Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

Hussain, Hasnain; Chong, Nikson Fatt-Ming

doi:10.1155/2016/8041532

Cited by 33 publications

(23 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The pGL3-basic firefly (Photinus pyralis) luciferase plasmid was co-transfected as a normalization control. The MDM2 mutant promoter constructs were synthesized using the combined overlap extension PCR method [20]. All assays were performed 24 h after transfection using the dual luciferase assay (Promega), according to the manufacturer's protocol.…”

Section: Promoter-luciferase Assaymentioning

confidence: 99%

Early Growth Response 1-Dependent Downregulation of Matrix Metalloproteinase 9 and Mouse Double Minute 2 Attenuates Head and Neck Squamous Cell Carcinoma Metastasis

Kim

Kang

et al. 2018

Cell Physiol Biochem

View full text Add to dashboard Cite

Background/Aims: The functional relevance of early growth response-1 (EGR1) on cancer invasion remains controversial. The effect of EGR1 on the expression of MMP9, which is important for HNSCC invasion, is still disputed. There is no previous data showing the effect of EGR1 on mouse double minute 2 (MDM2), an enhancer of matrix metalloproteinase 9 (MMP9) expression. Our aim is to clarify the negative correlation between EGR1 expression and head and neck squamous cell carcinoma (HNSCC) metastasis. Methods: EGR1 mRNA and protein expressions were compared in normal and HNSCC tissues using The Cancer Genome Atlas (TCGA) dataset analysis or immunohistochemistry (IHC), respectively. In vitro cell invasion was evaluated Matrigel invasion assay. EGR1-dependent inhibition of MDM2 transcription was assessed by promoter–luciferase assay and chromatin immunoprecipitation (ChIP). Results: TCGA data showed that EGR1 mRNA levels are significantly higher in normal oral tissues as compared with HNSCC tumor tissues (adjusted P = 1.64x10^-16). In addition, nonmetastatic HNSCC tissues showed significantly higher EGR1 mRNA levels as compared with metastatic tissues (adjusted P = 0.023). IHC analysis showed that primary tumor tissues expressed significantly higher levels of nuclear EGR1 compared with paired metastatic lymph node tissues (P < 0.05). EGR1 overexpression downregulated MMP9 and MDM2 protein expression. Consistent with these observations, TCGA data analysis found significantly fewer metastatic patients among a subgroup of population presenting higher EGR1 expressions with lower MMP9 and/or MDM2. Conclusion: Our data suggests that EGR1 prevents HNSCC metastasis through downregulation of MMP9 and MDM2. EGR1 might be a potential candidate to attenuate HNSCC metastasis.

show abstract

Section: Promoter-luciferase Assaymentioning

confidence: 99%

Early Growth Response 1-Dependent Downregulation of Matrix Metalloproteinase 9 and Mouse Double Minute 2 Attenuates Head and Neck Squamous Cell Carcinoma Metastasis

Kim

Kang

et al. 2018

Cell Physiol Biochem

View full text Add to dashboard Cite

show abstract

“…The BRCA2 c.79A>G (p.Ile27Val), c.91T>C (p.Trp31Arg), c.91T>G (p.Trp31Gly), c.93G>T (p.Trp31Cys) and the PALB2 c.2816T>G (p.Leu939Trp), c.3073G>A (p.Ala1025Arg), c.3266C>T (p.Pro1088Ser) variants were obtained by direct mutagenesis of pET11a-NfrGFP-BRCA2 and of pMRBAD-PALB2-CfrGFP using the QuickChange XL Site-directed Mutagenesis Kit (Stratagene) according to the manufacturer's instruction. The PALB2 c.3064AT>GC (p.Met1022Ala) was obtained by the overlap extension PCR mutagenesis method ( 25 ). The presence of variants in recombinant clones was verified by DNA sequencing (Eurofins Genomics).…”

Section: Methodsmentioning

confidence: 99%

Two Missense Variants Detected in Breast Cancer Probands Preventing BRCA2-PALB2 Protein Interaction

et al. 2018

View full text Add to dashboard Cite

PALB2 (partner and localizer of BRCA2) was initially identified as a binding partner of BRCA2. It interacts also with BRCA1 forming a complex promoting DNA repair by homologous recombination. Germline pathogenic variants in BRCA1, BRCA2 and PALB2 DNA repair genes are associated with high risk of developing breast cancer. Mutation screening in these breast cancer predisposition genes is routinely performed and allows the identification of individuals who carry pathogenic variants and are at risk of developing the disease. However, variants of uncertain significance (VUSs) are often detected and establishing their pathogenicity and clinical relevance remains a central challenge for the risk assessment of the carriers and the clinical decision-making process. Many of these VUSs are missense variants leading to single amino acid substitutions, whose impact on protein function is uncertain. Typically, VUSs are rare and due to the limited genetic, clinical, and pathological data the multifactorial approaches used for classification cannot be applied. Thus, these variants can only be characterized through functional analyses comparing their effect with that of normal and mutant gene products used as positive and negative controls. The two missense variants BRCA2:c.91T >G (p.Trp31Gly) and PALB2:c.3262C >T (p.Pro1088Ser) were detected in two breast cancer probands originally ascertained at Breast Cancer Units of Institutes located in Milan and Bergamo (Northern Italy), respectively. These variants were located in the BRCA2-PALB2 interacting domains, were predicted to be deleterious by in silico analyses, and were very rare and clinically not classified. Therefore, we initiate to study their functional effect by exploiting a green fluorescent protein (GFP)-reassembly in vitro assay specifically designed to test the BRCA2-PALB2 interaction. This functional assay proved to be easy to develop, robust and reliable. It also allows testing variants located in different genes. Results from these functional analyses showed that the BRCA2:p.Trp31Gly and the PALB2:p.Pro1088Ser prevented the BRCA2-PALB2 binding. While caution is warranted when the interpretation of the clinical significance of rare VUSs is based on functional studies only, our data provide initial evidences in favor of the possibility that these variants are pathogenic.

show abstract

“…For bacterial two-hybrid experiments, the complete ORFs of B. cereus flhF (GenBank ID: bc1670), hblC (GenBank ID: bc3104), bc1657, and the NG encoding fragment of flhF (flhF NG ) were directly amplified from genomic DNA of B. cereus ATCC 14579 using primer pairs flhFF1/flhFR1, bc1657F1/bc1657R1, L 2 F2/L 2 R1, NGF 1 /flhFR1, respectively. To produce flhF T253Q and flhF D391A mutant variants, site-directed mutagenesis by combined overlap extension PCR (COE-PCR) was performed (Hussain and Chong, 2016). For each variant, two primers pairs (BcflhF-T253Q-F/flhFR1 and flhFF1/BcflhF-T253Q-R, and BcflhF-D391A-F/flhFR1 and flhFF1/BcflhF-D391A-R, respectively), were used to generate two flhF mutated fragments with overlapping ends.…”

Section: Plasmids Constructionmentioning

confidence: 99%

GTP-Dependent FlhF Homodimer Supports Secretion of a Hemolysin in Bacillus cereus

et al. 2020

View full text Add to dashboard Cite

The multidomain (B-NG) protein FlhF, a flagellar biogenesis regulator in several bacteria, is the third paralog of the signal recognition particle (SRP)-GTPases Ffh and FtsY, which are known to drive protein-delivery to the plasma membrane. Previously, we showed that FlhF is required for Bacillus cereus pathogenicity in an insect model of infection, being essential for physiological peritrichous flagellation, for motility, and for the secretion of virulence proteins. Among these proteins, we found that the L 2 component of hemolysin BL, one of the most powerful toxins B. cereus produces, was drastically reduced by the FlhF depletion. Herein, we demonstrate that B. cereus FlhF forms GTP-dependent homodimers in vivo since the replacement of residues critical for their GTP-dependent homodimerization alters this ability. The protein directly or indirectly controls flagellation by affecting flagellin-gene transcription and its overproduction leads to a hyperflagellated phenotype. On the other hand, FlhF does not affect the expression of the L 2-encoding gene (hblC), but physically binds L 2 when in its homodimeric form, recruiting the protein to the plasma membrane for secretion. We additionally show that FlhF overproduction increases L 2 secretion and that the FlhF/L 2 interaction requires the NG domain of FlhF. Our findings demonstrate the peculiar behavior of B. cereus FlhF, which is required for the correct flagellar pattern and acts as SRP-GTPase in the secretion of a bacterial toxin subunit.

show abstract

Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

Cited by 33 publications

References 14 publications

Early Growth Response 1-Dependent Downregulation of Matrix Metalloproteinase 9 and Mouse Double Minute 2 Attenuates Head and Neck Squamous Cell Carcinoma Metastasis

Early Growth Response 1-Dependent Downregulation of Matrix Metalloproteinase 9 and Mouse Double Minute 2 Attenuates Head and Neck Squamous Cell Carcinoma Metastasis

Two Missense Variants Detected in Breast Cancer Probands Preventing BRCA2-PALB2 Protein Interaction

GTP-Dependent FlhF Homodimer Supports Secretion of a Hemolysin in Bacillus cereus

Contact Info

Product

Resources

About