Yeast mutant fusion

Similar techniques were used by Shinohara et al (71) to develop hybrids with increased production of fusel alcohols and esters. Protoplast fusion techniques have been used to confer amylolytic activity to brewery yeasts (22) and ethanol tolerance to wine yeasts (70) Farris et al (72) used protoplast fusion to produce hybrids with killer factor that is, the ability to secrete proteinic toxins. Kunkee and coworkers (25) utilized a leucine auxotrophic mutant strain of S. cerevisiae (UCD Montrachet 522) to produce base wine for brandy production the mutant strain produces less isoamyl alcohol, reducing the quantity of fusel alcohols in the subsequent brandy. And Thornton (48) discussed the progress in utilizing plasmid vectors to introduce new genes into wine yeasts he cautioned, however, that until the yeast genome is better understood that direct gene manipulation techniques will be of limited value. 

Yeast cells, like all eukaryotic cells, express more than 20 different related v-SNARE and t-SNARE proteins. Analyses of yeast sec mutants defective in each of the SNARE genes have identified the specific membrane-fusion event in which each SNARE protein participates. For all fusion events that have been examined, the SNAREs form four-helix bundled complexes, similar to the VAMP/syntaxin/SNAP-25 complexes that mediate fusion of secretory vesicles with the plasma membrane. However, in other fusion events (e.g., fusion of COPII vesicles with the c7s-Golgi network), each participating SNARE protein contributes only one a helix to the bundle (unlike SNAP-25, which contributes two helices) in these cases the SNARE complexes comprise one v-SNARE and three t-SNARE molecules. 

The assays described in this chapter are designed to quantify the relative rate with which an NLS green fluorescent protein (NLS-GFP) fusion protein moves in and out of the nucleus, in normal and mutant yeast cells, under a variety of physiological and nonphysiological conditions. The assay strategy circumvents the need to microinject substrates, which is not generally feasible in yeast. Al-... 

Figure 18.7 Results of the yeast-2-hybrid assay. PYRI and three different pyrabactin-insensitive mutants were constructed as binding domain (BD) fusion proteins and tested for their interaction with activation domain (AD)-fused HABl in the presence of...

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