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    Author(s): Dan Cullen
    Date: 2004
    Source: Biodiversity of fungi : inventory and monitoring methods. Amsterdam : Elsevier Academic Press, 2004: Pages 85-88
    Publication Series: Miscellaneous Publication
    PDF: View PDF  (358 KB)

    Description

    In contrast to DNA, messenger RNA (mRNA) in complex substrata is rarely analyzed, in large part because labile RNA molecules are difficult to purify. Nucleic acid extractions from fungi that colonize soil are particularly difficult and plagued by humic substances that interfere with Taq polymerase (Tebbe and Vahjen 1993 and references therein). Magnetic capture techniques have overcome these problems, allowing rapid and efficient purification of microbial DNA and RNA. Magnetic capture involves the use of magnetic beads that are covalently attached to single-stranded DNA, most commonly oligo (dT) chains. The beads are mixed with crude lysates, and hybrid molecules are removed by application of a magnetic field. In the case of beaded oligo (dT) chains, polyadenylated [Poly (A)] RNA, which is a suitable template for reverse-transcription-coupled PCR (RT-PCR), is obtained. When combined with competitive PCR techniques (Gilliland et al. 1990), quantitative assessment of transcript levels is possible. Quantitative transcript analysis is as sensitive and specific as conventional PCR amplification of DNA for identifying fungi. The competitive RT-PCR technique is particularly well suited for differentiating genes within complex gene families such as those encoding the peroxidases, laccases, and cellobiohydrolases of white-rot fungi. In addition, the technique provides a measure of fungal biomass and a glimpse of physiological activities of fungi in situ. The most thoroughly studied gene family includes 10 or more structurally related genes that encode isozymes of lignin peroxidases in Phanerochaete chrysosporium (reviewed in Gaskell et al. 1994; Cullen 1997). When the RT-PCR technique was applied to P. chrysosporium soil cultures, unusual patterns of peroxidase gene transcription were demonstrated (Lamar et al. 1995). For example, certain lignin peroxidase transcripts, abundant in defined media (Stewart and Cullen 1999), were not expressed in soil during organopollutant degradation. The absence of those peroxidase transcripts argues strongly against a significant role for those genes in the degradation of pentachlorphenol (PCP) and polycyclic aromatic hydrocarbons (Bogan et al. 1996a, 1996b).

    Publication Notes

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    Citation

    Cullen, Dan. 2004. Messenger RNA transcripts. Biodiversity of fungi : inventory and monitoring methods. Amsterdam : Elsevier Academic Press, 2004: Pages 85-88

    Keywords

    Messenger RNA, transcripts, Phanerochaete chrysosporium, decay fungi

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