Biomedical subjects

C J Bos

Publications and source records attributed to C J Bos.

At least 19 recordsLinked to original sources

PubMed2001

Genetic analysis in the asexual fungus Aspergillus niger.

The genetics of A. niger has been developed since 1980. An overview is presented of the advances in developing methods and collecting data. Important tools have been a) the application of essentially different methods to isolate mutants, b) the adaptation to A. niger ofA. nidulans methodology for analysis of the parasexual cycle, c) the choice of marker genes, and in some cases the artificial introduction of such genes, to select homozygous segregants arising from mitotic recombination. With the use of parasexual recombination, a genetic linkage map of A. niger has been established. In total, 110 nuclear and 1 cytoplasmic (mitochondrial) markers are available. The application of A. niger genetics in applied research is illustrated by examples.

Aspergillus niger↗

PubMed1994

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans.

A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 x 10(2) to 2.5 x 10(4) transformants per 10(6) viable protoplasts and microgram DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.

Aspergillus↗

PubMed1994

DOOR syndrome: additional case and literature review.

We report on a patient with sensorineural deafness, onycho- and osteodystrophy and mental retardation (DOOR syndrome) and review the literature. It appears that abnormal dermatoglyphics are a frequent feature of the DOOR syndrome, as all patients with DOOR syndrome in whom dermatoglyphic investigations were done, had multiple arches on their fingertips.

Bone Diseases↗

PubMed1993

Genetic maps of eight linkage groups of Aspergillus niger based on mitotic mapping.

This paper provides a genetic map of Aspergillus niger. At present 84 markers have been assigned to eight linkage groups. The chromosomal location of 60 markers is presented in this paper. The allocation of markers is based on recombination due to mitotic crossing over. Various methods for selection and analysis of homozygous recombinants were applied, using colour, auxotrophic and resistance markers. In addition, transformants carrying the heterologous Aspergillus nidulans gene coding for acetamidase (amdS) were used for mitotic mapping of markers in several linkage groups. In most of the transformants the amdS insert appeared to be centromere-distal to all known genetic markers, thus extending the genetic map. The linear order of the markers in the eight linkage groups has been determined. On the basis of these and earlier experiments tentative genetic maps for the eight linkage groups are presented. Genetic markers were found on both arms of the chromosomes, except for chromosomes II and IV. The genetic distance between markers and the centromere varies from about 10(-4) (LG I, II, V) up to more than 10(-2) (LG III, VI, VIII). The total frequency of mitotic recombination per genome in this fungus has been estimated to be at least 1.2 x 10(-1).

Amidohydrolases↗

PubMed1992

Arginine and proline genes of Aspergillus niger.

Aspergillus niger mutants defective in arginine or proline biosynthesis have been isolated and 12 genetic loci were identified. Mutation was induced by low doses UV, and mutants were isolated after filtration enrichment. The mutants were classified according to their phenotype in growth tests and were further characterized in complementation tests. The arginine auxotrophic mutants represent nine complementation groups. Three additional complementation groups were found for mutants that could grow on proline (two of them on arginine too). Linkage group analysis was done in somatic diploids obtained from a mutant and a master strain with genetic markers on six chromosomes. The arg genes belong to six different linkage groups and the pro genes to two. One arg-mutant could be complemented by transformation with the A. nidulans argB+ gene, and this A. niger gene thus appeared to be homologous to the A. nidulans argB. We isolated an A. niger strain with the argB gene tightly linked with the nicA1 marker. This strain is very suitable as acceptor for transformation with an argB-plasmid, because transformants with inserts on the homologous site can be recognized and analyzed genetically using the nicA1 marker gene.

Arginine↗

PubMed1991

Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function.

This study was prompted by the observation that an Aspergillus niger transformant with a multicopy bphA (benzoate-4-hydroxylase gene) insert did not grow on benzoate, whereas a transformant with only one extra copy could grow. Therefore, an extensive survey has been made for other genes involved in the conversion of benzoate into 4-hydroxy-benzoate. A transformant with two copies of the bphA gene was used in part of the mutation experiments in order to avoid the isolation of many bphA mutants. Filtration enrichment was used to isolate mutants defective in the conversion of benzoate. The Bph mutants that have been isolated belong to six complementation groups. Mutants with a defected structural gene (bphA) were again predominantly found but, in addition, five other groups of mutants that could not grow on benzoate were isolated. Genetic analysis of the mutants showed that the six genes were localized in different parts of the genome. This was used as an additional proof that some mutants involved different genes. Diploids with seven copies of the bphA gene and heterozygous for one of the other bph genes were constructed. No indication has been obtained that any one of the mutant classes is responsible for the growth-limiting factor in bphA multicopy transformants. This study shows that the p-hydroxylation of benzoate is very complex, although the metabolic pathway is straight forward.

Aspergillus niger↗

PubMed1990

Genetic analysis of Aspergillus niger: isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group.

This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers, form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9 x 10(-5) (cnxG13 in linkage group I) to 2.1 x 10(-2) (cnxD6 in linkage group III). The mean frequency of haploid chlorate resistant segregants was 1.3 x 10(-3). The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD+ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia+ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 x 10(-3) and 2.0 x 10(-5) respectively.

Aspergillus niger↗

PubMed1990

Isolation and molecular characterisation of the benzoate-para-hydroxylase gene (bphA) of Aspergillus niger: a member of a new gene family of the cytochrome P450 superfamily.

The gene coding for benzoate-para-hydroxylase (bphA) of Aspergillus niger was cloned using differential hybridisation techniques and complementation of mutants deficient in this enzyme activity. The nucleotide sequence of the gene was determined, the presence of two introns was shown and the transcription start and termination sites were determined. The structure of the mRNA upstream from the long open reading frame (ORF) is unusual. It contains two small, overlapping ORFs whose function is unknown. Comparison of the deduced amino acid sequence of the protein with the sequences present in the databanks, indicated a significant similarity of BPH to the superfamily of cytochrome P450 enzymes. Further analysis revealed that this protein is a member of a new P450 gene family designated P450LIII. The gene is designated CYP53. To increase the BPH activity of A. niger, multiple copies of the bphA gene were introduced into the genome of a recipient strain by transformation. Although increased intracellular levels of the BPH protein could be detected, the BPH enzyme activity was decreased, suggesting titration of another essential component.

Amino Acid Sequence↗

PubMed1990

An electrophoretic karyotype of Aspergillus niger.

An electrophoretic karyotype of Aspergillus niger was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosome-sized DNA was separated into four bands. Seven of the eight linkage groups could be correlated with specific chromosomal bands. For this purpose DNA preparations from seven transformant strains of A. niger each carrying the heterologous amdS gene of Aspergillus nidulans on a different chromosome were analysed. Some of the assignments were confirmed with linkage group-specific A. niger probes. The estimated sizes of the A. niger chromosome range from 3.5 to 6.6 Mb, based on gel migration relative to the chromosomes of Schizosaccharomyces pombe strains, Saccharomyces cerevisiae and A. nidulans. The total genome size of A. niger significantly exceeds that of A. nidulans and is estimated to be about 35.5-38.5 Mb. Electrophoretic karyotyping was used to allocate non-mutant rRNA genes and to estimate the number of plasmids integrated in a high copy number transformant.

Aspergillus niger↗

PubMed1990

Genetic analysis of amdS transformants of Aspergillus niger and their use in chromosome mapping.

The Aspergillus nidulans gene coding for acetamidase (amdS) was introduced into A. niger by transformation. Twelve Amd+ transformants were analysed genetically. The amdS inserts were located in seven different linkage groups. In each transformant the plasmid was integrated in only a single chromosome. Our (non-transformed) A. niger strains do not grow on acetamide and are more resistant to fluoroacetamide than the transformants. Diploids hemizygous for the amdS insert have the Amd+ phenotype. We exploited the opportunity for two-way selection in A. niger: transformants can be isolated based on the Amd+ phenotype, whereas counter-selection can be performed using resistance to fluoroacetamide. On this basis we studied the phenotypic stability of the heterologous amdS gene in A. niger transformants as well as in diploids. Furthermore, we mapped the plasmid insert of transformant AT1 to the right arm of chromosome VI between pabA1 and cnxA1, providing evidence for a single transformational insert. The results also show that the amdS transformants of A. niger can be used to localize non-selectable recessive markers and that the method meets the prerequisites for efficient mitotic mapping. We suggest the use of amdS transformants for mitotic gene mapping in other fungi.

Amidohydrolases↗

PubMed1989

Adenine and pyrimidine genes of Aspergillus niger and evidence for a seventh linkage group.

Mutants of Aspergillus niger requiring adenine and one mutant requiring cytosine were isolated after low-dose mutagenesis and enrichment. In addition we had mutants of two genes involved in the pyrimidine biosynthesis isolated as 5-fluoro-orotic acid-resistant mutants. The fifteen adenine-less mutants could be placed in seven complementation groups. From each group a representative mutant was analyzed in order to determine the linkage group by analysis of the mutants in a heterozygous diploid carrying markers in six linkage groups. AdeF could not be assigned to any one of these linkage groups and proved to be linked to nicB, oliC and cnxC, none of which could be placed in a linkage group. Thus, conclusive evidence was obtained for a seventh linkage group. As pyrA was used as selection marker for transformation, we constructed a pyrA strain with a linked marker which can be used in the genetic analysis of transformations.

Adenine↗

PubMed1989

Mitotic mapping in linkage group V of Aspergillus niger based on selection of auxotrophic recombinants by Novozym enrichment.

This paper describes a procedure which allows the quantitative selection of auxotrophs of the fungus Aspergillus niger by enzymatic killing of immobilized germinating prototrophic conidiospores. We have applied this procedure to linkage analysis on the basis of mitotic cross-over in this fungus. Starting with a heterozygous diploid strain, we could select auxotrophic homozygous diploid recombinants quantitatively. We estimated the frequency of crossing-over after correction for clonal distribution of recombinants, and localized four auxotrophic markers as well as the centromere on chromosome V of this fungus. The Novozym enrichment procedure proved to be useful in genetic analysis and for the construction of recombinant genotypes in the case of closely linked auxotrophic markers. The determination of gene order and the estimation of distances on the basis of benomyl-induced recombinant haploid segregants may lead to incorrect conclusions. Genetic analysis on the basis of homozygous recombinants, however, can provide reliable estimates of map distances.

Aspergillus niger↗

PubMed1988

Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger.

A start has been made on establishing a collection of Aspergillus niger colour and auxotrophic mutants with an isogenic background for use as a source of genetic markers. All strains have short conidiophores (csp A1), which makes them easy to handle on test plates. Genetic markers were combined stepwise by somatic recombination. Somatic diploids were obtained at frequencies of 10(-6) -10(-5) with conidiospores collected from a heterokaryon. The haploidization of heterozygous diploids was induced by benomyl. For unlinked markers, the frequency of recombinants varied from 35%-65%. Low frequencies of recombinants were found between markers on a same chromosome, but this was sometimes disturbed by mitotic crossing-over during an early stage of the diploid. Master strains were constructed having markers for six linkage groups.

Aspergillus niger↗

PubMed1988

Interpretation of UV-survival curves of Aspergillus conidiospores.

Semi-logarithmic dose-response curves for survival of UV-irradiated conidiospores of A. nidulans have an initial shoulder (at low doses) followed by a decline which becomes linear. To explain the initial shoulder and the resulting extrapolation number (log S intercept of the linear extrapolation line) a general model is presented, which includes multi-target (n) and multi-hit (h) effects and allows for the effect of initial repair and of a compound parameter k, which stands for inherent sensitivity of the spores and for dose received inside the spores. From experiments on (a) the modification of k (spore wall colour and shelter effects), (b) a repair-deficient strain (shoulderless) and (c) preincubation during which DNA-replication takes place, it is concluded that the shoulder is generated by initial repair rather than by a multi-hit nature of the cell-killing process. In experiments where k takes different values (sub a and c), notably the position of the point of intersection of the linear lines gives conclusive information. In general, the log S intercept of the linear extrapolation line cannot be used to estimate the target number.

Aspergillus nidulans↗

PubMed1987

Induction and isolation of mutants in fungi at low mutagen doses.

Since the yield of mutants per surviving cell increases in general with increasing dose of mutagen, it has often been concluded in the literature that it is the most efficient to apply high mutagen doses so that most spores are killed. As high doses of mutagen produce chromosome rearrangements and unnoticed mutations which disturb the genetic background, the relationship between mutant frequency and survival was analyzed with Aspergillus nidulans as a model. It is shown that for different types of mutants the highest mutant yield is obtained at low mutagen doses (20-50% survival). Mutant frequency increases with increasing dose of mutagen but levels off and even decreases at higher dosages. There is no simple linear relationship between mutant frequency and the logarithm of the mutagen dose or the logarithm of the surviving fraction. If appropriate enrichment procedures are also available auxotrophic mutants can best be isolated at low doses of mutagen. Taking into account the disturbance of the genetic background, mutation induction should be done preferentially at a survival level of at least 70%.

Acriflavine↗

PubMed1986

Characterization of Aspergillus nidulans mutants in carbon metabolism isolated after D-galacturonate enrichment.

A selective method for the isolation of Aspergillus nidulans mutants defective in the pyruvate dehydrogenase complex was devised. The essential steps in the procedure were a mutagenic treatment of conidia with X-rays to about 50% survival, followed by filtration enrichment in minimal medium with D-galacturonate as sole carbon source, and rescue on complete medium with acetate. The mutants thus isolated were phenotypically characterized on the basis of growth tests, and different genotypes were assigned on the basis of complementation tests. The majority of the mutants that were unable to utilize galacturonate were defective in one of the components of the pyruvate dehydrogenase complex. In addition, mutants defective in pyruvate carboxylase, mutants defective in glycerol catabolism and some novel mutants which were only unable to use D-galacturonate as carbon source were found. At least two genes were shown to be involved in D-galacturonate metabolism.

Aspergillus nidulans↗

PubMed1985

Gene amplification in Aspergillus nidulans by transformation with vectors containing the amdS gene.

Conidial protoplasts of an A. nidulans amdS deletion strain (MH1277) have been transformed to the AmdS+ phenotype with a plasmid carrying the wild type gene (p3SR2). Optimalisation of transformation and plating conditions now has resulted in frequencies of 300-400 transformants per microgram of DNA. Analysis of DNA from AmdS+ transformants of MH1277 showed that transformation had occurred by integration of vector DNA sequences into the genome. In virtually all these transformants multiple copies of the vector were present in a tandemly repeated fashion, not preferentially at the resident, partially deleted amdS gene. It is suggested that the observed integration phenomena are dependent on the genetic background of the A. nidulans strain, used for transformation. A model to explain the tandem type of integration is proposed.

Aspergillus nidulans↗

PubMed1985

Isolation and characterization of the Aspergillus niger trpC gene.

The Aspergillus niger trpC gene was isolated by complementation experiments with an Escherichia coli trpC mutant. Plasmid DNA containing the A. niger trpC gene transforms an Aspergillus nidulans mutant strain, defective in all three enzymatic activities of the trpC gene, to Trp+, indicating the presence of a complete and functional trpC gene. Southern blot analysis of DNA from these Trp+ transformants showed that plasmid DNA was present but that this DNA was not integrated at the site of the chromosomal trpC locus. The A. niger trpC gene was localized on the cloned fragment by heterologous hybridization experiments and sequence analysis. These experiments suggest that the organization of the A. niger trpC gene is identical to that of the analogous A. nidulans trpC and the Neurospora crassa trp-1 genes.

Aldose-Ketose Isomerases↗