ABSTRACT
CONTENTS 31.1 Introduction ............................................................................................................................. 513 31.2 Histological Organelle-Staining Methods and DNA-Binding Fluorochromes ....................... 514 31.3 Vital Dyes ................................................................................................................................. 514 31.4 Immunofl uorescence Microscopy ............................................................................................ 514 31.5 Fluorescent Protein Tagging .................................................................................................... 515
31.5.1 Labeling with Multiple Fluorochromes ..................................................................... 516 31.5.2 Additional Techniques Using Fluorescent Proteins ................................................... 517 31.5.3 Fluorescent Protein Tagging Approaches .................................................................. 518
31.5.3.1 Plasmid-Based Approaches ....................................................................... 518 31.5.3.2 Fusion PCR-Based Approaches ................................................................. 518 31.5.3.3 Use of Flexible Linkers Between the Target Protein and the
Fluorescent Protein .................................................................................... 521 31.5.3.4 Gene Targeting Using nkuA Deletion Strains ........................................... 521
31.6 Conclusions and Prospects ....................................................................................................... 522 Acknowledgments ............................................................................................................................... 522 References ........................................................................................................................................... 522
31.1 Introduction The sequencing of the genomes of Aspergillus nidulans, Aspergillus fumigatus, and Aspergillus oryzae has ushered in a new era in research with these organisms. In addition, the development of robust and effi cient gene targeting, and fusion PCR protocols has reduced to days or weeks tasks that formerly required weeks or months. Nowhere is this more apparent than in the imaging of intracellular structures and organelles. Tagging of proteins with fl uorescent moieties [green fl uorescent protein (GFP), monomeric red fl uorescent protein (mRFP) and others] is now rapid and effi cient. Genes encoding proteins of interest can be identifi ed from the genome sequence data and tagged using procedures that do not even require the gene to be cloned in the conventional sense. This allows proteins to be localized accurately in living cells and the movement of these proteins to be followed over time. Organelles can be observed by tagging proteins specifi c to those organelles with fl uorescent moieties. In addition, immunofl uorescence microscopy, which has been used for decades, benefi ts greatly from the ability to epitope-tag proteins rapidly.
