 + or - 0.03) whereas the enzyme excreted by the chaA1 pabaA1 palB7 mutant showed Michaelian kinetics (Km = 0.46 + or - 0.03 mM, n = 1.00 + or - 0.02). The apparent half-lives at 60§C, pH 5.5, of acid phosphatase excreted by the pabaA1 and chaA1 pabaA1 palB7 strains were 58.6 + or - 4.9 min and 21.5 + or - 1.8 min, respectively. Furthermore, the electrophoretic mobility of acid phosphatases excreted by the palA1, palB7, palC4, palE11 and palF15 mutants of A. nidulans was altered and differed from the electrophoretic mobility of the enzyme excreted by the wild-type strain. Also, the palB7 mutation altered the electrophoretic pattern of acid phosphatases synthesized on high-Pi medium. These results are compatible with the post-translational modifications in the Pi-repressible phosphatases rather than with the action of gene palB in controlling the transcription of structural genes of these enzymes

A new method to detect potential genotoxic agents using mitotic crossing-over in diploid strains of Aspergillus nidulans

This paper presents a new method for detecting mitotic crossing-over in Aspergillus nidulans, based on the "homozygosity index" (HI) of recessive genes originally present in hetrozygosis in diploid strains, which occurs after mitotic crossing-over between the marker in question and the centromere. Since homozygous diploids (-/-) for auxotrophic markers can not grow in MM, homozygotization can be demonstrated by distorted mitotic segregation of the alleles involved. Two similar diploid strains (UT 448/UT 184 and Z1//UT 184), which differ by a chromosomic duplicate segment transposed from chromosome II to I in the Z1 haploid strain, were used. This excess of genetic material confers to the Z1 mutant the uvs character and makes Z1//UT 184 more unstable and sensitive to genotoxic agents, as evidenced by its high spontaneous recombinational index.

A simplified method for the isolation of high molecular weight DNA from Aspergillus nidulans

We have developed a simple method for obtaining DNA from mycelium of the fungus Aspergillus nidulans. A single isopropanol preparation yields good quality high molecular weight DNA preparations that are not contaminated with proteins or salts, and that are easy to solubilize and to digest with restriction enzymes. High yields (approximately 1.6 mg DNA per gram of wet mycelium) are obtained. Contamination with RNA is minimal and there is no need to use RNase. It has been successfully used in our laboratory for many molecular biology experiments.

Isolation and characterization of an analogue-resistant aminoacyl-tRNA synthetase mutant in Aspergillus nidulans.

Six mutants resistant to p-fluorophenylalanine (FPA) were selected on a medium containing aspartate as the sole source of nitrogen using a phenylalanine-requiring (phenA)auxotroph of A. nidulans as the wild type. The mutants, on the basis of genetic characterization, were found to be alleilic and located on the left arm of the linkage group III, approximately 13 map unit left to meth H locus, henceforth assigned to the symbol fpaV. At a fixed concentration of phenylalanine (23 micrograms/ml), the LD50 value of FPA for all the six mutants was found to be about three times more than that for the wild type strain. Affinity chromatographic purification of the enzyme phenylalanyl-tRNA (Phe-tRNA) synthetase from the mutant as well as the wild type strains, revealed that the wild type enzyme had about 1.4-fold higher affinity for phenylalanine as compared to that for FPA, both in the affinity column and in the catalytic reaction. However, the mutant enzyme showed almost a similar affinity for both in columns but a greatly reduced affinity for FPA in the catalytic reaction.

Evaluación de la actividad genética de los medicamentos meprobamato y pirimetamina en Aspergillus nidulans / Evaluation of genetic activity of meprobamate and pirimetamine on Aspergillus nidulans

El objetivo de este trabajo es estudiar la actividad genética como inductores de recombinación mitótica y de delecciones, de dos medicamentos: meprobamato y pirimetamina, utilizando el ascomiceto Aspergillus nidulans como organismo de ensayo. Para el primer caso se utilizó una cepa diploide heterocigótica para marcadores del color de los conidios, y en el segundo caso, la cepa llevaba una duplicación de parte del genoma en el grupo de ligamento II. Para cada medicamento se probaron varias concentraciones diferentes; 6 para el meprobamato y 4 para la pirimetamina. Los resultados obtenidos indican que el meprobamato induce delecciones a las dosis de 10mM, 5mM y 2,5mM. La pirimetamina, a su vez, presentó un efecto positivo como inductora de recombinación mitótica a la dosis de 50*g/ml. Estos resultados indican que estos medicamentos, de amplio uso en nuestro país, presentan cierto tipo de actividad genética, detectada en las condiciones experimentales usadas por nosotros en este trabajo