Biology Reference
In-Depth Information
CHAPTER
22
Anna Luchniak
*
,1
, Yusuke Fukuda
{,1
and Mohan L. Gupta, Jr.
{
Structure-Function
Analysis of Yeast Tubulin
*
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA
{
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA
CHAPTER OUTLINE
Introduction ............................................................................................................ 356
22.1 Reagents and Equipment .................................................................................358
22.2 Introducing Tubulin Mutations Into Yeast..........................................................360
22.2.1 Preparation of Modified Tubulin DNA........................................... 360
22.2.2 Transformation of Modified Tubulin DNA Into Yeast ...................... 360
22.2.3 Verification of Tubulin Mutations in Yeast..................................... 362
22.3 Analysis of Tetrad Viability..............................................................................363
22.3.1 Sporulation ................................................................................ 364
22.3.2 Microdissection of Haploid Spores ............................................... 364
22.3.3 Genotype Analysis of Haploid Spores............................................ 365
22.3.4 Mating-type Analysis................................................................... 365
22.4 Assessing Microtubule Stability By Drug Sensitivity ..........................................366
22.4.1 Benomyl Plate Assay .................................................................. 366
22.5 Direct Analysis of Microtubule Dynamics In Vivo...............................................367
22.5.1 Yeast Strains for in Vivo Microtubule Dynamics Analysis ................ 367
22.5.2 Maintaining Comparable GFP-TUB1 Expression Levels.................. 367
22.5.3 Preparation of Samples for Microscopy......................................... 368
22.5.4 Microscopy and Analysis of Microtubule Dynamics ........................ 368
22.6 Localization of Microtubule-Associated Proteins In Vivo....................................370
22.6.1 Yeast Strains for Localization of Microtubule-Associated Proteins ... 370
22.6.2 Maintaining Equal Levels of YFP-fusion Proteins ........................... 371
22.6.3 Microscopy and Analysis of Microtubule-Associated Proteins.......... 371
Acknowledgments ................................................................................................... 372
References ............................................................................................................. 373
1
Equally contributing authors.
