Limitations on the recombinant plasmid selection by Lac(+)/Lac(-) colony phenotype detection.
Lac(+)/Lac(-) selection of recombinant plasmids based on the insertional inactivation of LacZalpha gene cannot differentiate recombinant clones in some cases. Several fragments of exon 11 of human brca1 gene were cloned in LacZalpha-containing plasmids so that frameshift appeared at the 5(')-end of the fragments tested but these fragments were in frame with the part of LacZalpha situated downstream of the polylinker. All plasmids except one caused blue colonies formation after being transformed in Escherichia coli LacZDeltaM15 cells in spite of the frameshift. The fact may be explained by reinitiation of translation within the mRNA transcribed from the inserted DNA fragments at in-frame AUG, GUG, and UUG. The data demonstrated limitations on the Lac(+)/Lac(-) selection of LacZalpha-based recombinant plasmids.
Gutkina
Nadezhda
N
Institute of Molecular Biology and Biophysics, Academy of Medical Sciences, Siberian Branch, Timakov str. 2, Novosibirsk 630117, Russia.
Varlakhanova
Natalia V
NV
Lysova
Marina V
MV
Kovalenko
Sergei P
SP
D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019398:Genes, BRCA1; D006801:Humans; D016254:Mutagenesis, Insertional; D010641:Phenotype; D010957:Plasmids; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics*
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase