Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Nuclear localisation of AM fungal genes by in situ hybridisation (repetitive sequences, metallothionein gene)

A fluorescent in situ hybridisation (FISH) protocol was optimised to localise target DNA sequences in interphase nuclei of G. mosseae, Gig. margarita and Gig. rosea.

Spores of the fungi were fixed in paraformaldehyde, crushed in NUC buffer, incubated in proteinase K and nuclei collected by filtering. The nuclei were then purified by centrifugation in a saccharose solution and resuspended in PBS buffer. FISH efficiency was confirmed by quantifying loci of tandem ribosomal DNA repeats in the nuclei using 18S or 25S digoxigenin-labelled probes and a three-step immunoreaction to amplify the fluorescence signal. Quantification of the rDNA loci performed on nuclei of Gig. margarita, Gig. rosea and G. mosseae subsequently provided a reference control for in situ hybridisation efficiency in experiments to localise other gene sequences.

Eight high copy number repetitive sequences isolated from Gig. margarita and Gig. rosea were also observed in nuclei. Their localisation using FISH showed that they were dispersed at numerous loci in nuclei making quantification unfeasible. The metallothionein gene from Gig. margarita (GmarMT1) gene was detected only at one or two loci in nuclei of Gig. margarita and detection of this low copy number gene necessitated further amplification of signal sensitivity. A similar distribution of the gene was observed also in Gig. rosea nuclei, which confirmed gene expression studies of the presence of a homologous gene to GmarMT1 in this fungus.

The protocol detected increased frequency of GmarMT1-homologous sequences in nuclei of spores bombarded with a GmarMT1 vector construct. Using the above protocol, extended DNA fibres were prepared from isolated nuclei of AM fungi for more detailed gene localisation by FISH.

More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/

Reported by

Institut national de la recherche agronomique
UMR 1088 PME, INRA-CMSE
21065 Dijon
France
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