Gene disruption by transformation in Neurospora crassa.

The regulatory gene cys-3+ controls the synthesis of a number of enzymes involved in sulfur metabolism. cys-3 mutants show a multiple loss of enzymes in different pathways of sulfur metabolism. The cys-3+ gene was isolated by transformation of an aro-9 qa-2 cys-3 inl strain with a clone bank followed by screening with the "sib selection" method. The library used (pRALl) contained inserts of Sau3a partial digest fragments of about 9 kilobases as well as the Neurospora qa-2+ gene. Double selection for qa-2+ and cys-3+ function was carried out. The transformants obtained with the isolated cys-3+ clone show recovery of the enzyme activities associated with the cys-3 mutation (e.g., arylsulfatase and sulfate permease). Restriction fragment length polymorphism experiments confirmed the identity of the clone. mRNA studies with Northern blots show that the expression of the cys-3+ gene is inducible. In contrast to cys-3+, the cys-3 (P22) mutant gene was not expressed at a higher level under sulfur-derepressed conditions.

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“…This result is in contrast to the rare homologous integrations with qa-2 and the low rate of homologous integration with am (17). 4).…”

Section: Discussioncontrasting

confidence: 49%

Molecular cloning and characterization of the cys-3 regulatory gene of Neurospora crassa.

Paietta¹,

Akins²,

Lambowitz³

et al. 1987

Mol. Cell. Biol.

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“…However, no marked effect on transformation was observed in filamentous fungi when vectors are linearized. Furthermore, analysis of the integration site did not reveal a targeting effect; most of the integrations of linear fragments occurred outside of the homologous locus (17).…”

Section: Resultsmentioning

confidence: 99%

Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila.

Perrot¹,

Barreau²,

Bégueret³

1987

Mol. Cell. Biol.

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The effect of the chromosomal ends of Tetrahymena thermophila on the stability of linear transforming molecules in the filamentous fungus Podospora anserina was tested. A derivative of an integrative vector for this fungus has been constructed, so that after linearization, the ends of the plasmid are the telomeric sequences of T. thermophila. After transformation, this linear molecule was maintained as an extrachromosomal plasmid with no integrated copies in about 50% of the transformants. Under selective conditions, there was approximately one linear molecule per 5 to 10 nuclei, and these extrachromosomal molecules were rapidly lost under nonselective conditions. The circular plasmid carrying an inverted repeat of T. thermophila telomeres could be linearized and processed in vivo.Transformation of mycelial fungi has been described previously, and common features emerge from the results observed for different species: the transformation efficiency is low and the vectors are integrative (11,25). In yeasts, integrative vectors were converted to autonomously replicating plasmids by the addition of sequences which act as replication origins. They are either autonomously replicating sequences (ARS) (10,22) or the replication origin of the natural 2,um plasmid (2). These autonomously replicating plasmids exhibit a high transformation efficiency and instability when the transformed strains are grown under nonselective conditions. Attempts to obtain such vectors in mycelial fungi were made by the addition of chromosomal sequences into the integrative vectors (5). The absence of positive results may be due to the coenocytic structure of these organisms, which leads to the possibility of complementation between different nuclei of the hyphae. The nuclei which lose the transforming gene can be maintained by the presence of a few nuclei which have retained the plasmid. Genomic sequences of Aspergillus nidulans which promote autonomous replication of yeast plasmids have been cloned (21). When they were introduced into A. nidulans, they did not confer replicating activity on the plasmids (1). One of these sequences greatly enhances the transformation efficiency of A. nidulans, but the vector containing this ansl sequence remains integrative. In Neurospora crassa, a recombinant pUC8 plasmid carrying the am gene has been reported to be able to replicate autonomously (8). However, the transforming vector is rearranged and is present as high-molecular-weight concatemers; furthermore, some plasmid sequences are integrated into the chromosomal DNA.For Podospora anserina, we have developed a system based on the transformation of an auxotrophic uraS-strain deficient for orotidylic acid pyrophosphorylase (OMPppase) by a recombinant vector (pPAura5-1) carrying the homologous uraS gene (3). This plasmid is integrative (19). We have tested the effect of the linearization of this vector on transformation. As it is known that broken DNA ends are highly * Corresponding author. reactive in recombination, the linearized vector was stabilized ...

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“…This was true regardless of the ade2 recipient or the type of mutated DNA, demonstrating the general utility of this method for introducing specific mutations into the genome of C. albicans. Recently, similar one-step gene disruptions have been carried out in both A. nidulans and N. crassa (19,22) but only a few of the Aspergillus transformants arose by a direct gene replacement, and none of the Neurospora transformants had fragments of the size expected for a simple gene replacement. Thus, recombination of linear molecules in C. albicans appears to be more similar to S. cerevisiae than to recombination in these filamentous fungi.…”

Section: Discussionmentioning

confidence: 99%

“…Other alternatives, such as integration at the ADE2 gene or multiple integrations at a nonhomologous site, may also account for the anomalous transformants. Nonhomologous integrations occur in A. nidulans and more frequently in N. crassa (7,19,22). Detailed studies are needed to establish the origin of the transformants with unusual hybridization patterns.…”

Section: Discussionmentioning

confidence: 99%

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Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants.

Kelly¹,

Miller²,

Kurtz³

et al. 1987

Mol. Cell. Biol.

162

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A method for introducing specific mutations into the diploid Candida albicans by one-step gene disruption and subsequent UV-induced recombination was developed. The cloned C. albicans URA3 gene was disrupted with the C. albicans ADE2 gene, and the linearized DNA was used for transformation of two ade2 mutants, SGY-129 and A81-Pu. Both an insertional inactivation of the URA3 gene and a disruption which results in a 4.0-kilobase deletion were made. Southern hybridization analyses demonstrated that the URA3 gene was disrupted on one of the chromosomal homologs in 15 of the 18 transformants analyzed. These analyses also revealed restriction site dimorphism of EcoRI at the URA3 locus which provides a unique marker to distinguish between chromosomal homologs. This enabled us to show that either homolog could be disrupted and that disrupted transformants of SGY-129 contained more than two copies of the URA3 locus. The A81-Pu transformants heterozygous for the ura3 mutations were rendered homozygous and Ura-by UV-induced recombination. The homozygosity of a deletion mutant and an insertion mutant was confimed by Southern hybridization. Both mutants were transformed to Ura+ with plasmids containing the URA3 gene and in addition, were resistant to 5-fluoro-orotic acid, a characteristic of Saccharomyces cerevisiae ura3 mutants as well as of orotidine-5'-phosphate decarboxylase mutants of other organisms.

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Gene disruption by transformation in Neurospora crassa.

Cited by 70 publications

References 12 publications

Molecular cloning and characterization of the cys-3 regulatory gene of Neurospora crassa.

Molecular cloning and characterization of the cys-3 regulatory gene of Neurospora crassa.

Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila.

Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants.

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