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Applied and Environmental Microbiology, December 2003, p. 7083-7090, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7083-7090.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Use of Multiplex Reverse Transcription-PCR To Study the Expression of a Laccase Gene Family in a Basidiomycetous Fungus

Tania González,{dagger} María C. Terrón, Ernesto J. Zapico,{ddagger} Alejandro Téllez,§ Susana Yagüe, José M. Carbajo,|| and Aldo E. González*

Centro de Investigaciones Biológicas, E-28040 Madrid, Spain

Received 30 June 2003/ Accepted 9 September 2003

Laccases produced by white rot fungi are involved in the degradation of lignin and a broad diversity of other natural and synthetic molecules, having a great potential for biotechnological applications. They are frequently encoded by gene families, as in the basidiomycete Trametes sp. strain I-62, from which the lcc1, lcc2, and lcc3 laccase genes have been cloned and sequenced. A multiplex reverse transcription-PCR method to simultaneously study the expression of these genes was developed in this study. The assay proved to be quick, simple, highly sensitive, and reproducible and is particularly valuable when numerous samples are to be analyzed and/or if the amount of initial mRNA is limited. It was used to analyze the effect of 3,4-dimethoxybenzyl alcohol (veratryl alcohol) and two of its isomers (2,5-dimethoxybenzyl alcohol and 3,5-dimethoxybenzyl alcohol) on differential laccase gene expression in Trametes sp. strain I-62. These aromatic compounds produced different induction patterns despite their chemical similarity. We found 2,5-dimethoxybenzyl alcohol to be the best inducer of laccase activity while also producing the highest increase in gene expression; 3,5-dimethoxybenzyl alcohol was the next best inducer. Transcript amounts of each gene fluctuated dramatically in the presence of these three inducers, while the total amounts of laccase mRNAs seemed to be modulated by a coordinated regulation of the different genes.

* Corresponding author. Mailing address: Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain. Phone: 34918373112. Fax: 34915360432. E-mail: aldo{at}cib.csic.es.

{dagger} Present address: Instituto Cubano de Derivados de la Caña de Azúcar, Havana, Cuba.

{ddagger} Present address: Biotechnology Department, University of Hamburg, Hamburg, Germany.

§ Present address: Departamento de Biotecnología, Universidad Autónoma Metropolitana Iztapalapa, Col. Vicentina, C.O. 09340 México D.F., Mexico.

|| Present address: INIA, 28040 Madrid, Spain.

Applied and Environmental Microbiology, December 2003, p. 7083-7090, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7083-7090.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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