Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi

Bainbridge, Brian W.; Spreadbury, Claire L.; Scalise, F; Cohen, Jonathan

doi:10.1111/j.1574-6968.1990.tb03981.x

Cited by 42 publications

(13 citation statements)

References 7 publications

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“…Both Aspergillus strains were obtained from the Fungal Genetics Stock Center (Kansas, USA). Genomic DNA from P. camemberti was isolated according to Bainbridge et al [12].…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers

Navarrete

Roa

Vaca

et al. 2009

Cellular and Molecular Biology Letters

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Genetic manipulation of the filamentous fungus Penicillium camemberti has been limited by a lack of suitable genetics tools for this fungus. In particular, there is no available homologous transformation system. In this study, the nitrate reductase (niaD) and orotidine-5'-monophosphate decarboxylase (pyrG) genes from Penicillium camemberti were characterized, and their suitability as metabolic molecular markers for transformation was evaluated. The genes were amplified using PCR-related techniques, and sequenced. The niaD gene is flanked by the nitrite reductase (niiA) gene in a divergent arrangement, being part of the putative nitrate assimilation cluster in P. camemberti. pyrG presents several polymorphisms compared with a previously sequenced pyrG gene from another P. camemberti strain, but almost all are silent mutations. Southern blot assays indicate that one copy of each gene is present in P. camemberti. Northern blot assays showed that the pyrG gene is expressed in minimal and rich media, and the niaD gene is expressed in nitrate, but not in reduced nitrogen sources. The functionality of the two genes as a pyrG-containing plasmid. This is the first study yielding a molecular and functional characterization of P. camemberti genes that would be useful as molecular markers for transformation, opening the way for the future development of a non-antibiotic genetic transformation system for this fungus.

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“…Both Aspergillus strains were obtained from the Fungal Genetics Stock Center (Kansas, USA). Genomic DNA from P. camemberti was isolated according to Bainbridge et al [12].…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers

Navarrete

Roa

Vaca

et al. 2009

Cellular and Molecular Biology Letters

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“…(17) and Cenis (18), respectively .E . cola genomic DNA was prepared as described by Sambrook et al (19) .…”

Section: Methodsmentioning

confidence: 99%

Specific Detection of Aspergillus and Penicillium Species From Respiratory Specimens by Polymerase Chain Reaction (Pcr)

Makimura

Murayama

Yamaguchi

1994

JJMSB

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SUMMARY: A polymerase chain reaction (PCR) method capable of detecting both Aspergillus fumigatus infections, pulmonary non-fumigatus Aspergillus species (spp.) and Penicillium spp. from clinical specimens was established.The primer pair was designed on the basis of the sequence of the 18S-ribosomal RNA gene of A. fumigatus and P. notatum. A 385 by product was successfully amplified by this PCR method from all of 12 medically important Aspergillus spp. and Penicillium spp. (38 strains), but not from human, calf, Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), any of 14 medically important yeastlike fungal species tested (32 strains) including Candida al bicans, several non-al bicans Candida and Saccharomyces cerevisiae, Cryptococcus neo formans, Mucor spp. or Pneumocystis carinii. This specificity was subsequently confirmed by Southern hybridization analysis. The established PCR can detect such a small amount as 1 pg of A, fumigatus DNA by staining the PCR product with ethidium bromide. With sputum specimens from clinically diagnosed aspergilloma patients, this PCR technique was demonstrated to be a more sensitive diagnostic method for Aspergillus infections than conventional culture techniques.

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“…Nucleic acid isolation and analysis N. crassa genomic DNA was isolated from lyophilized mycelial powders (Bainbridge et al 1990). Small-scale plasmid DNA preparations were obtained by the alkaline lysis method as previously described (Williams et al 1992).…”

Section: N Crassa Strains and Growth Conditionsmentioning

confidence: 99%

Cloning and expression of the S-adenosylmethionine decarboxylase gene of Neurospora crassa and processing of its product

et al. 2000

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S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the formation of decarboxylated AdoMetDC, a precursor of the polyamines spermidine and spermine. The enzyme is derived from a proenzyme by autocatalytic cleavage. We report the cloning and regulation of the gene for AdoMetDC in Neurospora crassa, spe-2, and the eect of putrescine on enzyme maturation and activity. The gene was cloned from a genomic library by complementation of a spe-2 mutant. Like other AdoMetDCs, that of Neurospora is derived by cleavage of a proenzyme. The deduced sequence of the Neurospora proenzyme (503 codons) is over 100 codons longer than any other AdoMetDC sequence available in genomic databases. The additional amino acids are found only in the AdoMetDC of another fungus, Aspergillus nidulans, a cDNA for which we also sequenced. Despite the conserved processing site and four acidic residues required for putrescine stimulation of human proenzyme processing, putrescine has no effect on the rate (t 0.5 10 min) of processing of the Neurospora gene product. However, putrescine is absolutely required for activity of the Neurospora enzyme (K 0.5 100 lM). The abundance of spe-2 mRNA and enzyme activity is regulated 2-to 4-fold by spermidine.

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Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi

Cited by 42 publications

References 7 publications

Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers

Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers

Specific Detection of Aspergillus and Penicillium Species From Respiratory Specimens by Polymerase Chain Reaction (Pcr)

Cloning and expression of the S-adenosylmethionine decarboxylase gene of Neurospora crassa and processing of its product

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