小鼠Lrp5基因启动子的克隆及功能分析

为分析小鼠LDL受体相关蛋白 5 (Lrp5 )基因启动子的结构与功能 ,采用DNA重组技术 ,构建了 7种含小鼠Lrp5基因启动子荧光素酶报告基因表达体系 ,分别为 :pGL3 10 3(- 10 3bp～ + 132bp) ,pGL3 30 3(- 30 3bp～ + 132bp) ,pGL3 4 99(- 499bp～ + 132bp) ,pGL3 70 8(- 70 8bp～ + 132bp) ,pGL3 90 9(- 90 9bp～ + 132bp) ,pGL3 10 34(- 10 34bp～ + 132bp ) ,pGL3 12 2 9(- 12 2 9bp～ + 132bp) .以pRL TK为内参照质粒 ,瞬时转染成骨细胞株 (U2OS )及非成骨细胞株 (COS 7) ,收集细胞测定荧光素酶相对表达活性 .7种荧光素酶表达质粒在 2种细胞中表达无显著差异 ,即在所分析的小鼠Lrp5基因的 136 1bp(- 12 2 9bp～ + 132bp)范围内 ,不存在成骨细胞特异的表达元件 ;而且 7种表达质粒在 2种细胞中呈现相似的变化趋势 ,pGL3 10 3表达活性最高 ,pGL3 12 2 9表达活性显著降低 .表明小鼠Lrp5基因转录所必需的基本启动子序列在 - 10 3bp～ + 1bp范围内 ,- 10 34bp～ - 12 2 9bp之间的 195bp片段内可能含有负调控元件 .     

Isolation and expression of an IFN-responsive Ly-6C chromosomal gene

The Ly-6 locus controls the expression of genes whose products in lymphoid cells are involved in the process of Ag-independent T cell activation. The Ly-6 locus contains multiple tightly linked genes which have been mapped to a specific region on murine chromosome 15. The present approach to further define the Ly-6 Ag is based on the transfection of cloned genes and identification of the expressed products by using mAb. Screening of Ly-6 related chromosomal clones revealed one that contains a gene that is closely related to yet distinct from that of the previously characterized Ly-6E.1 protein. Transfection of this chromosomal clone into COS cells shows that it contains the gene encoding Ly-6C.1 determinants. The expression of the transfected Ly-6C.1 gene is enhanced in COS cells following treatment with mouse IFN. Characterization of the DNA sequence of the Ly-6C.1 gene has established that it consists of four exons, the first of which is untranslated. Several possible regulatory elements have been identified in the putative promoter region of this gene (5' to the first exon), including a 28-base sequence closely resembling the consensus IFN-responsive sequence found in the promoter regions of other IFN-responsive genes.     

Functional dissection of the promoter of the interphotoreceptor retinoid-binding protein gene: the cone-rod-homeobox element is essential for photoreceptor-specific expression in vivo.

The essential control elements in the interphotoreceptor retinoid-binding protein gene (IRBP) promoter are located between -156 and +19. The -156/-109 sequence contains a retina-specific DNAse I footprint and shows a positive regulatory activity in transiently transfected retinoblastoma cells. The -105/-85 sequence is G/C rich, shows a non-tissue specific DNAse I hypersensitivity, and a negative regulatory activity in retinoblastoma cells. The -76/-42 sequence shows a retinal-specific footprint and contains a "cone-rod-homeobox element" (CRXE) and a "photoreceptor conserved element" (PCE). IRBP promoter fragments with mutations in either CRXE, PCE or in both were linked to reporter genes and analyzed both by transient transfection and in transgenic mice. In retinoblastoma cells, the mutated CRXE-containing promoter shows a 60% repression of the CAT activity whereas the mutated PCE-containing promoter shows a 30% repression. In HeLa cells transfected with these promoters, co-transfection of a Crx expression vector with wild-type, but not with CRXE mutant promoter, activates CAT activity 20-fold over the background activity. Mutation of PCE alone or conversion of CRXE to PCE reduces this Crx-activated CAT activity to only 4-fold over the background activity. In the transgenic mouse experiments, none of the 12 lines with CRXE mutant promoter show significant expression of lacZ in the retina. In contrast, 9 of the 17 transgenic lines with PCE mutant promoter show photoreceptor-specific lacZ expression. Thus the Crx interaction with CRXE is essential for the photoreceptor-specific activity of the IRBP promoter in vivo. This interaction does not appear to require PCE, but is enhanced when PCE is present.     

Analysis of the promoter region of the gene encoding mouse cholesterol side-chain cleavage enzyme

The cholesterol side-chain cleavage enzyme (SCC) catalyzes the initial and rate-limiting step in the synthesis of steroid hormones. The mouse gene encoding SCC was cloned and the nucleotide sequence of its 5'-flanking region determined. This sequence includes an AP-1 motif at -319 and two motifs, AGGTCA at -70 and AGCCTTG at -40, that match elements proposed to be important in the expression of steroid 21-hydroxylase. When transfected into mouse Y1 adrenocortical tumor cells, 1.5 kilobase pairs of 5'-flanking region of the SCC gene directed high levels of expression of a growth hormone reporter gene; treatment of the transfected Y1 cells with 8-bromo-cAMP increased this expression by 5-fold. In contrast, transfected mouse MA-10 Leydig cells showed appreciably lower expression, suggesting that SCC expression in Leydig cells requires additional elements not contained in the 5'-flanking region of the SCC gene used in these experiments. Deletion experiments showed that 424 base pairs of 5'-flanking sequences were sufficient for regulated expression in Y1 cells and mapped two regulatory regions: one from -424 to -327 and a second from -219 to -77. DNase I footprinting and gel mobility shift analyses of these 424 base pairs defined several interactions between nuclear proteins and the SCC promoter, including footprints centered over the AP-1 motif, over a sequence at -120, and over the sequences (-70 and -40) that resemble 21-hydroxylase promoter elements. Finally, site-selected mutagenesis of the potential elements at -40, -70, or -120 decreased SCC promoter activity in transfected Y1 adrenocortical cells, thus establishing their importance in SCC expression.     

Conserved regulatory elements in the promoter region of the N-CAM gene.

Genomic clones containing 5'-flanking sequences, the first exon, and the entire first intron from the chicken N-CAM gene were characterized by restriction mapping and DNA sequencing. A > 600-bp segment that includes the first exon is very G + C-rich and contains a large proportion of CpG dinucleotides, suggesting that it represents a CpG island. SP-1 and AP-1 consensus elements are present, but no TATA- or CCAAT-like elements were found within 300 bp upstream of the first exon. Comparison of the chicken promoter region sequence with similar regions of the human, rat, and mouse N-CAM genes revealed that some potential regulatory elements including a "purine box" seen in mouse and rat N-CAM genes, one of two homeodomain binding regions seen in mammalian N-CAM genes, and several potential SP-1 sites are not conserved within this region. In contrast, high CpG content, a homeodomain binding sequence, an SP-1 element, an octomer element, and an AP-1 element are conserved in all four genes. The first intron of the chicken gene is 38 kb, substantially smaller than the corresponding intron from mammalian N-CAM genes. Together with previous studies, this work completes the cloning of the chicken N-CAM gene, which contains at least 26 exons distributed over 85 kb.     

Molecular cloning and characterization of the promoter region of the mouse regulatory subunit RII beta of type II cAMP-dependent protein kinase

The promoter and exon 1 of the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a mouse genomic library. The 5'-flanking DNA lacked TATA and CAAT sites but contained GC rich regions typically found in constitutively expressed house keeping genes. Fusion gene constructs, containing RII beta 5'-flanking sequences and the bacterial CAT structural gene, were transfected into NB2a neuroblastoma cells and CHO cells. The NB2a cells expressed high levels of CAT activity. CHO cells expressed CAT activity at 5% of the level seen in the NB2a cells. Transfection of deletion constructs into both cell lines was used to define the core promoter and enhancer elements. The core promoter was situated between bp -291/-121. An enhancer element was located between bp -1426/-1018.     

Novel cAMP regulatory elements in the promoter region of bovine P-450(11 beta) gene

In order to elucidate the cAMP regulatory elements in the promoter region of bovine P-450(11 beta) genes, we analyzed the promoter region using chloramphenicol acetyltransferase (CAT) gene as the reporter. Various deletion plasmids were constructed using the promoter region of CB11 beta-7, which is one of the two normal genes. Examination of the effects of Bt2cAMP on the CAT activities of mouse adrenal tumor cells (Y-1 cells) transfected with these deletion plasmids suggested that two elements named Ad3 and Ad4 play major roles in the induction by cAMP. Ad3(AAGATAAGGCACCCATCCATCTT) is located at -306 bp to -284 bp and Ad4 (CCAAGGTC) is located at -331 bp to -324 bp in the promoter region of the P-450(11 beta) gene. Deletions of both Ad3 and Ad4 resulted in a large decrease of the induction ratio from 9- to 3-fold. Coexistence of Ad3 and Ad4 is essential for their function, because any mutations introduced into either one of them resulted in a decrease of the cAMP induction ratio. These two elements are highly conserved among bovine, mouse, and human P-450(11 beta) genes and have no similarity with known cAMP regulatory elements. DNase I footprint analysis indicated that factors which specifically bind to the two elements exist in the nuclear extract of bovine adrenal cortex cells. Ad3 and Ad4 showed different patterns in gel shift analysis using probes which contained Ad3 or Ad4 sequence, suggesting their interaction with different nuclear factors. We also found two other protected regions by DNase I footprint analysis of the promoter regions of P-450(11 beta) gene, and named them Ad5 and Ad6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcriptional activation of the human hematopoietic prostaglandin D synthase gene in megakaryoblastic cells. Roles of the oct-1 element in the 5'-flanking region and the AP-2 element in the untranslated exon 1

The human hematopoietic prostaglandin D synthase (H-PGDS) gene is highly expressed in human megakaryoblastic cells, in which phorbol ester induces its expression. We characterized the promoter activity of the 5'-flanking region and the untranslated exon 1 (-1044 to +290) of the human H-PGDS gene in human megakaryoblastic Dami cells. Transient expression analysis using the luciferase reporter gene revealed that the 5'-flanking region and the untranslated exon 1 were sufficient for efficient expression of the H-PGDS gene in Dami cells, but not in monocytic U937 cells. Deletion and site-directed mutagenesis of the Oct-1 element in the 5'-flanking region decreased the promoter activity by approximately 30% compared with that of the entire region from -1044 to +290. An electrophoretic mobility shift assay demonstrated that Oct-1 specifically bound to the promoter region. Interestingly, even only untranslated exon 1 (+1 to +290) showed approximately 60% of the promoter activity of the entire region from -1044 to +290. Site-directed mutagenesis of the AP-2 element within the untranslated exon 1 abolished the basal promoter activity as well as its phorbol ester-mediated up-regulation. In AP-2-deficient HepG2 cells, the H-PGDS promoter activity was enhanced by coexpression with AP-2alpha. These findings indicate that the Oct-1 element in the 5'-flanking region acts as a positive cis-acting element and that the AP-2 element in the untranslated exon 1 is crucial for both basal and phorbol ester-mediated up-regulation of human H-PGDS gene expression in megakaryoblastic Dami cells.     

Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells.

We sought to determine the cis-acting elements responsible for the pattern of tissue specific expression of the mouse alpha 2(I) collagen gene. Using an RNase protection assay we first verified that expression of the alpha 2(I) collagen gene is mainly confined to tendons, bone, and skin in mice. Both transgenic mice and DNA transfection of tissue culture cells were used as experimental approaches. Transgenic mice lines were generated harboring chloramphenicol acetyltransferase (CAT) chimeric genes that contained either (a) 2000 base pairs (bp) of 5'-flanking sequences of the mouse alpha 2(I) collagen gene plus additional sequences between +418 and +1524 of the first intron of this gene or (b) the same promoter sequences without intron sequences or (c) the 350-bp proximal promoter sequences. Transgenic mice containing both types of 2000-bp promoters showed a pattern of CAT expression that was tissue specific. The presence of sequences of the first intron in the transgene did not increase the level of promoter activity. Transgenic mice harboring the 350-bp alpha 2(I) collagen promoter also showed a pattern that was tissue-specific except that high level expression also occurred in the brain. This suggests that negative regulation is an important component of tissue-specific expression. In order to analyze the first 350 bases in detail, we performed transient expression experiments, using promoter fragments attached to the luciferase reporter gene. Fibroblasts, which show a high level expression of the endogenous alpha 2(I) collagen gene, and B cells, in which the gene is silent, were transfected with a series of deletions and substitution mutations within the proximal 350-bp promoter. These experiments were unable to define unique cell-specific cis-acting elements. However, when the sequence between -315 and -284 was tandemly repeated upstream of a minimal alpha 2(I) collagen promoter (-41 to +54), the activity of this construction was considerably higher in fibroblasts than in B cells when compared with the minimal promoter itself. In gel retardation assays, the levels of complexes that bind to this sequence were higher in fibroblast nuclear extracts than in myeloma nuclear extracts. Our results are consistent with the hypothesis that the -315 to -284 DNA sequence participates in the cell-specific control of the alpha 2(I) collagen gene in fibroblasts.     

Expression of PiM-and PiZ-mutated forms of the human alpha 1-antitrypsin gene in transfected monkey COS1 cells

The PiZ mutation of the gene coding for alpha 1-antitrypsin results in a serum deficiency of this protein leading to early onset emphysema and liver disease. The PiZ gene has a Z-specific point mutation in exon V together with a point mutation in exon III which is also present in some normal (PiM) individuals. There has thus far been no system to study the effects of PiZ point mutations in tissue culture. We constructed plasmids containing alpha 1-antitrypsin cDNA synthetically altered at either exon III or exon V mutation sites and linked to simian virus 40 promoter sequences. Such constructs with the exon V mutation were transfected into monkey COS1 cells followed by analysis of expression of alpha 1-antitrypsin gene products. COS1 cells normally synthesize virtually no alpha 1-antitrypsin mRNA or protein. alpha 1-Antitrypsin mRNA is transcribed at high levels in cells transfected with either M or Z plasmids. Immunologic staining of COS1 cells within 48 h of transfection localizes alpha 1-antitrypsin protein to specific regions of the cytoplasm. This extranuclear localization is also observed with human HepG2 hepatoma cells, which synthesize alpha 1-antitrypsin at high levels, and with human SK-Hep1 hepatoma cells transfected with an M plasmid. The cloned synthetically altered alpha 1-antitrypsin genes provide a system for dissecting contributions of distinct point mutations to the pathological effects of the PiZ protein.     

A cAMP-responsive element regulates expression of the mouse steroid 11 beta-hydroxylase gene

In Y1 mouse adrenocortical tumor cells, expression of steroid 11 beta-hydroxylase (11 beta-OHase) is stimulated by cAMP following a delay of 4-6 h. Our results demonstrate that a cAMP-responsive element (CRE) within the 11 beta-OHase promoter region is a major determinant of this induction. The 5'-flanking sequences from the mouse 11 beta-OHase gene were placed in front of a human growth hormone reporter gene and transfected into Y1 cells. Treatment of transfected cells with 8-bromo-cAMP increased expression directed by the 11 beta-OHase 5'-flanking region by 3.8-fold. In 5'-deletion analyses, 123 base pairs of 5'-flanking sequences were sufficient for cAMP induction, whereas cAMP treatment did not affect expression of a plasmid with only 40 base pairs of 5'-flanking sequence. Within these 123 base pairs, a region from -56 to -49 matched 7 of 8 bases comprising the consensus sequence for the CRE. 11 beta-OHase 5'-flanking sequences from -65 to -42, including the CRE-like sequence, conferred cAMP inducibility to promoters from the thymidine kinase and chorionic gonadotropin alpha-subunit genes. DNase I footprinting and Southwestern blotting analyses demonstrated that the protein which interacted with the CRE in the 11 beta-OHase promoter region was similar to the CRE-binding protein associated with other cAMP-regulated genes. Together, these results suggest that an interaction between the 11 beta-OHase CRE and CRE-binding protein mediates cAMP induction of the 11 beta-OHase gene.