与人PC-1同源的鼠PC-1基因的启动子的分子克隆和特性分析

为了鉴定鼠mPC-1基因表达的调控元件,克隆并分析了该基因的启动子.构建了一系列mPC-1基因启动子的截短序列.通过荧光素酶报道基因,分析了它们在前列腺癌细胞和其它细胞中的表达.结果表明,在AR阳性细胞系中,mPC-1基因启动子活性远远高于SV40和p61-PSA 启动子,mPC-1基因启动子 599 bp 至449bp 可能含有一个负调控元件; mPC-1 1.1 kb 启动子控制的表达主要在前列腺癌细胞系中; 雄激素可调控mPC-1 1.1kb 启动子表达.mPC-1 1.1kb 序列是一个有前列腺癌细胞特异性和较强的启动子,经过进一步的修饰有可能作为一种有用的前列腺癌基因治疗元件.     

Multiple elements for negative regulation of the rat catalase gene expression in dedifferentiated hepatoma cells

Like such hepatic genes as those for albumin and aldolase B, the rat catalase gene shows markedly reduced expression in carcinogenesis of hepatocytes. Strong silencer activity has been widely observed in the 5'-flanking region of the gene, downstream from the G-rich sequence identified in a previous study. In this study, we identified and characterized multiple elements involved in negative regulation of catalase gene expression by reporter assay and gel shift assay. One of the silencer elements is located 3 kb upstream of the gene and has GATATCCCGATATC as core sequence. The observation that protein binding to the element is abundantly expressed in dedifferentiated hepatoma cell lines, but scarcely in well-differentiated cell lines suggests that this element is involved in negative regulation of the catalase gene expression in hepatocarcinogenesis. This element was targeted by a novel 20-kDa nuclear protein, which is designated HNRF (hepatocarcinogenesis-related negative regulatory factor).     

Structure and myofiber-specific expression of the rat muscle regulatory gene MRF4.

We have cloned an 11.3-kb rat genomic DNA fragment encompassing the muscle regulatory factor 4 (MRF4) protein-coding sequence, 8.5 kb of 5'-flanking sequence, and 1.0 kb of 3'-flanking sequence. In order to study MRF4 gene expression, the rat myogenic cell line, L6J1-C, which expresses the endogenous MRF4 gene only in differentiated myofibers, was transfected stably with the full-length genomic clone and various 5' deletions. RNase protection assays demonstrated that MRF4 genes containing as little as 430 bp of 5'-flanking sequence exhibited an increase in expression as the cells differentiated into myofibers, indicating that elements responsible for fiber-specific expression are contained within this cloned DNA fragment. Similar up-regulation was observed with genes containing 1.5 kb of 5'-flanking sequence. Interestingly, MRF4 genes containing 5.0 kb and 8.5 kb of 5'-flanking sequence were up-regulated to even higher levels, suggesting that additional myofiber-specific regulatory elements located between 1.5 and 5.0 kb upstream from the coding region play a role in regulating the expression of this muscle-specific gene.     

The mast cell-specific expression of a protease gene, RMCP II, is regulated by an enhancer element that binds specifically to mast cell trans-acting factors

The rat mast cell protease gene, RMCP II, is specifically expressed in the mucosal subclass of rat mast cells. We show here that the 5'-flanking region of this gene contains a mast cell-specific enhancer that directs preferential expression of a linked reporter gene (human growth hormone) transfected into rat basophilic leukemia cells. A DNA fragment containing the enhancer sequence is capable of binding specifically to mast cell nuclear trans-acting factors. The sequence of this enhancer element contains a region of homology to a consensus core sequence present in the enhancer region of the pancreatic protease genes.     

Cloning and characterization of ovine alphaS1-casein gene promoter: a transfection study in rat mammary gland cell line.

Promoter regions of milk protein genes are frequently used to produce pharmaceutically and medically important proteins in the mammary gland of transgenic animals and also can be used for the construction of an inducible eukaryotic expression vector. The aim of the present study was to clone, sequence and characterize the regulatory elements in ovine alphaS1-CSNGP. For the first time we have cloned and sequenced region extending from - 2136 to +49 bp containing 5'-flanking region and exon I. Computational analysis of the sequence showed presence of core promoter elements viz., TATA box, CAAT box and initiator sequence. Mammary gland specific sequences included MGF/STAT 5, MPBF, Yu Lee 2, 4 and 5, Oka box C and hormone responsive elements (HRE) viz., GRE, PRE, PRL, IRE and also Polyoma enhancer 3 sequences. Computational analysis data is validated by following the reporter gene expression studies in rat breast cell line. Six reporter gene constructs under the control of full length, proximal, distal, minimal and proximal-distal fused promoter segments were constructed to assess the effect of presence or absence of few selected regulatory elements on expression ability of the promoter. Based on qualitative evaluation of fluorescence, the pGFP-F/VspI showed highest fluorescence followed by pGFP-P, pGFP-F/SpeI, pGFPminimal and pGFP-D.     

The 5'-flanking sequence and regulatory elements of the cystatin S gene.

The gene encoding rat cystatin S (Cys S), a salivary gland-specific secretory protein, has CAAT and TATA boxes upstream of the inititation codon (Cox and Shaw, 1992), and contains regions that resemble those of other hormonally responsive eukaryotic genes. The 5'-flanking sequence of the rat Cys S gene has a potential CREB/AP-1 binding site (Rupp et al., 1990; Trejo et al., 1992), two potential glucocorticoid responsive elements (GREs, Drouin et al., 1989), and a possible GR/PR (glucocorticoid/progesterone) responsive element (Forman and Samuels, 1990). One of these potential GREs is adjacent to a potential AP-2 binding site, and another is typical of the glucocorticoid and progesterone receptor binding site. In this report, we have identified three regions in the 5'-flanking region of the Cys S gene that are found in salivary gland-specific genes (Ting et al., 1992) with a GT-rich region located between conserved elements II and III. Transfection experiments described in this paper suggest that a 281-bp DNA fragment from the Cys S gene promoter region with conserved elements II and III, the GT-rich region, and a possible GR/PR responsive element contains a negative regulatory element. In addition, our experiments suggest that the GT-rich region by itself is acting as a positive regulatory element.     

Structure, organization, and expression of the rat cardiac myosin light chain-2 gene. Identification of a 250-base pair fragment which confers cardiac-specific expression

The present study characterized the structure, organization, and expression of the rat cardiac myosin light chain (MLC) -2 gene. The rat cardiac MLC-2 gene has seven exons which display complete conservation with the exon structure of the rat fast twitch skeletal MLC-2 gene. A 250-base pair (bp) sequence of the 5'-flanking region contains CArG motifs and additional cis elements, each greater than 10 bp in length, which were conserved in sequence and relative position with the chick cardiac MLC-2 gene. A series of MLC-2/luciferase fusion genes consisting of nested 5' deletions of the MLC-2 5'-flanking region were constructed and transfected into primary neonatal rat myocardial cells and a non-myocardial cell line (CV-1), demonstrating that this 250 bp of the MLC-2 5'-flanking region was sufficient to confer cardiac specific expression on a luciferase reporter gene. This study suggests the presence of important proximal regulatory sequences in the MLC-2 5'-flanking region which are capable of directing the cardiac specific expression of the rat cardiac myosin light chain-2 gene.