Metallothionein Genes in the Nematode Caenorhabditis elegans and Metal Inducibility in Mammalian Culture Cells

Genomic DNAs of metallothionein Ⅰ and Ⅱ in Caenorhabditis elegans (CeMT-Ⅰand CeMT-Ⅱ) were isolated by YAC library/polytene filter hybridization followed by subcloning of corresponding cosmid clones. Both genes are mapped at chromosome V. Although the similarities of 5'-flanking regions and coding regions have shown only 55-58%, the introns are split at the same position in both genes, indicating that these two genes are originally from the same gene. While several metal responsive elements are conserved among eukaryotes, only one metal responsive element was found in the promoter region in CeMT-Ⅱ and not in CeMT-Ⅰ. Indced, neither of 5'-flanking regions of CeMT-Ⅰ nor CeMT-Ⅱ connected to chloramphenicol acetyltransferase reporter gene is responsive to heavy metals in mammalian culture cells by transient transfection analysis. These results would suggest that the metal regulatory factors in C.elegans might be different from those conserved in invertebrates and vertebrates, although the MTs in C elegans revealed the similarities to mammalian MTs in several points

Molecular cloning and functional analyses of low-temperature induced genes from Ammopiptanthus mongolicus

Studies on the cold-responsive genes and cold signaling of woody species drop far behind in comparison to herbaceous plants.Due to similar lignified structure,perennial characteristic,and enhanced tolerance,it seems much easier to find strongly antifreeze genes and obtain effective results in transgenic woody plants.In this study,Ammopiptanthus mongolicus,an evergreen,broadleaf and cold-resist leguminous shrub growing in the desert of Inner Mongolia,was used as a material for low-temperature induced gene isolation.Through differential expression analysis induced by low-temperature,thirteen up-regulated cDNAs were identified.One of them,AmEBP1,(accession number:DQ519359)confers enhanced cold-tolerance to both transgenic E.coli and transgenic Arabidopsis.Results suggest that AmEBP1 can stimulate the synthesis of ribosome and the dephosphyration of the α-subunit of initiation factor 2(eIF2α),and subsequently promote the translation process.By which the transgenic plants obtained increased cold-resistant ability.

Anti-tumor effects induced by gene vaccines co-expressing truncated human prostate specific membrane antigen gene and mouse 4-1BBL

Objective To investigate the influence of m4-1BBL on anti-tumor effects induced by truncated human prostate specific membrane antigen ( tPSMA ) gene in mice. Methods A eukaryotic expression plasmid encoding tPSMA and m4-1BBL ( pDC316-tPSMA-IRES m4-1BBL) ,pDC316-tPSMA and pDC316 were constructed.

Identification of a candidate elongation related gene in rice induced by GA_3

We identified a gibberellin-induced gene frag-ment in rice elongation by using differentialdisplay(DD)of mRNA.The rice seedlingscarried the eui(elongated)gene,named Zhen-chang A,were used,which were sensitive toGAand elongated rapidly after application of

Cloning and characterization of a novel barley gene,HvORG4,induced by Fusarium graminearum infection

Barley Fusarium head blight(FHB),caused by species of the Fusarium fungus,is a devastating disease that is reemerging worldwide in recent years.In this study,a novel gene,HvORG4,was cloned from barley by using cDNA library and suppression subtractive hybridization(SSH) library strategies.The SSH library and cDNA library were constructed from the Chinese barley cultivar Jing02-461(resistance to FHB) infected by Fusarium graminearum isolate Huanggang-1.For the SSH analysis,more than 120 differentially expressed cDNAs were identified and sequenced.One of them showed high homology to the AtORG4 gene and was used as a probe to screen the cDNA library of Jing02-461.Six positive clones were identified and one of them contained a full-length cDNA,which was named HvORG4.Sequence analysis showed that HvORG4 encoded a deduced basic protein of 197 amino acids.Northern blotting analysis showed that HvORG4 was constitutively expressed in root and stalk,not in leaf or spike,and strongly induced in barley spikelets in response to infection with F.graminearum isolate Huanggang-1.Its homology and expression profile suggest that the HvORG4 might function as a transcription factor,playing an important role in signal transduction pathway for defense against FHB in barley.

The bHLH transcription factor GhPAS1 mediates BR signaling to regulate plant development and architecture in cotton

Cotton (Gossypium spp.) is the most important natural textile fiber crop in the world.The ideal plant architecture of cotton is suitable for mechanical harvesting and productivity in modern agricultural production.However,cotton genes regulating plant development and architecture have not been fully identified.We identified a basic helix-loop-helix (b HLH) transcription factor,GhPAS1 (PAGODA1 SUPPRESSOR1) in G.hirsutum (Upland cotton).GhPAS1 was located in the nucleus and showed a strong transcription activation effect.Tissue-specific expression patterns showed that GhPAS1 was highly expressed in floral organs,followed by high expression in early stages of ovule development and rapid fiber elongation.GhPAS1 overexpression in Arabidopsis and BRZ (brassinazole,BR biosynthesis inhibitor) treatment indicated that GhPAS1 positively regulates and responds to the BR (brassinosteroid) signaling pathway and promotes cell elongation.GhPAS1 overexpression in Arabidopsis mediated plant development in addition to increasing plant biomass.Virus-induced gene silencing of GhPAS1 indicated that down-regulation of GhPAS1 inhibited cotton growth and development,as plant height,fruit branch length,and boll size of silenced plants were lower than in control plants.Fiber length and seed yield were also lower in silenced plants.We conclude that GhPAS1,a b HLH transcription factor,regulates plant development and architecture in cotton.These findings may help breeders and researchers develop cotton cultivars with desirable agronomic characteristics.

Transcriptomic Analysis of the Tolerance Response to Dehydration and Rehydration in Wheat Seedlings

Drought is the main abiotic stress that restricts wheat production.The rapid development of sequencing technology and its widespread application to various fields have revealed the structural characteristics and regulation of related genes through gene expression analysis.Here,we studied responses of wheat plants under drought and rewatering conditions,using morphological and physiological indicators.Moreover,a transcriptome analysis was conducted on Jingmai 12,a drought-resistant wheat strain,to explore the mechanism underlying the response of drought-resistant wheat seedlings to drought stress at the transcriptome level.Drought stress caused morphological and physiological changes in both drought-resistant and-sensitive varieties,but to a greater extent in the drought-sensitive specimen.After re-watering,the drought-resistant wheat showed greater ability to recover than the drought-sensitive wheat.Transcriptome sequencing of Jingmai 12 revealed 97,422 genes,including 80,373 known genes and 17,049 newly predicted genes.The observed upregulation of genes was mostly involved in hormone and signal transduction,carbon metabolism,amino acid synthesis,small molecule production,transmembrane transport,ROS detoxification and defense,drought response protein,and protective enzyme activity.Downregulated genes were mostly involved in photosynthesis,lipid metabolism,signaling,and auxin response.Upon rehydration,these genes and metabolic pathways returned to normal.Our results suggest that all these changes are adaptations to drought stress.Through morphological adaptation,physiological regulation,and the expression of drought-induced genes,normal growth of drought-resistant varieties under drought stress can be promoted.These results increase our understanding of the transcriptomic changes taking place in drought-resistant wheat seedlings under drought stress,and provide a direction for future investigations.

Identification of a Herbicide Safener AD-67 Inducible cDNA in Rice

A herbicide safener AD-67 inducible cDNA was identified in an indica rice variety 9311 by mRNA differential display. The transcript was increased 6 h after sprayed with the safener solution, and 4 days later, the expression still could be detected. The fragment was recycled from the polygel and sequenced, and homologous analysis revealed the cDNA was 100% identical to some ESTs and cDNAs in rice database, and the amino acid sequence was 60-84% homologous to those of the Yippee genes in several eukaryotes. The fragment was extended to the whole long cDNA, and thus a primer pair was designed. RT-PCR analysis for the designed primer supported the induction result.

A New β-Estradiol-lnducible Vector Set that Facilitates Easy Construction and Efficient Expression of Transgenes Reveals CBL3-Dependent Cytoplasm to Tonoplast Translocation of CIPK5

Transient and stable expression of transgenes is central to many investigations in plant biology research. Chemical regulation of expression can circumvent problems of plant lethality caused by constitutive overexpression or allow inducible knock （out/down） approaches. Several chemically inducible or repressible systems have been described and successfully applied. However, cloning and application-specific modification of most available inducible expression systems have been limited and remained complicated due to restricted cloning options. Here we describe a new set of 57 vectors that enable transgene expression in transiently or stably transformed cells. All vectors harbor a synthetically optimized XVE expression cassette, allowing I^-estradiol mediated protein expression. Plasmids are equipped with the reporter genes GUS, GFP, mCherry, or with HA and Strepll epitope tags and harbor an optimized multiple cloning site for flexible and simple clon- ing strategies. Moreover, the vector design allows simple substitution of the driving promoter to achieve tissue-specificity or to modulate expression ranges of inducible transgene expression. We report details of the kinetics and dose-dependence of expression induction in Arabidopsis leaf mesophyll protoplasts, transiently transformed Nicotiana benthamiana leaves, and stably transformed Arabidopsis plants. Using these vectors, we investigated the influence of CBL （Calcineurin B-like） protein expression on the subcellular localization of CIPKs （Calcineurin B-like interacting protein kinases）. These analyses uncovered that induced co-expression of CBL3 is fully sufficient for dynamic translocation of CIPK5 from the cytoplasm to the tonoplast. Thus, the vector system presented here facilitates a broad range of research applications.

Isolation and Expression Analysis of Two Cold-Inducible Genes Encoding Putative CBF Transcription Factors from Chinese Cabbage （Brassica pekinensis Rupr）

Two homologous genes of the Arabidopsis C-repeat/dehydration-responsive element binding factors （CBF/ DREB1） transcriptional activator were isolated by RT-PCR from Chinese cabbage （Brassica pekinensis Rupr. cv. Qinbai 5） and were designated as BcCBF1 and BcCBF2. Each encodes a putative CBF/DREB1 protein with an AP2 （Apetal2） DNA-bindlng domain, a putative nuclear localization signal, and a possible acidic activation domain. Deduced amino acid sequences show that BcCBF1 is very similar to the Arabidopsis CBF1, whereas BcCBF2 Is different in that it contains two extra regions of 24 and 20 amino acids in the acidic domain. The mRNA accumulation profiles indicated that the expression of BcCBF1 and BcCBF2 is strongly induced by cold treatment, but does not respond similarly to dehydration or abscisic acid （ABA） treatment. However, the cold-induced accumulation of BcCBF2 mRNA was rapid but short-lived compared with that of BcCBFI. The mRNA levels of both BcCBF1 and BcCBF2 were higher in leaves than in roots when plants were exposed to cold, whereas, salt stress caused higher accumulation of BcCBF2 mRNA in roots than in leaves, suggesting that the organ specificity of the gene expression of the BcCBFs is probably stress dependent. In addition, the accumulation of BcCBF1 and BcCBF2 mRNAs was greatly enhanced by light compared with darkness when seedlings were exposed to cold. It is concluded that the two BcCBF proteins may be involved in the process of plant response to cold stress through an ABA-independent pathway and that there is also a cross-talk between the light signaling conduction pathway and the cold response pathway in B. pekinensis as in Arabidopsis.

Gene mutation in patients with primary open angle glaucoma in a pedigree in China

Objective To investigate the genetic basis of the pathogenesis of a Guangzhou (GZ 1) pedigree with primary open angle glaucoma (POAG) Methods DNA fragments of the trabecular meshwork inducible glucocorticoid response protein (TIGR) gene from 4 typical POAG patients and 2 normal subjects were amplified by polymerase chain reaction (PCR) The amplified PCR fragment was cloned into a pT Adv vector, and direct sequencing was carried out on an ABI 373 automated DNA sequencer using dye terminator chemistry to detect the mutation Results The TIGR gene mutation was identified in the selected subjects of this pedigree This mutation is a “C to T” transition at position 370, different from that of western countries and equivalent to the position change found in Japanese patients with familial POAG No mutation was found in the TIGR gene fragment in 2 normal subjects of the pedigree Conclusions These preliminary results provide insights into the pathogenesis of POAG by the TIGR gene mutation, and into the underlying action of the different mutations in oriental and western peoples

Dexamethasone-Inducible Green Fluorescent Protein Gene Expression in Transgenic Plant Cells

Genomic research has made a large number of sequences of novel genes orexpressed sequence tags available. To investigate functions of these genes, a system for conditionalcontrol of gene expression would be a useful tool. Inducible trans-gene expression that uses greenfluorescent protein gene (gfp) as a reporter gene has been investigated in transgenic cell lines ofcotton (COT; Gossypium hirsutum L.), Fraser fir [FRA; Abies fraseri (Pursh) Poir], Nordmann fir(NOR; Abies nord-manniana Lk.), and rice (RIC; Oryza sativa L. cv. Radon). Transgenic cell lineswere used to test the function of the chemical inducer dexamethasone. Inducible transgene expressionwas observed with fluorescence and confocal microscopy, and was confirmed by northern blotanalyses. Dexamethasone at 5 mg/L induced gfp expression to the nearly highest level 48 h aftertreatment in COT, FRA, NOR, and RIC. Dexamethasone at 10 mg/L inhibited the growth of transgeniccells in FRA and NOR, but not COT and RIC. These results demonstrated that concentrations of inducerfor optimum inducible gene expression system varied among transgenic cell lines. The inducible geneexpression system described here was very effective and could be valuable in evaluating thefunction of novel gene.

Supramolecular Vesicles Based on Gold Nanorods for Precise Control of Gene Therapy and Deferred Photothermal Therapy

In spite of being a promising therapeutic modality,gene therapy has limited clinical applications,mostly due to the lack of spatiotemporal resolution and inadequate efficacy.Herein,we present a facile strategy to remotely control intracellular gene expression by using gold nanorod-(Au NR)derived,host-guest interaction-mediated supramolecular vesicles as a gene carrier and photothermal transducer.Upon pulsed laser irradiation,mild photothermal conditions dissociate supramolecular vesicles to release gene and simultaneously activate heat shock protein-70 promoter(Hsp70)for spatiotemporally initiating gene expression inside cancer cells.Subsequently,upon introducing a polymeric guest species specifically into cancer cells,the dissociated Au NRs functionalized with macrocyclic host molecules could re-aggregate rapidly in cells to retard exocytosis of these NRs,thereby allowing deferred photothermal therapy to enhance the overall therapeutic outcome.

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era.Streptomyces phage-derived phiC31 integrase,which mediates an irreversible site-specific cassette exchange between the phage attachment site(attP)and the bacterial attachment site(attB),provides a promising option for the construction of a controllable gene-expression system.Here,we report a phiC3I integrase-mediated promoter flip system(FLIP)for the inducible expression of target genes in silk-worm(Bombyx mori).First,we constructed a FLIP reporter system,in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene.The coexpression of a C-terminal modified phiC3 I-NLS integrase carrying a simian virus 40(SV40)nuclear localization signal(NLS)effectively flipped the BmAct4 promoter through an attBlattP exchange,thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line,BmE.Subsequently,the FLIP system,together with a system continuously expressing the phiC3 I-NLS integrase,was used to construct binary transgenic silkworm lines.Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter,with an approximately 39%heritable transformation efficiency in silkworm offispring,leading to the constitutive and high-level expression of DsRed in silkworms,which accounted for approximately 0.81%of the silkworm pupal weight.Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.