P. Christopher LaRosa

Structure, Regulation and Function of the Osmotin Gene

Over the past years several genes have been reported to be osmotically regulated (Storey and Storey, 1981; Holtum and Winter, 1982; Singh et al., 1985; Singh et al., 1987a;Bedford et al., 1987; Close et al., 1989; Cushman et al., 1989; Singh et al., 1989a; 1989b; Delauney and Verma, 1990; Perez-Prat et al., 1990; Skriver and Mundy, 1990; Bartels et al., 1991; Dhindsa, 1991, Estragarcia et al., 1991; Narasimhan et al., 1991; Perez-Prat et al., 1992; Kononowicz et al., 1992; Nelson et al., 1992; Niu et al., 1993; Zhu et al., 1993b). These studies have been rationalized on the assumption that amongst these genes are molecular determinants of osmotic tolerance. Although the products of many of these genes still remain unidentified there are a number that have been well characterized and are of interest of several laboratories. One of these genes is osmotin (Singh et al., 1987a; LaRosa et al., 1989, Meeks-Wagner et al., 1989; Grosset et al., 1990; Roberts and Selitrennikoff, 1990; Stintzi et al., 1991; Woloshuk et al., 1991; Casas et al., 1992; LaRosa et al., 1992, Kononowicz et al., 1993).

Cloning of a Dna Fragment Encoding γ-Glutamyl Kinase and γ-Glutamyl Phosphate Reductase from a Tomato cDNA Library

We cloned the gene encoding the first enzyme of proline synthesis from tomato (L. esculentum) by complementation of a proB mutation in E. coli with a λgt-11 cDNA library of tomato fruit. We obtained seven phages which were able to restore proline prototrophy to the proB mutant. The insert from one of the complementing phages, PRO1, was subcloned into plasmid pBluescript IIKS+ When transferred into a mutant ΔproBA (which is deficient in both the first and second enzymes of proline biosynthesis, γ-glutamyl kinase and γ-glutamyl phosphate reductase), the PRO1 gene was able to complement this deletion mutation. Assays of the coupled γ-glutamyl kinase/γ-glutamyl phosphate reductase activity of E. coli proB or ΔproBA mutant strains revealed that these strains have elevated levels of both γ-glutamyl kinase and γ-glutamyl phosphate reductase. These results indicate that the PRO1 gene is a hybrid locus which specifies both enzymatic activities.