Arabidopsis U6 small nuclear RNA （snRNA） promoters are those transcribed by RNA polymerase Ⅲ, but all the core elements for transcriptional initiation are located in the 5＇ promoter region. Previously, three Arabi...Arabidopsis U6 small nuclear RNA （snRNA） promoters are those transcribed by RNA polymerase Ⅲ, but all the core elements for transcriptional initiation are located in the 5＇ promoter region. Previously, three Arabidopsis U6 snRNA genes （U6-1, U6-26, and U6-29） were identified. Herein, we have further identified three new U6 loci （U6-4, U6-5, and U6-6） in the Arabidopsis genome. Alignment of these revealed that the upstream sequence element and TATA elements were contained in six U6 promoters. In addition, a unique, highly conserved element named the ＂CAT＂ element was observed in the promoter region. To understand the expression patterns of these U6 genes in Arabidopsis, we fused these promoters to the DNA segment of β-glucuronidase and then transferred these six constructs into Arabidopsis. Real-time reverse transcription-polymerase chain reaction analysis of these fused transcripts indicated that the newly identified U6 genes are active in Arabidopsis and that the U6-26 promoter seems to have higher transcriptional activity in leaf, stem, flower and silique. These results help to understand the function of these U6 snRNAs in Arabidopsis.展开更多
Breast cancer-specific gene 1 (BCSG1), also referred as synuclein γ, was originally iso-lated from a human breast cancer cDNA library and the protein is mainly localized to presynaptic terminals in the nervous system...Breast cancer-specific gene 1 (BCSG1), also referred as synuclein γ, was originally iso-lated from a human breast cancer cDNA library and the protein is mainly localized to presynaptic terminals in the nervous system. BCSG1 is not expressed in normal or benign breast lesions, but expressed at an extremely high level in the vast majority of the advanced staged breast carcinomas and ovarian carcinomas. Overexpression of BCSG1 in cancer cells led to significant increase in cell proliferation, motility and invasiveness, and metastasis. To elucidate the molecular mechanism and regulation for abnormal transcription of BCSG1, a variety of BCSG1 promoter luciferase reporters were constructed including 3′ end deleted sequences, Sp1 deleted, and activator protein-1 (AP1) domains mutated. Transient transfection assay was used to detect the transcriptional activation of BCSG1 promoters. Results showed that the Sp1 sequence in 5′-flanking region was involved in the basal transcriptional activities of BCSG1 without cell-type specificity. In comparison to pGL3-1249, the reporter activities of pGL3-1553 in BCSG1-negative MCF-7 cells and pGL3-1759 in HepG2 cells were notably decreased. Mutations at AP1 sites in BCSG1 intron 1 significantly reduced the promoter ac-tivity in all cell lines. Transcription factors, c-jun, c-fos and cyclin AMP-responsive element binding (CREB) protein, could markedly enhance the promoter activities. Thus, our results suggest that the abnormal expression of BCSG1 in breast cancer cells is likely regulated by multiple mechanisms. The 5′ flanking region of BCSG1 provides the basal transcriptional activity without cell type specificity. A critical promoter element involved in abnormal expression of BCSG1 presents in the first exon. The cell type specificity of BCSG1 transcription is probably affected through intronic cis-regulatory se-quences. AP1 domains in the first intron play an important role in control of BCSG1 transcription.展开更多
Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin i...Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.展开更多
β-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) β-g...β-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) β-galactosidase gene, designated GhGal1, has previously been identified and its transcripts are highly abundant at the elongation stage of the cotton fiber. To examine the temporal and spatial control of GhGal1 expression, a transcriptional fusion of the GhGal1 promoter region (1770 bp) with the β-glucuronidase (GUS) reporter gene was introduced into tobacco plants by the Agrobacterium infection method. The resulting transgenic plants showed higher GUS activity of fruit in the transgenic plants than that in the negative and positive controls. Histochemical localization of GUS activity demonstrated that the expression of the GUS gene could be found in the meristem zones of roots, cotyledons, vascular tissues, fruit and trichomes in transgenic tobacco plants. Additionally, se-quence analysis of the regulatory region also revealed several conserved motifs among which some were shared with previously reported fruit/seed-specific elements and the others were related with trichome expression. These results indicated the temporal and spatial expression characterization of the GhGal1 promoter in transgenic tobacco plants and provided an important insight into the roles of GhGal1 in cotton fiber development.展开更多
基金Supported by the State Key Basic Research and Development Plan of China （2001CB109002）, the National Natural Science Foundation of China （30370893）, Shanghai Municipal Committee of Science and Technology （03JC14061）, the Program for New Century Excellent Talents in University （NCET-04-0403）, and the ShuGuang Scholarship （04SG15）.
文摘Arabidopsis U6 small nuclear RNA （snRNA） promoters are those transcribed by RNA polymerase Ⅲ, but all the core elements for transcriptional initiation are located in the 5＇ promoter region. Previously, three Arabidopsis U6 snRNA genes （U6-1, U6-26, and U6-29） were identified. Herein, we have further identified three new U6 loci （U6-4, U6-5, and U6-6） in the Arabidopsis genome. Alignment of these revealed that the upstream sequence element and TATA elements were contained in six U6 promoters. In addition, a unique, highly conserved element named the ＂CAT＂ element was observed in the promoter region. To understand the expression patterns of these U6 genes in Arabidopsis, we fused these promoters to the DNA segment of β-glucuronidase and then transferred these six constructs into Arabidopsis. Real-time reverse transcription-polymerase chain reaction analysis of these fused transcripts indicated that the newly identified U6 genes are active in Arabidopsis and that the U6-26 promoter seems to have higher transcriptional activity in leaf, stem, flower and silique. These results help to understand the function of these U6 snRNAs in Arabidopsis.
文摘Breast cancer-specific gene 1 (BCSG1), also referred as synuclein γ, was originally iso-lated from a human breast cancer cDNA library and the protein is mainly localized to presynaptic terminals in the nervous system. BCSG1 is not expressed in normal or benign breast lesions, but expressed at an extremely high level in the vast majority of the advanced staged breast carcinomas and ovarian carcinomas. Overexpression of BCSG1 in cancer cells led to significant increase in cell proliferation, motility and invasiveness, and metastasis. To elucidate the molecular mechanism and regulation for abnormal transcription of BCSG1, a variety of BCSG1 promoter luciferase reporters were constructed including 3′ end deleted sequences, Sp1 deleted, and activator protein-1 (AP1) domains mutated. Transient transfection assay was used to detect the transcriptional activation of BCSG1 promoters. Results showed that the Sp1 sequence in 5′-flanking region was involved in the basal transcriptional activities of BCSG1 without cell-type specificity. In comparison to pGL3-1249, the reporter activities of pGL3-1553 in BCSG1-negative MCF-7 cells and pGL3-1759 in HepG2 cells were notably decreased. Mutations at AP1 sites in BCSG1 intron 1 significantly reduced the promoter ac-tivity in all cell lines. Transcription factors, c-jun, c-fos and cyclin AMP-responsive element binding (CREB) protein, could markedly enhance the promoter activities. Thus, our results suggest that the abnormal expression of BCSG1 in breast cancer cells is likely regulated by multiple mechanisms. The 5′ flanking region of BCSG1 provides the basal transcriptional activity without cell type specificity. A critical promoter element involved in abnormal expression of BCSG1 presents in the first exon. The cell type specificity of BCSG1 transcription is probably affected through intronic cis-regulatory se-quences. AP1 domains in the first intron play an important role in control of BCSG1 transcription.
文摘Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.
文摘β-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) β-galactosidase gene, designated GhGal1, has previously been identified and its transcripts are highly abundant at the elongation stage of the cotton fiber. To examine the temporal and spatial control of GhGal1 expression, a transcriptional fusion of the GhGal1 promoter region (1770 bp) with the β-glucuronidase (GUS) reporter gene was introduced into tobacco plants by the Agrobacterium infection method. The resulting transgenic plants showed higher GUS activity of fruit in the transgenic plants than that in the negative and positive controls. Histochemical localization of GUS activity demonstrated that the expression of the GUS gene could be found in the meristem zones of roots, cotyledons, vascular tissues, fruit and trichomes in transgenic tobacco plants. Additionally, se-quence analysis of the regulatory region also revealed several conserved motifs among which some were shared with previously reported fruit/seed-specific elements and the others were related with trichome expression. These results indicated the temporal and spatial expression characterization of the GhGal1 promoter in transgenic tobacco plants and provided an important insight into the roles of GhGal1 in cotton fiber development.
