Working within the interest of the laboratory, I initiated a study to determine why a specific antibiotic resistance (R) plasmid, pAM alpha 1, does not transform Streptococcus sanguis cells. This is a fundamental problem of gene function and gene control. pAM alpha 1 is a relatively small (6 MDal; 9 kb) plasmid that carries resistance to tetracycline (Tc). It was originally described in Streptococcus faecalis, strain DS5. A working hypothesis as to why pAM alpha 1 does not transform cells is that the plasmid, for some reason, either does not express Tc resistance or it cannot replicate in its new environment. To test these, I transformed cells with a hybrid plasmid that I constructed from the Tc resistant EcoRI restriction fragment from pAM alpha 1 and the replication-control genes contained on an EcoRI restriction fragment from pAM beta 1. pAM beta 1 is another plasmid originally described in S. faecalis DS5 cells that does replicate in S. sanguis cells. Since I have isolated a number of Tc resistant transformants, the hypothesis that Tc does not express has been ruled out. To date, I examined 6 out of 30 Tc-resistant clones. All contained plasmid DNA derived from pAM alpha 1 and pAM beta 1, as expected. This was demonstrated by standard Southern blot analysis using pAM alpha 1 and pAM beta 1 radioactive probes. The approximately 8 kb plasmid present in one strain of S. sanguis has been physically mapped. A surprising feature of the map is a loss of one EcoRI site. This loss permits the new plasmid, pJR4, to act as a potential S. sanguis cloning vehicle (not yet tested). The minimal inhibitory concentration for Tc in strains containing the hybrid ranged from 2.5 to 10 Mug/ml and the resistance is constitutive. Control cells do not grow in 0.5 Mug/ml Tc. Work will continue trying to establish the cause for the non-replication (non-establishment) of pAM alpha 1 in S. sanguis cells.