The Arabidopsis ELF3 gene regulates vegetative photomorphogenesis and the photoperiodic induction of flowering.

Flowering in Arabidopsis thaliana is promoted by longday (LD) photoperiods such that plants grown in LD flower earlier, and after the production of fewer leaves, than plants grown in short-day (SD) photoperiods. The early-flowering 3 (elf3) mutant of Arabidopsis, which is insensitive to photoperiod with regard to floral initiation has been characterized elf3 mutants are also altered in several aspects of vegetative photomorphogenesis, including hypocotyl elongation. When inhibition of hypocotyl elongation was measured, elf3 mutant seedlings were less responsive than wild-type to all wavelengths of light, and most notably defective in blue and green light-mediated inhibition. When analyzed for the flowering-time phenotype, elf3 was epistatic to mutant alleles of the blue-light receptor encoding gene, HY4. However, when elf3 mutants were made deficient for functional phytochrome by the introduction of hy2 mutant alleles, the elf3 hy2 double mutants displayed the novel phenotype of flowering earlier than either single mutant while still exhibiting photoperiod insensitivity, indicating that a phytochrome-mediated pathway regulating floral initiation remains functional in elf3 single mutants. In addition, the inflorescences of one allelic combination of elf3 hy2 double mutants form a terminal flower similar to the structure produced by tfk1 single mutants. These results suggest that one of the signal transduction pathways controlling photoperiodism in Arabidopsis is regulated, at least in part, by photoreceptors other than phytochrome, and that the activity of the Arabidopsis inflorescence and floral meristem identity genes may be regulated by this same pathway.

Identification of genes expressed in the tobacco shoot apex during the floral transition.

The shoot apex of higher plants contains undifferentiated meristematic cells that serve as the origin of post-embryonic organs. The transition from vegetative to reproductive growth results in the commitment of the apical meristem to floral organ formation. To identify the molecular signals that initiate floral development, we have pursued the isolation of genes that are transcriptionally active in the shoot apex of tobacco during the transition from vegetative to floral growth. The small size of the apex led us to utilize polymerase chain reaction shoot apices. This approach enabled the isolation of the apex-specific and floral apex-specific cDNA clones described in this paper. One clone, A3, detected an equivalent level of transcript in the shoot apex during all developmental stages observed. The second clone, FA2, detected a unique transcript that increased in abundance in the shoot apex during the transition to flowering and showed high levels of expression in developing petals, stamens, and pistils.

The lethal lambda S gene encodes its own inhibitor.

The 107 codon reading frame of the lambda lysis gene S begins with the codon sequence Met1-Lys2-Met3..., and it has been demonstrated in vitro that both Met codons are used for translational starts. Furthermore, the partition of initiation events at the two start codons strongly affects the scheduling of lysis. We have presented a model in which the longer product, S107, acts as an inhibitor of the shorter product, S105, the lethal lysis effector, despite the fact that the two molecules differ only in the Met-Lys residues at the amino terminus of S107. Using immunological and biochemical methods, we show in this report that the two predicted protein products, S105 and S107, are detectable in vivo as stable, membrane-bound molecules. We show that S107 acts as an inhibitor in trans, and that its inhibitory function is entirely defined by the positively charged Lys2 residue. Moreover, our data show that energy poisons abolish the inhibitory function of S107 and simultaneously convert S107 into a lysis effector. We propose a two step model for the lethal action of gene S: first, induction of the S gene results in the accumulation of S105 and S107 molecules in mixed oligomeric patches in the cytoplasmic membrane; second, S monomers rearrange by lateral diffusion within the patch to form an aqueous pore. The R gene product, a transglycosylase, is released through the pore to the periplasm, resulting in destruction of the peptidoglycan and bursting of the cell. According to this model, the lateral diffusion step is inhibited by the energized state of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Oligomerization of the bacteriophage lambda S protein in the inner membrane of Escherichia coli.

Western blot (immunoblot) analysis of cell extracts from induced bacteriophage lambda lysogens probed with S-protein-specific antibody (raised against an S--beta-galactosidase fusion protein) demonstrated that the bacteriophage lambda S protein begins to appear 10 min after phage induction and is localized to the inner membrane at all times during the lytic cycle. Between 100 and 1,000 molecules of S protein per cell were present at the time of phage-induced lysis. Western blots of chemically cross-linked membranes from induced lysogens showed a ladder of bands at 18, 24, 32, and 42 kilodaltons (the S-protein monomer ran at 8 kilodaltons) that reacted with anti-S-protein antibody. Thus, the S protein appears to reside in the inner membrane as a multimer, and the molecular weights of the cross-linked species are consistent with those of S-protein homopolymers. Sodium dodecyl sulfate-resistant dimers were also detected when S protein was purified by immunoprecipitation.

Conservation of a dual-start motif in P22 lysis gene regulation.

Gene 13 of bacteriophage P22 is functionally equivalent to lambda lysis gene S. Gene S codes for two products, the polypeptides S105 and S107, produced from translational initiation events at the third and first codon, respectively. We have shown that the two polypeptides have opposing functions in lysis: S105 is the lethal lysis effector, and S107 acts as an inhibitor of lysis (U. Bläsi, K. Nam, D. Hartz, L. Gold, and R. Young, EMBO J. 11:3501-3510, 1989). Gene 13 has a 108-codon reading frame and its product begins with a similar motif: Met-1-Lys-2-Lys-3-Met-4. Here, we present in vivo and in vitro evidence for the expression of a 13(108) and a 13(105) product and show that the lambda lysis control mechanisms is evolutionarily conserved in phage P22. In this case 13(108), like S107 in lambda, functions as the inhibitor of the lysis effector 13(105). Although the DNA sequences upstream of the S and 13 gene starts showed less homology, the same structural characteristics, i.e., stem-loop structures immediately upstream and about 10 codons downstream of the start region, were present in both reading frames. Using in vitro mutagenesis and toeprinting, we show that the upstream stem-loop structures of genes 13 and S, containing the Shine-Dalgarno sequence for initiations at Met-1, are interchangeable. Moreover, our data indicate that the stability of the secondary structures present in the translational initiation regions of genes S and 13 is set to create a particular ratio of initiation events at Met-1 and Met-3 or Met-4. The ratio of effector to inhibitor was much higher in P22 than in lambda. We propose that this reflects less transcriptional readthrough at the late terminator t(R) and suggests that the dual-start motif in genes 13 and S may be important for establishment of maintenance of the lysogenic state.

Peptide mapping of bovine pancreatic ribonuclease A by reverse-phase high-performance liquid chromatography. I. Application to the reduced and S-carboxymethylated protein.

Complete peptide maps of reduced and S-carboxymethylated ribonuclease A were obtained by reverse-phase high-performance liquid chromatography with the following peptide-chain cleavage techniques: cyanogen bromide cleavage, limited and extensive Staphylococcus aureus protease digestion, tryptic digestion, and tryptic followed by chymotryptic digestion. Commercial samples of S. aureus protease exhibited a broader specificity than had previously been reported, as demonstrated by its ability to cleave after glutamine residues. Cleavage after asparagine and serine residues was also strongly implicated. The procedures developed require roughly 0.1 to 1 mg of ribonuclease A for the peptide mapping of this protein. These procedures will be useful for the identification of the sites of a chemical modification and also for the isolation of a variety of peptides for further studies.