A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.
Bacteriophage lambda ; genetics ; DNA, Recombinant ; genetics ; Escherichia coli ; genetics ; Genetic Engineering ; Plasmids ; genetics ; Rec A Recombinases ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo analyze sequences of the housekeeping genes including recA, dnaE, and mdh in different serogroups or different biotypes of Vibrio cholerae (VC) strains isolated from China.

METHODS AND RESULTSrecA, dnaE, and mdh genes of Vibrio cholerae were obtained by PCR, sequenced and analyzed. Forty-four variable bases were identified in the 500 bases of recA gene of 18 VC strains, and the mutation of only 3 variable bases could result in changes of 2 amino acids. In the 600 bases of dnaE genes of 18 strains, 32 variable bases were found and only 1 contributed to an amino acid change. In the 367 bases of mdh genes of 18 strains, only 1 variable base was identified whose mutation involved an amino acid convertion. Toxic EI Tor biotype (EVC) strains and toxic O139 serogroup strains were closely related. Non-toxic strains of different serogroups or types were lowly related. Non-toxic and toxic strains of different serogroups or types were lowly related.

CONCLUSIONToxic EVC and toxic O139 serogroup strains isolated from different areas of China may evolve from a common original strain, and toxic O139 VC strains may come from toxic EVC strains.

Bacterial Proteins ; genetics ; Base Sequence ; China ; DNA Polymerase III ; genetics ; Gene Expression Profiling ; Humans ; Molecular Sequence Data ; Phylogeny ; Rec A Recombinases ; genetics ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Vibrio cholerae ; classification ; genetics ; isolation & purification

OBJECTIVETo construct a red fluorescent shuttle vector controlled by recA operon promoter to transform Streptococcus mutans.

METHODSThe promoter of recA was amplified from Streptococcus mutans UA159, and connected to plasmid pDsRed2-N1 to construct pRred with a red fluorescent coding gene, which was then inserted into the shuttle vector pDL276 to construct pLRred.

RESULTSpLRred was successfully constructed, and Escherichia coli transformed with the pLRred plasmid could express reporter gene DsRed.

CONCLUSIONSThe recombination plasmid pLRred can be used in the further research of the expression of cariogenic virulence factor gene by Streptococcus mutans in biofilm.

Escherichia coli ; genetics ; metabolism ; Fluorescent Dyes ; Genes, Essential ; Genes, Reporter ; Genetic Vectors ; Luminescent Proteins ; genetics ; Operon ; Plasmids ; Promoter Regions, Genetic ; Rec A Recombinases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Streptococcus mutans ; genetics ; Transformation, Bacterial

Red/ET recombination, a powerful homologous recombination system based on the Red operon of lambda phage or RecE/ RecT from Rac phage, provides an innovative approach for DNA engineering. Deletion, insertion and mutation can be quickly and precisely performed on the target gene mediated by Red/ET recombination with PCR derived DNA fragments or oligonucleotides. This technical platform has extensive applications in biomedical field including bacterial artificial chromosome modification, gene knock-out construction and genetic modification of E. coli strains as well as some other kinds of microorganisms. Recently, Red/ET recombination was improved in several aspects so that it becomes more powerful and maneuverable. The characteristic and development of Red/ET recombination and its biomedical applications were described in this review.
Bacteriophage lambda ; genetics ; DNA, Recombinant ; genetics ; DNA-Binding Proteins ; genetics ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; Exodeoxyribonucleases ; genetics ; Genetic Engineering ; methods ; Rec A Recombinases ; genetics ; Recombination, Genetic ; genetics

OBJECTIVETo investigate the relationship between RAD51-G135C and XRCC3-C241T single nucleotide polymorphisms and onset of acute myeloid leukemia (AML).

METHODSThe study was performed in 2 groups: AML patient group and normal person group as control group. Genomic DNA was extracted from peripheral blood cells of 545 AML patients and 1 034 normal persons. Genotypes of RAD51-G135C and XRCC3-C241T were analyzed by TaqMan probe technology and the ralatienship between RAD51-G135C/XRCC3-C241T polymorphisms and onset of acute myeloid leukemia was investigated.

RESULTSCompared with the control group, RAD51-G135C homozygous mutant (CC) could significantly increase the risk of AML patients (OR=3.07), and there was no statistical relationship between heterozygous mutant (GC) of RAD51-G135C and onset of AML. There was no statistical relationship between homozygous mutant (TT) of XRCC3-C241T and onset of AML, and the XRCC3-C241T heterozygous mutation type (CT) increased the risk of AML patients (OR=0.66).

CONCLUSIONRAD51-G135C homozygous mutant and XRCC3-C241T heterozygous mutation significantly increase the risk of the AML onset, which can provide more predictive value for incidence of AML.

DNA-Binding Proteins ; Heterozygote ; Homozygote ; Humans ; Leukemia, Myeloid, Acute ; Polymorphism, Single Nucleotide ; Rad51 Recombinase

OBJECTIVETo clarify the regulatory elements of Rad51 gene in its 5'flanking region.

METHODSVarious constructs were obtained by cloning different DNA fragments into pGL3 reporter vector. These constructs were then introduced into osteosarcoma cell line U2-OS by calcium phosphate method for transient expression of reporter gene, and luciferase activities were measured by luciferase assay.

RESULTSCells transfected with pGL3 constructs containing fragment -964 to +1430 and -733 to +1430 showed high luciferase activities. Obvious elevation of luciferase activities was also observed in cells transfected with pGL3 constructs containing four shorter derivative fragments -964 to -412, -746 to -412, -651 to -412 and -536 to -412. The highest luciferase activities were measured in transfected cells with plasmids containing fragment -964 to -412, and the lowest were in transfected cells with plasmids containing fragment -536 to -412. Luciferase activities in transfected cells with plasmids containing fragment -651 to -412 were higher than that in transfected cells with plasmids containing fragment -746 to -412.

CONCLUSIONIt is believable that the basic transcription-promoting element (promoter) for Rad51 gene resides between -536 to -412, and two transcription-enhancing elements (enhancer) or binding sites of positive transcription factors reside between -651 to -536 and -964 to -746, whereas one transcription-inhibiting element (silencer) or binding site of negative transcription factor may reside between -746 to -651.

5' Flanking Region ; DNA Repair ; DNA-Binding Proteins ; genetics ; Humans ; Promoter Regions, Genetic ; Rad51 Recombinase

The genomic instability may lead to an initiation of cancer in many organisms. Homologous recombination repair (HRR) is vital in maintaining cellular genomic stability. RAD51 associated protein 1 (RAD51AP1), which plays a crucial role in HRR and primarily participates in forming D-loop, was reported as an essential protein for maintaining cellular genomic stability. However, recent studies showed that RAD51AP1 was significantly overexpressed in various cancer types and correlated with poor prognosis. These results suggested that RAD51AP1 may play a significant pro-cancer effect in multiple cancers. The underlying mechanism is still unclear. Cancer stemness-maintaining effects of RAD51AP1 might be considered as the most reliable mechanism. Meanwhile, RAD51AP1 also promoted resistance to radiation therapy and chemotherapy in many cancers. Thus, researches focused on RAD51AP1, and its regulatory molecules may provide new targets for overcoming cancer progression and treatment resistance. Here, we reviewed the latest research on RAD51AP1 in cancers and summarized its differential expression and prognostic implications. In this review, we also outlined the potential mechanisms of its pro-cancer and drug resistance-promoting effects to provide several potential directions for further research.
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Humans ; DNA-Binding Proteins/metabolism* ; RNA-Binding Proteins/metabolism* ; Lung Neoplasms ; DNA Repair ; Genomic Instability ; Rad51 Recombinase/metabolism*

Ovarian carcinoma is the most lethal gynecologic malignancy. Resistance to platinum is considered the major problem affecting prognosis. Our recent study established that microRNA-506 (miR-506) expression was closely associated with progression-free survival and overall survival in two independent patient cohorts totaling 598 epithelial ovarian cancer cases. Further functional study demonstrated that miR-506 could augment the response to cisplatin and olaparib through targeting RAD51 and suppressing homologous recombination in a panel of ovarian cancer cell lines. Systemic delivery of miR-506 in an orthotopic ovarian cancer mouse model significantly augmented the cisplatin response, thus recapitulating the clinical observation. Therefore, miR-506 plays a functionally important role in homologous recombination and has important therapeutic value for sensitizing cancer cells to chemotherapy, especially in chemo-resistant patients with attenuated expression of miR-506.
Animals ; Antineoplastic Agents ; Cell Line, Tumor ; Cisplatin ; Disease-Free Survival ; Drug Resistance, Neoplasm ; Drug Therapy, Combination ; Female ; Homologous Recombination ; Humans ; Mice ; MicroRNAs ; Ovarian Neoplasms ; Piperazines ; Prognosis ; Rad51 Recombinase

OBJECTIVEThe functional characters of MCF7 and HCC1937 cell lines were compared through the activity of BRCA1 and p53 following DNA damage in order to provide more research evidence for the related studies in both breast cancer cell lines.

METHODSThe protein level of BRCA1 and p53 in two breast cancer cell lines and the protein level of BRCA1 in MCF7, HCC1937 and HCC1937 wtBRCA1 breast cancer cell lines treated with 10Gy after 1 h, 4 h or 8 h were detected by western blotting analysis. The distribution and foci formation of BRCA1 in the cells were observed through immunostaining assay and the percentage of BRCA1 or Rad51 foci formation after ionizing radiation was calculated. Cell cycle profiling was analyzed using flow cytometry.

RESULTSMost of BRCA1 and p53 localized in nucleus, and both proteins responded to DNA damage in MCF7 cells. In MCF7 cells,BRCA1 and Rad51 foci formation respectively increased to (59.40 ± 3.66)% from (11.80 ± 3.51)% (t = 16.26, P < 0.05) and (73.90 ± 8.66) % from (16.70 ± 3.76) % (t = 10.49, P < 0.05) after 10 Gy 8 h ; p53 and p21 protein level was further separately induced and enhanced to (82.54 ± 1.04) from (23.75 ± 0.51) (t = 87.90, P < 0.05) and (90.95 ± 1.13) from (50.19 ± 0.89) ( t = 49.11, P < 0.05) after 10 Gy 8 h; and the cells were accumulated in G1 phase. In contrast to MCF7, in HCC1937 cell line, both of BRCA1 and p53 were defective in nucleus since both proteins were mutated; in response to DNA damage, BRCA1 foci formation was not found, p53 and p21 was not induced; there was no cell accumulation in both of G1-S and G2-M phases. However, after complementation of wild-type BRCA1 in HCC1937 cells, DNA damage-induced Rad51 foci formation increased to (61.70 ± 4.03) % from (6.22 ± 2.27) % (t = 20.78, P < 0.05) and accumulation of cells in G2-M phase was also restored after 10 Gy 8h , which was similar to that of in MCF7 cells.

CONCLUSIONSWe have identified that BRCA1 and p53 have dramatically different functions in MCF7 and HCC1937 cell lines in response to DNA damage.

Cell Cycle ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; DNA Damage ; Humans ; MCF-7 Cells ; Rad51 Recombinase ; Radiation, Ionizing ; Tumor Suppressor Protein p53 ; Ubiquitin-Protein Ligases

OBJECTIVE: To investigate the expression and clinical significance of RAD51 in laryngocarcinoma. METHOD: The expression of RAD51 in laryngocarcinoma and polyp of vocal cord tissues were determined by immunohistochemical staining. The results were analyzed and compared with the clinical stage, lymph node metastasis and pathologic grade. RESULT: (1) The expression of RAD51 in laryngocarcinoma group was extremely stronger than that in polyp of vocal cord group (P < 0.01). (2) There was significant difference for RAD51 expression in cancer cells between earlier clinical stage group and advanced clinical stage group (P < 0.01). (3) There was significant difference for RAD51 expression in different pathologic grades (P < 0.05). (4) There was also significant difference for RAD51 expression between groups with and without lymph node metastasis. CONCLUSION RAD51 may play a critical role in tumorigenesis of laryngocarcinoma. RAD51 may be a potential marker for clinical diagnosis and treatment of Laryngocarcinoma. It may be significant in predicting clinical stage, pathologic grade and metastasis.
Female ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; Polyps ; metabolism ; pathology ; Prognosis ; Rad51 Recombinase ; metabolism ; Vocal Cords ; pathology