Abstract
Biochemistry is an important experimental tool in the study of protein functions. Biochemical studies frequently involve overexpression of a cloned gene and purification of the recombinant protein. The yeast Saccharomyces cerevisiae provides an effective system for expression and purification of recombinant proteins owing to the ease of applying molecular techniques and obtaining large quantities of cells with a low cost. Additionally, complex biochemical processes such as transcription and DNA repair can be studied in yeast cell-free extracts in vitro, which benefit greatly from a large collection of well-defined mutant strains. Controlled gene expression and preparation of cell-free extracts are important techniques in the yeast system. Two commonly used inducible gene expression systems, the GAL1 promoter and the CUP1 promoter, are described. Protocols of preparing yeast whole cell extracts and nuclear extracts are presented, each of which is designed for specific applications.
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References
Yuan, F., Zhang, Y., Rajpal, D. K., et al. (2000) Specificity of DNA lesion bypass by the yeast DNA polymerase η. J. Biol. Chem. 275, 8233–8239.
Zhang, Y., Yuan, F., Wu, X., and Wang, Z. (2000) Preferential incorporation of G opposite template T by the low fidelity human DNA polymerase ι. Mol. Cell. Biol. 20, 7099–7108.
Zhang, Y., Yuan, F., **n, H., Wu, X., Rajpal, D., Yang, D., and Wang, Z. (2000) Human DNA polymerase κ synthesizes DNA with extraordinarily low fidelity. Nucleic Acids Res. 28, 4147–4156.
Zhang, Y., Yuan, F., Wu, X., Rechkoblit, O., Taylor, J.-S., Geacintov, N. E., and Wang, Z. (2000) Error-prone lesion bypass by human DNA polymerase η. Nucleic Acids Res. 28, 4717–4724.
Lue, N. F., Flanagan, P. M., Edwards, A. M., and Kornberg, R. D. (1991) RNA polymerase II transcription in vitro. Methods Enzymol. 194, 545–550.
Wang, Z., Wu, X., and Friedberg, E. C. (1992) Excision repair of DNA in nuclear extracts from the yeast Saccharomyces cerevisiae. Biochemistry 31, 3694–3702.
Wang, Z., Wu, X., and Friedberg, E. C. (1993) Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 90, 4907–4911.
Wang, Z., Wu, X., and Friedberg, E. C. (1996) A yeast whole cell extract supports nucleotide excision repair and RNA polymerase II transcription in vitro. Mutat. Res. 364, 33–41.
He, Z., Wong, J. M. S., Maniar, H. S., Brill, S. J., and Ingles, C. J. (1996) Assessing the requirements for nucleotide excision repair proteins of Saccharomyces cerevisiae in an in vitro system. J. Biol. Chem. 271, 28,243–28,249.
Schultz, M. C., Choe, S. Y., and Reeder, R. H. (1991) Specific initiation by RNA polymerase I in a whole-cell extract from yeast. Proc. Natl. Acad. Sci. USA 88, 1004–1008.
Wang, Z., Svejstrup, J. Q., Feaver, W. J., Wu, X., Kornberg, R. D., and Friedberg, E. C. (1994) Transcription factor b (TFIIH) is required during nucleotide-excision repair in yeast. Nature 368, 74–76.
Wang, Z., Buratowski, S., Svejstrup, J. Q., et al. (1995) Yeast TFB1 and SSL1 genes, which encode subunits of transcription factor IIH, are required for nucleotide excision repair and RNA polymerase II transcription. Mol. Cell. Biol. 15, 2288–2293.
Wang, Z., Wei, S., Reed, S. H., et al. (1997) The RAD7, RAD16 and RAD23 genes of S. cerevisiae: requirement for transcription-independent nucleotide excision repair in vitro and interactions between the gene products. Mol. Cell. Biol. 17, 635–643.
Wang, Z., Wu, X., and Friedberg, E. C. (1997) Molecular mechanism of base excision repair of uracil-containing DNA in yeast cell-free extracts. J. Biol. Chem 272, 24064–24071.
Lombaerts, M., Tijsterman, M., Verhage, R. A., and Brouwer, J. (1997) Saccharomyces cerevisiae mms19 mutants are deficient in transcription-coupled and global nucleotide excision repair. Nucleic Acids Res. 25, 3974–3979.
Fields, S. and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–246.
Gietz, R. D. and Sugino, A. (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527–534.
Wang, Z., Wu, X., and Friedberg, E. C. (1991) Nucleotide excision repair of DNA by human cell extracts is suppressed in reconstituted nucleosomes. J. Biol. Chem. 266, 22472–22478.
Lue, N. F., Flanagan, P. M., Sugimoto, K., and Kornberg, R. D. (1989) Initiation by yeast RNA polymerase II at the adenoviral major late promoter in vitro. Science 246, 661–664.
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© 2006 Humana Press Inc., Totowa, NJ
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Wang, Z. (2006). Controlled Expression of Recombinant Genes and Preparation of Cell-Free Extracts in Yeast. In: **ao, W. (eds) Yeast Protocol. Methods in Molecular Biology, vol 313. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-958-3:317
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DOI: https://doi.org/10.1385/1-59259-958-3:317
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-437-1
Online ISBN: 978-1-59259-958-5
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