ABSTRACT
The gene encoding for bacterio-opsin (bop gene) from Halobacterium halobium has been introduced in a yeast expression vector. After transformation in Schizosaccharomyces pombe, bacterio-opsin (BO) is expressed and was detected by antisera. The precursor protein of BO (pre-BO) is processed by cleavage of amino acids at the N-terminal end as in H. halobium. Addition of the chromophore, retinal, to the culture medium results in a slight purple colour of the yeast cells indicating the in vivo regeneration of BO to bacteriorhodopsin (BR) and its incorporation into membranes. Therefore, in contrast to the expression in E. coli, isolation of the membrane protein and reconstitution in lipid vesicles is not necessary for functional analysis. The kinetics of the ground state signal of the photocycle BR in protoplasts is demonstrated by flash spectroscopy and is comparable to that of the natural system. The present investigation shows for the first time the transfer of an energy converting protein from archaebacteria to eukaryotes by genetic techniques. This is a basis for further studies on membrane biogenesis, genetics, and bioenergetics by analysis of in vivo active mutants.
Subject(s)
Bacteriorhodopsins/genetics , Halobacterium/genetics , Saccharomycetales/genetics , Schizosaccharomyces/genetics , Transfection , Bacteriorhodopsins/biosynthesis , Bacteriorhodopsins/physiology , Blotting, Western , Genes, Bacterial , Genetic Vectors , Membrane Proteins/genetics , Photochemistry , Plasmids , Protein Conformation , Protein Precursors/metabolism , Spectrum Analysis/methodsABSTRACT
Isolated Acetabularia crenulata nuclei were injected with Mengo virus RNA solution and then implanted into anucleate posterior Acetabularia mediterranea cell fragments or fused with Acetabularia ryukyuensis cytoplasts. The injected animal virus RNA was actively translated in the plant cell cytoplasm. Mengo virus proteins were detected and localized in Acetabularia cytoplasts by use of an immunofluorescence method on the first to fifth day after injection.
Subject(s)
Acetabularia/metabolism , Chlorophyta/metabolism , Mengovirus/metabolism , Protein Biosynthesis , RNA, Viral/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , Fluorescent Antibody Technique , Species Specificity , Viral Proteins/biosynthesisABSTRACT
Protoplasts were isolated from cells of Acetabularia cliftonii, which are presumed to be haploid. The release of the protoplasts occurred after treatment of the cells with papain or proteinase K. They are genuine protoplasts since they contain a nucleus. Fusion was initiated by mechanically pushing together two protoplasts. Under these conditions, the efficiency of fusion was more than 90% within 30 minutes at room temperature. Haploid cells from one cyst, i.e., cells which eventually would have formed gametes of the same mating type, exhibit a greater propensity for fusion as compared to haploid cells from different cysts.
