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  • 1
    Electronic Resource
    Electronic Resource
    Effect of spermine on peptide-bond formation, catalyzed by ribosomal peptidyltransferase (1992)
    Springer
    Molecular and cellular biochemistry 115 (1992), S. 19-26 
    Details
    ISSN: 1573-4919
    Keywords: spermine ; puromycin reaction ; peptidyltransferase ; peptide bond ; ribosome
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of spermine on the binding of AcPhe-tRNA to poly(U)-programmed ribosomes (step 1) and on the puromycin reaction (step 2) has been studied in a cell-free system, derived from E. coli. In the absence of ribosomal wash (FWR fraction) and at suboptimal concentration of Mg++ (6 mM), spermine stimulated the binding of AcPhe-tRNA at least five fold, while at 10 mM Mg++ there was a three fold stimulation. The above stimulatory effect was decreased at 6 mM Mg++, or was abolished at 10 mM Mg++ by the presence of FWR during the binding. Beside the stimulatory effect, spermine enhanced the stability of initiation complex AcPhe-tRNA-poly(U)-ribosome. In step 2, spermine affected the final degree of puromycin reaction and the activity status of peptidyltransferase. Both stimulatory and inhibitory effects have been observed, depending on the experimental conditions followed during the binding of the donor and during the peptide bond formation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00229091
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  • 2
    Electronic Resource
    Electronic Resource
    Positive selection for Dictyostelium discoideum mutants lacking UMP synthase activity based on resistance to 5-fluoroorotic acid (1991)
    Springer
    Molecular genetics and genomics 225 (1991), S. 492-500 
    Details
    ISSN: 1617-4623
    Keywords: Dictyostelium discoideum ; Gene disruption ; 5-Fluoroorotic acid ; UMP synthase ; Uracil auxotrophy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In the cellular slime mould Dictyostelium discoideum the two enzymatic activities of the pyrimidine pathway, orotidine-5′-phosphate decarboxylase (EC 4.1.1.23; OMPdecase) and orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase), are encoded by a single gene (DdPYR5-6). As in higher eukaryotes the bifunctional enzyme is referred to as UMP synthase. Here we present a method that allows efficient generation and selection of mutants lacking UMP synthase. D. discoideum cells are transformed with either of two different types of plasmids. One plasmid type contains no sequences homologous to the UMP synthase gene whereas the other type contains at least parts of this gene. UMP synthase− mutants, which were positively selected for in the presence of 5-fluoroorotic acid (5-FOA), were obtained with both plasmids. However, mutation rates were at least one order of magnitude higher if plasmids containing various portions of the UMP synthase gene were used as opposed to plasmids that lack any homology to the UMP synthase locus. Several mutant strains were extensively characterized. These strains lack OMPdecase activity and exhibit in addition to 5-FOA resistance a ura − phenotype. All mutants carry UMP synthase loci with deletions of various extents but integration of transforming plasmids was not detected. This efficient generation of 5-FOA resistance is part of a proposed complex selection scheme which allows multiple rounds of transformation of D. discoideum.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00261692
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  • 3
    Electronic Resource
    Electronic Resource
    Positive selection for Dictyostelium mutants lacking uridine monophosphate synthase activity based on resistance to 5-fluoro-orotic acid (1990)
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 11 (1990), S. 396-402 
    Details
    ISSN: 0192-253X
    Keywords: Transformation ; Dictyostelium discoideum ; UMP-synthase/ura-complementation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Whereas transformation ofDictyostelium discoideum now can be done routinely and reliably, there is increasing demand for a system that allows selection of cells that have gone through repeated transformation cycles. Such a system is presented here. Selection is based on resistance to 5-fluoro-orotic-acid (5-FOA). D. discoi-deum is highly sensitive to this drug, and cells can survive only in the presence of 5-FOA if they carry a defective UMP-synthase gene. After transformation with a plasmid carrying a cloned version of the UMP-synthase gene, 5-FOA resistant cells are obtained at high frequency. Because 5-FOA-resistant cells depend on exogenously added uracil, these cells can be taken through a second round of transformation. A plasmid-borne UMP-synthase gene renders 5-FOA-resistant cells phenotypically ura +. Finally, cells can be further transformed using the standard G418 selection system.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/dvg.1020110512
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    Paper (German National Licenses)
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