The thermostable properties of the DNA polymerase activity from Thermus aquaticus (Taq) have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction method for amplifying DNA. We report the cloning and expression of Taq DNA polymerase in Escherichia coli. From a lambda gt11:Taq library we identified a Taq DNA fragment encoding an epitope of Taq DNA polymerase via antibody probing. The fusion protein from the lambda gt11:Taq candidate selected an antibody from an anti-Taq polymerase polyclonal antiserum which reacted with Taq polymerase on Western blots. We used the lambda gt11 clone to identify Taq polymerase clones from a lambda Ch35:Taq library. The complete Taq DNA polymerase gene has 2499 base pairs. From the predicted 832-amino acid sequence of the Taq DNA polymerase gene, Taq DNA polymerase has significant similarity to E. coli DNA polymerase I. We subcloned and expressed appropriate portions of the insert from a lambda Ch35 library candidate to yield thermostable, active, truncated, or full-length forms of the protein in E. coli under control of the lac promoter.
Download full-text PDF |
Source |
|---|
Publication Analysis
Top Keywords
Similar Publications
Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts.
J Vis Exp
January 2025
Institute of Biochemistry and Molecular Biology, Hengyang Medical School, University of South China; National Health Commission Key Laboratory of Birth Defect Research and Preventio, Hunan Provincial Maternal and Child Health Care Hospital;
Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation.
View Article and Find Full Text PDFEstablishment of a human ovarian endometrioid carcinoma cell line by constitutive expression of cyclin-dependent kinase 4, cyclin D1 and telomerase reverse transcriptase.
Hum Cell
January 2025
Department of Tumor Pathology, Faculty of Medical Sciences, University of Fukui, 23-3 Matsuoka-Shimoaizuki, Eiheiji, Fukui, 910-1193, Japan.
Only a few human ovarian endometrioid carcinoma cell lines are currently available, partly due to the difficulty of establishing cell lines from low-grade cancers. Here, using a cell immortalization strategy consisting of i) inactivation of the p16-pRb pathway by constitutive expression of mutant cyclin-dependent kinase 4 (R24C) (CDK4) and cyclin D1, and ii) acquisition of telomerase reverse transcriptase (TERT) activity, we established a human ovarian endometrioid carcinoma cell line from a 46-year-old Japanese woman. That line, designated JFE-21, has proliferated continuously for over 6 months with a doubling time of ~ 55 h.
View Article and Find Full Text PDFComprehensive evaluation of genomic and functional assays for homologous recombination deficiency with high-grade epithelial ovarian cancer: Platinum sensitivity and prognosis.
Int J Gynecol Cancer
January 2025
Fudan University Shanghai Cancer Center, Department of Gynecologic Oncology, Shanghai, China; Fudan University, Shanghai Medical College, Department of Oncology, Shanghai, China. Electronic address:
Objective: Homologous recombination deficiency assays, guiding treatment of poly (adenosine diphosphate ribose) polymerase inhibitors, are increasingly applied in clinics. This study aimed to evaluate the predictive performance of homologous recombination deficiency status at genomic and functional perspective on the efficacy of platinum-based chemotherapy in ovarian cancer.
Methods: Between 2016 and 2019, 134 patients with high-grade ovarian cancer were retrospectively analyzed.
Clinicopathologic stratification demonstrates survival differences between endometrial carcinomas with mismatch repair deficiency and no specific molecular profile: a cohort study.
Int J Gynecol Cancer
January 2025
Helsinki University Hospital and University of Helsinki, Department of Obstetrics and Gynecology, Helsinki, Finland; University of Helsinki, Faculty of Medicine, Helsinki University Hospital and Research Program in Applied Tumor Genomics, Department of Pathology, Helsinki, Finland.
Objective: Endometrial carcinomas with mismatch repair deficiency (MMRd) and no specific molecular profile (NSMP) are considered to have intermediate prognoses. However, potential prognostic differences between these molecular subgroups remain unclear due to the lack of standardized control for clinicopathologic factors. This study aims to evaluate outcomes of MMRd and NSMP endometrial carcinomas across guideline-based clinicopathologic risk groups.
View Article and Find Full Text PDFLiquid biopsy for diagnosing epithelial ovarian cancer: quantification of cell-free DNA and p53 mutational analysis.
Int J Gynecol Cancer
January 2025
All India Institute of Medical Sciences, Department of Obstetrics and Gynecology (Gynecologic Oncology), Rishikesh, Uttarakhand, India. Electronic address:
Objective: To isolate and quantify cell-free DNA, analysis for p53 mutations, and correlation with tumor burden in women with epithelial ovarian cancer compared with benign and borderline epithelial ovarian tumors.
Methods: In this case-control study, plasma samples of eligible women collected 1 hour before surgery and based on final histopathology, women with epithelial ovarian cancer recruited as cases and borderline, and benign ovarian tumors as controls. Cell-free DNA extracted from plasma serum and quantified using Nanodrop Spectrophotometer.
Want AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!