A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences.

Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x(2)-H-x(11)-C-x(2)-C-x((4-5))-C-x(2)-C-x((3-7))-H-x(2)-H and C-x(2)-C-x((10-11))-C-x(3)-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with (65)ZnCl(2) showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression.

Chloroplast-encoded protein as a subunit of acetyl-CoA carboxylase in pea plant.

The gene product of an open reading frame of the chloroplast genome, accD, that has sequence similarity with a subunit of acetyl-CoA carboxylase from Escherichia coli was detected immunochemically in pea chloroplasts. The apparent molecular mass of the accD protein was 87 kDa on SDS-polyacrylamide gel electrophoresis. The protein was acidic and had less mobility than the calculated value, 67,116. Acetyl-CoA carboxylase activity solubilized from pea chloroplasts was inhibited by antibodies against recombinant accD protein. The antibodies precipitated a polypeptide of 35 kDa containing biotin and a polypeptide of 91 kDa together with the 87-kDa-accD protein. The accD protein formed a complex with the molecular mass of about 700 kDa, probably with the 35- and 91-kDa proteins. These results indicate that the chloroplast-encoded polypeptide, accD protein, is a component of a functional acetyl-CoA carboxylase in chloroplasts and this enzyme is a multi-subunit complex, like that from E. coli. The synthesis of accD protein was not induced by light.

Isolation and characterization of cDNAs that encode eleven small GTP-binding proteins from Pisum sativum.

Eleven cDNA clones (pra1 to pra9A, pra9B, and pra9C) were isolated from a pea (Pisum sativum) leaf cDNA library which were similar to small GTP-binding proteins. These cDNAs encoded proteins of 22-25 kDa, which exhibited 45-92% identity to one another at the amino acid level. The putative proteins included the characteristic sequences of ras-related small GTP-binding proteins: four conserved domains involved in binding of GTP/GDP; an effector domain; and cysteine residues at the COOH-terminus. Indeed, the pra6 protein, expressed in Escherichia coli, clearly showed GTP-binding activity. Phylogenetic analysis showed that these clones can be classified into two subgroups: proteins encoded by pra1 to pra7 being related to ypt3 and rab11 proteins; and proteins encoded by pra8, pra9A, pra9B, and pra9C being related to YPT1 and rab1 proteins. The effector sequences in these two subgroups were different. RNA gel blot analysis showed that most of the corresponding genes are differentially expressed in pea leaves and roots. Variations in expression were also observed for structurally related genes.

Chloroplast envelope protein encoded by chloroplast genome.

The gene product of an open reading frame of chloroplast genome, ORF 231 in pea, was immunochemically detected in chloroplast and etioplast envelopes. This is the first protein of a Chloroplast Envelope Membrane encoded by a chloroplast genome. It was named CEM A and the gene, cem A. CEM A is an acidic protein having an apparent molecular mass of 34 kDa on SDS-PAGE, and a minor component detected in the fractionated inner envelope.

Sequence and transcriptional analysis of the gene cluster trnQ-zfpA-psaI-ORF231-petA in pea chloroplasts.

A 5.1 kb segment of pea chloroplast DNA containing the upstream region of petA was sequenced. RNAs produced from this DNA were characterized. This region encodes putative genes for psbK, trnQ, zfpA, psaI, ORF231, and petA. These genes are all on the same reading strand except for psbK. The gene organization is somewhat different from that of tobacco, rice, and liverwort, which lack the psbK-trnQ genes in this region and contain ORF184/185. Northern blot and primer extension analysis show that the pea transcript covers the zfpA-psaI-ORF231-petA gene cluster and trnQ. These results indicated that the psbK-trnQ genes have been rearranged and a new transcription unit was formed.

Detection of small GTP-binding proteins in the outer envelope membrane of pea chloroplasts.

We found small GTP-binding proteins in the outer envelope membrane of pea chloroplasts. The proteins in this membrane were separated by SDS-PAGE, transferred to a nitrocellulose filter, and incubated with [alpha-32P]GTP. Three GTP-binding proteins with the molecular weight of 24,000 were found. Binding was prevented by 10(-8)-10(-7) M GTP or by 10(-7) M guanosine 5'-[gamma-thio]triphosphate or GDP; binding was unaffected by 10(-8)-10(-6) M ATP. Thermolysin treatment of intact chloroplasts resulted in the loss of GTP-binding activity, suggesting that these proteins were in the cytosolic side of the outer envelope membrane.

Nucleotide sequence and expression of the ribosomal protein L2 gene in pea chloroplasts.

We sequenced the nucleotides around the rpl2 gene, encoding the ribosomal protein L2, in pea (Pisum sativum cv. Alaska) chloroplasts and analyzed the expression of the rpl2 gene. During deetiolation, accumulation of the rpl2 transcript did not require de novo protein synthesis on chloroplastic ribosomes, in contrast to that of most chloroplast-encoded genes. This suggested that the mechanism involved in the expression of rpl2 differed from that of most chloroplast-encoded genes.

A chloroplast gene encoding a protein with one zinc finger.

We have sequenced a pea chloroplast gene encoding a protein with a zinc finger (zfp A). The putative protein is conserved in chloroplast DNA and shows sequence homology to the E. coli protein controlling fol C expression. Exposure of etiolated pea leaves to light leads to the accumulation of zfp A transcripts. The accumulation of these transcripts does not require de novo protein synthesis in the chloroplast, although the expression of highly inducible genes such as rbc L requires it. These properties suggest that zfp A encodes a protein involved in the regulation of chloroplast gene expression.

Prevention of atherosclerotic progression in Watanabe rabbits by probucol.

The foam cell has been recognized as a characteristic feature of xanthomas in skin and tendons, and also of atheromas. Many foam cells in these lesions share properties characteristic of the macrophages. Therefore macrophages may be the progenitor of certain foam cells that are involved in atherogenesis. Several investigators demonstrated in vitro that macrophages can ingest large amounts of certain chemically modified lipoproteins, such as acetylated low-density lipoprotein (LDL) and malondialdehyde-treated LDL, through the process of receptor-mediated endocytosis. By this process, macrophages become foam cells. But this process has not been demonstrated in vivo. Recently, oxidized LDL has been suggested to play an important role in atherogenesis by facilitating the accumulation of lipids in macrophages in vitro. Probucol, originally developed as an antioxidant, prevents this oxidative modification of LDL in vitro. Moreover, there are some clinical reports that probucol induces regression of cutaneous and tendon xanthomas in patients with homozygous familial hypercholesterolemia. A question was posed whether in vivo probucol could prevent the progression of atherosclerosis in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia. At age 2 months, 8 WHHL rabbits were classified into 2 groups: group A rabbits were controls and group B rabbits were treated with 1% probucol. After 6 months of treatment, average plasma concentrations of cholesterol were 704 +/- 121 mg/dl in group A and 584 +/- 61 mg/dl in group B. The percentage of surface area of total thoracic aorta with visible plaques in group A vs group B was 54.2 +/- 18.8% vs 7.0 +/- 6.3%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)