Plasmid construction by homologous recombination in yeast

Ma, Hong; Kunes, Sam; Schatz, Peter J.; Botstein, David

doi:10.1016/0378-1119(87)90376-3

Cited by 539 publications

(383 citation statements)

References 22 publications

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“…These initial PCR products were then individually reamplified with a common 52-base forward primer and a common 60-base reverse primer. This second round of PCR converted the vaccinia virus ORFs into DNA fragments with common flanking sequences that allow efficient homologous recombination into linearized yeast vectors (17). Cotransformation of these fragments with the vector pOAD (12) into yeast strain PJ69-4A (14) generated a set of 266 activation domain hybrid proteins.…”

Section: Resultsmentioning

confidence: 99%

Genome-wide analysis of vaccinia virus protein–protein interactions

McCraith

Holtzman²,

Moss³

et al. 2000

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

266

205

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To detect interactions between proteins of vaccinia virus, we carried out a comprehensive two-hybrid analysis to assay every pairwise combination. We constructed an array of yeast transformants that contained each of the 266 predicted viral ORFs as Gal4 activation domain hybrid proteins. The array was individually mated to transformants containing each ORF as a Gal4 -DNAbinding domain hybrid, and diploids expressing the two-hybrid reporter gene were identified. Of the Ϸ70,000 combinations, we found 37 protein-protein interactions, including 28 that were previously unknown. In some cases, e.g., late transcription factors, both proteins were known to have related roles although there was no prior evidence of physical associations. For some other interactions, neither protein had a known role. In the majority of cases, however, one of the interacting proteins was known to be involved in DNA replication, transcription, virion structure, or host evasion, thereby providing a clue to the role of the other uncharacterized protein in a specific process.

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Section: Resultsmentioning

confidence: 99%

Genome-wide analysis of vaccinia virus protein–protein interactions

McCraith

Holtzman²,

Moss³

et al. 2000

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

266

205

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“…pRG216 was assembled by gap repair in yeast (Ma et al, 1987). The MCS, bacterial origin of replication and bla gene originated from pBluescript II KS + (Stratagene, USA) and the CEN6-ARSH4 sequences from pRS313 (Sikorski and Hieter, 1989).…”

Section: Plasmid Constructionmentioning

confidence: 99%

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Gnügge

Liphardt

Rudolf

2016

Yeast

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Shuttle vectors allow for an efficient transfer of recombinant DNA into yeast cells and are widely used in fundamental research and biotechnology. While available shuttle vectors are applicable in many experimental settings, their use in quantitative biology is hampered by insufficient copy number control. Moreover, they often have practical constraints, such as limited modularity and few unique restriction sites. We constructed the pRG shuttle vector series, consisting of single-and multi-copy integrative, centromeric and episomal plasmids with marker genes for the selection in all commonly used auxotrophic yeast strains. The vectors feature a modular design and a large number of unique restriction sites, enabling an efficient exchange of every vector part and expansion of the series. Integration into the host genome is achieved using a double-crossover recombination mechanism, resulting in stable single-and multi-copy modifications. As centromeric and episomal plasmids give rise to a heterogeneous cell population, an analysis of their copy number distribution and loss behaviour was performed. Overall, the shuttle vector series supports the efficient cloning of genes and their maintenance in yeast cells with improved copy number control.

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“…First, the AIP1 gene was mutated through a PCR reaction in which a low fidelity Taq polymerase was used to incorporate random mutations into the gene. These mutated PCR products were introduced by recombination (Ma et al, 1987) into a two-hybrid DNA activation domain vector and differential interaction screening was used to isolate separation-of-function mutants that had the ability to interact with actin but not cofilin in two-hybrid assays. From this we recovered aip1-56, which replaces Trp261 with a stop codon, ending translation of Aip1p ϳ80% into the N-terminal propeller.…”

Section: Confirmation Of Two Independent Actin Interaction Sitesmentioning

confidence: 99%

A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

Clark

Teply

Haarer

et al. 2006

MBoC

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Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal ␤-propeller and a secondary actin binding site lies in a comparable location on its C-terminal ␤-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding-and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N-and C-terminal propeller domains.

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Plasmid construction by homologous recombination in yeast

Cited by 539 publications

References 22 publications

Genome-wide analysis of vaccinia virus protein–protein interactions

Genome-wide analysis of vaccinia virus protein–protein interactions

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

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