Molecular cloning of a murine type III sodium-dependent phosphate cotransporter (Pit-2) gene promoter.

We report the novel cloning and preliminary characterization of a murine type III sodium-dependent phosphate cotransporter (Pit-2) gene promoter. Five promoter/luciferase reporter gene constructs, -1816/+61, -1620/+61, -1223/+61, -600/+61 and -225/+61, showed significant luciferase activity (6-14-fold over background) when transfected into human colon carcinoma (Caco-2) and opossum kidney (OKP) cells.     

Molecular cloning of murine sodium-phosphate cotransporter type IIb (Na/P(i)-IIb) gene promoter and characterization of gene structure

We report the cloning of the murine Na/P(i)-IIb cotransporter gene, which spans more than 18 kilobases and consists of 12 introns and 13 exons. Three promoter/reporter gene constructs, -159/+73, -429/+73 and -954/+73, showed significant luciferase activity (22-82-fold over background) when transfected into in rat intestinal epithelial (RIE-1) cells.     

Promoter sequences targeting tissue-specific gene expression of hypothalamic and ovarian gonadotropin-releasing hormone in vivo.

Molecular mechanisms directing tissue-specific expression of gonadotropin-releasing hormone (GnRH) are difficult to study due to the paucity and scattered distribution of GnRH neurons. To identify regions of the mouse GnRH (mGnRH) promoter that are critical for appropriate tissue-specific gene expression, we generated transgenic mice with fragments (-3446/+23 bp, -2078/+23 bp, and -1005/+28 bp) of mGnRH promoter fused to the luciferase reporter gene. The pattern of mGnRH promoter activity was assessed by measuring luciferase activity in tissue homogenates. All three 5'-fragments of mGnRH promoter targeted hypothalamic expression of the luciferase transgene, but with the exception of the ovary, luciferase expression was absent in non-neural tissues. High levels of ovarian luciferase activity were observed in mice generated with both -2078 and -1005 bp of promoter. Our study is the first to define a region of the GnRH gene promoter that directs expression to both neural and non-neural tissues in vivo. We demonstrate that DNA sequences contained within the proximal -1005 bp of the mGnRH promoter are sufficient to direct mGnRH gene expression to both the ovary and hypothalamus. Our results also suggest that DNA sequences distal to -2078 bp mediate the repression of ovarian GnRH.     

Enhancer-independent promoter activity of the mouse alphaB-crystallin/small heat shock protein gene in the lens and cornea of transgenic mice

The alphaB-crystallin/small heat shock protein gene is expressed very highly in the mouse eye lens and to a lesser extent in many other nonocular tissues, including the heart, skeletal muscle and brain. Previously we showed in transgenic mice that lens-specific alphaB-crystallin promoter activity is directed by a proximal promoter fragment (-164/+44) and that non-lens promoter activity depends on an upstream enhancer (-427/-259) composed of at least 5 cis-control elements. Here we have used truncated alphaB-crystallin promoter-CAT transgenes to test by biphasic CAT assays and/or histochemistry for specific expression in the cornea and lens. Deletion either of 87 bp (-427/-340) from the 5' end of the alphaB-crystallin enhancer or of the whole enhancer (-427/-258) abolished alphaB-crystallin promoter activity in all tissues except the lens and corneal epithelium when examined by the biphasic CAT assay in 4-5-week-old transgenic mice. These truncations also lowered promoter strength in the lens. The -426/+44-CAT, -339/+44-CAT and -164/+44-CAT (previously thought to be lens-specific in transgenic mice) transgenes were all expressed in the 4-6-week-old corneal epithelium when examined histochemically. Immunohistochemical staining confirmed the presence of endogenous alphaB-crystallin in the mature corneal epithelial cells. CAT gene expression driven by the alphaB-crystallin promoter with or without the enhancer was evident in the embryonic and 4-6-week-old lens. By contrast, activity of the alphaB-crystallin promoter/enhancer-CAT transgene was not detectable in the corneal epithelium before birth. Taken together, these results indicate that the intact enhancer of the alphaB-crystallin/small heat shock protein gene is required for promoter activity in all tissues tested except the lens and cornea.     

12-O-tetradecanoylphorbol-13-acetate activation of the MDR1 promoter is mediated by EGR1.

P-glycoprotein, the product of the MDR1 gene (multidrug resistance gene 1), is an energy-dependent efflux pump associated with treatment failure in some hematopoietic malignancies. Its expression is regulated during normal hematopoietic differentiation, although its function in normal hematopoietic cells is unknown. To identify cellular factors that regulate the expression of MDR1 in hematopoietic cells, we characterized the cis- and trans-acting factors mediating 12-O-tetradecanoylphorbol-13-acetate (TPA) activation of the MDR1 promoter in K562 cells. Transient-transfection assays demonstrated that an MDR1 promoter construct containing nucleotides -69 to +20 conferred a TPA response equal to that of a construct containing nucleotides -434 to +105. TPA induced EGR1 binding to the -69/+20 promoter sequences over a time course which correlated with increased MDR1 promoter activity and increased steady-state MDR1 RNA levels. The -69/+20 promoter region contains an overlapping SP1/EGR site. The TPA-responsive element was localized to the overlapping SP1/EGR site by using a synthetic reporter construct. A mutation in this site that inhibited EGR protein binding blocked the -69/+20 MDR1 promoter response to TPA. The expression of a dominant negative EGR protein also blocked the TPA response of the -69/+20 promoter construct. Finally, the expression of EGR1 was sufficient to activate a construct containing tandem MDR1 promoter SP1/EGR sites. These data suggest a role for EGR1 in modulating MDR1 promoter activity in hematopoietic cells.     

一种单核苷酸多态性的单倍型分析技术

运用多步PCR和测序技术,完成基因组中相距较远的单核苷酸多态位点的单倍型构建。通过设计2条等位基因特异性引物,扩增大片段DNA(10kb左右),以此大片段DNA作为下一轮PCR反应的模板,再在该片段中设计待检测区域的PCR引物,进行第2轮PCR。对PCR产物进行测序分析,确定其多态位点处的等位基因。结合第1轮PCR中的等位基因特异性引物,即可确定该大片段DNA中不同单核苷酸多态性构成的单倍型。以脂蛋白脂酶基因为例,应用其启动子区以及第4外显子区的等位基因特异性引物扩增约16kb的DNA片段,然后检测位于该片段中第2、3外显子的多态性。在￡-尸￡-基因第2内含子中发现了 13557G→A多态性。经分析确定出-421G／ 13557G／ 15222A、-421A／ 13557G／ 15222A、-421G／ 13557G／ 15222G、-421G／ 13557A／ 15222A等4种单倍型。等位基因特异性PCR结合小片段测序是一种快捷高效的对相距较远的多个SNP进行单倍型构建的新策略。