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Selective amplification of DNA fragments coding for the G domain of factors IF2 and EF-Tu, two G proteins from the myxobacterium Stigmatella aurantiaca.

Institut de Biologie Moléculaire et d'Ingénierie Génétique, Centre National de la Recherche Scientifique (CNRS) URA1172, Université de Poitiers, France.

Abstract

Two DNA fragments of the genome of the myxobacterium Stigmatella aurantiaca were selectively amplified by PCR. These fragments encode a segment of the G domain of translational initiation factor IF2 and elongation factor EF-Tu, two GTP-binding proteins. This was made possible by carefully designing the primers for this reaction to avoid the amplification of every G-domain-encoding region of the genome. The sequence of two pairs of primers was deduced from highly conserved regions, namely G1 and G3 and/or their vicinal amino acids, within each subfamily (initiation and elongation factors, respectively) of GTP-binding proteins. On the basis of the expected size, one band was selected in each experiment, cloned into a vector, and sequenced. This showed unambiguously after comparison analysis that they belong to the IF2 and EF-Tu genes, respectively. This strategy seems suitable for the amplification of a segment of any gene coding for a G protein from any origin.

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