Objective: To analyze the genomic structure of SNC6, a progesterone\|receptor associated protein gene and its regulatory elements in its 5'\|flanking region. Methods: Genomic sequence from GenBank database (access...Objective: To analyze the genomic structure of SNC6, a progesterone\|receptor associated protein gene and its regulatory elements in its 5'\|flanking region. Methods: Genomic sequence from GenBank database (accession number: Z98048) covering the whole SNC6 gene was used to analyze the genomic structure of SNC6 and design primers for PCR amplification of its 5'\|flanking region. A 1894 bp fragment of the 5'\|flanking region \{(-1814\} to +75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments (1423 bp, 632 bp and 416 bp, which correspond to -1344 to +75, -552 to +75 and -337 to +75 respectively), were subcloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luciferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All transfected SW620 cells with the above 5\|flanking region\|containing constructs showed luciferase activities. The highest luciferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luciferase activities were measured in transfected cells with vectors containing 416 bp fragments. Luciferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in transfected cells with vectors containing 1423 bp fragments. Conclusion: The basic transcription\|promoting element (promoter) for SNC6 expression resides between 0 to -337, and two transcription\|enhancing elements (enhancer) resides between -337 to -552 and -1344 to -1814, whereas one transcription\|inhibiting element (silencer) exists between -552 to -1344.展开更多
The complex transformation of a tadpole to a frogduring amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previouslyi...The complex transformation of a tadpole to a frogduring amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previouslyisolated many genes which are up-regulated by T3 in theintestine of Xenopus laevis tadpoles. We have now cloneda full- length cDNA for one such gene (IU12). Sequenceanalysis shows that the IU12 cDNA encodes a plasmamembrane protein with 12 transmembrane domains andhomologous to a mammalian gene associated with cell activation and organ development. Similarly, we have foundthat IU12 is activated during intestinal remodeling whenboth cell death and proliferation take place. Furthermore,IU12 is an early T3-response gene and its expression in theintestine during T3-induced metamorphosis mimics thatduring normal development. These results argue for a roleof IU12 in the signal transduction pathways leading to intestinal metamorphosis.展开更多
The cDNA molecule encoding the mouse GABA transporter gene (GAT-1) was used as probe for selecting GAT-1 gene from mouse genomic library. A positive clone, harboring the whole open reading frame of the GAT-1 protein a...The cDNA molecule encoding the mouse GABA transporter gene (GAT-1) was used as probe for selecting GAT-1 gene from mouse genomic library. A positive clone, harboring the whole open reading frame of the GAT-1 protein and designated as MGABAT-G, was fished out from the library, the 5’ proximal region and nitron 1 were sequenced and analysed, and low homology was found in the above region between GAT-1 genes from mouse and human except some short conserved sequences. The DNA-protein interactions between DNA fragments containing the conserved sequences in the 5’ proximal region and nuclear proteins from different tissues of mouse were studied by means of gel-shift assay, and Southern-Western blot. The results indicate a possible positive-negative regulation mode controlling the expression of the mouse GAT-1 gene.展开更多
The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-globin gene have been examined. Using in vitro DNA-matrix binding assay,it has ...The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-globin gene have been examined. Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII, -446bp-419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating thatε-PREII mad be an erythroidspecilic facultstive MAR. In gel mobility shift assay and Southwestern blotting assal an eothroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (E-PREII). Furthermore, we demonstrated that the silencer (-392hp -177bp) uP8tream of the humanε-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nucleax matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element linght be a constitutive nuclear mains attachment region (constitutive MAR). Our results demonstrated that the nucleax madrid and nuclear mains proteins lxilght play an important role in the regulation of the human 5-globin gene expression.展开更多
文摘Objective: To analyze the genomic structure of SNC6, a progesterone\|receptor associated protein gene and its regulatory elements in its 5'\|flanking region. Methods: Genomic sequence from GenBank database (accession number: Z98048) covering the whole SNC6 gene was used to analyze the genomic structure of SNC6 and design primers for PCR amplification of its 5'\|flanking region. A 1894 bp fragment of the 5'\|flanking region \{(-1814\} to +75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments (1423 bp, 632 bp and 416 bp, which correspond to -1344 to +75, -552 to +75 and -337 to +75 respectively), were subcloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luciferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All transfected SW620 cells with the above 5\|flanking region\|containing constructs showed luciferase activities. The highest luciferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luciferase activities were measured in transfected cells with vectors containing 416 bp fragments. Luciferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in transfected cells with vectors containing 1423 bp fragments. Conclusion: The basic transcription\|promoting element (promoter) for SNC6 expression resides between 0 to -337, and two transcription\|enhancing elements (enhancer) resides between -337 to -552 and -1344 to -1814, whereas one transcription\|inhibiting element (silencer) exists between -552 to -1344.
文摘The complex transformation of a tadpole to a frogduring amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previouslyisolated many genes which are up-regulated by T3 in theintestine of Xenopus laevis tadpoles. We have now cloneda full- length cDNA for one such gene (IU12). Sequenceanalysis shows that the IU12 cDNA encodes a plasmamembrane protein with 12 transmembrane domains andhomologous to a mammalian gene associated with cell activation and organ development. Similarly, we have foundthat IU12 is activated during intestinal remodeling whenboth cell death and proliferation take place. Furthermore,IU12 is an early T3-response gene and its expression in theintestine during T3-induced metamorphosis mimics thatduring normal development. These results argue for a roleof IU12 in the signal transduction pathways leading to intestinal metamorphosis.
文摘The cDNA molecule encoding the mouse GABA transporter gene (GAT-1) was used as probe for selecting GAT-1 gene from mouse genomic library. A positive clone, harboring the whole open reading frame of the GAT-1 protein and designated as MGABAT-G, was fished out from the library, the 5’ proximal region and nitron 1 were sequenced and analysed, and low homology was found in the above region between GAT-1 genes from mouse and human except some short conserved sequences. The DNA-protein interactions between DNA fragments containing the conserved sequences in the 5’ proximal region and nuclear proteins from different tissues of mouse were studied by means of gel-shift assay, and Southern-Western blot. The results indicate a possible positive-negative regulation mode controlling the expression of the mouse GAT-1 gene.
文摘The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-globin gene have been examined. Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII, -446bp-419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating thatε-PREII mad be an erythroidspecilic facultstive MAR. In gel mobility shift assay and Southwestern blotting assal an eothroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (E-PREII). Furthermore, we demonstrated that the silencer (-392hp -177bp) uP8tream of the humanε-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nucleax matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element linght be a constitutive nuclear mains attachment region (constitutive MAR). Our results demonstrated that the nucleax madrid and nuclear mains proteins lxilght play an important role in the regulation of the human 5-globin gene expression.
