Protoplast fusion, increased

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. 

During the fusion process the relative surface area decreases with increasing volume indicating a loss of membrane material (about 22% in Fig. 51). In analogy to the fusion process of protoplasts it can be assumed that the excess lipid is removed in form of small, submicroscopic vesicles (Fig. 52). The electric breakdown in the membrane contact zone leads to the formation of several pores in which lipid molecules are randomly oriented (Fig. 52 b). The molecules reorient forming submicroscopic vesicles and the new membrane of the fused vesicle (Fig. 52c). Thus, fused giant liposomes should contain small, submicroscopic vesicles. This could possibly be proven by using fluorescence-labelled lipids for liposome fusion. 

Recently, a photoactivatable variant from Aequoria victoria green fluorescent protein (pa-GFP) was reported (Patterson and Lippincott-Schwartz 2002), yielding an increase in fluorescence emission intensity (at k 520 nm) by a factor of 100 when excited at k 488 nm after spectral activation at A. 408 nm. This phenomenon is due to an internal photoconversion process in the protein and allows spectral photoactivation of this protein in a very local way such as in the nucleus of a living cell (Post et al. 2005). In tobacco BY-2 protoplasts, we transiently co-expressed pa-GFP or pa-GFP fusion proteins and red-fluorescent protein (DsRed)-tagged prenylated Rab acceptor 1 (Pral At2g38360), a membrane protein that localizes in speckles around the nuclear envelope. The DsRed transfection allows proper cell identification and visualization before activation (via Pral -DsRed fluorescence). After pa-GFP... 

When each of two point-adherent protoplasts was stimulated separately and successively with a time interval t, , the percentage of fusion decreased with increasing t and eventually no fusion was induced if, for instance, t. > 30s for R.serpentina protoplasts. This result indicates that the fusion-susceptible state, to which the membrane or protoplast is transferred by electric impulse, decays within about 30 s in this case. 

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