ABSTRACT
To understand the function of basic Krüppel-like factor (BKLF), it was confirmed by direct fluorescence and indirect fluorescence observation that hBKLF was localized in nucleus, and distributed throughout nucleoplasm in a speckled pattern, except the nucleoli. This pattern is similar to many but not all transcription factors. To clarify the specific sequence responsible for its nuclear localization, a series of deletion mutants of GFP/hBKLF were constructed. By observing their subcellular localization, it was found that the three zinc fingers of hBKLF and the N-terminal aside from the fingers all served as nuclear localization signals (NLS); the sub-NLS of hBKLF was located in the N-terminus, including the CtBP-binding motif and the proline rich domain. These results provided a basis for further clarifying the function of BKLF.
Subject(s)
Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , Animals , COS Cells , DNA-Binding Proteins/genetics , Green Fluorescent Proteins , Humans , Kruppel-Like Transcription Factors , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Zinc Fingers/geneticsABSTRACT
To study the transcription regulatory function of basic Krüppel-like factor (BKLF)on gamma- and epsilon-globin genes, recombinant expression vectors containing the full-length human BKLF gene, and a deletion mutant that lost N-terminal 40 amino acids, were constructed and used, respectively, to transiently transfect COS7 cells in order to assay their reporter activities. Results showed that hBKLF was able to repress the activity of gamma- and epsilon-globin gene promoters, while the antisense nucleic acid specific for hBKLF activated the transcription of these promoters. Deleting 40 amino acids from N-terminus did not influence the transcriptional repression of hBKLF. The stimulatory function of FKLF on gamma- and epsilon-globin gene promoters was also significantly reduced by hBKLF. In addition, BKLF bound the CACCC element in the SHP1 (SH2-containing protein tyrosine phosphatase 1) gene promoter. These results suggest that gamma- and epsilon-globin genes may be transcriptional targets of BKLF, providing evidence for further studies on the role of BKLF in participating the transcriptional regulation of haemocyte-specific genes.
Subject(s)
DNA-Binding Proteins/genetics , Globins/genetics , 3T3 Cells , Animals , Base Sequence , Binding Sites/genetics , COS Cells , DNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , Gene Expression Regulation , Humans , Intracellular Signaling Peptides and Proteins , Kruppel-Like Transcription Factors , Mice , Mutation , Oligonucleotide Probes/genetics , Oligonucleotide Probes/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Protein Phosphatase 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatases/genetics , Sequence Deletion , Transcription, Genetic/genetics , TransfectionABSTRACT
AIM:To partially isolate and identify hepatic stimulator substance mRNA from human fetal liver tissues.METHODS:The poly (A)mRNA was extracted from human fetal liver tissues of 4-5 month gestation, fractionated by size on sucrose gradient centrifugation, translated into protein from each fraction in vitro and then its products were tested for HSS activity.RESULTS:Twenty-two 500 total RNA was obtained from human fetal liver tissues and pooled.mRNA of 420 was yielded,processed by oligo(dT)-cellulose column chromatography, then was size-fractionated by ultracentrifution on a continuous sucrose density gradient (5%-25%), and separated into 18 fractions.Translated products of mRNA in fraction 8 and 9 could produce a two-fold increase in the incorporation of( 3)H-TdR into DNA of SMMC-7721 hepatoma cells and in a heat resistant and organ-specific way.CONCLUSION:The partially purified HSS mRNA was obtained and this would facilitate the cloning of HSS using expression vectors.
