A rice promoter containing both novel positive and negative cis-elements for regulation of green tissue-specific gene expression in transgenic plants

The tissue-specific expression of transgenes is essential in plant breeding programmes to avoid the fitness costs caused by constitutive expression of a target gene. However, knowledge on the molecular mechanisms of tissue-specific gene expression and practicable tissue-specific promoters is limited. In this study, we identified the cis -acting elements of a tissue-specific promoter from rice, P_D54O , and tested the application of original and modified P_D54O and its cis -elements in the regulation of gene expression. P_D54O is a green tissue-specific promoter. Five novel tissue-specific cis -elements (LPSE1, LPSE2, LPSRE1, LPSRE2, PSE1) were characterized from P_D54O . LPSE1 activated gene expression in leaf and young panicle. LPSRE2 suppressed gene expression in leaf, root, young panicle and stem, and PSE1 suppressed gene expression in young panicle and stem. LPSRE1 and LPSE2 had dual roles in the regulation of tissue-specific gene expression; both functioned as activators in leaf, but LPSRE1 acted as a repressor in stem and LPSE2 as a repressor in young panicle and root. Transgenic rice plants carrying cry1Ac encoding Bacillus thuringiensis endotoxin, regulated by P_D54O , were resistant to leaf-folders, with no Cry1Ac protein found in endosperm or embryo. A reporter gene regulated by a series of truncated P_D54O showed various tissue-specific expression patterns. Different fragments of P_D54O fused with the constitutive cauliflower mosaic virus 35S promoter suppressed 35S -regulated gene expression in various tissues. P_D54O , truncated P_D54O and the tissue-specific cis -elements provide useful tools for the regulation of tissue-specific gene expression in rice breeding programmes.     

The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements

Experimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis -elements similar to other eukaryotes. Using a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii , we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite- and bradyzoite-specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis -acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite-specific NTPase during bradyzoite development were fine mapped to a 6–8 bp resolution and these minimal cis -elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel-shift experiments show that mapped cis -elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a 'poised' chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.     

Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping

The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) "domain swapping," wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using beta-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.     

ASF-2: a factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in Cab promoters.

We have used nuclear extracts prepared from tobacco leaf tissue to characterize a factor binding site, designated as-2 (activating sequence-2), at the -100 region of the cauliflower mosaic virus 35S promoter. The activity of this factor, called ASF-2 (activating sequence factor-2), is not detected in tobacco root extracts. as-2 includes two GT motifs with sequence homology to the SV40 enhancer core A element and the Box II element of pea rbcS. Nevertheless, oligomers of these sequence elements do not compete for ASF-2 binding in gel retardation assays, indicating that the GT motifs may not be involved. Methylation interference studies identify two guanines (G93 and G98) that are required for interaction with ASF-2. Sequences surrounding these two critical guanines display homologies to a GATA repeat conserved among several light-responsive promoters. One such sequence from a petunia Cab promoter is able to compete with as-2 for factor binding. In transgenic plants, a tetramer of as-2 is able to confer leaf expression when fused 5' to the -90 derivative of the 35S promoter. The expression is not dependent on light and, thus, the as-2 tetramer does not function as a light-responsive element in this context. Histochemical localization of the reporter gene product suggests that the as-2 tetramer directs expression in trichomes, vascular elements, and epidermal and mesophyll cells.     

Bioluminescence imaging of hepatitis B virus enhancer and promoter activities in mice

By bioluminescence imaging and hydrodynamic gene transfer technology, the activities of hepatitis B virus (HBV) promoters and the effects of HBV enhancers on these promoters in mice under true physiological conditions have been assessed. Our studies reveal that either of the two HBV enhancers can stimulate HBV major promoter activity in hepa 1-6 cells (in vitro) and in mouse liver (in vivo), and the enhancer effects on the three promoters (S1, S2 and X promoter) are markedly greater in vivo than in vitro. The two HBV enhancers have no cooperative action on HBV promoters in vitro or in vivo.     

用于植物的铜诱导基因表达系统的改进

铜诱导基因表达系统已被用于植物转基因表达的时空调控.它由两部分构成:(1)组成型或器官专一性表达的acel基因编码铜反应性的转录因子;(2)融合启动子控制下的目的基因,融合启动子由含ACE1结合位点的金属反应元件(MRE)连接CaMV 35S(-90～+8)启动子组成.本实验测试了来自酵母金属硫蛋白基因5′调控区的两个不同的ACE1结合区域在转基因烟草(Nicotiana tabacum L. cv.W38)中的效果.结果表明,与MRE(-148～-105)相比,使用MRE(-210～-126)的铜诱导系统效率增加50%到100%.在植物生物技术中使用这一系统控制基因性状是很有潜力的.     

Identification of cis-elements regulating the expression of an Arabidopsis histone H4 gene

Protein—DNA interactions in the proximal region of an Arabidopsis H4 histone gene promoter were analyzed by DMS in vivo footprinting combined with LMPCR amplification. Interactions were identified over six particular sequence motifs, five of which were previously shown to bind proteins in maize histone H3 and H4 promoters and are commonly found in the corresponding regions of other plant histone gene promoters. These motifs are located within a 126 bp fragment which was previously shown to confer preferential expression in meristems of transgenic plants. The contribution of each cis -element to the overall expression level and specificity was investigated by testing individual or combined mutations in transgenic Arabidopsis plants. All five motifs behaved as positive cis -elements of unequal strength. The GCCAAT-like sequence GCCACT behaved as a strong positive cis -element but had no influence on the specificity. In contrast, the nonamer AGATCGACG and to a lesser extent the closely linked hexamer CCGTCG proved to be essential for meristem-specific expression. Involvement of the highly conserved histone-specific octamer CGCGGATC in specific expression was revealed at some stages of meristem development. Importance of these three cis -elements, nonamer, hexamer, and octamer, was further confirmed by the fact that combining mutations of two of them either abolished the promoter activity or completely modified the promoter specificity. Mutation of the fifth cis -element, a degenerate copy of the octamer, little perturbed the promoter function. However disruption of both octamers had a dramatic negative effect, thus suggesting that the two copies cooperate to achieve maximal function in the wild-type promoter, possibly by mobilizing the proliferation-specific factors binding to the nonamer and CCGTCG cis -elements.