Nucleotide sequence of the PR-1 gene of Nicotiana tabacum

A gene encoding one of the pathogenesis-related proteins, PR1a, and two related pseudogenes were isolated from Nicotiana tabacum. The cloned PR1a gene (pPR-gamma) and one of the pseudogenes (pPR-alpha) were sequenced and found to have similar structures. The sequence of pPR-gamma was quite similar to that of the cDNA clone of PR1a. The plasmid pPR-gamma did not contain an intron and had a typical promoter sequence in the 5'-flanking region.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Ca2+ transport through lipid membrane by diastereomer cyclic octapeptides

Effects of the nature and orientation of a side chain in cyclic octapeptides on Ca2+ transport were examined by using cyclo[L-Lys(Z)-Sar-L-Leu-Sar]2 (C8-L), cyclo[L-Lys(Z)-Sar]4 (C8KS), and their diastereomer cyclic octapeptides, cyclo[L-Lys(Z)-Sar-D-Leu-Sar]2 (C8-D) and cyclo[L-Lys(Z)-Sar-D-Lys(Z)-Sar]2 (C8Kk). All these cyclic octapeptides were found to take a single conformation in CDCl3, and the conformation was C2-symmetric for C8-L and C8-D, and C4-symmetric for C8KS and C8Kk. They formed a complex with Ca2+. Upon complexation, C8KS accompanied isomerization of peptide bonds, but C8-D retained the arrangement of peptide bonds. The amount of Ca2+ extracted from an aqueous solution to a chloroform solution by all L cyclic octapeptide C8-L or C8KS was about twice that of Na+, but 6-8-fold smaller than that by C8-D or C8Kk including D units. These cyclic octapeptides were capable of transporting Ca2+ through a lipid membrane above the phase transition temperature, and the transport rate decreased in the order of C8Kk-C8KS greater than C8-D greater than C8-L.     

Inhibition by gossypol of tumor promoter-induced arachidonic acid metabolism in rat peritoneal macrophages

Rat peritoneal macrophages were prelabeled with [3H]arachidonic acid. The release of radioactivity into the medium was increased by treatment with TPA-type tumor promoters, such as TPA, teleocidin and aplysiatoxin, and the non-TPA-type tumor promoter, thapsigargin. Gossypol, at concentrations of 3 and 10 microM, inhibited the release of radioactivity stimulated by both types of tumor promoter, although the mechanism of stimulation of arachidonic acid metabolism is different in the two types of tumor promoter. Stimulation of prostaglandin E2 production by these tumor promoters was also inhibited by treatment with gossypol. Calcium ionophore A23187-stimulated release of radioactivity and prostaglandin E2 production were also inhibited by gossypol treatment. The mechanism of inhibition by gossypol of prostaglandin E2 production is discussed.     

Primary structure of hemorrhagic protein, HR2a, isolated from the venom of Trimeresurus flavoviridis

The complete amino acid sequence and disulfide bridge location of HR2a, one of the hemorrhagic proteins isolated from the snake venom of Trimeresurus flavoviridis, have been determined by analysis of peptides derived from digests with cyanogen bromide, lysyl endopeptidase, trypsin, and Staphylococcus aureus V8 protease. Peptides were purified by gel filtration followed by reversed-phase HPLC. HR2a has the amino-terminal sequence of less than Glu-Gln-Arg- and consists of a total of 202 residues with a calculated molecular weight of 23,015. Sequence analysis indicates the presence of another isoform which lacks the amino-terminal residue, making 201 amino acid residues with a molecular weight of 22,887. Three disulfide bridges of HR2a link Cys-118 to Cys-197, Cys-159 to Cys-181, and Cys-161 to Cys-164. HR2a contains a segment which is similar to the zinc-chelating sequences found in thermolysin and several mammalian metalloproteinases, suggesting that HR2a is a metalloproteinase with limited substrate specificity. However, there is no other significant sequence homology with thermolysin except for the zinc-ligand region.     

Nucleotide sequence of Marchantia polymorpha chloroplast DNA: a region possibly encoding three tRNAs and three proteins including a homologue of E. coli ribosomal protein S14.

The nucleotide sequence of a region of Marchantia polymorpha chloroplast DNA was determined. On this DNA sequence (3.38kb), three open reading frames (ORFs) and three putative tRNA genes were detected in the following order: -ORF701-tRNASer(UGA)-ORF702-tRNAGly(GCC)-initiator tRNAMet(CAU)-ORF703-. The ORF703 is composed of 100 codons in which those for lysine (15%) and arginine (11%) are abundant, and could be accounted for as a counterpart of E. coli ribosomal protein S14 since they share 45% homology in the amino acid sequences. The ORF701 appears to code for a membrane protein, showing a periodic appearance of seven clusters of hydrophobic amino acids. Although the mechanisms remain unknown, the ORF701 causes a streptomycin-sensitive phenotype in resistant mutants of E. coli. The ORFs and tRNA genes are separated from each other by extremely AT-rich spacers containing sequences of dyad symmetry. The third letter positions of the codons in the ORFs are also rich in A and T residues.     

Identification of transfer RNA suppressors in Escherichia coli. I. Amber suppressor su+2, an anticodon mutant of tRNA2Gln.

In order to isolate the gene for amber suppressor su⁺2 (SupE) in Escherichia coli, a non-defective su⁺2-transducing phage lambda was isolated in three steps: first, deletion derivatives of F′su⁺2 gal (λ) were selected, linking su⁺2 to the right-hand prophage attachment site, att^λPB′; second, these F′-factors were relysogenized by λ and defective transducing phages, λdsu⁺2, were produced by induction; and third, non-defective λpsu⁺2 transducing phages were produced by recombination of λdsu⁺2 isolates with λ. Upon infection by λpsu⁺2, the production of transferRNAs accepting glutamine and methionine was markedly stimulated. Fingerprint analysis of these tRNAs revealed that they consisted of normal tRNA₂^Gln, mutant tRNA₂^Gln and tRNA_m^Met. The mutant tRNA₂^Gln carried a singlebase alteration from G to A at the 3′-end of the anticodon. The production of tRNA₁^Gln was not stimulated by the infection of λpsu⁺2. We conclude that the wild-type allele of su⁺2 (SupE) is the structural gene for tRNA₂^Gln, and the su⁺2 amber suppressor was derived by a single base mutation, changing the anticodon from CUG to CUA, in one of the multi-copy genes for tRNA₂^Gln. The fact that λpsu⁺2 also induces the production of tRNA_m^Met suggests that this tRNA is encoded in the same chromosomal region of E. coli as is tRNA₂^Gln.