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  • 1
    ISSN: 1432-0983
    Keywords: Key words Bleomycin hydrolase ; Saccharomyces cerevisiae ; Thiol proteases ; Protein amphitropism ; Processing of glycosyl-phosphatidylinositol (GPI) anchor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Bleomycin hydrolase, Blh1p, from yeast was co-purified with Gce1p, a cAMP-binding ectoprotein, anchored to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Blh1p is a hydrophilic thiol protease lacking transmembrane domains. We have used polyclonal antibodies to study the topology of the over-expressed protein in yeast and have found that it is amphitropic. Part of Blh1p is associated with plasma membranes, and most of the rest occurs in the cytosol. Both the growth conditions and calcium were found to have minor influences on the topology of Blh1p, in that glucose and the earth-alkali ion slightly enhanced recruitment to the membrane. We have examined the possibility that co-purification of Blh1p with Gce1p has a functional basis, and have observed that over-expression of BLH1 in yeast leads to an acceleration of the glucose-induced amphiphilic to hydrophilic conversion of Gce1p, wherein Blh1p could either directly catalyse the proteolytic removal of the polar headgroup of the GPI anchor subsequent to an initial lipolytic cleavage by a GPI-specific phospholipase C or indirectly modulate the reaction. The data show that a thiol protease is involved, but point to an indirect role of Blh1p in GPI processing. Proteases with similar or overlapping substrate specificity are likely to exist, since deletion of BLH1 neither entails a growth defect on any carbon source tested, nor the loss of proteolytic processing of the GPI anchor of Gce1p. Reduced proteolytic GPI processing is, however, observed in the blh1 mutant and the corresponding acceleration in the respective BLH1 multi-copy transformant.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-2242
    Keywords: Gliadin ; HMW glutenin ; SDS electrophoresis ; Biochemical marker ; 1A-1R, 1B–1R wheatrye double translocation ; Doubled haploids ; Triticum aestivum L.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Eighteen available doubled haploid wheat lines with a cytologically proven 1A–1R, 1B–1R double translocation, which where derived via anther culture from four crosses of the 1A–1R wheat-rye translocation cv “Amigo” with several 1B–1R wheat-rye translocation forms, were subjected to electrophoretic seed protein analysis. Besides, the five parents used in the crosses and some other wheat cultivars and doubled haploid lines (19 with a 1B–1R single translocation, 10 with a 1A–1R translocation and 7 without any 1R translocation) were also included in the investigation. It was found that the gliadin patterns visualized after SDS polyacrylamide gel electrophoresis of alcohol-soluble seed protein extracts can differentiate not only 1B–1R and 1A–1R translocation forms from wheats without any 1R-translocation chromosome, but also 1B–1R and 1A–1R wheats from each other. Moreover, 1A–1R, 1B–1R double translocation lines can be distinguished as well due to characteristic differences revealed between 1A–1R and 1B–1R translocation forms. Thus, all of tested dh1- and dh2-grains of the double translocation lines showed the expected doublet: the 1A–1R translocation (“Amigo”)-typical rye band and the 1B–1R translocation (“Kawkas”)-typical rye band. Consequently, gliadin patterns estimated after SDS electrophoresis may be used as markers for the fast detection of the desired 1A–1R, 1B–1R double translocation forms among 1A–1R single translocation lines, 1B–1R single translocation lines and lines without any 1R-translocation in the progenies of appropriate crosses. Furthermore, by means of gliadin tests on the dh2-generation the excellent stability of the double translocation 1A–1R, 1B–1R during more than one propagation phase has been proven. Estimations of high-molecular weight (HMW) glutenin subunits coded by 1A and 1B chromosomes are compatible with the double translocation constitution. A few deviating results can be explained by crossing-over events. Seed protein analysis revealed that it is possible to produce 1A–1R, 1B–1R double translocation lines with good glutenin compositions provided that adequate favourable parents are used.
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