ABSTRACT
CONTENTS 30.1 Introduction ............................................................................................................................. 493 30.2 General Considerations for Gene Manipulations ................................................................... 494
30.2.1 Effi cient Gene Targeting Requires >500 bp Homologous DNA in A. nidulans ......... 494 30.2.2 Making Gene Targeting Constructs Using Fusion PCR .......................................... 494 30.2.3 Recent Advances in Improvement of Gene Targeting-The Ku Story ................... 495 30.2.4 Types of Transformation Markers for the Aspergilli ............................................... 497
30.2.4.1 Auxotrophic Markers .............................................................................. 497 30.2.4.2 Markers Providing Both Positive and Negative Selection ...................... 498 30.2.4.3 Drug Resistance Markers ....................................................................... 498 30.2.4.4 Markers that can be Recycled for Multiple Gene Modifi cations ............ 498 30.2.4.5 Considerations Regarding Accuracy of Gene Calling and Gene
Manipulations ......................................................................................... 499 30.2.5 Confi rming Gene Targeting ..................................................................................... 500 30.2.6 Nonintegrative Gene Expression Utilizing the AMA1 Sequence ........................... 501
30.3 Specifi c Types of Gene Manipulations ................................................................................... 502 30.3.1 Gene Deletion and Promoter Rundown ................................................................... 502
30.3.1.1 Gene Deletion and the Heterokaryon Rescue Technique ........................ 503 30.3.1.2 Promoter Rundown .................................................................................. 503
30.3.2 Fluorescent Protein Tagging .................................................................................... 505 30.3.3 Affi nity Tags for Protein Purifi cations and Proteomics .......................................... 505 30.3.4 Two-Step Site-Specifi c Mutation ............................................................................. 506
30.4 Conclusions ............................................................................................................................. 508 Acknowledgments .............................................................................................................................. 508 References .......................................................................................................................................... 508
30.1 Introduction The value and utility of any model genetic organism relies on many factors, including the basic biological features of the system and the ease with which the organism can be experimentally manipulated. Aspergillus nidulans has proved to be a highly valued model fungus not only because of its natural biology (e.g., it can undergo self-and out-crosses) but also because over 50 years of development of classical1 and molecular genetic methodologies2 have provided a multitude of techniques by which this organism can be experimentally manipulated. Since the advent of gene transformation over twenty years ago3 gene
manipulations in A. nidulans have been routine because homologous recombination occurs at a reasonable frequency in this organism.4 However, recent technical advances have greatly improved gene targeting. These advancements were spurred by the availability of high-quality genome sequences and the desire to easily target all A. nidulans genes. In this chapter, we review the types of approach that have been developed to manipulate A. nidulans for global functional gene analysis.
