No abstract

Kinetically improved diacylglycerol acyltransferase (DGAT) variants were created to favorably alter carbon partitioning in soybean (Glycine max) seeds. Initially, variants of a type 1 DGAT from a high-oil, high-oleic acid plant seed, Corylus americana, were screened for high oil content in Saccharomyces cerevisiae. Nearly all DGAT variants examined from high-oil strains had increased affinity for oleoyl-CoA, with S 0.5 values decreased as much as 4.7-fold compared with the wild-type value of 0.94 mM. Improved soybean DGAT variants were then designed to include amino acid substitutions observed in promising C. americana DGAT variants. The expression of soybean and C. americana DGAT variants in soybean somatic embryos resulted in oil contents as high as 10% and 12%, respectively, compared with only 5% and 7.6% oil achieved by overexpressing the corresponding wildtype DGATs. The affinity for oleoyl-CoA correlated strongly with oil content. The soybean DGAT variant that gave the greatest oil increase contained 14 amino acid substitutions out of a total of 504 (97% sequence identity with native). Seed-preferred expression of this soybean DGAT1 variant increased oil content of soybean seeds by an average of 3% (16% relative increase) in highly replicated, single-location field trials. The DGAT transgenes significantly reduced the soluble carbohydrate content of mature seeds and increased the seed protein content of some events. This study demonstrated that engineering of the native DGAT enzyme is an effective strategy to improve the oil content and value of soybeans.

Oligomers based on amino acids conserved between known plant w-3 and cyanobacterium w -6 fatty acid desaturases were used to screen an Arabidopsis cDNA library for related sequences. An identified clone encoding a novel desaturase-like polypeptide was used to isolate its homologs from Glycine mar and Brassica napus. The plant deduced amino acid sequences showed less than 27% similarity to known plant w -6 and w-3 desaturases but more than 48% similarity to cyanobacterial w-6 desaturase, and they contained putative plastid transit sequences. Thus, we deduce that the plant cDNAs encode the plastid w-6 desaturase. l h e identity was supported by expression of the B. napus cDNA in cyanobaderium.Synechococcus transformed with a chimeric gene that contains a prokaryotic promoter fused to the rapeseed cDNA encoding all but the first 7 3 amino acids partially converted its oleic acid fatty acid to linoleic acid, and the 161(9c) fatty acid was converted primarily to 162(9c,12) in vivo. lhus, the plant w -6 desaturase, which utilizes 16:1(7c) in plants, can utilize 161(9c) in the cyanobaderium. l h e plastid and cytosolic homologs of plant w-6 desaturases are much more distantly related than those of w-3 desaturases.In leaf tissue there are two distinct pathways for the biosynthesis of the polyunsaturated fatty acids 1 8 2 and 18:3, one located in cytosolic membranes and the other located in plastid membranes. In Arabidopsis thaliana, cytosolic and plastid w-6 fatty acid desaturations that result in the production of diene fatty acids are controlled by the FAD 2 and FAD 6 loci, respectively. Cytosolic and plastid w-3 desaturations that result in the production of triene fatty acids are controlled by FAD 3 and FAD 7, respectively (Lemieux et al., 1990;Browse and Somerville 1991). It has been postulated that these loci correspond to structural genes for the desaturase enzymes. For FAD 3, proof of this postulate has come from the cloning of a cDNA encoding a desaturase corresponding to the FAD 3 locus (Arondel et al., 1992; Yadav et al., 1993).Cytosolic w-3 fatty acid desaturase cDNAs have been used to isolate their plastid homologs by screening plant cDNA libraries under low-stringency hybridization conditions, and the amino acid sequences of the higher-plant 0-3 desaturases show significant similarity (68% or greater) both among different plant species and between the microsomal and plastid homologs within each species (Iba et al., 1993; Yadav et al., 1993). However, this screening did not result in the isolation of cDNAs encoding other than w-3 desaturases.* Corresponding author; fax 1-302-695-4296.Recently, the T-DNA tagging method was used to isolate the Arabidopsis microsomal w-6 fatty acid desaturase (FAD 2) and it showed only 36% similarity at the amino acid leve1 with the w-3 desaturases (Okuley et al., 1994). In contrast to the results with w-3 desaturases, low-stringency screening of an Arabidopsis cDNA library with the Arabidopsis microsomal w-6 desaturase cDNA did not result in the identification of its plastid...

Autoimmune hemolysis due to IgA antibodies alone in rare, with red cell destruction occurring through mechanisms similar to those for IgG. Most commonly, IgA acts synergistically with other immunoglobulins (usually IgG) and complement; the hemolysis may be severe. Whether IgA autoantibodies alone can activate complement remains controversial, but increasing evidence suggests that they can, possibly via the alternative pathway, and that this activation may result in intravascular hemolysis.