HEL cells, a human erythroleukemia cell line, mainly express the fetal (r)globin gene and trace amount of the embryonic (E)globin gene, but not adult (B) globin gene. Here we show that hydroxyurea (HU) can induce HEL ...HEL cells, a human erythroleukemia cell line, mainly express the fetal (r)globin gene and trace amount of the embryonic (E)globin gene, but not adult (B) globin gene. Here we show that hydroxyurea (HU) can induce HEL cells to express adult (B) globin gene and lead these cells to terminal differentiation. Results showed in Gel mobility shift assays that GATA factors could specifically bind to the regulatory elements of human B- globin gene, including the proximal regulatory element (the B- promoter) and the distal regulatory elements (the DNase I hypersensitive sites in the LCR, HS2-HS4 core sequences). However, the DNA binding patterns of GATA factors were quite different between HU-induced and uninduced HEL cells. Western-blot analysis of nuclear extracts from both the uninduced and HU- induced HEL cells revealed that the level of GATA-2 transcription factor decreased, whereas the level of GATA-1 transcription factor increased following the time of hydroxyurea induction. Furthermore, using RT-PCR analysis the expression of human B-globin gene in HU-induced HEL cells could be blocked again when HEL cells were incubated in the presence of antisense oligonucleotides for hGATA-1, suggesting that the upregulation of hGATA-1 transcription factor might be critical for the expression of human β- globin gene in HU-induced HEL cells.展开更多
An Amh promoter driving expression of a reporter gene (d2EGFP) has been used to analyze the role of two specific promoter transcription factor binding elements. In addition a downstream (3’) enhancer (DE) was also in...An Amh promoter driving expression of a reporter gene (d2EGFP) has been used to analyze the role of two specific promoter transcription factor binding elements. In addition a downstream (3’) enhancer (DE) was also investigated. The transcription factors GATA-1 and GATA-4 had opposite effects, the former being incremental and the latter decremental. The quantitative balance between these two factors may provide a degree of control over the level of gene expression.展开更多
The transcriptional factor GATA-6 gene produces two translational isoforms from a single mRNA through ribosomal leaky scanning. L-type GATA-6 has an extension of 146 amino acid residues at its amino terminus. In the e...The transcriptional factor GATA-6 gene produces two translational isoforms from a single mRNA through ribosomal leaky scanning. L-type GATA-6 has an extension of 146 amino acid residues at its amino terminus. In the extension, there is a unique PEST sequence (Glu31-Cys46), which is composed of an amino terminal Pro-rich segment and a carboxyl terminal Ser-cluster. Substitution of either half of the PEST sequence with Ala residues by cassette mutagenesis reduced the apparent molecular size of L-type GATA-6 on SDS-polyacrylamide gel-electrophoresis. However, the effect of substitution of the Pro-rich segment was much more significant;the mobility increase of the Pro-rich segment on the gel was 13% while that of the Ser-cluster was 8%. Substitution of each amino acid residue demonstrated that the effect of Pro substitution is greater than that of the Ser and Thr residues. Such increased mobility of L-type GATA-6 in the presence of a detergent may apparently correlate with the decrease in transcription activity in vivo as determined by means of luciferase reporter gene assay. The activity of ΔAla (with Ala residues instead of the PEST sequence) was reduced to one fifth of that of ΔA (with the PEST sequence). These results suggest that the PEST sequence of L-type GATA-6 does not function as a constitutive protein degradation signal, but rather plays structural and functional roles in the activation of gene expression on the GATA responsive promoter.展开更多
本研究旨在分析和鉴定辣椒GATA转录因子的家族成员的生物信息学特征,为今后进一步的实验研究奠定基础。利用生物信息学手段,通过HMMER和SMART软件对辣椒GATA转录因子进行鉴定。利用Clustal X 1.83、MEGA5.05、GSDS、MEME等软件对辣椒GAT...本研究旨在分析和鉴定辣椒GATA转录因子的家族成员的生物信息学特征,为今后进一步的实验研究奠定基础。利用生物信息学手段,通过HMMER和SMART软件对辣椒GATA转录因子进行鉴定。利用Clustal X 1.83、MEGA5.05、GSDS、MEME等软件对辣椒GATA转录因子进行生物信息学分析。本研究鉴定出24个辣椒GATA转录因子家族成员。系统进化树的结果显示,24个GATA家族成员分为4个亚族。GSDS软件结果表明辣椒GATA转录因子的内含子、外显子分布情况。DNAMAN软件和MEME软件结果显示辣椒的大部分GATA转录因子是保守的。染色体定位软件Map Inspect分析表明,辣椒GATA转录因子在辣椒12条染色体上分布并不均匀。本研究鉴定并分析了辣椒GATA转录因子的生物信息学的特征,为以后继续研究其功能提供了一定的理论基础与现实意义。展开更多
文摘HEL cells, a human erythroleukemia cell line, mainly express the fetal (r)globin gene and trace amount of the embryonic (E)globin gene, but not adult (B) globin gene. Here we show that hydroxyurea (HU) can induce HEL cells to express adult (B) globin gene and lead these cells to terminal differentiation. Results showed in Gel mobility shift assays that GATA factors could specifically bind to the regulatory elements of human B- globin gene, including the proximal regulatory element (the B- promoter) and the distal regulatory elements (the DNase I hypersensitive sites in the LCR, HS2-HS4 core sequences). However, the DNA binding patterns of GATA factors were quite different between HU-induced and uninduced HEL cells. Western-blot analysis of nuclear extracts from both the uninduced and HU- induced HEL cells revealed that the level of GATA-2 transcription factor decreased, whereas the level of GATA-1 transcription factor increased following the time of hydroxyurea induction. Furthermore, using RT-PCR analysis the expression of human B-globin gene in HU-induced HEL cells could be blocked again when HEL cells were incubated in the presence of antisense oligonucleotides for hGATA-1, suggesting that the upregulation of hGATA-1 transcription factor might be critical for the expression of human β- globin gene in HU-induced HEL cells.
文摘An Amh promoter driving expression of a reporter gene (d2EGFP) has been used to analyze the role of two specific promoter transcription factor binding elements. In addition a downstream (3’) enhancer (DE) was also investigated. The transcription factors GATA-1 and GATA-4 had opposite effects, the former being incremental and the latter decremental. The quantitative balance between these two factors may provide a degree of control over the level of gene expression.
文摘The transcriptional factor GATA-6 gene produces two translational isoforms from a single mRNA through ribosomal leaky scanning. L-type GATA-6 has an extension of 146 amino acid residues at its amino terminus. In the extension, there is a unique PEST sequence (Glu31-Cys46), which is composed of an amino terminal Pro-rich segment and a carboxyl terminal Ser-cluster. Substitution of either half of the PEST sequence with Ala residues by cassette mutagenesis reduced the apparent molecular size of L-type GATA-6 on SDS-polyacrylamide gel-electrophoresis. However, the effect of substitution of the Pro-rich segment was much more significant;the mobility increase of the Pro-rich segment on the gel was 13% while that of the Ser-cluster was 8%. Substitution of each amino acid residue demonstrated that the effect of Pro substitution is greater than that of the Ser and Thr residues. Such increased mobility of L-type GATA-6 in the presence of a detergent may apparently correlate with the decrease in transcription activity in vivo as determined by means of luciferase reporter gene assay. The activity of ΔAla (with Ala residues instead of the PEST sequence) was reduced to one fifth of that of ΔA (with the PEST sequence). These results suggest that the PEST sequence of L-type GATA-6 does not function as a constitutive protein degradation signal, but rather plays structural and functional roles in the activation of gene expression on the GATA responsive promoter.
文摘本研究旨在分析和鉴定辣椒GATA转录因子的家族成员的生物信息学特征,为今后进一步的实验研究奠定基础。利用生物信息学手段,通过HMMER和SMART软件对辣椒GATA转录因子进行鉴定。利用Clustal X 1.83、MEGA5.05、GSDS、MEME等软件对辣椒GATA转录因子进行生物信息学分析。本研究鉴定出24个辣椒GATA转录因子家族成员。系统进化树的结果显示,24个GATA家族成员分为4个亚族。GSDS软件结果表明辣椒GATA转录因子的内含子、外显子分布情况。DNAMAN软件和MEME软件结果显示辣椒的大部分GATA转录因子是保守的。染色体定位软件Map Inspect分析表明,辣椒GATA转录因子在辣椒12条染色体上分布并不均匀。本研究鉴定并分析了辣椒GATA转录因子的生物信息学的特征,为以后继续研究其功能提供了一定的理论基础与现实意义。
