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The active site of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been probed using site-directed mutagenesis and inhibitor binding techniques. Replacement of a specific glycyl with an alanyl or a prolyl with a seryl residue in a highly conserved region confers glyphosate tolerance to several bacterial and plant EPSPS enzymes, suggesting a high degree of structural conservation between these enzymes. The glycine to alanine substitution corresponding to Escherichia coli EPSPS G96A increases the Ki(app) (glyphosate) of petunia EPSPS 5000-fold while increasing the Km(app)(phosphoenolpyruvate) about 40-fold. Substitution of this glycine with serine, however, abolishes EPSPS activity but results in the elicitation of a novel EPSP hydrolase activity whereby EPSP is converted to shikimate 3-phosphate and pyruvate. This highly conserved region is critical for the interaction of the phosphate moiety of phosphoenolpyruvate with EPSPS.

The kinetics of synthesis and incorporation of the photosynthetic pigments and several of the major oxidative and photosynthetic electron transport components of Rhodopseudomonas sphaeroides have been studied during synchronous and asynchronous phototrophic growth. The photosynthetic pigments and cytochromes c and b, measured spectroscopically, exhibited continuous patterns of synthesis and incorporation into the membrane particulate fraction in both synchronous and asynchronous cultures. Succinic dehydrogenase and NADH-oxidase activities, present at low levelnous growth. In a previous paper, Leuking, D.R., Fraley, R.T., and Kaplan, S. ((1978) J. Biol. Chem. 253, 451-457) have shown that total cellular phospholipid is also accumulated discontinuously during synchronous growth. A continuously incorporated membrane component is thus subject to a wide variation in the membrane protein/lipid ratio. The significance of this ratio in regulating the activity of membrane proteins is discussed and the distinction between protein incorporation and function is drawn with particular reference to the photosynthetic pigments and cytochrome components and the oxidative activities measured. It is suggested that a dependence of membrane protein activity on the membrane protein to lipid ratio in vivo is of possible significance in the control of membrane synthesis and cell division.

Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 × 10−5 to 2 × 10−6) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.

A nonspecific density labeling technique has been employed to monitor the synthesis of intracytoplasmic membrane in synchronously dividing populations of Rhodopseudomonas sphaeroides. The intracytoplasmic membranes of cells synchronized in D2O-based medium were found to undergo discontinuous decreases in specific density during synchronous cell growth following transfer to H2O-based medium. These abrupt decreases in membrane specific density occurred immediately prior to cell division and were not observed with intracytoplasmic membranes prepared from asynchronously dividing cells (see also Kowakowski, H., and Kaplan, S. (1974) J. Bacteriol. 118, 1144-1157). Discontinuous increases in the net accumulation of cellular phospholipid were also observed during the synchronous growth of R. sphaeroides. This is to be contrasted to the continuous insertion of protein and the photopigment components of the photosynthetic apparatus into the intracytoplasmic membrane during the cell division cycle (Fraley, R.T., Lueking, D.R., and Kaplan, S. (1978) J. Biol. Chem. 253, 458-464; Wraight, C.A., Lueking, D.R., Fraley, R.T., and Kaplan, S. (1978) J. Biol. Chem. 253, 465-471). Further, examination of the protein/phospholipid ratios of purified intracytoplasmic membrane preparations revealed that this ratio undergoes cyclical changes of 35 to 40% during a normal cycle of cell division. In contrast to the results of Ferretti and Gray ((1968) J. Bacteriol, 95, 1400-1406), DNA synthesis was found to occur in a stepwise manner in synchronously dividing cell populations of R. sphaeroides.

The development of efficient methods for introducing nucleic acids into plant cells is critical to the successful application of molecular genetic approaches to plant systems. Two methods have been developed for this purpose: 1) liposome-mediated delivery and 2) in vitro transformation of protoplasts by co-cultivation with Agrobacterium tumefaciens cells.

Recently, it was reported that transgenic tobacco plants that express the coat protein gene of tobacco mosaic virus (TMV) exhibit a degree of resistance to infection by TMV (11). To determine whether the expression of other viral sequences would also confer resistance, transgenic plants were produced which expressed TMV nucleotides (nt) 3335 to 6395 (representing approximately 52% of the viral genome), their complement (antisense), or nucleotides that encode the 30-kDa protein. Plants that expressed the coat protein gene or other viral RNA sequences were inoculated with TMV and observed for development of disease symptoms. Symptoms were delayed only if transgenic plants expressed the coat protein gene; i.e., plants that express other sequences of TMV, of either the sense or antisense polarity, developed disease symptoms simultaneous with nontransgenic plants. The results indicate that the resistance to TMV infection is greater in transgenic plants that express the viral coat protein gene than in plants that express other sequences of TMV RNA.

The kinetics of synthesis and incorporation of the photosynthetic pigments and several of the major oxidative and photosynthetic electron transport components of Rhodopseudomonas sphaeroides have been studied during synchronous and asynchronous phototrophic growth. The photosynthetic pigments and cytochromes c and b, measured spectroscopically, exhibited continuous patterns of synthesis and incorporation into the membrane particulate fraction in both synchronous and asynchronous cultures. Succinic dehydrogenase and NADH-oxidase activities, present at low levelnous growth. In a previous paper, Leuking, D.R., Fraley, R.T., and Kaplan, S. ((1978) J. Biol. Chem. 253, 451-457) have shown that total cellular phospholipid is also accumulated discontinuously during synchronous growth. A continuously incorporated membrane component is thus subject to a wide variation in the membrane protein/lipid ratio. The significance of this ratio in regulating the activity of membrane proteins is discussed and the distinction between protein incorporation and function is drawn with particular reference to the photosynthetic pigments and cytochrome components and the oxidative activities measured. It is suggested that a dependence of membrane protein activity on the membrane protein to lipid ratio in vivo is of possible significance in the control of membrane synthesis and cell division.

A nonspecific density labeling technique has been employed to monitor the synthesis of intracytoplasmic membrane in synchronously dividing populations of Rhodopseudomonas sphaeroides. The intracytoplasmic membranes of cells synchronized in D2O-based medium were found to undergo discontinuous decreases in specific density during synchronous cell growth following transfer to H2O-based medium. These abrupt decreases in membrane specific density occurred immediately prior to cell division and were not observed with intracytoplasmic membranes prepared from asynchronously dividing cells (see also Kowakowski, H., and Kaplan, S. (1974) J. Bacteriol. 118, 1144-1157). Discontinuous increases in the net accumulation of cellular phospholipid were also observed during the synchronous growth of R. sphaeroides. This is to be contrasted to the continuous insertion of protein and the photopigment components of the photosynthetic apparatus into the intracytoplasmic membrane during the cell division cycle (Fraley, R.T., Lueking, D.R., and Kaplan, S. (1978) J. Biol. Chem. 253, 458-464; Wraight, C.A., Lueking, D.R., Fraley, R.T., and Kaplan, S. (1978) J. Biol. Chem. 253, 465-471). Further, examination of the protein/phospholipid ratios of purified intracytoplasmic membrane preparations revealed that this ratio undergoes cyclical changes of 35 to 40% during a normal cycle of cell division. In contrast to the results of Ferretti and Gray ((1968) J. Bacteriol, 95, 1400-1406), DNA synthesis was found to occur in a stepwise manner in synchronously dividing cell populations of R. sphaeroides.

The kinetics of accumulation of phospholipids into the intracytoplasmic membrane of Rhodopseudomonas sphaeroides have been examined. We have previously demonstrated that accumulation of phospholipids in the intracytoplasmic membrane is discontinuous with respect to the cell cycle. In this study we demonstrated a sevenfold increase in the rate of phospholipid incorporation into the intracytoplasmic membrane concurrent with the onset of cell division. Pulse-chase labeling studies revealed that the increase in the rate of phospholipid accumulation into the intracytoplasmic membrane results from the transfer of phospholipid from a site other than the intracytoplasmic membrane, and that the transfer of phospholipid, rather than synthesis of phospholipid, is most likely subject to cell cycle-specific regulation. The rates of synthesis of the individual phospholipid species (phosphatidylethanolamine, phosphatidyglycerol, and an unknown phospholipid) remained constant with respect to one another throughout the cell cycle. Similarly, each of these phospholipid species appeared to be transferred simultaneously to the intracytoplasmic membrane. We also present preliminary kinetic evidence which suggested that phosphatidylethanolamine may be converted to phosphatidycholine within the intracytoplasmic membrane.