Isolation of a ras gene from the basidiomycete Coriolus hirsutus and use of its promoter for the expression of Pleurotus ostreatus manganese(II) peroxidase cDNA in C. hirsutus

Abstract

A ras gene homologue (named Ch.ras) was cloned from the basidiomycete Coriolus hirsutus. Ch.ras has a coding capacity of 215 amino acids (aa) interrupted by six small introns. The deduced Ch.Ras protein exhibited significant homology (86.5% or 86.0% identical) to the basidiomycete Coprinus cinereus Ras (215 aa) and Lentinula edodes Ras (217 aa) proteins. The 5Ј-upstream region of Ch.ras contains two GC boxlike sequences, one TATA boxlike sequence, one CCAAT box, and three CT-rich sequences. Primer extension analysis showed the presence of three transcriptional initiation sites: one is located in the most upstream CT-sequence and the other two just after it. By using the 1.4-kb fragment containing the promoter elements and transcriptional initiation sites, we have constructed the chromosome-integrating vector pHRP, which is useful for the expression of foreign genes in C. hirsutus. The Pleurotus ostreatus manganese(II) peroxidase (MnP) cDNA (designated mnpc) was inserted into the downstream of the Ch.ras promoter elements of pHRP, yielding pHRP-mnp. We obtained, with pHRP-mnp, C. hirsutus strains that show high levels of enzymatic activity of MnP and efficiently degrade pentachlorophenol (PCP), a chlorinated aromatic toxic compound.

Isolation, characterization, and expression analysis of a class IV chitin synthase gene from the edible basidiomycetous mushroom Pleurotus ostreatus

“…This result is consistent with the previous observation that the transcription starting points of various basidiomycetous fungal genes are often found in or near the CTstretches (Yamazaki et al 2004). Two polyadenylation signal-like sequences were found in the 3′-fl anking region.…”

Specific distribution in homobasidiomycete hymenophores of the transcripts of Ras protein and G-protein α-subunit genes

“…For amplification of the L. shimeji genomic and cDNA fragments encoding the Ras conserved region, the primers 3 and 4 (see Section 2) were used, which were designed based on the amino acid sequence conserved among the Ras proteins of homobasidiomycetes L. edodes [8], Coprinus cinereus [17] and Coriolus hirsutus [18]. Conventional PCR amplified 157-bp genomic DNA fragment and RT-PCR amplified 106-bp cDNA fragment.…”

Intron-Dependent Accumulation of mRNA inCoriolus hirsutusof Lignin Peroxidase Gene the Product of Which Is Involved in Conversion/Degradation of Polychlorinated Aromatic Hydrocarbons

“…For assessment of LiP activity, PCP conversion, and 2,7-DCDD degradation of C. hirsutus strains, mycelial cells were cultivated at 25 C in BK medium containing 25 g brewer's grains and 100 ml Kirk Basal III medium per liter. 17) Brewer's grains were obtained from Kirin Brewery (Tokyo) and Kirk basal III medium was prepared according to the method of Tien and Kirk. 18) Construction, propagation, and amplification of recombinant plasmids were carried out in E. coli JM109.…”