Intracellular mechanisms of gonadotropin-stimulated gene expression in granulosa cells.

Previous studies have shown that the gonadotropins follicle-stimulating hormone and luteinizing hormone stimulate proopiomelanocortin (POMC) promoter activity and mRNA levels in ovarian granulosa cells. The objective of these studies was to determine the role of cAMP-dependent protein kinases (pKA) in gonadotropin-stimulated gene expression. Primary cultures of rat granulosa cells were transfected with a gene construct consisting of the POMC promoter (-150 to +63; designated pOMC-CAT) fused to the chloramphenicol acetyltransferase (CAT) reporter gene either alone or cotransfected with an expression plasmid (designated mutant RI), which overexpresses a mutant form of the murine RI subunit incapable of binding cAMP and serving as an irreversible inhibitor of the catalytic subunit of pKA. Follicle-stimulating hormone or isoproterenol caused a significant stimulation of pOMC-CAT activity in transfected cells. Cotransfection of pOMC-CAT with mutant RI caused a significant inhibition of basal pOMC-CAT activity and abolished the gonadotropin stimulation. As a control, transfection of the SV-40 viral enhancer-promoter fused to CAT (pSV2-CAT) was unresponsive to follicle-stimulating hormone stimulation and cotransfection with mutant RI had no significant effect on pSV2-CAT activity. These studies suggest that gonadotropin regulation of the POMC promoter is mediated by pKA and that promoter activity is stringently controlled by pKA.     

纤维介素基因hfgl2启动子的SARS冠状病毒核心蛋白反应元件初步分析

 SARS冠状病毒核心蛋白对hfgl2纤维介素基因有激活作用,采用实时荧光定量PCR和Western印迹已验证了hfgl2基因在mRNA和蛋白水平的表达.为进一步明确在SARS冠状病毒核心蛋白刺激下,hfgl2纤维介素基因5′端非编码区对转录激活起重要作用的转录调控序列,构建了一系列hfgl2 基因启动子荧光素酶报告基因质粒,将其与SARS冠状病毒核心蛋白真核表达质粒共转染CHO细胞.结果表明,转染前3个质粒hfgl2p(－1334)LUC、hfgl2p(－997)LUC、hfgl2p(－816)LUC的细胞的相对荧光素酶活性无显著改变;但转染hfgl2p( 468)LUC 质粒的细胞荧光素酶的活性较前明显降低,说明在hfgl2 基因启动子－816位至－468位（相对于转录起始点）之间存在着激活该基因的调控序列.本研究在一定程度上从分子水平揭示了SARS冠状病毒蛋白与宿主纤维介素基因之间的关系.     

The use of the luciferase reporter system forin planta gene expression studies

The properties of the firefly luciferase (LUC) make it a very good nondestructive reporter to quantify and image transgene promoter activity in plants. The short half-life of the LUC mRNA and protein, and the very limited regeneration of the LUC protein after reacting with luciferin, enables monitoring of changes in gene activity with a high time resolution. However, the ease at which luciferase activity is measuredin planta, using a light sensitive camera system (2D-luminometer), contrasts sharply with the complications that arise from interpreting the results. A variegated pattern of luciferase activity, that is often observed inin planta measurements, might either be caused by differences in influx, availability of the substrates (luciferin, oxygen, ATP) or by local differences in reporter gene activity. Here we tested the possible contribution of differences in the availability of each substrate to the variegatedin planta luciferase activity, and we show whenin planta luciferase activity is measured under substrate equilibrium conditions and can be related to the promoter activity of the reporter gene. Furthermore, we demonstrate the effects of protein stability, apparent half-life of luciferase activity, regeneration of luciferase and pH on thein vivo andin vitro luciferase measurements. The combined results give the prerequisites for the correct utilisation of the luciferase reporter system, especially forin vivo gene expression studies in plant research.     

Regulating genes with electromagnetic response elements

A 900 base pair segment of the c-myc promoter, containing eight nCTCTn sequences, is required for the induction of c-myc expression by electromagnetic (EM) fields. Similarly, a 70 bp region of the HSP70 promoter, containing three nCTCTn sequences, is required for the induction of HSP70 expression by EM fields. Removal of the 900 base pair segment of the c-myc promoter eliminates the ability of EM fields to induce c-myc expression. Similarly, removal of the 70 bp region of the HSP70 promoter, with its three nCTCTn sequences, eliminates the response to EM fields. The nCTCTn sequences apparently act as electromagnetic field response elements (EMRE). To test if introducing EMREs imparts the ability to respond to applied EM fields, the 900 bp segment of the c-myc promoter (containing eight EMREs) was placed upstream of CAT or luciferase reporter constructs that were otherwise unresponsive to EM fields. EMREs-reporter constructs were transfected into HeLa cells and exposed to 8 microT 60 Hz fields. Protein extracts from EM field-exposed transfectants had significant increases in activity of both CAT and luciferase, compared with identical transfectants that were sham-exposed. Transfectants with CAT or luciferase constructs lacking EMREs remained unresponsive to EM fields, i.e., there was no increase in either CAT or luciferase activity. These data support the idea that EMREs can be used as switches to regulate exogenously introduced genes in gene therapy.     

An improved CAT assay for promoter analysis in either transgenic mice or tissue culture cells.

We have developed an improved method for determining CAT activity directed by stably (transgenic mice) or transiently (tissue culture cell lines) introduced CAT reporter gene constructs. The procedure is based on the use of a new buffer system which considerably increases the stability of the CAT enzyme during the preparation of the crude cell extracts. When compared to other procedures, our method enables an increase of up to 100-fold in the sensitivity of the assay, depending on the transgenic tissue tested. Furthermore, a strong increase (up to 23-fold) was also observed with various promoter/CAT constructs transiently transfected in established tissue culture cell lines. This increase in sensitivity provides a significant reduction in the time required to perform the CAT assay when strong promoters are studied (from 18 to 1 hr) and is also very useful for the analysis of CAT gene expression driven by weak promoters.     

Regulation of the human interleukin-2 receptor alpha chain promoter: activation of a nonfunctional promoter by the transactivator gene of HTLV-I

We have characterized regulatory regions of the human IL-2 receptor alpha chain (IL2R alpha) promoter. 5' deletion constructs extending to -327 directed CAT expression in HTLV-I-infected T cells, which express IL2R alpha constitutively, and in Jurkat cells, which express IL2R alpha only after induction. Deletions to -267 and -265 were active only in HTLV-I-transformed T cells, but their activity in Jurkat cells was restored by cotransfection of a construct expressing the HTLV-I transactivator protein (tat-I). However, HTLV-I-infected human osteosarcoma cells do not express IL2R alpha-CAT constructs. Thus cell-type-specific factors are required for IL2R alpha expression, and direct or indirect interaction(s) between tat-I and a specific region of the IL2R alpha promoter may cause altered regulation. Tat-I also augments IL2-CAT expression under some conditions, suggesting possible autocrine or paracrine mechanisms for HTLV-I-induced leukemogenesis.     

Quantification of Atlantic salmon type-I interferon using an Mx1 promoter reporter gene assay

We here describe an assay for the detection of interferon-like activity in Atlantic salmon based on the transient transfection of chinook salmon embryo cells (CHSE-214 cells) with a rainbow trout Mx1 promoter linked to a luciferase reporter. A beta-galactosidase gene under the control of a constitutively expressed beta-actin promoter was used as a transfection standard, and luciferase and beta gal expression were measured by a commercially available kit. Interferon containing supernatants from poly I:C- or CpG-stimulated leucocytes added to transfected CHSE-cells induced high luciferase expression (>60-fold induction compared to supernatants from non-stimulated cells). There was no response to supernatants from LPS- and ConA/PMA-stimulated leucocytes, demonstrating the specificity for type I interferon-like activity. Duplicate samples analysed using a cell protection assay for detection of antiviral activity correlated well with levels obtained by the Mx1 promoter reporter gene assay (R2=0.97), confirming the reporter assay as a reliable substitute for the standard antiviral assay. The Mx reporter gene assay also has advantages in terms of sensitivity, high dynamic range and reliability over the conventional cell protection assay.     

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 the murine PHEX gene promoter

We report the novel cloning of the murine PHEX promoter, the gene that is mutated in X-linked hypophosphatemic rickets (XLH). Four promoter/reporter gene constructs, -133/+104, -542/+104, -1061/+104, and -2866/+104, showed significant luciferase activity (4.9-13.2-fold over background) when transfected into rat osteogenic sarcoma (UMR-106) cells.     

Herpes simplex virus thymidine kinase enzymatic assay in transient transfection experiments using thymidine kinase-deficient cells.

An enzymatic assay for herpes virus simplex type 1 thymidine kinase (HSV-TK) that was sensitive enough to quantitate intracellular levels of enzyme transiently expressed after transfection of HSV-TK vectors into TK-deficient cells using the DNA-calcium phosphate coprecipitation technique is described. TK activity in extracts of transfected cells was determined by binding of [methyl-3H]thymidylate product to thin layers of polyethyleneimine (PEI)-impregnated cellulose. The assay used high-specific-activity [methyl-3H]thymidine as substrate, which required removal of anionic material on a column of PEI-cellulose to enhance the signal-to-noise ratio. The assay was linear over a wide range with respect to the amount of HSV-TK plasmid transfected or content of HSV-TK enzyme in cell extracts. To validate the assay in transient expression experiments, HSV-TK and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected into NIH/3T3 tk- fibroblasts. Transient TK and CAT levels were concordant in cell extracts prepared from replicate plates of transfected cells. Normalizing the transient TK activity for CAT activity from the cotransfected "internal standard" CAT plasmid improved precision significantly, reducing the sample-to-sample coefficient of variation from 41 to 19%. CAT normalization reduced experimental variability mostly by correcting outlying results in transfection efficiency. The HSV-TK reporter gene system based on TK enzymatic assay was thus subject to experimental variation similar to that of the well-established CAT reporter function, demonstrating its utility in transient gene expression analysis.     

Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes

As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.