Bacterial Proteins ; genetics ; metabolism ; Biofilms ; DNA-Binding Proteins ; genetics ; metabolism ; Flagella ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Transcription Factors ; genetics ; metabolism ; Vibrio parahaemolyticus ; cytology ; genetics ; growth & development ; physiology

Antigens, Neoplasm ; genetics ; metabolism ; Breast Neoplasms ; enzymology ; genetics ; metabolism ; DNA Topoisomerases, Type II ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; physiology ; Humans

In this new era of the genome, the complete sequences of various organisms (from the simplest to the most complex such as human) are now available, which provides new opportunities to study biology and to develop therapeutic strategies. But the paucity of research tools that manipulate specific genes in vivo represents a major limitation of functional genomic studies. In nature, the expression of genes is regulated at the transcriptional level primarily by proteins that bind to nucleic acids. Many of these proteins, which are termed transcription factors, are typically consist of two essential yet separable modules: DNA-binding domain (DBD) and effector domain (ED). Attempts to control the gene expression by artificial transcription factors are based on the application of this rule. Among the many naturally occurring DNA-binding domains, the Cys2-His2 zinc-finger domain has demonstrated the greatest potential for the design of novel sequence-specific DNA-binding proteins. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. Ideally, these artificial DNA binding proteins could be designed to specifically target and regulate one single gene within a genome as complex as that found in human. Such proteins would be powerful tools in basic and applied research.
DNA ; chemistry ; genetics ; metabolism ; DNA-Binding Proteins ; metabolism ; Gene Expression Regulation ; Humans ; Transcription Factors ; chemistry ; genetics ; metabolism ; Zinc Fingers ; genetics ; physiology

The low dose hyper-radiosensitivity (HRS) in human lung cancer cell line A549 was investigated, the changes of ATM kinase, cell cycle and apoptosis of cells at different doses of radiation were observed, and the possible mechanisms were discussed. A549 cells in logarithmic growth phase were irradiated with (60)Co gamma-rays at doses of 0-2 Gy. Together with flow cytometry for precise cell sorting, cell survival fraction was measured by means of conventional colony-formation assay. The expression of ATM1981Ser-P protein was examined by Western blot 1 h after radiation. Apoptosis was detected by Hoechst 33258 fluorescent staining, and Annexin V-FITC/PI staining flow cytometry 24 h after radiation. Cell cycle distribution was observed by flow cytometry 6, 12 and 24 h after radiation. The results showed that the expression of ATM1981Ser-P protein was observed at 0.2 Gy, followed by an increase at >0.2 Gy, and reached the peak at 0.5 Gy, with little further increase as the dose exceeded 0.5 Gy. Twenty-four h after radiation, partial cells presented the characteristic morphological changes of apoptosis, and the cell apoptosis curve was coincident with the survival curve. As compared with control group, the cell cycle almost had no changes after exposure to 0.1 and 0.2 Gy radiation (P>0.05). After exposure to 0.3, 0.4 and 0.5 Gy radiation, G(2)/M phase arrest occurred 6 and 12 h after radiation (P<0.05), and the ratio of G(2)/M phase cells was decreased 24 h after radiation (P<0.05). It was concluded that A549 cells displayed the phenomenon of HRS/IRR. The mode of cell death was mainly apoptosis. The activity of ATM and cell cycle change may take an important role in HRS/IRR.
Cell Cycle Proteins/genetics ; Cell Cycle Proteins/metabolism ; Cell Cycle Proteins/physiology ; Cell Line, Tumor ; DNA-Binding Proteins/antagonists & inhibitors ; DNA-Binding Proteins/metabolism ; DNA-Binding Proteins/*physiology ; Dose-Response Relationship, Radiation ; Lung Neoplasms/*pathology ; Protein-Serine-Threonine Kinases/*metabolism ; Radiation Dosage ; Radiation Tolerance/*physiology ; Tumor Suppressor Proteins/metabolism

It has been suggested that non-structural protein 5A (NS5A) of hepatitis C virus (HCV) may have a regulatory role similar to other RNA viruses in RNA replication. In order to investigate the replication function of NS5A, we tried to purify recombinant His(6) tagged NS5A expressed in Escherichia coli by a denature-renaturing method. The native lysis buffer was used to remove most of the soluble non-specific proteins. His-NS5A protein was solublized with the denaturing lysis buffer containing 8 mol/L Urea, and then bound to Ni2+ -NTA resin. The protein bound resin was successively washed with buffer containing reducing concentrations of Urea in the presence of NaCl and DTT to renature the protein. The renatured His-NS5A protein was eluted from the resin and it was capable of interacting with glutathione S-transferase fused form NS5Bt (GST-NS5Bt). The purified His-NS5A exhibited an inhibitory effect on RNA-dependent RNA Polymerase (RdRP) activity of GST-NS5Bt in vitro. Conclusively, in this study, we have established a purification method of bacterial recombinant HCV NS5A, and the results support the notion that NS5A may involve in the regulation of HCV replication through direct interaction with NS5B.
DNA Primers ; Escherichia coli ; genetics ; Hepacivirus ; genetics ; physiology ; Plasmids ; Protein Binding ; RNA Replicase ; metabolism ; Recombinant Proteins ; metabolism ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; Virus Replication

OBJECTIVETo investigate telomerase gene expression in precancerous mammary lesion, such as atypical ductal hyperplasia and breast cancer and to study the relationship between expression and malignant transformation.

METHODSExpression of human telomerase genes (hTR) and human reverse transcriptase gene (hTRT) in 76 cases of mammary tissue was evaluated using in situ hybridization and included 50 cases of mammary hyperplasia, 6 of which were benign hyperplasia, 9 were mild atypical hyperplasia, 12 were moderate atypical hyperplasia, 23 were severe atypical hyperplasia and 26 were mammary cancer.

RESULTSThe expressions of hTR and hTRT mRNA were much weaker or negative in benign hyperplasia (16.6%, 0), weak to mild moderate in atypical hyperplasia (22.2%, 11.1%, 33.3%, 25.0%), strong in severe atypical hyperplasia (60.9%, 52.1%), and significantly strong in mammary cancer (88.5%, 80.8%). The difference between mild-moderate atypical hyperplasia, invasive ductal carcinoma and severe atypical hyperplasia was significant (P < 0.05) and the difference between severe atypical hyperplasia and intraductal carcinoma was not significant (P > 0.05).

CONCLUSIONTelomerase genes (hTR and hTRT) expressions are related to the transformation of atypical hyperplasia. Activated telomerase may play a role in mammary cancer development.

Breast ; metabolism ; pathology ; Breast Neoplasms ; genetics ; pathology ; DNA-Binding Proteins ; Female ; Gene Expression ; Humans ; Precancerous Conditions ; genetics ; pathology ; RNA ; genetics ; physiology ; RNA, Messenger ; analysis ; Telomerase ; genetics ; physiology

Vernalization makes Arabidopsis and other cruciferous plants flowering earlier. During this process, an important plant homeodomain-finger(PHD-finger) protein named VIN3 is involved. The PHD domain was a conserved zinc-finger domain in eukaryotic organism. It used to take part in the interaction between proteins, especially the modification on histone of nucleosome, such as methylation, acetylation and phosphorylation. In vernaliazation pathway, the proteins translated by VERNALIZATION INSENSITIVE 3(VIN3) and homologous genes could result in methylation on H3K9 and H3K27 and deacetylation on H3K9 and H3K14 on chromatin histone of FLOWERING LOCUS C, a gene that inhibited flowering. The structure state of FLC would be changed from relaxation into compression. Then the transcription activity of FLC could be restrained and it couldn't inhibit flowering any more, so it would induce flowering earlier. This paper reviewed the function of PHD-finger proteins in vernalization pathway in Arabidopsis and other cruciferous plants, and overviewed the vernalization mechanism.
Amino Acid Sequence ; Arabidopsis ; genetics ; metabolism ; Arabidopsis Proteins ; genetics ; metabolism ; physiology ; Brassicaceae ; genetics ; DNA-Binding Proteins ; genetics ; metabolism ; physiology ; Gene Expression Regulation, Plant ; genetics ; physiology ; Histones ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; physiology ; MADS Domain Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Transcription Factors ; genetics ; metabolism ; physiology ; Zinc Fingers

OBJECTIVETo explore the protein factors that could interact with the testis-specific protein encoded by HSD-3.8 gene (GenBank Accession Number AF311312) related with female fertilization.

METHODSYeast two-hybrid system was used to screen the human ovary MATCHMAKER cDNA library with constructed "bait plasmid" containing the 0.7 kb fragment (HSD-0.7) of HSD-3.8. The interaction with the positive fragments using a series of truncated bait plasmids was investigated.

RESULTSOne positive gene fragment was obtained, which coded for 144 amino acids of the C-terminus of human G protein beta subunit 1. Truncated bait plasmids couldn't interact with the fish protein fragment in yeast.

CONCLUSIONSThe protein encoded by HSD-3.8 gene may function through G protein signal transduction pathway and the interaction depends on the integration of the bait protein.

Adenosine Triphosphate ; metabolism ; Adult ; Antigens, Surface ; DNA-Binding Proteins ; genetics ; Female ; GTP-Binding Proteins ; genetics ; physiology ; Gene Library ; Humans ; Male ; Protein Biosynthesis ; Proteins ; genetics ; Spermatozoa ; chemistry ; physiology ; Synaptophysin ; Testis ; chemistry ; Two-Hybrid System Techniques ; Yeasts ; genetics

OBJECTIVETo explore the molecular mechanism of the differences in biofilm formation abilities of Candida albicans isolated from the respiratory tract.

METHODSThe biofilms formed by Candida albicans isolates from the respiratory tract and the standard strain ATCC90028 were examined for bacterial proliferation using XTT reduction assay. The mRNA expression of CPH1, EFG1, ALS3 and HWP1 in the isolates were measured with fluorescent quantitative RT-PCR.

RESULTSXTT reduction assay demonstrated a strong ability of biofilm formation in 8 clinical isolates, and a relatively low biofilm formation ability in 7 clinical isolates and ATCC90028 strain. The strong and weak biofilm formers showed significant differences in ALS3 and HWP1 mRNA expressions (P<0.05) but not in EFG1 or CPH1 mRNA expressions (P>0.05).

CONCLUSIONThe clinical isolates from the respiratory tract have different biofilm formation abilities under regulation by genes other than the transcription factors CPH1 and EFG1.

Biofilms ; Candida albicans ; classification ; genetics ; physiology ; DNA-Binding Proteins ; metabolism ; Exons ; Fungal Proteins ; metabolism ; Humans ; Membrane Glycoproteins ; metabolism ; Phenotype ; Respiratory System ; microbiology ; Transcription Factors ; metabolism

OBJECTIVETo investigate the role of Ser 219 phosphorylation of TRF1 (telomere repeat binding factor 1) in regulation of cell cycle.

METHODSThe mimicking phosphorylation mutant (TRF1S219D-GFP) and the non-phosphorylatable mutant (TRF1S219A-GFP) were constructed; the mutant genes and corresponding proteins were checked by sequencing and Western blot, respectively. Immunofluorescence staining was performed to detect the localization of mutants in HeLa cells. Cell cycle was analyzed by flow cytometry and ATM level was evaluated by immunoblotting.

RESULTSThe mutant genes were verified by direct sequencing and protein expression of GFP-tagged mutants was confirmed by immunoblotting.TRF1S219A-GFP and TRF1S219D-GFP were both localized in telomere of HeLa cells. Moreover, overexpression of TRF1-GFP or TRF1S219A-GFP resulted in an accumulation of HeLa cells in G2/M (P<0.05). The protein level of ATM was increased when overexpression the wide type or mutants.

CONCLUSIONThe Ser 219 phosphorylation of TRF1 by ATM could result in cell cycle arrest in G2/M, which is related to overexpression of TRF1.

Ataxia Telangiectasia Mutated Proteins ; Cell Cycle ; genetics ; physiology ; Cell Cycle Proteins ; metabolism ; DNA-Binding Proteins ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; HeLa Cells ; Humans ; Immunoblotting ; Microscopy, Fluorescence ; Mutation ; Phosphorylation ; Protein-Serine-Threonine Kinases ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Serine ; genetics ; metabolism ; Telomeric Repeat Binding Protein 1 ; genetics ; metabolism ; physiology ; Transfection ; Tumor Suppressor Proteins ; metabolism