DNA Cloning and Engineering by Uracil Excision

Bitinaite, Jurate; Nichols, Nicole M.

doi:10.1002/0471142727.mb0321s86

Cited by 32 publications

(41 citation statements)

References 14 publications

(24 reference statements)

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“…After slow cooling to 30°C, the annealed oligonucleotides were ligated as a SpeI-KpnI fragment into the vector pGEM1 containing the Lep construct. Alternatively, uracil excision-based cloning was used (9). All inserts and mutagenesis were confirmed by sequencing of plasmid DNA at Eurofins MWG Operon (Ebersberg, Germany).…”

Section: Methodsmentioning

confidence: 99%

Efficient Glycosylphosphatidylinositol (GPI) Modification of Membrane Proteins Requires a C-terminal Anchoring Signal of Marginal Hydrophobicity

Galián

Björkholm

Bulleid

et al. 2012

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Efficient Glycosylphosphatidylinositol (GPI) Modification of Membrane Proteins Requires a C-terminal Anchoring Signal of Marginal Hydrophobicity

Galián

Björkholm

Bulleid

et al. 2012

Journal of Biological Chemistry

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“…All PCR amplifications were performed using the uracil read-through Pfu Turbo Cx Hotstart DNA polymerase (Agilent Technologies). The three purified fragments were fused into pX6U-GFP by USER cloning (Bitinaite and Nichols, 2009). All primers used are listed in Supplemental Table S8.…”

Section: Cloning Of the Ago1 Constructmentioning

confidence: 99%

mRNA Decay of Most Arabidopsis miRNA Targets Requires Slicer Activity of AGO1

2016

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“…The vector pFB008 was constructed using uracil excisionbased cloning (52). The pUG72 plasmid was amplified in two fragments using primer pairs Pr50/53 and Pr51/52, respectively.…”

Section: Methodsmentioning

confidence: 99%

A Plasma Membrane Association Module in Yeast Amino Acid Transporters

Popov‐Čeleketić¹,

Bianchi²,

Ruiz

et al. 2016

Journal of Biological Chemistry

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Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake of amino acids and involved in regulation of their cellular levels. Here, we report on a strong and complex module for PM association found in the C-terminal tail of AAPs. Using in silico analyses and mutational studies we found that the C-terminal sequences of Gap1, Bap2, Hip1, Tat1, Tat2, Mmp1, Sam3, Agp1, and Gnp1 are about 50 residues long, associate with the PM, and have features that discriminate them from the termini of organellar amino acid transporters. We show that this sequence (named PM asseq ) contains an amphipathic ␣-helix and the FWC signature, which is palmitoylated by palmitoyltransferase Pfa4. Variations of PM asseq , found in different AAPs, lead to different mobilities and localization patterns, whereas the disruption of the sequence has an adverse effect on cell viability. We propose that PM asseq modulates the function and localization of AAPs along the PM. PM asseq is one of the most complex protein signals for plasma membrane association across species and can be used as a delivery vehicle for the PM.Yeast transport amino acids across the plasma membrane (PM), 4 the vacuolar membrane (VM), and to a lesser extent the mitochondrial membrane (1). In the plasma and vacuolar membranes, there are 22 and 11 secondary amino acid transporters, respectively. These transporters are polytopic membrane proteins with 10 -14 transmembrane segments. They belong to three superfamilies: amino acid/polyamine/organocation (APC), major facilitator superfamily (MFS), and amino acid/ polyamine transporter II (AAPTII) (2). The amino acid transporters belonging to the APC superfamily are often referred to as amino acid permeases (AAPs). They are mainly localized in the PM and can be highly specific, e.g. transport only one (enantiomer) amino acid or have a broad range of substrates, like the general amino acid permease Gap1.Alongside the transporter function, additional roles have been proposed for some AAPs. The most prominent example is Gap1, which has a receptor function whereby it signals the protein kinase A (PKA) pathway (3). This so-called transceptor function has also been described for the phosphate transporter Pho84 and the ammonium transporter Mep2 but not for any other AAPs (4, 5). On the other end of the spectrum is Ssy1, an endoplasmic reticulum (ER)-resident AAP member, that plays a role in amino acid sensing but has no transport function (6 -8). The levels of AAPs at the PM are modulated by several transcriptional and post-translational control mechanisms, e.g. nitrogen catabolite repression, general amino acid control, and substrate/stress-induced endocytosis (9 -12). Although for several AAPs many details of these control mechanisms have been elucidated, quite a few questions concerning the spatiotemporal regulation of AAPs remain unanswered. Which mechanism ensures that the function is performed in the right organelle and what determines the positioning of transporters within diff...

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DNA Cloning and Engineering by Uracil Excision

Cited by 32 publications

References 14 publications

Efficient Glycosylphosphatidylinositol (GPI) Modification of Membrane Proteins Requires a C-terminal Anchoring Signal of Marginal Hydrophobicity

Efficient Glycosylphosphatidylinositol (GPI) Modification of Membrane Proteins Requires a C-terminal Anchoring Signal of Marginal Hydrophobicity

mRNA Decay of Most Arabidopsis miRNA Targets Requires Slicer Activity of AGO1

A Plasma Membrane Association Module in Yeast Amino Acid Transporters

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