Endothelial-specific gene expression directed by the tie gene promoter in vivo

The tie gene encodes a receptor tyrosine kinase that is expressed in the endothelium of blood vessels, particularly during embryonic development and angiogenesis in adults. We have cloned and characterized the mouse tie gene and isolated the human and mouse tie promoters. The promoter activities of human and mouse tie were analyzed using luciferase reporter gene constructs in transfected cell lines and beta-galactosidase constructs in transgenic mice. In transfection assays of cultured cells, both human and mouse promoter DNA fragments showed activity that was not restricted to endothelial cells. In contrast, in transgenic mice both promoters directed expression of the reporter gene to endothelial cells undergoing vasculogenesis and angiogenesis. In adult mice, tie promoter activity in lung and many vessels of the kidney was as high as in the vessels of the corresponding embryonic tissues, whereas in the heart, brain and liver, tie promoter activity was downregulated and restricted to coronaries, cusps, capillaries, and arteries. Our results show that the endothelial cell-type specificity of the tie promoter in vivo can be transferred to heterologous genes by using relatively short promoter fragments. The tie promoter, thus, has useful properties for potential gene therapy.     

GnRH gene expression in neuronal cell lines.

Little is known about the mechanisms that subserve GnRH synthesis. The recent availability of clonal populations of GnRH neurons enables us for the first time to examine the structural organization of the GnRH locus. Studies are underway to dissect the manner in which neural specific expression and developmental regulation of GnRH are programmed. These studies will complement and provide direction to future animal studies designed to better understand the control of GnRH gene expression.     

Identification of long-range regulatory elements in the protocadherin-alpha gene cluster

The clustered protocadherins (Pcdh) are encoded by three closely linked gene clusters (Pcdh-alpha, -beta, and -gamma) that span nearly 1 million base pairs of DNA. The Pcdh-alpha gene cluster encodes a family of 14 distinct cadherin-like cell surface proteins that are expressed in neurons and are present at synaptic junctions. Individual Pcdh-alpha mRNAs are assembled from one of 14 "variable" (V) exons and three "constant" exons in a process that involves both differential promoter activation and alternative pre-mRNA splicing. In individual neurons, only one (and rarely two) of the Pcdh alpha1-12 promoters is independently and randomly activated on each chromosome. Thus, in most cells, this unusual form of monoallelic expression leads to the expression of two different Pcdh-alpha 1-12 V exons, one from each chromosome. The two remaining V exons in the cluster (Pcdh-alphaC1 and alphaC2) are expressed biallelically in every neuron. The mechanisms that underlie promoter choice and monoallelic expression in the Pcdh-alpha gene cluster are not understood. Here we report the identification of two long-range cis-regulatory elements in the Pcdh-alpha gene cluster, HS5-1 and HS7. We show that HS5-1 is required for maximal levels of expression from the Pcdh alpha1-12 and alphaC1 promoters, but not the Pcdh-alphaC2 promoter. The nearly cluster-wide requirement of the HS5-1 element is consistent with the possibility that the monoallelic expression of Pcdh-alpha V exons is a consequence of competition between individual V exon promoters for the two regulatory elements.     

Identification of upstream regulatory elements involved in the developmental expression of the Arabidopsis thaliana cab1 gene.

We studied cis regulatory elements controlling the light-dependent organ-specific expression of Arabidopsis thaliana chlorophyll a/b binding protein gene (cab1) by stably transforming tobacco plants using a tumor-inducing (Ti) plasmid vector system. The results from the 5' and internal deletion analyses indicate that there are at least three cis-acting elements that are involved in the light-dependent developmental expression of cab1 gene. Two such elements are located at the immediate upstream regulatory region and the other element is located at the further upstream region. The 1120-base-pair (bp) DNA fragment containing the immediate and far upstream region can confer light-inducible organ specificity on the truncated nos promoter. However, deletion of the 39-bp DNA fragment at the immediate upstream regulatory region from this hybrid promoter resulted in a nonfunctional promoter, revealing that the 39-bp region is important for the cab promoter specificity. Further analyses of this region suggest that a potential Z-DNA-forming sequence (ATACGTGT) is involved in light-dependent developmental expression of the cab1 gene. Two additional Z-DNA-forming sequences (ACACATAT) that are inverted repeats of this sequence are also found in the upstream region where the additional regulatory elements are expected.     

肝富集转录因子对HBV基因转录和复制的调控作用

目的建立乙型肝炎病毒（HBV）在非肝源细胞的复制体系,探讨肝富集转录因子对HBV基因转录和复制的调控作用。方法用复制型HBV重组质粒pHBV4.1与肝富集转录因子HNF3、HNF4和RXRα/PPARα的表达质粒分别共转染非肝源细胞株NIH3T3。用Northern吸印杂交检测HBV 3.5 kb、2.4/2.1 kb、0.7 kb mRNA的转录情况,Southern吸印杂交检测HBV DNA复制中间体水平。结果复制型HBV重组质粒pHBV4.1在肝癌细胞中能检测到3.5 kb HBV RNA转录产物和HBV DNA复制中间体;用pHBV4.1转染NIH3T3细胞后,在没有肝富集转录因子表达质粒共转染时未能检出3.5 kb HBV RNA转录产物,也无HBV DNA复制中间体;当用肝富集转录因子共转染时,HNF4和RXRα/PPARα的表达能够激活3.5 kb HBV RNA转录和HBV DNA复制,而HNF3未能激活HBV复制,但在HNF4或RXRα/PPARα介导的病毒复制体系中,均见3.5 kb HBV mRNA的转录和HBV DNA复制水平下降。结论利用肝富集转录因子成功地建立HBV在非肝源细胞中的转录与复制。其中,HNF4和RXRα/PPARα可支持HBV在非肝源细胞中转录与复制;HNF3抑制HBV肝特异性基因转录与复制。