Janis M. Keller

The FATTY ACID ELONGATION1 (FAE1) gene of Arabidopsis is required for the synthesis of very long chain fatty acids in the seed. The product of the FAE1 gene is presumed to be a condensing enzyme that extends the chain length of fatty acids from C18 to C20 and C22. We report here the cloning of FAE1 by directed transposon tagging with the maize element Activator (Ac). An unstable fae1 mutant was isolated in a line carrying Ac linked to the FAE1 locus on chromosome 4. Cosegregation and reversion analyses established that the new mutant was tagged by Ac. A DNA fragment flanking Ac was cloned by inverse polymerase chain reaction and used to isolate FAE1 genomic clones and a cDNA clone from a library made from immature siliques. The predicted amino acid sequence of the FAE1 protein shares homology with those of other condensing enzymes (chalcone synthase, stilbene synthases, and beta-ketoacyl-acyl carrier protein synthase III), supporting the notion that FAE1 is the structural gene for a synthase or condensing enzyme. FAE1 is expressed in developing seed, but not in leaves, as expected from the effect of the fae1 mutation on the fatty acid compositions of those tissues.

The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.

A series of seven carboxy-terminal deletion mutants of oat phytochrome A were stably expressed in transgenic tobacco to localize phytochrome domains involved in chromophore attachment, spectral integrity, photoreversibility between the red light (Pr)- and far-red light (Pfr)-absorbing forms, dimerization, and biological activity. Amino acids necessary for chromophore attachment in vivo were localized to the amino-terminal 398 residues because mutant proteins this small had covalently bound chromophore. Deletion mutants from the carboxy terminus to residue 653 were spectrally indistinguishable from the full-length chromoprotein. In contrast, further truncation to residue 399 resulted in a chromoprotein with a bleached Pfr absorbance spectrum, Pr and Pfr absorbance maxima shifted toward shorter wavelengths, and reduced Pfr to Pr phototransformation efficiency. Thus, residues between 399 ad 652 are required for spectral integrity but are not essential for chromophore attachment. The sequence(s) between residues 919 and 1093 appears to be necessary for dimerization. Carboxy-terminal mutants containing this region behaved as dimers under nondenaturing conditions in vitro, whereas truncations without this region behaved as monomers. None of the plants expressing high levels of deletion mutants lacking the 35 carboxy-terminal amino acids displayed the light-exaggerated phenotype characteristic of plants expressing biologically active phytochrome A, even when the truncated phytochromes were expressed at levels 6- to 15-fold greater than that effective for the full-length chromoprotein. Collectively, these data show that the phytochrome protein contains several separable carboxy-terminal domains required for structure/function and identify a domain within 35 residues of the carboxy terminus that is critical for the biological activity of the photoreceptor in vivo.