Improved method for high efficiency transformation of intact yeast cells

Gietz, D.; Jean, Annie; Woods, Robin A.; Schiestl, Robert H.

doi:10.1093/nar/20.6.1425

Cited by 3,187 publications

(2,252 citation statements)

References 3 publications

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“…This was sequentially transformed into the L40 yeast strain expressing the cytoplasmic domain of Tek (amino acids 780 to 1122) in a modi®ed pBTM116 vector using the lithium acetate method of transformation (Gietz et al, 1992). Approximately 10 7 transformants were plated on synthetic medium lacking tryptophan, histidine, uracil, leucine and lysine, supplemented with 20 mM 3-aminotriazole, and incubated at 308C for 5 days.…”

Section: Discussionmentioning

confidence: 99%

The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R

1998

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Tek/Tie2 is an endothelial cell-speci®c receptor tyrosine kinase that has been shown to play a role in vascular development of the mouse. Targeted mutagenesis of both Tek and its agonistic ligand, Angiopoietin-1, result in embryonic lethality, demonstrating that the signal transduction pathway(s) mediated by this receptor are crucial for normal embryonic development. In an attempt to identify downstream signaling partners of the Tek receptor, we have used the yeast two-hybrid system to identify phosphotyrosine-dependent interactions. Using this approach, we have identi®ed a novel docking molecule called Dok-R, which has sequence and structural homology to p62 dok and IRS-3. Mapping of the phosphotyrosine-interaction domain within Dok-R shows that Dok-R interacts with Tek through a PTB domain. Dok-R is coexpressed with Tek in a number of endothelial cell lines. We show that coexpression of Dok-R with activated Tek results in tyrosine phosphorylation of Dok-R and that rasGAP and Nck coimmunoprecipitate with phosphorylated Dok-R. Furthermore, Dok-R is constitutively bound to Crk presumably through the proline rich tail of Dok-R. The cloning of Dok-R represents the ®rst downstream substrate of the activated Tek receptor, and suggests that Tek can signal through a multitude of pathways.

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Section: Discussionmentioning

confidence: 99%

The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R

1998

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“…Transformation was carried out by the method of Gietz et al (1992). All growth was carried out at 308C.…”

Section: Yeast Two Hybrid Methodsmentioning

confidence: 99%

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair

et al. 1997

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Following genomic damage, the cessation of DNA replication is co-ordinated with onset of DNA repair; this co-ordination is essential to avoid mutation and genomic instability. To investigate these phenomena, we have analysed proteins that interact with PCNA, which is required for both DNA replication and repair. One such protein is p21 Cip1 , which inhibits DNA replication through its interaction with PCNA, while allowing repair to continue. We have identi®ed an interaction between PCNA and the structure speci®c nuclease, Fen1, which is involved in DNA replication. Deletion analysis suggests that p21 Cip1 and Fen1 bind to the same region of PCNA. Within Fen1 and its homologues a small region (10 amino acids) is su cient for PCNA binding, which contains an 8 amino acid conserved PCNA-binding motif. This motif shares critical residues with the PCNA-binding region of p21 Cip1 . A PCNA binding peptide from p21 Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA synthesis. Competition between homologous regions of Fen1 and p21 Cip1 for binding to the same site on PCNA may provide a mechanism to co-ordinate the functions of PCNA in DNA replication and repair.

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“…Transformation of this strain by a library of human cDNA fused to GAD (Durfee et al, 1993) was performed by a variation of the lithium acetate method (Gietz et al, 1992). The cDNAs originated from mRNA of Epstein ± Barr virus-transformed human peripheral lymphocytes and were inserted into l-ACT (Durfee et al, 1993).…”

Section: Yeast Two-hybrid Screenmentioning

confidence: 99%

The C-terminus of the HTLV-1 Tax oncoprotein mediates interaction with the PDZ domain of cellular proteins

et al. 1998

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Infection by HTLV-1 has been correlated with the appearance of various proliferative or degenerative diseases. Some of these disorders have been observed in transgenic mice expressing the Tax protein, which is known to transactivate various viral and cellular promoters through interactions with several transcription factors. In this study we show that the C-terminus of this viral oncoprotein represents a motif permitting binding of Tax to the PDZ domains of several cellular proteins. A two-hybrid screen with Tax as bait indeed yielded complementary DNAs coding for six proteins including PDZ domains. Two of them correspond to truncated forms of the PSD-95 and b1-syntrophin proteins, another clone codes for a protein homologous to the product of the C. elegans gene lin-7. The other three clones code for new human members of the PDZ family of cellular proteins. The interaction of Tax with the products of these clones was con®rmed by immunoprecipitation assays in mammalian cells, and analysis of various mutants of Tax established the importance of the Cterminal amino acids for several of these interactions. These data suggest that Tax could perturb the normal function of targeted cellular proteins by strongly interacting with their PDZ domains.

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Improved method for high efficiency transformation of intact yeast cells

Cited by 3,187 publications

References 3 publications

The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R

The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair

The C-terminus of the HTLV-1 Tax oncoprotein mediates interaction with the PDZ domain of cellular proteins

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