In vitro synthesis of novel genes: mutagenesis and recombination by PCR.

Vallejo, Abbe N.; Pogulis, Robert J.; Pease, Larry R.

doi:10.1101/gr.4.3.s123

Cited by 68 publications

(46 citation statements)

References 17 publications

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“…pSK mbfA was used as a template, and the PCR product was cloned to pSK to make pSK mbfA-HA and then subsequently cloned into pRK290X. SOEing PCR (17) was used to tailor the His 6 tag sequence, in-frame after the fifth amino acid of mbfA ORF. The final product was amplified using a reverse primer having a HA tag sequence in-frame with the mbfA ORF.…”

Section: Methodsmentioning

confidence: 99%

A Bacterial Iron Exporter for Maintenance of Iron Homeostasis

Sankari

O’Brian

2014

Journal of Biological Chemistry

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Background: Iron export has not been previously demonstrated in a bacterium. Results: A bacterial iron exporter was identified and shown to be important for iron-dependent processes. The N-terminal cytoplasmic domain is essential for function. Conclusion: Iron export activity participates in the maintenance of bacterial homeostasis. Significance: Iron export is likely to be a common homeostatic mechanism in prokaryotes.

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Section: Methodsmentioning

confidence: 99%

A Bacterial Iron Exporter for Maintenance of Iron Homeostasis

Sankari

O’Brian

2014

Journal of Biological Chemistry

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“…In order to make a DkatA mutant, oligonucleotide primer pairs KAT1/KATR1 and KATR2/KAT2 (Table 1) were used to PCR-amplify two 0.5 kb DNA fragments that flanked the 59 and 39 ends of katA, respectively, from chromosomal DNA of S. aureus 2386. Primers KATR1 and KATR2 carried complementary sequences (underlined in Table 1) that were used to fuse the two fragments by recombinant PCR (Vallejo et al, 1994). The resulting chimeric PCR product was inserted into the pCR2.1 vector, excised using SpeI and XbaI, and cloned into the XbaI site of the pE194ts thermosensitive vector to generate pERkat (Table 1).…”

Section: Methodsmentioning

confidence: 99%

Simultaneous lack of catalase and beta-toxin in Staphylococcus aureus leads to increased intracellular survival in macrophages and epithelial cells and to attenuated virulence in murine and ovine models

et al. 2009

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Staphylococcus aureus produces a variety of virulence factors that allow it to cause a wide range of infections in humans and animals. In the latter, S. aureus is a leading cause of intramammary infections. The contribution of catalase (KatA), an enzyme implicated in oxidative stress resistance, and beta-toxin (Hlb), a haemolysin, to the pathogenesis of S. aureus is poorly characterized. To investigate the role of these enzymes as potential virulence factors in S. aureus, we examined the intracellular survival of DkatA, Dhlb and DkatA Dhlb mutants in murine macrophages (J774A.1) and bovine mammary epithelial cells (MAC-T), and their virulence in different murine and ovine models. Catalase was not required for the survival of S. aureus within either J774A.1 or MAC-T cells. However, it was necessary for the intracellular proliferation of the bacterium after invasion of MAC-T cells. In addition, catalase was not needed for the full virulence of S. aureus in mice. Deletion of the hlb gene had no effect on the intracellular survival of S. aureus in J774A.1 cells but did cause a slight increase in survival in MAC-T cells. Furthermore, like catalase, beta-toxin was not required for complete virulence of S. aureus in murine models. Unexpectedly, the DkatA Dhlb mutant showed a notably increased persistence in both cell lines, and was significantly less virulent for mice than were the wild-type strain and single mutants. Most interestingly, it was also markedly attenuated in intramammary and subcutaneous infections in ewes and lambs.

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“…The ⌬518 deletion mutant has a stop codon inserted at amino acid 519 and a new MseI site (5Ј-TTATTCAGAAGTTTAATAGAAGTGAGCAT-3Ј). Point mutations were also introduced in the C-terminal region of the Bgp1 cytoplasmic domain using an overlap PCR technique (42). Two PCR fragments were generated for each mutant using a combination of common and specific oligonucleotides.…”

Section: Methodsmentioning

confidence: 99%

The Carboxyl-terminal Region of Biliary Glycoprotein Controls Its Tyrosine Phosphorylation and Association with Protein-tyrosine Phosphatases SHP-1 and SHP-2 in Epithelial Cells

Huber

Izzi

Grondin

et al. 1999

Journal of Biological Chemistry

156

155

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Biliary glycoprotein (Bgp, C-CAM, or CD66a) is an immunoglobulin-like cell adhesion molecule and functions as a tumor suppressor protein. We have previously shown that the Bgp1 isoform responsible for inhibition of colonic, liver, prostate, and breast tumor cell growth contains within its cytoplasmic domain two tyrosine residues positioned in immunoreceptor tyrosine-based inhibition motif (ITIM) consensus sequences. Moreover, we determined that these residues, upon phosphorylation, associate with the protein-tyrosine phosphatase SHP-1. In this report, we have further evaluated the structural bases of the association of Bgp1 with Tyr phosphatases. First, we demonstrate that Bgp1 also associates with the SHP-2 Tyr phosphatase, but not with an unrelated Tyr phosphatase, PTP-PEST. Association of Bgp1 and SHP-2 involves the Tyr residues within the Bgp1 ITIM sequences, Val at position ؉3 relative to the second Tyr (Tyr-515), and the SHP-2 N-terminal SH2 domain. In addition, our results indicate that residues ؉4, ؉5, and ؉6 relative to Tyr-515 in the Bgp1 cytoplasmic domain play a significant role in these interactions, as their deletion reduced Bgp1 Tyr phosphorylation and association with SHP-1 and SHP-2 by as much as 80%. Together, these results indicate that both SHP-1 and SHP-2 interact with the Bgp1 cytoplasmic domain via ITIM-like sequences. Furthermore, they reveal that the C-terminal amino acids of Bgp1 are critical for these interactions.

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In vitro synthesis of novel genes: mutagenesis and recombination by PCR.

Cited by 68 publications

References 17 publications

A Bacterial Iron Exporter for Maintenance of Iron Homeostasis

A Bacterial Iron Exporter for Maintenance of Iron Homeostasis

Simultaneous lack of catalase and beta-toxin in Staphylococcus aureus leads to increased intracellular survival in macrophages and epithelial cells and to attenuated virulence in murine and ovine models

The Carboxyl-terminal Region of Biliary Glycoprotein Controls Its Tyrosine Phosphorylation and Association with Protein-tyrosine Phosphatases SHP-1 and SHP-2 in Epithelial Cells

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