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

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.