Preferential translation mediated by Hsp81-3 5'-UTR during heat shock involves ribosome entry at the 5'-end rather than an internal site in Arabidopsis suspension cells

Translational inhibition of most mRNAs and preferential translation of mRNAs coding heat shock proteins (Hsps) occur in most cells under heat shock stress. For most Hsp mRNAs, preferential translation in heat-shocked cells is conferred by their 5'-untranslated regions (5'-UTRs). However, the preferential translation directed by 5'-UTRs during heat shock remains mostly unknown in plants. Here, we found that the mRNA of Hsp81-3, which is an Arabidopsis Hsp90 family gene, continued to be associated with polysomes in heat-shocked Arabidopsis suspension-cultured cells. The Hsp81-3 5'-UTR was found to contribute to the efficient translation of capped reporter mRNAs in heat-shocked Arabidopsis protoplasts using a transient expression assay. Further characterization of the Hsp81-3 5'-UTR revealed that the anterior half of the 5'-UTR is important for the efficient translation in heat-shocked protoplasts. Moreover, the Hsp81-3 5'-UTR was highly capable of enhancing translation from uncapped reporter mRNAs relative to the 5'-UTR of a housekeeping gene in both normal and heat-shocked protoplasts. These Hsp81-3 5'-UTR-directed translations both in capped and uncapped reporter mRNAs were substantially reduced by the insertion of an upstream AUG at the 5'-end of the 5'-UTR, indicating that ribosomes are recruited to the 5'-end of the Hsp81-3 5'-UTR regardless of temperature and the presence or absence of the cap structure. These results suggest that the preferential translation of Hsp81-3 mRNA in heat-shocked Arabidopsis cells involves a ribosome scanning from the 5'-end of the 5'-UTR rather than ribosome entry to the internal site.     

The 5'-untranslated region of the Oryza sativa alcohol dehydrogenase gene functions as a translational enhancer in monocotyledonous plant cells

The 5'-untranslated region (5'-UTR) functions as a translational enhancer in monocotyledonous plant cells is necessary to express a foreign gene efficiently. Here, we show that the 5'-UTR of the rice alcohol dehydrogenase gene contributes to efficient translation in not only dicotyledonous plant cells, but also in monocotyledonous rice cells.     

A novel gene delivery system in plants with calcium alginate micro-beads

We have produced micrometer-sized calcium alginate beads referred to as "bio-beads" that encapsulate plasmid DNA molecules carrying a reporter gene. In order to evaluate the efficiency of the bio-beads in mediating genetic transfection, protoplasts isolated from cultured tobacco cells (BY-2) were transfected with bio-beads containing a plasmid that carries the modified green fluorescent protein gene CaMV35S-sGFP. With the bio-beads treatment, approximately ten-fold higher GFP expression was observed after 24 h incubation compared to that with the conventional method using a naked plasmid solution. Transfection was up to 0.22% efficient. These results indicate that bio-beads have a possibility for efficient transformation in plants.     

烟草抗冷相关基因在两种不同育苗方式下的低温应答差异分析

为探究水旱两段式育苗技术抗低温胁迫的分子机制,利用生物信息学方法分析了烟草低温应答关键转录因子CBF（C-repeat binding factor）基因家族的进化关系、基因结构、保守结构域及其启动子顺式作用元件,进一步利用qRT-PCR分析两种育苗方式下（常规漂浮育苗和水旱两段式育苗）NtCBF基因家族、CBF-regulon基因和非CBF-regulon相关基因的低温应答模式。结果表明,NtCBF15~NtCBF21基因与拟南芥CBF5、CBF6基因进化关系相近,NtCBF1~NtCBF14基因与拟南芥CBF1~CBF4基因进化关系相近。NtCBF基因家族成员均具有motif 1/2/4/5保守蛋白结构域,表明该家族在进化过程中较为保守;其中,NtCBF3、NtCBF4、NtCBF5、NtCBF6、NtCBF10、NtCBF13、NtCBF14和NtCBF18等8个基因启动子区具有低温响应元件。低温处理后,NtCBF5、NtCBF12和NtCBF13及CBF-regulon基因中NtRD29A、NtRD29B和NtGOLS3的表达量均显著升高。推测NtCBF5、NtCBF12和NtCBF13及其下游调控基因NtRD29A、NtRD29B和NtGOLS3在水旱两段式育苗抗低温胁迫过程中起重要作用。      

Boron-dependent regulation of translation through AUGUAA sequence in yeast

Under high boron (B) conditions, nodulin 26-like intrinsic protein 5;1 (NIP5;1) mRNA, a boric acid channel, is destabilized to avoid excess B entry into roots of Arabidopsis thaliana. In this regulation, the minimum upstream open reading frame (uORF), AUGUAA, in its 5′-untranslated region (5′-UTR) is essential, and high B enhances ribosome stalling at AUGUAA and leads to suppression of translation and mRNA degradation. This B-dependent AUGUAA-mediated regulation occurs also in animal transient expression and reticulocyte lysate translation systems. Thus, uncovering the ubiquitousness of B-dependent unique regulation is important to reveal the evolution of translational regulation. In the present study, we examined the regulation in Saccharomyces cerevisiae. Reporter assay showed that in yeast, carrying ATGTAA in 5′-UTR of NIP5;1 upstream of the reporter gene, the relative reporter activities were reduced significantly under high B conditions compared with control, whereas deletion of ATGTAA abolished such responses. This suggests that AUGUAA mediates B-dependent regulation of translation in Saccharomyces cerevisiae. Moreover, the deletion of ATGTAA resulted in up to 10-fold increase in general reporter activities indicating the suppression effect of AUGUAA on translation of the main ORF. Interestingly, mRNA level of the reporter gene was not affected by B in both yeast cells with and without AUGUAA. This finding reveals that in yeast, unlike the case in plants, mRNA degradation is not associated with AUGUAA regulation. Together, results suggest that B-dependent AUGUAA-mediated translational regulation is common among eukaryotes.     

Stable expression of a thermostable xylanase of Clostridium thermocellum in cultured tobacco cells

Two distinct domains of the xynA gene from Clostridium thermocellum encoding a xylanase catalytic domain (XynAl) and a xylanase catalytic domain with a cellulose binding domain (XynA2) under the control of the cauliflower mosaic virus 35S promoter were electroporated into cultured tobacco BY-2 cells. Transgenic BY -2 calli expressing xylan-hydrolyzing activity were obtained at high frequency for both genes. Western blot analysis using an anti-XynA antibody indicated that XynAl and XynA2 were produced in these calli.     

Systematic transient assays of promoter activities for leaf-specific genes identified by gene-expression profiling with cDNA microarrays in Arabidopsis thaliana

We identified 85 genes highly expressed in leaves using an Arabidopsis cDNA microarray. A vector, pRAB5, was designed to allow cloning and assaying of the promoters. Fifty-one promoters from the selected genes were cloned and then assayed using a microprojectile bombardment and dual luciferase reporter assay system. This system allowed efficient systematic assays of promoter activity.     

5'-secondary structure formation, in contrast to a short string of non-preferred codons, inhibits the translation of the pyruvate kinase mRNA in yeast

The effects of poor codon bias and secondary structure formation upon the translation of the pyruvate kinase (PYK1) mRNA have been investigated in Saccharomyces cerevisiae. Following insertion mutagenesis at the 5'-end of the PYK1 coding region, the gene was transformed into yeast, and translation assessed directly in vivo by determining the distribution of the modified PYK1 mRNAs across polysomes fractionated by sucrose density gradient centrifugation. The chromosomally-encoded (wild-type) PYK1 mRNA, and the actin, ribosomal protein L3 and glyceraldehyde-3-phosphate dehydrogenase mRNAs were used to control for minor differences between polysome preparations. An insertion containing 13 non-preferred codons at the 5'-end of the coding region was found to have no significant effect upon PYK1 mRNA translation. In contrast, translation was inhibited by an insertion which increased the formation of secondary structures at the 5'-end of the mRNA (overall delta G = -36.6 kcal/mol). Control insertions were also analysed to exclude the possibility that alterations to the amino acid sequence of pyruvate kinase affect the translation of its mRNA. These insertions, which introduced preferred codons or restored wild-type levels of secondary structure formation, did not significantly influence PYK1 mRNA translation.     

Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast.     

The small heat-shock protein Hsp26 of Saccharomyces cerevisiae assembles into a high molecular weight aggregate.

Hsp26 is one of the major small heat-shock proteins (Hsp) of the yeast Saccharomyces cerevisiae, yet its cellular role remains to be discovered. To examine the cellular consequences of overexpression of Hsp26, the gene encoding this protein (HSP26) was overexpressed from a multicopy plasmid using either its own promoter or by coupling it to the efficient constitutive PGK promoter. The PGK promoter provided the opportunity to overexpress Hsp26 under non-stress conditions and such high level synthesis, prior to a lethal heat shock (50 degrees C), gave a small but reproducible elevation in thermotolerance. In transformed strains overexpressing Hsp26 under either stressed or non-stress conditions, the Hsp26 polypeptide was recovered almost exclusively as a high molecular weight aggregate. This high molecular weight aggregate (or heat-shock granule; HSG) was purified by differential centrifugation and sucrose gradient density centrifugation and shown, by electron microscopic analysis, to be of a uniform size (15-25 nm diameter). Analysis of the purified HSG demonstrated that it had a molecular weight of 550 kDa, yet contained no other integral polypeptides or other macromolecules.     

Molecular cloning and characterization of two inducible NAD⁺-adh genes encoding NAD⁺-dependent alcohol dehydrogenases from Acetobacter pasteurianus SKU1108

The cytosolic NAD?-dependent alcohol dehydrogenases (NAD?-ADHs) are induced in the quinoprotein ADH-(PQQ-ADH) defective Acetobacter pasteurianus SKU1108 mutant during growth in an ethanol medium. The adhI and adhII genes, which encode NAD?-ADH I and ADH II, respectively, of this strain have been cloned and characterized. Sequence analyses have revealed that the adhI gene consists of 1029 bp coding for 342 amino acids, which share 99.71% identity with the same protein from A. pasteurianus IFO 3283. Conversely, the adhII gene is composed of 762 bp encoding for a polypeptide of 253 amino acids, which exhibit 99.60% identity with the A. pasteurianus IFO 3283 protein. ADH I is a member of the group I Zn-dependent long-chain ADHs, while the ADH II belongs to the group II short-chain dehydrogenase/reductase NAD?-ADHs. The NAD?-adh gene disruptants exhibited a growth reduction when grown in an ethanol medium. In Escherichia coli, ethanol induced adhI and adhII promoter activities by approximately 1.5 and 2.0 times, respectively, and the promoter activity of the adhII gene exceeded that of the adhI gene by approximately 3.5 times. The possible promoter regions of the adhI and adhII genes are located at approximately 81-105 bp and 74-92 bp, respectively, from their respective ATG start codons. Their repressor regions might be located in proximity to these promoters and may repress gene expression in the wild-type, where the membrane-bound ADH effectively functions.     

菜热休克蛋白基因HSP81.1启动子的克隆与功能验证

本研究从甘蓝型油菜（Brassica napus）基因组中扩增获得热休克蛋白基因启动子,并将其克隆至双元载体pBI121的GUS报告基因上游,构成双元表达载体pBI121 HSP GUS。通过农杆菌（Agrobacterium tumefaciens）介导法转化烟草后获得转基因植株。对T1转基因植株研究表明,外源基因在42℃处理1h后开始表达,且即使用同样条件诱导,GUS基因的表达率和表达量在不同生长时期也有差异。在第50d左右的苗期,GUS基因的表达率和表达量高,但随植株老化而逐渐下降。研究显示,利用油菜的热休克蛋白启动子可以达到通过温度控制烟草基因表达的目的。