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1.
Chinese Journal of Biotechnology ; (12): 950-965, 2021.
Article in Chinese | WPRIM | ID: wpr-878606

ABSTRACT

Saccharomyces cerevisiae is one of the most important hosts in metabolic engineering. Advanced gene editing technology has been widely used in the design and construction of S. cerevisiae cell factories. With the rapid development of gene editing technology, early gene editing technologies based on recombinase and homologous recombination have been gradually replaced by new editing systems. In this review, the principle and application of gene editing technology in S. cerevisiae are summarized. Here, we first briefly describe the classical gene editing techniques of S. cerevisiae. Then elaborate the genome editing system of MegNs, ZFNs and TALENs based on endonuclease. The latest research progress is especially introduced and discussed, including the CRISPR/Cas system, multi-copy integration of heterologous metabolic pathways, and genome-scale gene editing. Finally, we envisage the application prospects and development directions of Saccharomyces cerevisiae gene editing technology.


Subject(s)
CRISPR-Cas Systems/genetics , Endonucleases/genetics , Gene Editing , Saccharomyces cerevisiae/genetics , Technology
2.
Chinese Medical Journal ; (24): 564-572, 2021.
Article in English | WPRIM | ID: wpr-878081

ABSTRACT

BACKGROUND@#The pathogenesis of osteosarcoma (OS) is still unclear, and it is still necessary to find new targets and drugs for anti-OS. This study aimed to investigate the role and mechanism of the anti-OS effects of miR-296-5p.@*METHODS@#We measured the expression of miR-296-5p in human OS cell lines and tissues. The effect of miR-296-5p and its target gene staphylococcal nuclease and tudor domain containing 1 on proliferation, migration, and invasion of human OS lines was examined. The Student's t test was used for statistical analysis.@*RESULTS@#We found that microRNA (miR)-296-5p was significantly downregulated in OS cell lines and tissues (control vs. OS, 1.802 ± 0.313 vs. 0.618 ± 0.235, t = 6.402, P < 0.01). Overexpression of miR-296-5p suppressed proliferation, migration, and invasion of OA cells. SND1 was identified as a target of miR-296-5p by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of SND1 abrogated the effects induced by miR-296-5p upregulation (miRNA-296-5p vs. miRNA-296-5p + SND1, 0.294 ± 0.159 vs. 2.300 ± 0.277, t = 12.68, P = 0.003).@*CONCLUSION@#Our study indicates that miR-296-5p may function as a tumor suppressor by targeting SND1 in OS.


Subject(s)
Humans , Bone Neoplasms/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Endonucleases/genetics , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , MicroRNAs/genetics , Osteosarcoma/genetics
3.
Chinese Journal of Biotechnology ; (12): 4342-4350, 2021.
Article in Chinese | WPRIM | ID: wpr-921510

ABSTRACT

The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type (V type) CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5'T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1 (AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3A-346 knockout vectors. The efficiency for editing the target genes BmATAD3A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.


Subject(s)
Animals , Bombyx/metabolism , CRISPR-Cas Systems/genetics , Endonucleases/genetics , Gene Editing , /genetics
4.
Clinics ; 73: e455, 2018. tab, graf
Article in English | LILACS | ID: biblio-974907

ABSTRACT

OBJECTIVES: To study the relationship between the Asp1104His polymorphism of the nucleotide excision repair gene ERCC5 and treatment sensitivity to oxaliplatin in patients with advanced colorectal cancer (CRC) in China. METHODS: A group of 226 patients in the Department of Gastrointestinal Oncology at Zhejiang Xiaoshan Hospital from July 2011∼December 2016 and a control group of 226 normal healthy individuals were involved in this study. All patients were first diagnosed with advanced CRC and were treated with oxaliplatin-based chemotherapy. The genotype of ERCC5 at the site of amino acid 1104 was determined by a TaqMan probe-based real-time PCR approach. RESULTS: There were no differences in age or gender between the groups, but the percentages of smokers and individuals with a family history of cancer were significantly higher in the patient group than in the control group. Analysis of the G/C polymorphism frequency among the patients and the healthy controls showed that the frequencies of the CC genotype and the CC+GC genotype were significantly related to CRC, but no significant difference in these frequencies was found between genders. The analysis of the relationship between the 5-year survival rate and different genotypes showed that in the total patient group, regardless of gender, the 5-year survival rate was significantly associated with the Asp1104His polymorphism of ERCC5. CONCLUSIONS: The Asp1104His polymorphism of ERCC5 was associated with the risk and 5-year survival rate of CRC as well as treatment sensitivity to oxaliplatin.


Subject(s)
Humans , Male , Female , Middle Aged , Nuclear Proteins/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/drug therapy , DNA-Binding Proteins/genetics , Endonucleases/genetics , Antineoplastic Agents/therapeutic use , Transcription Factors/genetics , Colorectal Neoplasms/mortality , Case-Control Studies , Polymorphism, Single Nucleotide/genetics , Kaplan-Meier Estimate , Oxaliplatin/therapeutic use , Genotype , Neoplasm Staging
5.
Electron. j. biotechnol ; 19(6): 32-37, Nov. 2016. ilus
Article in English | LILACS | ID: biblio-840310

ABSTRACT

Background: To identify the critical amino acid residues that contribute to the high enzyme activity and good thermostability of Yersinia enterocolitica subsp. palearctica (Y. NSN), 15 mutants of Y. NSN were obtained by site-directed mutagenesis in this study. And their enzyme activity and thermostability were assayed. Effect of several factors on the enzyme activity and thermostability of Y. NSN, was also investigated. Results: The results showed that the I203F and D264E mutants retained approximately 75% and 70% enzyme activity, respectively, compared to the wild-type enzyme. In addition to the I203F and D264E mutants, the mutant E202A had an obvious influence on the thermostability of Y. NSN. According to the analysis of enzyme activity and thermostability of Y. NSN, we found that Glu202, Ile203 and Asp264 might be the key residues for its high enzyme activity and good thermostability. Conclusions: Among all factors affecting enzyme activity and thermostability of Y. NSN, they failed to explain the experimental results well. One reason might be that the enzyme activity and thermostability of Y. NSN were affected not only by a single factor but also by the entire environment.


Subject(s)
Deoxyribonucleases/chemistry , Deoxyribonucleases/genetics , Yersinia enterocolitica/enzymology , Endonucleases/chemistry , Endonucleases/genetics , Enzyme Assays , Enzyme Stability , Hot Temperature , Mutagenesis, Site-Directed
6.
Journal of Forensic Medicine ; (6): 415-419, 2016.
Article in Chinese | WPRIM | ID: wpr-984869

ABSTRACT

OBJECTIVES@#To explore the expression of xeroderma pigmentosum complementation group G (XPG) gene in healthy Han population of different ages and to analysis the relationship between the mRNA and protein expression levels of XPG and age, which may provide a new molecular-biological indicator for forensic age determination.@*METHODS@#Total 150 samples of peripheral blood were collected from healthy Han population of different ages. Total RNA of peripheral blood mononuclear cell (PBMC) were extracted by TRIzol method, and the relative expression of XPG mRNA in PBMC was detected by quantitative real-time PCR, and the protein expression levels of XPG in plasma were detected by ELISA.@*RESULTS@#The mRNA and protein expression levels of XPG in ≤18 years old group were significantly different from 19-45 years old group and ≥46 years old group (P<0.05), while there was no significant difference between 19-45 years old group and ≥46 years old group (P>0.05). No significant sex differences were observed in mRNA and protein expression levels of XPG (P>0.05).@*CONCLUSIONS@#The relative expression level of XPG mRNA in PBMC declines with the increase of age in younger age, while the protein expression level in plasma increases with age, and XPG gene can be used as one of new markers for forensic age estimation.


Subject(s)
Adult , Humans , Middle Aged , Young Adult , Age Factors , Asian People , DNA-Binding Proteins/genetics , Endonucleases/genetics , Forensic Genetics , Leukocytes, Mononuclear , Nuclear Proteins/genetics , RNA, Messenger , Real-Time Polymerase Chain Reaction , Transcription Factors/genetics
7.
Einstein (Säo Paulo) ; 13(2): 249-254, Apr-Jun/2015. tab
Article in English | LILACS | ID: lil-751432

ABSTRACT

ABSTRACT Objective: To evaluate the change in respiratory function and functional capacity according to the type of preoperative fasting. Methods: Randomized prospective clinical trial, with 92 female patients undergoing cholecystectomy by laparotomy with conventional or 2 hours shortened fasting. The variables measured were the peak expiratory flow, forced expiratory volume in the first second, forced vital capacity, dominant handgrip strength, and non-dominant handgrip strength. Evaluations were performed 2 hours before induction of anesthesia and 24 hours after the operation. Results: The two groups were similar in preoperative evaluations regarding demographic and clinical characteristics, as well as for all variables. However, postoperatively the group with shortened fasting had higher values than the group with conventional fasting for lung function tests peak expiratory flow (128.7±62.5 versus 115.7±59.9; p=0.040), forced expiratory volume in the first second (1.5±0.6 versus 1.2±0.5; p=0.040), forced vital capacity (2.3±1.1 versus 1.8±0.9; p=0.021), and for muscle function tests dominant handgrip strength (24.9±6.8 versus 18.4±7.7; p=0.001) and non-dominant handgrip strength (22.9±6.3 versus 17.0±7.8; p=0.0002). In the intragroup evaluation, there was a decrease in preoperative compared with postoperative values, except for dominant handgrip strength (25.2±6.7 versus 24.9±6.8; p=0.692), in the shortened fasting group. Conclusion: Abbreviation of preoperative fasting time with ingestion of maltodextrin solution is beneficial to pulmonary function and preserves dominant handgrip strength. .


RESUMO Objetivo: Avaliar a alteração da função respiratória e da capacidade funcional, conforme o tipo de jejum pré-operatório. Métodos: Ensaio clínico prospectivo randomizado, com 92 pacientes do sexo feminino, submetidas à colecistectomia por laparotomia, observando jejum convencional ou abreviado de 2 horas com maltodextrina. As variáveis foram: pico de fluxo expiratório, volume expiratório no primeiro segundo, capacidade vital forçada, força de preensão palmar dominante e força de preensão palmar não dominante. As avaliações foram realizadas 2 horas antes da indução anestésica e 24 horas após a operação. Resultados: Os dois grupos foram semelhantes quanto às características demográficas, clínicas e em todas as variáveis estudadas, quando avaliadas no pré-operatório. No entanto, no pós-operatório, o grupo abreviado apresentou valores maiores que o grupo convencional para pico de fluxo expiratório (128,7±62,5 versus 115,7±59,9; p=0,040), volume expiratório no primeiro segundo (1,5±0,6 versus 1,2±0,5; p=0,040), capacidade vital forçada (2,3±1,1 versus 1,8±0,9; p=0,021), força de preensão palmar dominante (24,9±6,8 versus 18,4±7,7; p=0,001) e força de preensão palmar não dominante (22,9±6,3 versus 17,0±7,8; p=0,0002). Na avaliação intragrupo, houve diminuição nas variáveis ao se compararem os valores do pré-operatório em relação ao pós-operatório, exceto para força de preensão palmar dominante (25,2±6,7 versus 24,9±6,8; p=0,692) no grupo de jejum abreviado. Conclusão: A abreviação do tempo de jejum pré-operatório com solução contendo maltodextrina beneficia a função pulmonar e preserva a força de preensão palmar dominante. .


Subject(s)
Aged , Female , Humans , Male , Atrial Fibrillation/genetics , Body Height/genetics , Endonucleases/genetics , Genetic Predisposition to Disease , Polymorphism, Single Nucleotide , Black or African American/genetics , White People/genetics , Longitudinal Studies , Proportional Hazards Models , Risk Factors
8.
Indian J Biochem Biophys ; 2014 Dec ; 51 (6): 559-566
Article in English | IMSEAR | ID: sea-156537

ABSTRACT

In addition to well-known process of proteasome-mediated degradation of polyubiquitinated proteins, monoubiquitination of proteins is also an important post-translational modification that regulates various non-degradative cellular processes like protein trafficking, cellular signalling, DNA replication and DNA repair. We have previously characterized a multi-domain cycling sequence binding protein LdCSBP from Leishmania donovani, which binds specifically to a conserved CAUAGAAG octamer containing RNAs via its uniquely arranged CCCH type Zn-fingers and degrades them using its Smr endonuclease domain, indicative of its potential role in the turnover of the S-phase mRNAs. Remarkably, its riboendonuclease activity is inhibited due to the incorporation of a monoubiquitin residue in the ZnF domain, though the target Lys residue remains unknown. Here, we report through systematic mutation of Lys residue to Ala that Lys-413 in LdCSBP is the site of monoubiquitination. However, the amino acid motif around the target Lys in LdCSBP is not consensus with any previously known monoubiquitination site, though partial homology is observed with a subset of recently identified mammalian ubiquitination target sites. Interestingly, Lys-413 of LdCSBP is conserved in the homologous annotated proteins from the related kinetoplastida parasites, suggesting similar monoubiquitination-mediated regulation of RNA endonuclease activity in the organisms.


Subject(s)
Amino Acid Sequence , Binding Sites , Endonucleases/chemistry , Endonucleases/genetics , Endonucleases/metabolism , Leishmania donovani/cytology , Leishmania donovani/physiology , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Protozoan Proteins/metabolism , RNA-Binding Proteins , S Phase/physiology , Structure-Activity Relationship , Ubiquitination , Zinc Fingers
9.
Journal of Korean Medical Science ; : 352-359, 2011.
Article in English | WPRIM | ID: wpr-52140

ABSTRACT

We studied the expression of BRCA1, ERCC1, and RRM1 which play an important role in DNA repair systems in breast cancer. Immunohistochemical staining for EGFR, BRCA1, ERCC1, and RRM1 were performed by using a tissue microarray made from 230 breast cancer patients. Patients were classified into luminal A, luminal B, HER-2, and triple negative breast cancer (TNBC) types according to ER, PR, and HER-2 expression. The expression of ERCC1, RRM1, and BRCA1 were correlated (P < 0.05). The expression level of ERCC1 was the lowest in TNBC type (P = 0.031), ERCC1 negativity was more prominent in TNBC and luminal B groups than luminal A and HER-2 groups (P = 0.013). Cases with EGFR overexpression showed high expression of RRM1 and BRCA1 (P = 0.046, and 0.004, respectively). In conclusion, the expression of ERCC1 is particularly lower in TNBCs than other types of breast cancers.


Subject(s)
Adult , Female , Humans , Middle Aged , BRCA1 Protein/genetics , Breast Neoplasms/genetics , DNA Repair , DNA-Binding Proteins/genetics , Disease-Free Survival , Endonucleases/genetics , Gene Expression , Immunohistochemistry , Phenotype , Prognosis , Protein Array Analysis , ErbB Receptors/genetics , Biomarkers, Tumor/genetics , Tumor Suppressor Proteins/genetics
10.
Southeast Asian J Trop Med Public Health ; 2007 Nov; 38(6): 991-7
Article in English | IMSEAR | ID: sea-35480

ABSTRACT

Genomic DNA of Blastocystis isolates released into 0.1% Triton X-100 was suitable for amplification and yielded similar results as the genomic DNA extracted with standard kit. The specific B. hominis primers (BH1: GCT TAT CTG GTT GAT CCT GCC AGT and BH2: TGA TCC TTC CGC AGG TTC ACC TAC A) successfully produced the PCR product of about 1,770 bp with all the 7 Blastocystis isolates tested. The restriction fragment length polymorphism (RFLP) patterns yielded by 13 out of 25 restriction endonucleases showed that the 7 isolates could be grouped into 4 subgroups: subgroup-1 consisted of isolate C; subgroup-2 of isolates H4 and H7; subgroup-3 of isolates KP1, Y51 and M12; and subgroup-4 of isolate 27805. The differences between subgroups manifested as clear-cut RFLP patterns. A common band of 230 bp was revealed by Eco R1 in all the Blastocystis isolates tested. The band of about 180 bp was revealed by Alu I, differentiated symptomatic from asymptomatic isolates of this parasite, and might indicate the pathogenicity of this parasite.


Subject(s)
Animals , Blastocystis Infections/parasitology , Blastocystis hominis/genetics , DNA, Protozoan/analysis , Endonucleases/genetics , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length/genetics , Sequence Analysis, DNA
11.
Experimental & Molecular Medicine ; : 320-324, 2006.
Article in English | WPRIM | ID: wpr-51257

ABSTRACT

ERCC1 is a DNA repair gene and has been associated with resistance to DNA damaging agents. In this study we hypothesized that a polymorphism of ERCC1 Asn118Asn (C->T) might affect the platinum-resistance of epithelial ovarian cancer patients to platinum-taxane chemotherapy administered postoperatively. Using the SNapShot assay, we assessed this polymorphism in ERCC1 in 60 ovarian cancer patients. Platinum-resistance was defined as progression on platinum-based chemotherapy or recurrence within 6 months of completing therapy. Although not significant, platinum-resistance was less frequently observed in patients with the C/T+T/T genotype (P=0.064). Multivariate analysis showed that the C/T+T/T genotypes constituted an independent predictive factor of reduced risk of platinum-resistance in ovarian cancer (odds ratio 0.17, 95% confidence interval 0.04-0.74, P=0.018, Fisher's exact test). No significant correlation was observed between overall survival and the ERCC1 polymorphism. Our results suggest that genotyping of the ERCC1 polymorphism Asn118Asn may be useful for predicting the platinum-resistance of epithelial ovarian cancer patients. However, these findings require prospective confirmation.


Subject(s)
Middle Aged , Humans , Female , Aged , Adult , Adolescent , Survival Analysis , Polymorphism, Single Nucleotide/genetics , Ovarian Neoplasms/drug therapy , Multivariate Analysis , Linkage Disequilibrium , Genotype , Gene Frequency , Epithelial Cells/pathology , Endonucleases/genetics , Drug Resistance, Neoplasm/genetics , Disease Progression , DNA-Binding Proteins/genetics , DNA Repair , Codon/genetics , Cisplatin/therapeutic use , Antineoplastic Agents/therapeutic use
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