Protocol

Pichia Protocols

Volume 389 of the series Methods in Molecular Biology pp 189-201

Classical Genetics

  • Ilya TolstorukovAffiliated withKeck Graduate Institute of Applied Life Sciences
  • , James M. CreggAffiliated withKeck Graduate Institute of Applied Life Sciences

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Abstract

A significant advantage of Pichia pastoris as an experimental system is the ability to readily bring to bear both classical and molecular genetic approaches to a research problem. Although the advent of yeast molecular genetics has introduced new and exciting capabilities, classical genetics remains the approach of choice in many instances. These include the generation of mutations in previously unidentified genes (mutagenesis), the removal of unwanted secondary mutations (backcrossing), the assignment of mutations to specific genes (complementation analysis), and the construction of strains with new combinations of mutant alleles. This chapter describes these genetic manipulation methods for P. pastoris. In addition, certain yeast genes are essential for survival of the organism. However, determining whether a newly cloned gene is essential or not can be difficult with P. pastoris. In this chapter, we also describe a series of experiments to investigate the potential essential nature of a cloned gene in this yeast.

Key Words

Pichia pastoris molecular and classical genetics backcrossing complementation analysis strain construction