Promoter elements controlling developmental and environmental regulation of a tobacco ribosomal protein gene L34

The rpL34 gene, which encodes a cytoplasmic ribosomal protein with a high homology to the rat 60S r-protein L34, was isolated from a genomic library of tobacco (Nicotiana tabacum L. cv. Xanthi-nc). A 1500 bp upstream promoter fragment was fused to the chloramphenicol acetyltransferase (CAT) reporter gene or -glucuronidase (GUS) reporter gene and transferred into tobacco plants by the Agrobacterium-mediated leaf disk transformation method. Analysis of CAT activity in leaf tissues showed that mechanical wounding increased the rpL34 promoter activity about 5 times as compared to untreated controls and that the promoter activity was further enhanced by plant growth regulators, 2,4-dichlorophenoxyacetic acid and benzyladenine. Histochemical GUS staining patterns of the transgenic plants showed that the rpL34 promoter activity is high in actively growing tissues, including various meristems, floral organs, and developing fruits. A series of 5 deletion analyses of the rpL34 promoter indicated that a 50 bp region located between –179 and –129 is essential for wound, auxin and cytokinin responses. Deletion of this region reduced the promoter activity to an undetectable level. Insertion of the 50 nucleotide sequence into a minimal promoter restored the promoter activity and the promoter strength was proportional to the copy number of the upstream sequence. The role of TATA and CAAT box regions was studied by a series of 3 deletion analyses. A 3 deletion up to –28 did not significantly affect the promoter strength. However deletion of the promoter up to 70 bp, which deleted the TATA box region, significantly reduced promoter activity. Further deletion of the promoter up to –104, eliminating the CAAT box region, abolished the promoter activity. These results suggest that the TATA box and CAAT box regions are also important for the rpL34 promoter activity in addition to the 50 bp upstream region.     

Cell cycle-dependent regulation of telomerase activity by auxin, abscisic acid and protein phosphorylation in tobacco BY-2 suspension culture cells

Telomerase is a specialized RNA-directed DNA polymerase that adds telomeric repeats onto the ends of linear eukaryotic chromosomes. It was recently reported that the low, basal level of telomerase activity markedly increased at early S-phase of the cell cycle, and auxin further increased the S-phase-specific telomerase activity in tobacco BY-2 cells. In this study we show that abscisic acid (ABA), a phytohormone known to induce the cyclin-dependent protein kinase inhibitor, effectively abolished both the auxin- and S-phase-specific activation of telomerase in a concentration- and time-dependent fashion in synchronized tobacco BY-2 cells. These results suggest that there exists a hormonal cross-talk between auxin and ABA for the regulation of telomerase activity during the cell cycle of tobacco cells. Treatment of synchronized BY-2 cells with the protein kinase inhibitor staurosporine or H-7 effectively prevented the S-phase-specific activation of telomerase activity. By contrast, when okadaic acid or cantharidin, potent inhibitors of protein phosphatase 2A (PP2A), was applied to the cells, the S-phase-specific high level of telomerase activity was continuously maintained in the cell cycle for at least 14 h after release from M-phase arrest. Incubation of tobacco cell extracts with exogenous PP2A rapidly abrogated in vitro telomerase activity, while okadaic acid and cantharidin blocked the action of PP2A, effectively restoring in vitro telomerase activity. Taken together, these findings are discussed in the light of the suggestion that antagonistic functions of auxin and ABA, and reciprocal phosphorylation and dephosphorylation of telomerase complex, are necessarily involved in the cell cycle-dependent modulation of telomerase activity in tobacco cells.     

The role of cytokinins in responses to water deficit in tobacco plants over-expressing trans-zeatin O-glucosyltransferase gene under 35S or SAG12 promoters

The impact of water deficit progression on cytokinin (CK), auxin and abscisic acid (ABA) levels was followed in upper, middle and lower leaves and roots of Nicotiana tabacum L. cv. Wisconsin 38 plants [wild type (WT)]. ABA content was strongly increased during drought stress, especially in upper leaves. In plants with a uniformly elevated total CK content, expressing constitutively the trans -zeatin O-glucosyltransferase gene ( 35S::ZOG1 ), a delay in the increase of ABA was observed; later on, ABA levels were comparable with those of WT.
As drought progressed, the bioactive CK content in leaves gradually decreased, being maintained longer in the upper leaves of all tested genotypes. Under severe stress (11 d dehydration), a large stimulation of cytokinin oxidase/dehydrogenase (CKX) activity was monitored in lower leaves, which correlated well with the decrease in bioactive CK levels. This suggests that a gradient of bioactive CKs in favour of upper leaves is established during drought stress, which might be beneficial for the preferential protection of these leaves.
During drought, significant accumulation of CKs occurred in roots, partially because of decreased CKX activity. Simultaneously, auxin increased in roots and lower leaves. This indicates that both CKs and auxin play a role in root response to severe drought, which involves the stimulation of primary root growth and branching inhibition.     

Systematic chromosomal deletion of bacterial ribosomal protein genes

Detailed studies of ribosomal proteins (RPs), essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual RP genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, we found nine RPs (L15, L21, L24, L27, L29, L30, L34, S9, and S17) nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli RP genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA-protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function and may ultimately allow systematic substitution of RPs with RNA.     

Mapping of 12 bovine ribosomal protein genes using a bovine radiation hybrid panel

Twelve bovine ribosomal protein genes, for which sequence data had been acquired from complementary deoxyribonucleic acid (cDNA) clones isolated from a cattle skin cDNA library, were mapped. As ribosomal protein genes are a group of highly conserved house keeping genes, specific primers were designed to span the intron-exon splice sites and to amplify intronic sequences, in order to obtain bovine-specific polymerase chain reaction (PCR) products. Two of 12 ribosomal protein genes were genotyped in this way and the remaining 10 were mapped using additional primers designed from within the intron. Eleven previously unmapped ribosomal protein genes were localized and one previously reported ribosomal protein gene localization was confirmed. The 12 ribosomal protein genes mapped in this study are spread over 10 chromosomes, including the X chromosome. The locations show conservation of comparative map position in cattle and human.     

A cytokinin-binding protein complex from tobacco leaves

A cytokinin binding protein complex (CBP130) has been purified from tobacco leaves (Nicotiana sylvestris). It contains two protein species of 57 and 36 kDa (CBP57 and CBP36). The cDNAs encoding CBP57 have been isolated from a tobacco cDNA library. Their predicted amino acid sequences showed significant homology between CBP57 and S-adenosyl-L-homocysteine (SAH) hydrolase, which catalyzes the reversible hydrolysis of SAH, a methyltransferase inhibitor. A combination of gel filtration an western blot analysis revealed that both CBP57 and benzyladenine (BA)-binding activity were eluted at a peak of 130 kDa. A purified CBP130 fraction contains SAH hydrolase activity. We discuss possible CBP57 as a cytokinin receptor subunit and its possible role as a regulator of methylation.     

Translational regulation of ribosomal protein S15 drives characteristic patterns of protein‐mRNA epistasis

Do coding and regulatory segments of a gene co‐evolve with each‐other? Seeking answers to this question, here we analyze the case of Escherichia coli ribosomal protein S15, that represses its own translation by specifically binding its messenger RNA (rpsO mRNA) and stabilizing a pseudoknot structure at the upstream untranslated region, thus trapping the ribosome into an incomplete translation initiation complex. In the absence of S15, ribosomal protein S1 recognizes rpsO and promotes translation by melting this very pseudoknot. We employ a robust statistical method to detect signatures of positive epistasis between residue site pairs and find that biophysical constraints of translational regulation (S15‐rpsO and S1‐rpsO recognition, S15‐mediated rpsO structural rearrangement, and S1‐mediated melting) are strong predictors of positive epistasis. Transforming the epistatic pairs into a network, we find that signatures of two different, but interconnected regulatory cascades are imprinted in the sequence‐space and can be captured in terms of two dense network modules that are sparsely connected to each other. This network topology further reflects a general principle of how functionally coupled components of biological networks are interconnected. These results depict a model case, where translational regulation drives characteristic residue‐level epistasis—not only between a protein and its own mRNA but also between a protein and the mRNA of an entirely different protein.     

Purification and partial characterization of an acidic ribosomal protein kinase from maize

Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 m M KCl. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross-reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P₂). Its activity is inhibited by Ca²⁺ and Zn²⁺ and is activated by Mg²⁺, polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed.     

Cloning and expression of two genes encoding auxin-binding proteins from tobacco

Two genes encoding the auxin-binding protein (ABP1) of tobacco (Nicotiana tabacum L.), both of which possess the characteristics of a luminal protein of the endoplasmic reticulum (ER), were isolated and sequenced. These genes were composed of at least five exons and four introns. The two coding exons showed 95% sequence homology and coded for two precursor proteins of 187 amino acid residues with molecular masses of 21 256 and 21 453 Da. The deduced amino acid sequences were 93% identical and both possessed an amino-terminal signal peptide, a hydrophilic mature protein region with two potential N-glycosylation sites and a carboxyl-terminal sorting signal, KDEL, for the ER. Restriction mapping of the cDNAs encoding tobacco ABP1, previously purified by amplification of tobacco cDNA libraries by polymerase chain reaction (PCR) using specific primers common to both genes, indicated that both genes were expressed, although one was expressed at a higher level than the other. Genomic Southern blot hybridization showed no other homologous genes except for these two in the tobacco genome. The apparent molecular mass of the mature form of tobacco ABP1 was revealed to be 25 kDa by SDS polyacrylamide gel electrophoresis using affinity-purified anti (tobacco ABP1) antibodies raised against a fusion protein with maltose-binding protein. Expression of the recombinant ABP1 gene in transgenic tobacco resulted in accumulation of the 25 kDa protein. A single point mutation of an amino acid residue at either of the two potential N-glycosylation sites resulted in a decrease in the apparent molecular mass and produced a 22 kDa protein. Mutations at both sites resulted in the formation of a 19.3 kDa protein, suggesting that tobacco ABP1 is glycosylated at two asparagine residues.     

Evolutionary conservation of nuclear and nucleolar targeting sequences in yeast ribosomal protein S6A

Over 1 billion years ago, the animal kingdom diverged from the fungi. Nevertheless, a high sequence homology of 62% exists between human ribosomal protein S6 and S6A of Saccharomyces cerevisiae. To investigate whether this similarity in primary structure is mirrored in corresponding functional protein domains, the nuclear and nucleolar targeting signals were delineated in yeast S6A and compared to the known human S6 signals. The complete sequence of S6A and cDNA fragments was fused to the 5'-end of the LacZ gene, the constructs were transiently expressed in COS cells, and the subcellular localization of the fusion proteins was detected by indirect immunofluorescence. One bipartite and two monopartite nuclear localization signals as well as two nucleolar binding domains were identified in yeast S6A, which are located at homologous regions in human S6 protein. Remarkably, the number, nature, and position of these targeting signals have been conserved, albeit their amino acid sequences have presumably undergone a process of co-evolution with their corresponding rRNAs.     

Binding of human ribosomal protein p40 and its truncated mutants to the small ribosomal subunit

Ribosomal protein SA (rpSA), or p40, is a structural element of the small subunit of the eukaryotic ribosome. The N-terminal and central parts of rpSA are homologous to prokaryotic S2, whereas its C-terminal part is specific to eukaryotes. Preparations of 40S ribosomal subunits isolated from full-term human placenta proved to be deficient in SA to a varying extent. To study the rpSA binding to human 40S subunits, recombinant rpSA and its mutant forms with N-and C-terminal deletions were synthesized. The full-size and N-truncated rpSA variants bound to 40S subunits, while deletion of the C-terminal domain completely abolished the binding.     

Different effects of two brassinosteroids on growth,auxin and cytokinin content in tobacco callus tissue

Two synthetic brassinosteroids, 24-epibrassinolide (24-epiBR) and 2,3, 17-trihydroxy-5-androstan-6-one (THA-BR), exhibit different effects on growth of tobacco callus tissue. When added to a culture medium containing growth-limiting amounts of auxin, 24-epiBR reduced and THA-BR increased the fresh weight yield of tissue up to 53% and 207%, respectively, after 6 weeks of cultivation. The stimulatory and inhibitory effects of the two brassinosteroids on tissue growth occurred over a broad range of concentrations without a pronounced maximum corresponding to the yes or no type of response. Different effects of 24-epiBR and THA-BR on tissue growth were inversely proportional to the content of endogenous cytokinins. Maximum contents of predominant cytokinins N⁶-(²-isopentenyl)adenine (iP) and trans-zeatin (Z) in tissues supplied with 24-epiBR in growth-inhibiting concentrations were up to 3.7 fold and 3.4 fold higher, respectively, as compared to tissues grown on media containing growth-stimulating concentrations of THA-BR. Stimulation of tissue growth by THA-BR correlated with content of endogenous IAA and an inverse correlation was found between the content of endogenous IAA and cytokinins in tissues supplied with 24-epiBR. THA-BR exhibited weak cytokinin-like activity in a bioassay based on stimulation of growth of lateral buds of pea while 24-epiBR was inactive. Results indicate that the qualitatively different effects of the two brassinosteroids on growth of tobacco tissue may reflect their different influence on content of endogenous cytokinin.Abbreviations BR(s) brassinosteroid(s) - 24-epiBR 24-epibrassinolide - THA-BR 2,33, 17-trihydroxy-5-androstan-6-one - CK(s) cytokinin(s) - iP N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - Z trans-zeatin - [9R]Z ribosyl-trans-zeatin - ABA abscisic acid - IAA indole-3-acetic acid - NAA naphtalene-1-acetic acid - DEAE cellulose diethylaminoethyl cellulose - HPLC high performance liquid chromatography - ELISA enzyme linked immuno-sorbent assay