Insertion and Deletion Mutagenesis by Overlap Extension PCR

Lee, Jehan; Shin, Myeong Kyun; Ryu, Dong Kyun; Kim, Seahee; Ryu, Wang Shick

doi:10.1007/978-1-60761-652-8_10

Cited by 59 publications

(43 citation statements)

References 12 publications

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“…Once the sequence of the ORF in the destination vector was validated, large amounts of the plasmid were grown and purified from E. coli using an EndoFree Plasmid Maxi kit (Qiagen). The Isl1β ORF was generated using deletion PCR, to remove the 69 nucleotides from Isl1α that are not present in Isl1β (Lee et al, 2010). Once isolated, the Isl1β ORF was also cloned into the same destination vector described above, and large amounts were purified.…”

Section: Experimental Methodsmentioning

confidence: 99%

Alternative splicing of the LIM-homeodomain transcription factor Isl1 in the mouse retina

Whitney

Kautzman

Reese

2015

Molecular and Cellular Neuroscience

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Islet-1 (Isl1) is a LIM-homeodomain (LIM-HD) transcription factor that functions in a combinatorial manner with other LIM-HD proteins to direct the differentiation of distinct cell types within the central nervous system and many other tissues. A study of pancreatic cell lines showed that Isl1 is alternatively spliced generating a second isoform, Isl1β, which is missing 23 amino acids within the C-terminal region. This study examines the expression of the canonical and alternative Isl1 transcripts across other tissues, in particular, within the retina, where Isl1 is required for the differentiation of multiple neuronal cell types. The alternative splicing of Isl1 is shown to occur in multiple tissues, but the relative abundance of Isl1α and Isl1 β expression varies greatly across them. In most tissues, Isl1α is the more abundant transcript, but in others the transcripts are expressed equally, or the alternative splice variant is dominant. Within the retina, differential expression of the two Isl1 transcripts increases as a function of development, with dynamic changes in expression peaking at E16.5 and again at P10. At the cellular level, individual retinal ganglion cells vary in their expression, with a subset of small-to-medium sized cells expressing only the alternative isoform. The functional significance of the difference in protein sequence between the two Isl1 isoforms was also assessed using a luciferase assay, demonstrating that the alternative isoform forms a less effective transcriptional complex for activating gene expression. These results demonstrate the differential presence of the canonical and alternative isoforms of Isl1 amongst retinal ganglion cell classes. As Isl1 participates in the differentiation of Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Competing interestsThe authors have no competing interests for this manuscript. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript multiple cell types within the CNS, the present results support a role for alternative splicing in the establishment of cellular diversity in the developing nervous system.

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Section: Experimental Methodsmentioning

confidence: 99%

Alternative splicing of the LIM-homeodomain transcription factor Isl1 in the mouse retina

Whitney

Kautzman

Reese

2015

Molecular and Cellular Neuroscience

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“…P ced-1 piki-1::gfp was constructed by cloning the piki-1 cDNA into the multi-cloning sites of pZZ829, a plasmid carrying P ced-1 , a 3′ gfp tag, and the unc-54 3′ UTR [20]. The overlap extension PCR method [56] was used to delete the DNA sequence encoding the PI kinase domain from P ced-1 piki-1::gfp and generate P ced-1 piki-1(Δkinase)::gfp . To generate P ced-1 piki-1(K1059A)::gfp , the K1059A mutation was introduced into P ced-1 piki-1::gfp using the QuickChange Site-directed Mutagenesis Kit (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

Two PI 3-Kinases and One PI 3-Phosphatase Together Establish the Cyclic Waves of Phagosomal PtdIns(3)P Critical for the Degradation of Apoptotic Cells

et al. 2012

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“…The resulting plasmid was named pPAF (for phoA fusion). The DNA fragment coding for the PhoA-LacZ␣ fusion protein was obtained by overlap extension PCR of the set of fragments (the phoA fragment without the signal sequence and stop codon and the lacZ␣ fragment encoding amino acid residues 4 to 60 amplified from the chromosome of E. coli MG1655) (34). The phoA-lacZ␣ gene fragment was ligated into pPAF in place of the phoA gene fragment.…”

Section: Methodsmentioning

confidence: 99%

pqiABC and yebST , Putative mce Operons of Escherichia coli, Encode Transport Pathways and Contribute to Membrane Integrity

Nakayama

Zhang-Akiyama

2017

J Bacteriol

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The membranes of single-cell organisms are crucial as the first line of defense. The outer membrane of Gram-negative bacteria is an asymmetric bilayer in which lipopolysaccharides (LPSs) and phospholipids are localized in the outer and inner leaflet, respectively. This asymmetry is important for membrane integrity. In Escherichia coli, the Mla transport pathway maintains this asymmetry by removing phospholipids from the outer leaflet. The MlaD component of this system is a mammalian cell entry (MCE) domain protein, and E. coli has two other MCE domain proteins of unknown function (PqiB and YebT). Here, we show that these two proteins are components of novel transport pathways that contribute to membrane integrity. The pqiAB operon is regulated by SoxS and RpoS. The yebST operon contains pqiAB homologues. Here, we found a third member of the pqi operon, ymbA (pqiC). A PqiB-PqiC complex bridges the inner and the outer membrane, and in other bacteria, pqiBC genes are located in operons together with transporter proteins. We show here that simultaneous deletion of pqiABC and yebST operons in an Δmla background rendered cells more sensitive to SDS-EDTA, and the SDS-EDTA sensitivity of mla mutants was rescued by additional copies of pqiABC. We also found that the yebST operon was induced by a defect in LPS molecules. In conclusion, PqiABC and YebST are novel transport pathways related to the Mla transport pathway and important for membrane integrity.IMPORTANCE Membranes of bacteria are crucial for stress resistance. The composition of the E. coli outer membrane is asymmetric, with asymmetry maintained by the Mla ABC transport pathway. We propose that the stress-inducible pqiABC operon and homologous yebST operon, both of previously unknown function, encode transport pathway proteins related to the Mla transport pathway. Deletion of these operons rendered cells more sensitive to membrane stress, and additional copies of pqiABC suppressed the SDS-EDTA sensitivity of mla mutant strains. We found that yebS=-=lacZ fusion was activated in mutant strains with defective LPS molecules.KEYWORDS SoxS, membranes, oxidative stress, pqiABC, yebST E scherichia coli, a Gram-negative bacterium, has an inner membrane (IM) and an outer membrane (OM). Since these membranes separate essential cellular components from the environment, they are important for stress resistance (1, 2). The OM of E. coli is an asymmetric bilayer in which lipopolysaccharides (LPSs) and phospholipids (PLs) are localized in the outer and the inner leaflet, respectively (3). Lipophilic molecules easily penetrate a bilayer of PLs. Because of the low fluidity of lipid A (the lipid portion of LPSs), the asymmetric OM performs a barrier function against lipophilic molecules (3). Mutant strains with defective LPSs are also sensitive to hydrophilic molecules, presumably due to transient cracks in the OM (3). Therefore, disruption of

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Insertion and Deletion Mutagenesis by Overlap Extension PCR

Cited by 59 publications

References 12 publications

Alternative splicing of the LIM-homeodomain transcription factor Isl1 in the mouse retina

Alternative splicing of the LIM-homeodomain transcription factor Isl1 in the mouse retina

Two PI 3-Kinases and One PI 3-Phosphatase Together Establish the Cyclic Waves of Phagosomal PtdIns(3)P Critical for the Degradation of Apoptotic Cells

pqiABC and yebST , Putative mce Operons of Escherichia coli, Encode Transport Pathways and Contribute to Membrane Integrity

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