High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier

Schiestl, Robert H.; Gietz, R. Daniel

doi:10.1007/bf00340712

Cited by 2,046 publications

(1,423 citation statements)

References 35 publications

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“…Yeast two-hybrid screen and cDNA library screen Yeast strain Y190 (Harper et al, 1993), containing`bait' plasmid pPC97-p107, encoding the GAL4 DNA binding domain (DBD) fused to the pocket region of p107, was transformed with a day 14.5 CD1 mouse embryo library (Chevray and Natans, 1992) by the lithium acetate method (Schiestl and Gietz, 1989). Transformants were selected for growth on plates lacking histidine and supplemented with 25 mM 3-aminotriazole.…”

Section: Methodsmentioning

confidence: 99%

Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein

1999

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The proteins of the retinoblastoma family are potent inhibitors of cell cycle progression. It is well documented that their growth-inhibitory activity can be abolished by phosphorylation on serine and threonine residues by cyclin dependent kinases. In contrast, very little is known about the dephosphorylation of retinoblastoma-family proteins. We report here the isolation, by virtue of its ability to associate with p107, of a novel Protein Phosphatase 2A (PP2A) regulatory subunit, named PR59. PR59 shares sequence homology with a known regulatory subunit of PP2A, PR72, but di ers from PR72 in its expression pattern and its functional properties. We show that PR59 co-immunoprecipitates with the PP2A catalytic subunit, indicating that PR59 is a genuine component of PP2A holo-enzymes. In vivo, PR59 associates speci®cally with p107, but not with pRb. Elevated expression of PR59 results in dephosphorylation of p107, but not of pRb, and inhibits cell proliferation by causing cells to accumulate in G1. These data support a model in which the distinct PP2A regulatory subunits act to target the PP2A catalytic subunit to speci®c substrates and suggest a role for PP2A in regulation of p107.

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Section: Methodsmentioning

confidence: 99%

Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein

1999

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“…Yeast was transformed using a modi®ed version of the lithium acetate method of Schiestl and Gietz (1989).…”

Section: Yeast Strainmentioning

confidence: 99%

APETALA1 and SEPALLATA3 interact to promote flower development

et al. 2001

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SummaryIn Arabidopsis, the closely related APETALA1 (AP1) and CAULIFLOWER (CAL) MADS-box genes share overlapping roles in promoting¯ower meristem identity. Later in¯ower development, the AP1 gene is required for normal development of sepals and petals. Studies of MADS-domain proteins in diverse species have shown that they often function as heterodimers or in larger ternary complexes, suggesting that additional proteins may interact with AP1 and CAL during¯ower development. To identify proteins that may interact with AP1 and CAL, we used the yeast two-hybrid assay. Among the ®ve MADS-box genes identi®ed in this screen, the SEPALLATA3 (SEP3) gene was chosen for further study. Mutations in the SEP3 gene, as well as SEP3 antisense plants that have a reduction in SEP3 RNA, display phenotypes that closely resemble intermediate alleles of AP1. Furthermore, the early¯owering phenotype of plants constitutively expressing AP1 is signi®cantly enhanced by constitutive SEP3 expression. Taken together, these studies suggest that SEP3 interacts with AP1 to promote normal¯ower development.

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“…Yeast cells were transformed using the lithium acetate method (15). Cells carrying plasmids were grown in synthetic medium containing adenine and amino acid additives, but lacking uracil to select for plasmid maintenance (16).…”

Section: Experimental Procedures Strains and Mediamentioning

confidence: 99%

Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

Choi

Konopka

2006

Biochemistry

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The yeast α-factor pheromone receptor (Ste2) belongs to the family of G protein-coupled receptors (GPCRs) that contain seven transmembrane domains. To define the residues that are accessible to the cytoplasmic G protein, Cys scanning mutagenesis was carried out in which each of the residues that span the intracellular loops and the cytoplasmic end of transmembrane domain 7 were substituted with Cys. The 90 different Cys-substituted residues were then assayed for reactivity with MTSEAbiotin (2-([biotinoyl] amino) ethyl methanethiosulfonate), which reacts with solvent accessible sulfhydryl groups. As part of these studies we show that adding free Cys to stop the MTSEA-biotin reactions has potential pitfalls in that Cys can rapidly undergo disulfide exchange with the biotinylated receptor proteins at pH ≥7. The central regions of the intracellular loops of Ste2 were all highly accessible to MTSEA-biotin. Residues near the ends of the loops typically exhibited a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. Interestingly, these boundary residues were enriched in hydrophobic residues, suggesting that they may form a hydrophobic pocket for interaction with the G protein. Comparison with accessibility data from a previous study of the extracellular side of Ste2 indicates that the transmembrane domains vary in length, consistent with some transmembrane domains being tilted relative to the plane of the membrane as they are in rhodopsin. Altogether, these results define the residues that are accessible to the G protein and provide an important structural framework for the interpretation of the role of Ste2 residues that function in G protein activation.The S. cerevisiae α-factor pheromone receptor (Ste2) belongs to the large family of Gproteincoupled receptors (GPCRs) that transduce the signals for light, taste, olfaction, and many biomedically important hormones. Although GPCRs are quite diverse in sequence, they function in a similar manner to activate the α subunit of heterotrimeric G proteins to bind GTP and they also show similar structural architecture in that they are composed of a bundle of seven transmembrane domains (TMDs) connected by extracellular loops and intracellular loops (1,2). Ste2 shows overall similarity to many mammalian GPCRs in that the core region containing the TMDs is involved in signal transduction and the C terminal tail is a target for † This work was supported by National Institutes of Health Grant GM55107 awarded to J.B.K. post-translational modifications that regulate receptor desensitization and endocytosis (3). Ste2 activates a heterotrimeric G protein in which the α subunit shows about 45% identity with mammalian Gα proteins and is most closely related to the Gi subfamily (4). In addition, comparison of Ste2 with rhodopsin, a member of the large class A subfamily of mammalian GPCRs, indicates that there are similar microdomains in these divergent receptors (5). These results suggest that there are underlying ...

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High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier

Cited by 2,046 publications

References 35 publications

Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein

Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein

APETALA1 and SEPALLATA3 interact to promote flower development

Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

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