Do Bacteria Have the Transcription Factors? The Formation and Development of the Concept of “Transcription Factor” / 中国生物化学与分子生物学报

Over the past 20 years, there have been substantial divergences in academia around the world on the issue of whether bacteria have transcription factors (TFs). The traditional view is that bacteria do not have TFs, and their transcriptional activators and/or repressors regulate the transcription initiation, and TFs only bind to eukaryotic promoters. The typical representative of the traditional view is the mainstream international textbooks of Biochemistry and Molecular biology edited by some academic authorities. However, the new idea is that DNA binding transcriptional activators and repressors in bacteria are TFs, and the content and importance of which are no less than those of eukaryotes. Although the new idea has long been common in academic papers published in international academic journals, many scholars still have doubts. The concept of " transcription factor", like many terms of molecular biology, is constantly updated with the development of sciences, from narrowly-defined sense to broadly-defined sense. In the beginning, people thought that TFs were only necessary for the transcription initiation of eukaryotic genes, and bacteria did not need TFs. It was understandable that bacteria were excluded from the TFs’ scope of application at that time. The rich scientific research achievements in the past 40 years have proved that a large number of transcriptional activators and repressors bind to cis-regulatory elements other than promoters, including enhancers, silencers, and insulators in eukaryotes, as well as a variety of positive and negative regulatory elements in bacteria. These transcriptional regulatory proteins conform to all the basic characteristics of TFs, which make them worthy of the name " transcription factors". Therefore, the new idea is scientific, reasonable, and should be widely accepted and adopted by the academic community. In the future, whether the concept of " transcription factor " will be further expanded to chromatin-modifying proteins such as histone acetyltransferase (HAT) and ncRNAs, and even to " elongation factors" and " termination factors" of transcription, we should be open to this issue.

Bio panning of human stem cell factor(2) mimetic peptides from phage displayed random peptide library / 中国医学科学院学报

<p><b>OBJECTIVE</b>To screen human stem cell factor (hSCF) mimetic peptides in vitro with a phage-display random peptide library.</p><p><b>METHODS</b>Phage clones with high hSCF receptor (rc-kit/Ig 1-3)-binding activity was screened from phage-displayed random hepta/dodecapeptide library by phage enzyme-linked immunosorbent assay (ELISA). Phage single DNA was extracted and sequenced. Four kinds of peptide with higher c-Kit/Ig 1-3 binding activity were chosen for synthesis and characterized by using cell proliferation assay with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) method in UT-7 cells.</p><p><b>RESULTS</b>Eleven Ph.D.-C7C clones and eight Ph.D-12 phage clones with high hSCF receptor-binding activity were selected from phage-displayed random hepta/dodecapeptide library, respectively. Sequence analysis showed there were no homologous sequence between hSCF and these screened mimetic peptides except one homologous sequence DPSPHTH found in heptapeptide library. All these four synthesized peptides (CE3, CE16, LE4, and LE20), particularly CE16 and LE20, stimulated UT-7 cell proliferation.</p><p><b>CONCLUSION</b>Four hSCF mimetic peptides were successfully isolated from phage-displayed random peptide library..</p>

Expression, purification, and characterization of the first three immunoglobulin-like domains of human stem cell factor receptor / 中国医学科学院学报

<p><b>OBJECTIVE</b>To express the first three immunoglobulin-like domains of human stem cell factor receptor (c-Kit/Ig1-3) in E. coli and HEK293 ET cells and study their binding activity for stem cell factor (SCF).</p><p><b>METHODS</b>In prokaryotic expression system, a double mutant form of c-Kit /Ig1-3 (c-Kit /Ig1-3(DM) was produced by overlap PCR and cloned into pET16b. The recombinant protein was expressed in E. coli BL21 (DE3) and refolded by dilution. In eukaryotic expression system, the gene of c-Kit/Igl13 with eight histidine segments was cloned into pEAK12 and the recombinant plasmid was transfected into HEK293 ET cells. The fusion protein was harvested from the growth medium and purified on Ni-NTA agarose column. The recombinant protein was tested for the receptor binding activity with his-tag pull-down and enzyme-linked immunosorbent binding assay.</p><p><b>RESULTS</b>In E. coli c-Kit /Ig1-3(DM) as produced as an inclusion body and showed low binding activity for SCF after refolding. Two HEK293 ET cell clones that express high levels of c-Kit/Ig1-3 were produced and each clone secreted 2p micro/ml of recombinant protein, whose relative molecular mass was about 58,000. Eukaryotically expressed c-Kit/Ig1-3 had specific binding activity for SCF, and the dissociation constant (Kd) was 9.39 nmol/L.</p><p><b>CONCLUSION</b>c-Kit/Ig1-3 with high receptor binding activity is successfully produced in HEK293 ET cells.</p>

Expression of human HSF in E. coli and its effects on mobilization of hematopoietic stem cells in rhesus monkeys / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expression of hHSF in E. coli and its effect on the mobilization of hematopoietic stem/progenitor cells.</p><p><b>METHODS</b>The hHSF gene was obtained by overlapping PCR and cloned into the vector pET30a to yield pET30a-hHSF, which was transformed into E. coli BL21(DE3) and expressed with IPTG induction. Subsequently, rhHSF was purified by gel filtration and cation exchange chromatography and subjected to refolding. Molecular weight of hHSF was measured by MALDI-TOF Mass Spectroscopy. The N terminal amino acid sequence rhHSF was determined by protein sequencing. rhHSF was profiled in rhesus monkey for mobilization of peripheral blood stem cells. Eight rhesus monkeys were equally divided into two groups. The first group was administered single subcutaneous injection of 500 microg/kg hHSF, while the other one was administered 10 microg.kg(-1).d(-1) G-CSF for 4 days followed by a single subcutaneous injection of 500 microg/kg rhHSF.</p><p><b>RESULTS</b>The sequence coding hHSF was confirmed by sequencing and the induced-expression level was about 30% of total cell proteins. The purity of target protein was over 95%. The sequence of N terminal 10 amino acids and the amino acid composition were consistent with the theoretical parameters; molecular weight of rhHSF was 7540. The peripheral CD34(+) cells, CFU-GM yields, and neutrophils peaked at 3 h (16.3-folds increase compared with baseline), 1 h (1.9-folds increase) and 45 min (4.4-folds increase) respectively after the single injection of rhHSF. The addition of rhHSF after the last dose of G-CSF boosted these levels to 25.8-folds, 8.7-folds and 8.3-folds respectively.</p><p><b>CONCLUSION</b>hHSF is highly expressed in E. coli and rapidly mobilizes the hematopoietic stem/progenitor cells and neutrophils in rhesus monkeys. hHSF shows distinct synergistic effect with G-CSF.</p>

The transcriptional regulation study on human delta globin gene with CAAT box C-->T point mutation in its promoter / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the transcriptional regulation of human delta globin gene with C-->T point mutation at -64 in its promoter.</p><p><b>METHODS</b>Human delta globin genes including wild CAAT box and mutant CAAT box (-64C-->T) were separately cloned into eukaryotic expression vector pcDNA3.1 (-)/Myc-His A which was cut out the strong promoter CMV, transfected MEL cells, and induced by DMSO to express. The transcriptional regulation of human delta globin gene was analysed using semi-quantitative RT-PCR.</p><p><b>RESULTS</b>The expression level of human delta globin gene with mutant CAAT box was 2.2-fold as high as that with wild CAAT box.</p><p><b>CONCLUSION</b>The defective CAAT box of human delta globin gene promoter region may be one of the major reasons for its low expression level.</p>