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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Current genetics 19 (1991), S. 317-321 
    ISSN: 1432-0983
    Keywords: Algae ; Transformation ; Luciferase ; Chlorella ellipsoidea
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We report here on the development of a transient expression system for Chlorella ellipsoidea using a heterologous gene, firefly luciferase. Cells of this unicellular green alga were converted to protoplasts and treated with plasmid pDO432, which bears luciferase under the control of the CaMV 35S promoter. This treatment resulted in detectable luciferase activity in cell extracts. Expression required Cellulysin treatment, active cell metabolism, and the addition of carrier DNA and polyethylene glycol. Linearization of the luciferase plasmid did not significantly alter the activity. A time course of expression showed that luciferase is made rapidly, within about 7 h after addition of DNA, but that the activity disappears over the course of a few days. These experiments represent an important first step in the development of a Chlorella transformation system.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1617-4623
    Keywords: Yeast ; Transcription ; a- and α-specific genes ; MCM1 ; STE12
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have examined the relative contributions of MCM1 and STE12 to the transcription of the a-specific STE2 gene by using a 367 by fragment from the STE2 5′-noncoding region to drive expression of a reporter lacZ gene. Mutation of the MCM1 binding site destroyed MCM1 · α2-mediated repression in α cells and dramatically reduced expression in a cells. The residual expression was highly stimulated by exposure of cells to pheromone. Likewise, the loss of STE12 function reduced lacZ expression driven by the wild-type STE2 fragment. In the absence of both MCM1 and STE12 functions, no residual expression was observed. Thus, the STE2 fragment appears to contain two distinct upstream activation sequences (UASs), one that is responsible for the majority of expression in cells not stimulated by pheromone, and one that is responsible for increased expression upon pheromone stimulation. In further support of this idea, a chemically synthesized version of the STE2MCM1 binding site had UAS activity, but the activity was neither stimulated by pheromone nor reduced in ste12 mutants. Although transcription of aspecific genes also requires both MCM1 and STE12, these genes differ from a-specific genes in that they have a single, MCM1-dependent UAS system. The activity of the minimal 26 by UAS from the α-specific STE3 gene was both stimulated by pheromone and reduced in ste12 mutants. These data suggest that at α-specific genes STE12 and MCM1 exert their effects through a single UAS.
    Type of Medium: Electronic Resource
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