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  • 1
    ISSN: 1432-0983
    Keywords: E. coli trpC ; Schizophyllum TRP1 ; Complementation ; Gene isolation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A Schizophyllum gene library was made in plasmid pRK9, Plasmids from this library were tested for their ability to complement several auxotrophic mutations of Escherichia coli. The goal was to isolate a Schizophyllum auxotrophic gene that could be used to transform a corresponding Schizophyllum auxotrophic mutant to prototrophy. Complementation was observed only for E. coli trpC indole 3-glycerol phosphate synthetase (IGPS) and phosphoribosyl-anthranilate isomerase (PRAI) mutations. Plasmids with a Schizophyllum sequence coding for both IGPS and PRAI activities were recovered from E. coli transformants. Expression of the Schizophyllum gene (TRP1) in E. coli is probably dependent on the Serratia marcescens promoter of plasmid pRK9. The DNA sequence containing the Schizophyllum TRP1 gene was not obviously rearranged in cloning.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-0983
    Keywords: Schizophyllum commune ; Transformation ; Gene isolation ; Basidiomycetes ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have developed a routine way to isolate genes directly from the basidiomycete fungus, Schizophyllum commune. Plasmid DNA from a genomic gene library was used to isolate five specific genes by complementation of Schizophyllum mutations via transformation. The mutant strains were deficient in the ability to synthesize either adenine (ade2 and ade5), uracil (ural, encoding orotidine-5′-phosphate decarboxylase; OMPdecase), tryptophan (rpl, encoding indole-3-glycerol phosphate synthetase; IGPS) or para aminobenzoic acid (pab1). In each case, Southern analysis revealed that transformation to prototrophy was concomitant with the integration of vector sequence into the genome of the S. commune mutant. Total DNA from transformants was restricted, religated, and used to transform E. coli. Ampicillin resistant plasmids were recovered from E. coli and tested for their ability to transform the corresponding mutant of S. commune. Plasmids complementing the ade2, adeS, pabl, trpl, and ural mutations were recovered.
    Type of Medium: Electronic Resource
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