Splicing together different regions of a gene by modified polymerase chain reaction-based site-directed mutagenesis

Li, Xiangdong; Qiu, Yafeng; Shen, Yang; Ding, Chan; Liu, Peihong; Zhou, Jinping; Ma, Zhiyong

doi:10.1016/j.ab.2007.10.021

Cited by 30 publications

(28 citation statements)

References 7 publications

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“…The mutated expression construct of BR 120–395 (BR* 120–395 ) with R165S and R168S substitutions was generated following previously described protocols41. All coding sequences of the protein-expression constructs were confirmed by DNA sequencing and are listed in the supplemental information.…”

Section: Methodsmentioning

confidence: 99%

The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation

Schulte

Mikaelsson

Beaussart

et al. 2016

Sci Rep

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The major human pathogen Streptococcus pneumoniae is a leading cause of disease and death worldwide. Pneumococcal biofilm formation within the nasopharynx leads to long-term colonization and persistence within the host. We have previously demonstrated that the capsular surface-associated pneumococcal serine rich repeat protein (PsrP), key factor for biofilm formation, binds to keratin-10 (KRT10) through its microbial surface component recognizing adhesive matrix molecule (MSCRAMM)-related globular binding region domain (BR187–385). Here, we show that BR187–385 also binds to DNA, as demonstrated by electrophoretic mobility shift assays and size exclusion chromatography. Further, heterologous expression of BR187–378 or the longer BR120–378 construct on the surface of a Gram-positive model host bacterium resulted in the formation of cellular aggregates that was significantly enhanced in the presence of DNA. Crystal structure analyses revealed the formation of BR187–385 homo-dimers via an intermolecular β-sheet, resulting in a positively charged concave surface, shaped to accommodate the acidic helical DNA structure. Furthermore, small angle X-ray scattering and circular dichroism studies indicate that the aggregate-enhancing N-terminal region of BR120–166 adopts an extended, non-globular structure. Altogether, our results suggest that PsrP adheres to extracellular DNA in the biofilm matrix and thus promotes pneumococcal biofilm formation.

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Section: Methodsmentioning

confidence: 99%

The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation

Schulte

Mikaelsson

Beaussart

et al. 2016

Sci Rep

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“…The L-Meq variant was amplified by PCR from MSB-1 cells and subcloned into the expression vector p3xFLAG-CMV-7.1. The Meq deletion mutant (Meq-Δp53BD) that lacked the p53 binding domain (Figure 1A), and several variants of Meq including S-Meq, VS-Meq and ∆Meq (Figure 4A), were generated by PCR-based site-directed mutagenesis [30] using Flag-Meq as the template. The primers used are shown in Table 1.…”

Section: Methodsmentioning

confidence: 99%

The Meq oncoprotein of Marek's disease virus interacts with p53 and inhibits its transcriptional and apoptotic activities

Deng

Shen

et al. 2010

Virol J

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BackgroundMarek's disease virus (MDV) is an oncogenic herpesvirus, which causes malignant lymphoma in chickens. The Meq protein of MDV, which is expressed abundantly in MDV-infected cells and in Marek's disease (MD) tumor cells, functions as a transcriptional activator and has been proposed to play an important role in oncogenic transformation. Preliminary studies demonstrated that Meq is able to bind p53 in vitro, as demonstrated using a protein-binding assay. This observation prompted us to examine whether the interaction between Meq and p53 occurs in cells, and to investigate the biological significance of this interaction.ResultsWe confirmed first that Meq interacted directly with p53 using a yeast two-hybrid assay and an immunoprecipitation assay, and we investigated the biological significance of this interaction subsequently. Exogenous expression of Meq resulted in the inhibition of p53-mediated transcriptional activity and apoptosis, as analyzed using a p53 luciferase reporter assay and a TUNEL assay. The inhibitory effect of Meq on transcriptional activity mediated by p53 was dependent on the physical interaction between these two proteins, because a Meq deletion mutant that lacked the p53-binding region lost the ability to inhibit p53-mediated transcriptional activity and apoptosis. The Meq variants L-Meq and S-Meq, but not VS-Meq and ∆Meq, which were expressed in MD tumor cells and MDV-infected cells, exerted an inhibitory effect on p53 transcriptional activity. In addition, ∆Meq was found to act as a negative regulator of Meq.ConclusionsThe Meq oncoprotein interacts directly with p53 and inhibits p53-mediated transcriptional activity and apoptosis. These findings provide valuable insight into the molecular basis for the function of Meq in MDV oncogenesis.

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“…Site-directed mutagenesis (SDM) has a variety of applications and is extensively used in molecular biology. Over the last three decades, various SDM methods have been described (Nagy et al, 2004;Zheng et al, 2004;Seyfang and Jin, 2004;An et al, 2005;Wei et al, 2004;Jin et al, 2007;Heckman and Pease, 2007;Tseng et al, 2008;Li et al, 2008;Chapnik et al, 2008) and some commercial SDM kits based on these techniques are available. The SDM techniques can be grouped into two major categories: polymerase chain reaction (PCR)-based and non-PCR-based.…”

mentioning

confidence: 99%

An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

Zhang

et al. 2009

J. Zhejiang Univ. Sci. B

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Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonuclease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5′-phosphorylated, circularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step. Site-directed mutagenesis (SDM) has a variety of applications and is extensively used in molecular biology. Over the last three decades, various SDM methods have been described (Nagy et al., 2004;Zheng et al., 2004;Seyfang and Jin, 2004;An et al., 2005;Wei et al., 2004;Jin et al., 2007;Heckman and Pease, 2007;Tseng et al., 2008;Li et al., 2008;Chapnik et al., 2008) and some commercial SDM kits based on these techniques are available. The SDM techniques can be grouped into two major categories: polymerase chain reaction (PCR)-based and non-PCR-based. The PCR-based SDM methods are used more frequently than the non-PCR-based methods. Rabhi et al.(2004) have introduced an inverse PCR-based SDM method with forward and reverse primers to amplify the full-length plasmid. The blunt-ended amplification products are 5′-phosphorylated, self-ligated, and transformed into Escherichia coli. The design of this protocol is straightforward and the procedures are brief. However, hybridization has to be conducted for mutant screening because the difference between the original sequence and the target sequence is only one or a few base pairs, which makes the most common screening method, i.e., restriction digestion, not applicable. The laborious hybridization step with hazardous isotope deters researchers from adopting this simple mutagenesis method. Here we present a novel mutagenesis strategy, designed restriction endonuclease-assisted

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Splicing together different regions of a gene by modified polymerase chain reaction-based site-directed mutagenesis

Cited by 30 publications

References 7 publications

The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation

The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation

The Meq oncoprotein of Marek's disease virus interacts with p53 and inhibits its transcriptional and apoptotic activities

An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

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