Live cell fluorescence imaging for early expression and localization of RIB1 and RIB3 genes in Ashbya gossypii

Ashbya gossypii is a riboflavin overproducing filamentous fungus. RIB1 and RIB3 genes encode GTP‐cyclohydrolase II (GCH II) and DHBP synthase, respectively, the two rate limiting enzymes of the riboflavin biosynthetic pathway. The genes encoding yeast enhanced green fluorescent protein (yEGFP) and m...

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Bibliographic Details
Published in:Journal of basic microbiology 2014-01, Vol.54 (1), p.81-87
Main Authors: Sengupta, Sudeshna, Kaufmann, Andreas, T. S., Chandra
Format: Article
Language:English
Subjects:
Ashbya gossypii
Fluorescent imaging
Fungal Proteins - genetics
Fungal Proteins - metabolism
Gene expression
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
GTP Cyclohydrolase - genetics
GTP Cyclohydrolase - metabolism
Intramolecular Transferases - genetics
Intramolecular Transferases - metabolism
Microscopy, Fluorescence
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
RIB1 and RIB3 genes
Riboflavin - biosynthesis
Saccharomycetales - genetics
Saccharomycetales - metabolism
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Summary:Ashbya gossypii is a riboflavin overproducing filamentous fungus. RIB1 and RIB3 genes encode GTP‐cyclohydrolase II (GCH II) and DHBP synthase, respectively, the two rate limiting enzymes of the riboflavin biosynthetic pathway. The genes encoding yeast enhanced green fluorescent protein (yEGFP) and mCherry were fused with RIB1 and RIB3 genes, under their native promoters by PCR‐based gene tagging method for their early in vivo expression and cellular localization in A. gossypii. In the integrative transformants, the fusion proteins were expressed as cytoplasmic proteins from the germ bubble stage, in all the cells throughout the hypha. This was evident from the observation that mCherry fusion proteins were seen outside the vacuoles in the cytoplasm. The older matured cells of 14 h hyphae developed large vacuoles which showed green autofluorescence due to riboflavin. It is concluded that RIB1 and RIB3 genes are constitutively expressed in all the cells of this multicellular multinucleate fungus.
ISSN:0233-111X
1521-4028
DOI:10.1002/jobm.201200292