A reporter transgene based on a human keratin 6 gene promoter is specifically expressed in the periderm of mouse embryos

We report the developmental regulation of a lacZ reporter transgene fused to the promoter region of the human keratin 6a gene. In mouse embryos, the transgene is expressed in the periderm (the outermost layer of embryonic epidermis), as are the endogenous keratin 6 alpha and beta genes. A subset of periderm cells, localized to temporary epithelial fusions, is known to contain keratin 6 protein, and we find that these cells also harbor LacZ enzymatic activity.     

The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse

The regulation of the mouse tyrosinase gene expression is controlled by a highly conserved element at -100 bp, the M-box, and an enhancer at -12 kb. In most vertebrates, the length of intergenic sequences makes it difficult to analyze the whole gene and the complete regulatory region. We took advantage of the compact Fugu genome to identify regulatory regions involved in pigment cell-specific expression. We isolated the Fugu tyrosinase gene, and identified putative cis-acting regulatory elements within the promoter. We then asked whether the Fugu promoter sequence functions in mouse pigment cells. We showed that E11.5 transgenic embryos bearing 6 kb or 3 kb of Fugu tyrosinase 5' sequence fused to the reporter gene lacZ revealed melanoblast and RPE-specific expression. This is the first evidence that the tyrosinase promoter is active at midgestation in melanoblasts, long before the onset of pigmentation.     

Pigment cell-specific expression of the tyrosinase gene in ascidians has a different regulatory mechanism from vertebrates

Tyrosinase is the key enzyme required for the synthesis of melanin pigments. Sequence comparison and functional analysis of the 5' upstream regions of vertebrate tyrosinase genes have revealed the importance of conserved E-box motifs in regulating their specific expression in pigment cells, optic cup-derived retinal pigment epithelium (RPE) and neural crest-derived melanocytes. In ascidians (more basal protochordates), two pigment cells that resemble vertebrate RPE cells are formed and specifically express the orthologous tyrosinase gene (HrTyr) in the cerebral vesicle located at the anterior end of the neural tube. To define regulatory sequences required for pigment cell-lineage-specific expression of HrTyr during embryogenesis, a series of mutations of the 5' upstream region of HrTyr were fused to the lacZ reporter gene and were microinjected into fertilized eggs. We found that the -152bp upstream of the translational start site is essential for expression in pigment cell precursors of tailbud-stage embryos. Further, additional positive and unique restriction elements were identified in the region up to -1.8kb. Surprisingly, in the -152bp minimal promoter or in other regions with regulatory activities, there are no E-box motifs or sequences correlating with other conserved elements regulating vertebrate tyrosinase promoters. The possibility that Pax proteins regulate HrTyr expression is also discussed.     

Unexpected position-dependent expression of H-2 and beta 2-microglobulin/lacZ transgenes.

In order to study sequences involved in the developmentally regulated and tissue-specific expression of the class I Major Histocompatibility Complex (MHC) genes, we have constructed several H-2/lacZ transgenic lines in which the 5' regulatory sequences of the H-2Kb gene are linked to the Escherichia coli beta-galactosidase (lacZ) gene. In five H-2/lacZ lines, the pattern of lacZ expression, detected histochemically varied greatly from line to line. None of the H-2/lacZ transgenes were transcribed in cells normally expressing a high level of endogenous H-2 molecules, although these H-2 regulatory sequences have been shown to be sufficient to drive tissue-specific expression of other reporter genes. Interestingly, when constructs containing 5' beta 2-microglobulin (beta 2m) regulatory sequences linked to lacZ were used to derive transgenic lines, similar results were obtained. A survey of lacZ labeling in H-2/lacZ and beta 2m/lacZ transgenic mice strongly suggests that these transgenes are very sensitive to position effect, lacZ expression being controlled by endogenous chromosomal regulatory elements specific for each insertion site. Here we describe the complex pattern of lacZ expression in the different transgenic lines during development; we discuss the unusual properties of these transgenes and underline their potential use for developmental studies and characterization of genomic sequences involved in spatiotemporal gene expression.     

Structural elements required for the localization of ASH1 mRNA and of a green fluorescent protein reporter particle in vivo

The sorting of the Ash1 protein to the daughter nucleus of Saccharomyces cerevisiae in late anaphase of the budding cycle correlates with the localization of ASH1 mRNA at the bud tip [1] [2]. Although the 3' untranslated region (3' UTR) of ASH1 is sufficient to localize a reporter mRNA, it is not necessary, a result which indicates that other sequences are involved [1]. We report the identification of three additional cis-acting elements in the coding region. Each element alone, when fused to a lacZ reporter gene, was sufficient for the localization of the lacZ mRNA reporter to the bud. A fine-structure analysis of the 3' UTR element showed that its function in mRNA localization did not depend on a specific sequence but on the secondary and tertiary structure of a minimal 118 nucleotide stem-loop. Mutations in the stem-loop that affect the localization of the lacZ mRNA reporter also affected the formation of the localization particles, in living cells, composed of a green fluorescent protein (GFP) complexed with lacZ-ASH1-3' UTR mRNA [3]. A specific stem-loop in the 3' UTR of the ASH1 mRNA is therefore required for both localization and particle formation, suggesting that complex formation is part of the localization mechanism. An analysis on one of the coding-region elements revealed a comparable stem-loop structure with similar functional requirements.     

Regulatory elements of copia retrotransposons, controlling the level of its expression in Drosophila melanogaster testes

Expression of the lacZ reporter gene under the control of five deletion derivatives of the copia regulatory region including the 5' long terminal repeat (LTR) and the 5' untranslated region (UTR) was assayed in the testes of transgenic Drosophila melanogaster males (larvae and imago). The full-length copia regulatory region (LTR + UTR) ensured expression of the reporter gene in testes of both larvae and adult males. Deletion of UTR or 3' end of LTR increased lacZ expression in the testes, whereas deletion of the 5' end of LTR increased it. This indicated that a positive regulator of copia expression is at the 5' end of LTR and that negative regulators are at the 3' end of LTR and in UTR. The effects of the fragments of the copia regulatory region on reporter gene expression in the testes in vivo did not completely coincide with the effects observed earlier in cultured cells. We suggest that this difference is due to different regulation of expression of the fusion constructs integrated into chromatin as compared to their transient expression.     

Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L ssp.)

In plant genetic engineering, using tissue-specific promoters to control the expression of target gene is an effective way to avoid potential negative effects of using constitutive promoter, such as metabolic burden and so on. However, until now, there are few tissue-specific promoters with strong and reliable expression that could be used in crop biotechnology application. In this study, based on microarray and RT-PCR data, we identified a rice green tissue-specific expression gene DX1 (LOC_Os12g33120). The expression pattern of DX1 gene promoter was examined by using the β-glucuronidase (GUS) reporter gene and analyzed in transgenic rice plants in different tissues. Histochemical assays and quantitative analyses of GUS activity confirmed that P (DX1):GUS was highly expressed in green tissues. To identify the regulatory elements controlling the expression of the DX1 gene, a series of 5' and 3' deletions of DX1 promoter were fused to GUS gene and stably introduced into rice plants. In addition, gel mobility shift assays and site-directed mutagenesis studies were used, allowing for the identification of two novel tissue-specific cis-acting elements (GSE1 and GSE2) within P(DX1). GSE1 acted as a positive regulator in all green tissues (leaf, sheath, stem and panicle). Compared with GSE1, GSE2 acted as a positive regulator only in sheath and stem tissue, and had a weaker effect on gene expression. In addition, P(DX1):GUS was not expressed in anther and seed, this characteristic reduced the potential ecological risk and potential food safety issues. Taken together, our results strongly suggest that the identified promoter, P(DX1), and its cis regulatory elements, GSE1 and GSE2, are potentially useful in the field of rice transgenic breeding. KEY MESSAGE: We have isolated and characterized the rice green tissue-specific promoter P(DX1), and identified two novel positive cis-acting elements in P(DX1).     

毛白杨PtSEP2基因启动子克隆和瞬时表达特性分析

利用PCR技术从毛白杨基因组DNA中扩增获得花器官发育相关的SEPALLATA2类似基因PtSEP25′侧翼约2.3kb的一段序列,经PlantCARE序列分析表明,该序列中含有启动子特征的保守序列及多种光应答元件,初步推测其为PtSEP2基因启动子.进一步以GUS为报告基因,构建了pPtSEP2 promoter::...