Objective To elucidate physiological functions of human PIF1 helicase at the molecular level,purify N-terminal truncated PIF1 helicase,PIF1△N,and assay its biochemical properties.Methods The N-terminal cDNA sequence of PIF1 helicase was amplified by PCR using the Hela cell cDNA library as template.The cDNA with a histidine tag at the N-terminus was inserted into the pET20b vector to produce recombinant plasmid.The recombinant PIF1△N was successfully expressed by co-transforming a plasmid encoding rare rRNA.At 4 ℃ through a series of affinity column the recombinant PIF1△N protein was purified by fast protein liquid chromatograph.The biochemical activity of PIF1△N was assayed.Results The cDNA fragment of human PIF1 from 540～1 923 was cloned from Hela cDNA library,and the recombinant PIF1△N protein was successfully overexpressed in E.coli.The purification procedure of PIF1△N protein was established and its biochemical activity was identified.Conclusion N-terminal truncated PIF1 helicase,PIF1△N,has ATPase activity,which is DNA and Mg2+ dependent.

Objective To evaluate the value of detection of interleukin 28B (IL28B) rs12979860 by allele-specific PCR (AS-PCR) for the prediction of antiviral treatment hepatitis C patients.Methods One hundred seventy-four blood samples were random collected from hospitalized patients with hepatitis C,who came from department of infectious diseases,Renmin Hospital of Wuhan University from May 2011 to May 2012.Two pairs of specific primers were designed for rs12979860 gene polymorphisms,and one mutated base was introduced to the second or third site of the end of 3' with the reverse primer.rs12979860 gene polymorphism of 30 cases with hepatitis C was detected by AS-PCR,and gene sequencing was further used to verify the consistency of the two methods in parallel.Then,the frequency distribution of different rs12979860 genotypes with 174 cases were analyzed by the AS-PCR method in the population.Results The genotype CC,CT or TT of rs12979860 with 30 cases could be well identified by both AS-PCR and gene sequencing,and the coincidence rate was 100％ (x2 =60.0,P ＜ 0.01).Compared to gene sequencing,both of the sensitivity and specificity of AS-PCR were 100％.Compared to the control (CC genotype),TT genotype detection sensitivity by AS-PCR was 10-5,while sequencing sensitivity was 2 × 10-1.rs12979860 polymorphism in the TT,CC and CT genotype distribution in the Chinese population frequencies were 3.45％ (6/174),13.2％ (23/174) and 83.3％ (145/174),respectively.Conclusion AS-PCR can quickly,accurate,reliable,economic and efficiently detect IL28B rs12979860 gene polymorphism of hepatitis C in patients,which could predict the effect of antiviral therapy on patients with hepatitis C.

To identify Salvia shandongensis and its relatives at molecular level, the psbA-trnH intergenic region of three species including Salvia shandongensis, Salvia miltiorrhiza and S. miltiorrhiza f. alba were amplified and sequenced. Sequences were assembled with CodonCode Aligner. The K2P genetic distances between Salvia shandongensis and its relatives were calculated and UPGMA tree was performed by MEGA5.0. The results indicated that the lengths of psbA-trnH regions of Salvia shandongensis were about 391 bp, while the lengths of psbA-trnH regions of Salvia miltiorrhiza and S. miltiorrhiza f. alba were about 386 bp. The psbA-trnH sequences showed considerable variations between species and thus were revealed as a promising candidate for barcoding of Salvia shandongensis and its relatives. The intra-specific genetic distances of Salvia shandongensis were 0, while the intra-specific genetic distances of Salvia miltiorrhiza and S. miltiorrhiza f. alba were 0.002 and 0.001 respectively. Additionally, the genetic distance of Salvia shandongensis and Salvia miltiorrhiza ranged from 0.034 to 0.04, and the genetic distance of Salvia shandongensis and S. miltiorrhiza f. alba ranged from 0.005 to 0.008, the intra-specific genetic distances of Salvia shandongensis were much smaller than that of Salvia miltiorrhiza and S. miltiorrhiza f. alba; clustering results showed that there were obvious differences between Salvia shandongensis, Salvia miltiorrhiza and S. miltiorrhiza f. alba, which was consistent with morphological characteristics. This study not only firstly provides the scientific basis for establishing the taxonomy position in molecular level and revealing their genetic relationships of S. shandongensis, S. miltiorrhiza and S. miltiorrhiza f. alba; but also provides DNA molecular identification scientific basis for the development of new medicinal plant resources of Salvia shandongensis. Our results suggest that the psbA-trnH intergenic spacer region can be used as a barcoding to identify Salvia shandongensis, Salvia miltiorrhiza and S. miltiorrhiza f. alba.

Objective To investigate the expression profile of human genes in response to acute sodium arsenite treatment by cDNA microarray. Methods The RNA was purified from the L-02 cells without and with arsenite sodium induction for 2 hours, 15 hours and 24 hours, respectively. Results The hybridization patterns were different between every interval of arsenite induction. Expression of hCYR61 increased after 2 hours' induction, but decreased after 15 hours and 24 hours. Expression of metallothionein Ⅳ and Ⅲ elevated at the whole induction phase. HSP86 was up-regulated after 15 hours and 24 hours' induction, but it did not alter at two hours' induction. Conclusion When exposed to arsenite, the cells are under a meet-an-emergency situation to synthesize the most necessary protein and inhibit synthesis of unessential proteins.

Objective To evaluate and analyze the clinical application value of detection of Warfarin-related gene polymorphisms,cytochrome P450 2C9 (CYP2C9) and Vitamin K epoxide reductase complex subunit 1 (VKORC1) polymorphisms.Methods From July of 2011 to July of 2012,the blood samples were randomly collected from 140 lung cancer patients from Department of Oncology in Renmin Hospital of Wuhan University.These lung cancer patients were diagnosed through imaging examination and pathological examination.CYP2C9 and VKORC1 polymorphisms were detected in 70 patients (studied group) but not detected in the other 70 patients (control group) before they used warfarin.According to known gene sequences of CYP2C9 and VKORC1,specific primers were designed to genotype the CYP2C9 *2 and CYP2C9 * 3 alleles as well as the VKORC1-1639G ＞ A polymorphism through PCR amplification and DNA sequencing.Meanwhile,the distribution of these alleles in the studied group was analyzed.The clinical significance of detection of these polymorphisms was evaluated by comparing the proportion of patients within the therapeutic INR (International Normalized Ratio) range between control and genotype-guided dosing groups using Chi square test after 2 and 4 weeks of Warfarin therapy.Results Based on the results of agarose gel electrophoresis of PCR products and DNA sequencing,the primers for CYP2C9 and VKORC1 polymorphisms were indeed specific to these SNPs (CYP2C9 * 1,CYP2C9 * 2 and CYP2C9 * 3 ;VKORC1-1639GG,VKORC1-1639AG and VKORC1-1639AA) and both of the specificity and sensitivity of these primers are 100％,thus contributiug for genotyping these alleles.The distribution of CYP2C9 * 1/* 1 was 100％,CYP2C9 * 1/* 2,CYP2C * 1/* 3,CYP2C9 * 2/* 2,CYP2C9 * 3/* 3 and CYP2C9 * 2/* 3 were 0％.The distribution of VKORC1-1639AG,VKORC1-1639AA and VKORC1-1639 GG were 10％,90％ and 0％ respectively.2 weeks after the treatment of Warfarin,85.7％ patients in the genotype-guided dosing group reached the stable therapeutic INR range,which was significantly higher than that in the control group (48.6％,x2 =21.9,P ＜ 0.01); 4 weeks later,all patients (100％) were inside the stable therapeutic INR range whereas only 65 patients (92.9％) in the control group reached the therapeutic INR range.No haemorrhage or thromboembolic events occurred in both groups.Conclusions CYP2C9 and VKORC1 polymorphisms can be accurately detected by PCR reaction with the designed primers and the subsequent DNA sequencing in patients with lung cancer.This method is validated to be reliable.The genotyping of the Warfarin-related genes detective method can effectively guide Warfarin-dosing.

Pif1 subfamily helicase is conserved from yeast to humans with a lot of cellular functions. In order to elucidate the function of human PIF1 helicase from biochemical level, we cloned human PIF1 gene by PCR from HeLa cell cDNA library. We co-transformed a pMStRNA1 plasmid encoding rare tRNA codons and a plasmid encoding molecular chaperon to greatly enhance the overexpression of human PIF1 protein. Finally we purified full-length PIF1 helicase by column chromatograph carried out at 4 degrees C using fast protein liquid chromatograph (FPLC) system. The human PIF1 protein was purified in enough quantity for detailed biochemical analysis. Biochemical assay showed that PIF1 had ATPase activity and helicase activity. The purification and biochemical properties analysis of human PIF1 helicase will allow us to understand how, at the molecular and mechanistic level, this conserved helicase operates in the cell.
DNA Helicases ; biosynthesis ; genetics ; metabolism ; HeLa Cells ; Humans ; RNA, Transfer ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

Objective To investigate the effect of salt-induced kinase 2 (SIK2) in the G2/M checkpoint in response to ionizing radiation and the possible mechanism.Methods HeLa cells were irradiated with 60Co γ-rays.The cell model of knockdown SIK2 expression was constrcuted by transfecting HeLa cells with a pSicoR-based lentivirus vector of expressing SIK2 shRNA by lipofectamin 2000.Western blot and flow cytometry were performed to measure the changes of SIK2 protein level and cell cycle distribution.The phosphorylated histone protein H3 on Ser 10 was used as a molecular marker of mitotic cells for detecting the function of G2/M checkpoint.Results The expression level of SIK2 protein increased in HeLa cells after 60Co γ-ray irradiation.A cell model of knockdown SIK2 expression was successfully generated by transfecting the specific shRNA against SIK2.Depression of SIK2 significantly increased the cellular sensitivity at 1,2,4,6 Gy post-irradiation (t =-3.445,-2.581,-3.251,-2.553,P ＜0.05),and led cells to release earlier from the G2/M boundary arrest compared to control cells at 5,6 h post-irradiation (t =4.341,6.500,P ＜ 0.05).Western blot analysis indicated that the irradiation-induced phosphorylated CHK2/T68 in SIK2 knock-down cells was earlier than that in control cells.Conclusions salt-induced kinase 2 (SIK2) participates in the regulation of G2/M checkpoint induced by ionizing radiation and affects cellular radiosensitivity.

Objective To construct the recombinant eukaryotic expression plasmids of human adenylyl cyclase-associated protein 1 (CAP1)and to explore its intracellular location and functions.Methods By using Hela cDNA as the template,the cDNAs encoding CAP1 was amplified by PCR and inserted into pCMV-Myc vector to construct the recombinant plasmid.The recombinant plasmid was transfected into 293 cells using lipofectamine 2000.The protein expression and the intracellular location of the inserted gene were confirmed by Western blotting and immunofluorescence,respectively.Scratch-repair experiment was used to detect the cancer cells’ migration ability.Results The recombinant eukaryotic expression plasmid of human CAP1 was successfully constructed and transfected into eukaryote cells.The recombinant plasmid was successfully expressed in eukaryote cells.CAP1 was located in the cytoplasm.The results of scratch-repair experiment showed that the overexpression of CAP1 could significantly inhibit the cells’ migration.Conclusion CAP1 recombinant plasmid was successfully expressed in eukaryotic cells.CAP1 protein was located in the cytoplasm.The overexpression of CAP1 inhibited cell migration. The present study provides important experimental evidence for further study on CAP1.

Objective To construct recombinant plasmids of SIK2 cDNA and its truncated mutants and induce its expression in E.coil.Methods We designed primers of SIK2 and its truncated mutants.The gene fragments of SIK2,SIK2-Δ1 (280-926),SIK2-Δ2 (400-926),SIK2-Δ3 (1-400),and SIK2-Δ4 (700-926)were amplified by polymerase chain reaction (PCR)and cloned into pGEX-4T-2 vector to construct recombinant plasmids with GST. The plasmids were transformed into E.coil BL2 1 respectively,and induced with IPTG to express fusion protein. The results were confirmed by Coomassie blue staining and Western blot.Results We successfully constructed recombinant plasmid of SIK2 cDNA and its truncated mutants.Coomassie blue staining and Western blot resutls showed that these plasmids were induced to be expressed in E.coil BL21.Conclusion SIK2 cDNA and its truncated mutants were overexpressed in E.coil BL2 1 ,which lays expereimental foundation for further study on the function of each domain of SIK2 .

Objective To observe the effect of PIF1 knockdown on cell growth and cell cycle arrest induced by ionizing radiation.Methods HeLa cell lines that consistently down-regulated PIF1 were prepared by the lentivirus granules interfering technology and confirmed by real-time PCR and Western blotting.The effect of down-regulation of PIF1 on cell growth and cell cycle arrest induced by ionizing radiation was evaluated by cell counting and flow cytometry.Results HeLa cell lines consistently down-regulating PIF1 were established.The growth of HeLa that down-regulated PIF1 was inhibited greatly after 4 Gy of γ-ray irradiation.There was little cell proliferation until the 5th day post 4 Gy γ-ray.Moreover, the S phase block and G2/M phase block of PIF1 knock-downed cell lines were significantly delayed after 8 Gy γ-ray irradiation.Conclusion Knockdown of PIF1 can significantly enhance the radiation sensitivity and delayes the S phase block and G 2 /M phase block induced by ionizing radiation.