Gene Splicing by Overlap Extension

Horton, Robert M.; Ho, Steffan N.; Pullen, Jeffrey K.; Hunt, Henry D.; Cai, Zeling; Pease, Larry R.

doi:10.1016/b978-0-12-765561-1.50029-1

Cited by 183 publications

(216 citation statements)

References 10 publications

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“…Various site-directed aliA mutants were generated by overlap extension PCR (Horton et al, 1993). The full-length mutated versions of aliA were cloned into the pET-16b expression vector.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

Use of the Rhodopseudomonas palustris genome sequence to identify a single amino acid that contributes to the activity of a coenzyme A ligase with chlorinated substrates

Samanta¹,

Harwood²

2005

Molecular Microbiology

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SummaryRhodopseudomonas palustris strain RCB100 degrades 3-chlorobenzoate (3-CBA) anaerobically. We purified from this strain a coenzyme A ligase that is active with 3-CBA and determined its N-terminal amino acid sequence to be identical to that of a cyclohexanecarboxylate-CoA ligase encoded by aliA from the R. palustris strain (CGA009) that has been sequenced. Strain CGA009 differs from strain RCB100 in that it does not use 3-CBA as a sole carbon source.

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“…Various site-directed aliA mutants were generated by overlap extension PCR (Horton et al, 1993). The full-length mutated versions of aliA were cloned into the pET-16b expression vector.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

Use of the Rhodopseudomonas palustris genome sequence to identify a single amino acid that contributes to the activity of a coenzyme A ligase with chlorinated substrates

Samanta¹,

Harwood²

2005

Molecular Microbiology

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“…The 3' primer (AII) was 5'-CGCGCTCTAGA TTA CTT GAG GAG AAA GAG C-3', the reverse complement of the region encoding the C-terminal residues of A1 (LFLLK) followed by an XbaI restriction site (underlined). A1 G87E was generated from the wt cDNA by PCR and splice-overlapextension, 54 in which two overlapping PCR products carrying the point mutation are joined together by another round of PCR. The first PCR product (A) was generated using the 5' primer AI described above and a 3' primer (AIII) which corresponds to residues 83 to 93 (G I I N W E R I V T I F), with the point mutation in bold.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Survival activity of Bcl-2 homologs Bcl-w and A1 only partially correlates with their ability to bind pro-apoptotic family members

et al. 1999

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Certain Bcl-2 family members promote cell survival, whereas others promote apoptosis. To explore further how heterodimerization of opposing members affects survival activity, we have compared the abilities of the anti-apoptotic Bcl-w and A1 to bind to the pro-apoptotic Bax, Bak, Bad and Bik and to protect cells from their cytotoxic action. Bcl-w co-immunoprecipitated from cell lysates with Bax, Bak, Bad and Bik, but A1 bound only Bak and Bik. Mutation of A1 at a highly conserved glycine within the BH1 domain prevented binding, but the comparable Bcl-w mutant still bound Bak, Bad and Bik, indicating that the glycine is not essential for all heterodimerization. Bcl-w and A1 protected against apoptosis induced by over-expression of Bax or Bad but not that induced by Bak or Bik. With several gene pairs, binding and protection were discordant. The results may reflect critical threshold affinities but also suggest that certain pro-apoptotic proteins may also contribute to apoptosis by a mechanism independent of binding pro-survival proteins.

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“…This vector contains neomycin resistance gene allowing stably transfected cells to be selected using G-418 (Mediatech, Herndon, VA). Chimeric molecules containing the extracellular domains of either mGluR2 or mGluR3 and the transmembrane domains and C-terminus of mGluR1a were prepared using the PCR-based overlap extension method (Horton et al, 1993). The primers, which shared complementary sequence on the strands to be joined were designed as follows: 5′-GTACATCCGCTGGGGTGAT-ATAGAATCTATCATAGCC-3′ (primer A, mGluR2-mGlur1a), 5′-GGCTATGATAGATTCTATATCACCCCAGCGGATGTAC-3′ (primer B, mGluR1a-mGluR2), and 5′-TTACATCAAATGGGAAGAC-ATAGAATCTATCATAGCC-3′ (primer C, mGluR3-mGluR1a), and 5′-GGCTATGATAGATTCTATGTCTTCCCATTT GATGTAA-3′ (primer D, mGluR1a-mGluR3).…”

Section: Preparation Of Transfection Vectorsmentioning

confidence: 99%

Cyclothiazide selectively inhibits mGluR1 receptors interacting with a common allosteric site for non-competitive antagonists

et al. 2007

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Metabotropic glutamate receptors mGluR1 and mGluR5 stimulate phospholipase C, leading to an increased inositol triphosphate level and to Ca 2+ release from intracellular stores. Cyclothiazide (CTZ), known as a blocker of AMPA receptor desensitization, produced a non-competitive inhibition of [Ca 2+ ] i increases induced by mGluR agonists in HEK 293 cells transfected with rat mGluR1a but had no effect on the [Ca 2+ ] i signals in cells expressing rat mGluR5a. In cells expressing mGluR1, CTZ also inhibited phoshoinositide hydrolysis, as well as cAMP accumulation and arachidonic acid release induced by mGluR1 agonists, indicating a direct inhibition of the receptor and not of a particular signal transduction system. However, CTZ failed to antagonize cAMP inhibition stimulated by rat mGluR2, -3, -4, -6, -7 and -8 receptors confirming its selectivity for mGluR1. The use of chimeric receptors with substituted N-terminal domains showed that CTZ did not interact with the N-terminal mGluR1a domain. Instead, mutation analysis revealed that CTZ interacts with the Thr-815 and Ala-818 residues, located at the 7 th transmembrane domain, similarly as the mGluR1-selective antagonist CPCCOEt. In primary cultures of cerebellar granule neurons, expressing native metabotropic and ionotropic glutamate receptors, the final outcome of CTZ effects depended on its combined ability to potentiate AMPA receptors and inhibit mGluR1a receptors.

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Gene Splicing by Overlap Extension

Cited by 183 publications

References 10 publications

Use of the Rhodopseudomonas palustris genome sequence to identify a single amino acid that contributes to the activity of a coenzyme A ligase with chlorinated substrates

Use of the Rhodopseudomonas palustris genome sequence to identify a single amino acid that contributes to the activity of a coenzyme A ligase with chlorinated substrates

Survival activity of Bcl-2 homologs Bcl-w and A1 only partially correlates with their ability to bind pro-apoptotic family members

Cyclothiazide selectively inhibits mGluR1 receptors interacting with a common allosteric site for non-competitive antagonists

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