Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol protocol

Agatep, Ronald; Kirkpatrick, Robert; Parchaliuk, Debra; Woods, Robin A.; Gietz, R. Daniel

doi:10.1016/s1366-2120(08)70121-1

Cited by 131 publications

(100 citation statements)

References 13 publications

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“…PCB was obtained by methanolysis of soluble proteins from Spirulina as described (48). Yeast was transformed as described (49,50). an aliquot of the supernatant and mixed for 40 min at 4°C in R. Subsequently, protein G-coupled magnetic beads (NEB) were added and mixed further.…”

Section: Methodsmentioning

confidence: 99%

A phytochrome–phototropin light signaling complex at the plasma membrane

Jaedicke

Lichtenthäler

Meyberg

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

102

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Phytochromes are red/far-red photochromic photoreceptors central to regulating plant development. Although they are known to enter the nucleus upon light activation and, once there, regulate transcription, this is not the complete picture. Various phytochrome effects are manifested much too rapidly to derive from changes in gene expression, whereas others seem to occur without phytochrome entering the nucleus. Phytochromes also guide directional responses to light, excluding a genetic signaling route and implying instead plasma membrane association and a direct cytoplasmic signal. However, to date, no such association has been demonstrated. Here we report that a phytochrome subpopulation indeed associates physically with another photoreceptor, phototropin, at the plasma membrane. Yeast two-hybrid methods using functional photoreceptor molecules showed that the phytochrome steering growth direction in Physcomitrella protonemata binds several phototropins specifically in the photoactivated Pfr state. Split-YFP studies in planta showed that the interaction occurs exclusively at the plasma membrane. Coimmunoprecipitation experiments provided independent confirmation of in vivo phy-phot binding. Consistent with this interaction being associated with a cellular signal, we found that phytochrome-mediated tropic responses are impaired in Physcomitrella phot − mutants. Split-YFP revealed a similar interaction between Arabidopsis phytochrome A and phototropin 1 at the plasma membrane. These associations additionally provide a functional explanation for the evolution of neochrome photoreceptors. Our results imply that the elusive phytochrome cytoplasmic signal arises through binding and coaction with phototropin at the plasma membrane.

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Section: Methodsmentioning

confidence: 99%

A phytochrome–phototropin light signaling complex at the plasma membrane

Jaedicke

Lichtenthäler

Meyberg

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

102

View full text Add to dashboard Cite

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“…Sequence-verified yeast two-hybrid Gateway destination vectors were a gift from the Finley laboratory (Wayne State University; Serebriiskii et al, 2001). The reporter plasmid pSH18-34 was first transformed into yeast stain EGY48 using the TRAFO Protocol (Agatep et al, 1998), followed sequentially by the bait and prey vectors. Transformed yeast were selected and maintained on dropout medium (2His/uracil/Trp; BioShop Canada).…”

Section: Ethylene Production Measurementsmentioning

confidence: 99%

Arabidopsis RING E3 Ligase XBAT32 Regulates Lateral Root Production through Its Role in Ethylene Biosynthesis

et al. 2010

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XBAT32, a member of the RING domain-containing ankyrin repeat subfamily of E3 ligases, was previously identified as a positive regulator of lateral root development. Arabidopsis (Arabidopsis thaliana) plants harboring a mutation in XBAT32 produce fewer lateral roots that wild-type plants. We found that xbat32 mutants produce significantly more ethylene than wild-type plants and that inhibition of ethylene biosynthesis or perception significantly increased xbat32 lateral root production. XBAT32 interacts with the ethylene biosynthesis enzymes AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE4 (ACS4) and ACS7 in yeast-two-hybrid assays. XBAT32 is capable of catalyzing the attachment of ubiquitin to both ACS4 and ACS7 in in vitro ubiquitination assays. These results suggest that XBAT32 negatively regulates ethylene biosynthesis by modulating the abundance of ACS proteins. Loss of XBAT32 may promote the stabilization of ACSs and lead to increased ethylene synthesis and suppression of lateral root formation. XBAT32 may also contribute to the broader hormonal cross talk that influences lateral root development. While auxin treatments only partially rescue the lateral root defect of xbat32, they completely restore wild-type levels of xbat32 lateral root production when coupled with ethylene inhibition. Abscisic acid, an antagonist of ethylene synthesis/signaling, was also found to stimulate rather than inhibit xbat32 lateral root formation, and abscisic acid acts synergistically with auxin to promote xbat32 lateral root production.

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“…All mutant derivatives of the Mtccs52A were cloned in pREP1 and verified by sequencing. These pREP1 plasmids as well as the empty vector were transformed into S. pombe SP-Q01 (Stratagene) according to Agatep et al (1998) and plated on Edinburgh minimal medium containing 50 mM thiamine. Single colonies from each transformation were grown in liquid medium in the presence of thiamine overnight, and then gene expression was induced by culturing the cells for 8 h in EMM medium without thiamine.…”

Section: Dna Constructs and Genetic Manipulationsmentioning

confidence: 99%

Two Classes of the Cdh1-Type Activators of the Anaphase-Promoting Complex in Plants: Novel Functional Domains and Distinct Regulation[W]

et al. 2004

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The Cdc20 and Cdh1/Fzr proteins are the substrate-specific activators of the anaphase-promoting complex (APC). In Medicago truncatula, the MtCcs52A and MtCcs52B proteins represent two subgroups of the Cdh1-type activators, which display differences in their cell cycle regulation, structure, and function. The ccs52A transcripts are present in all phases of the cell cycle. By contrast, expression of ccs52B is restricted to late G2-phase and M-phase, and its induced overexpression in BY2 cells inhibited mitosis. MtCcs52A is active in Schizosaccharomyces pombe and binds to the S. pombe APC, whereas MtCcs52B does not because of differences in the N-terminal region. We identified a new functional domain, the Cdh1-specific motif conserved in the Cdh1 proteins that, in addition to the C-box and the terminal Ile and Arg residues, was essential for the activity and required for efficient binding to the APC. Moreover, we demonstrate that cyclin-dependent kinase phosphorylation sites adjacent to the C-box may regulate the interaction with the APC. In the different plant organs, the expression of Mtccs52A and Mtccs52B displayed differences and indicated the involvement of the APC in differentiation processes.

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Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol protocol

Cited by 131 publications

References 13 publications

A phytochrome–phototropin light signaling complex at the plasma membrane

A phytochrome–phototropin light signaling complex at the plasma membrane

Arabidopsis RING E3 Ligase XBAT32 Regulates Lateral Root Production through Its Role in Ethylene Biosynthesis

Two Classes of the Cdh1-Type Activators of the Anaphase-Promoting Complex in Plants: Novel Functional Domains and Distinct Regulation[W]

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