An improved protocol for the preparation of yeast cells for transformation by electroporation

Thompson, John R.; Register, Elizabeth; Curotto, James E.; Kurtz, Myra B.; Kelly, Rosemarie

doi:10.1002/(sici)1097-0061(19980430)14:6<565::aid-yea251>3.0.co;2-b

Cited by 187 publications

(119 citation statements)

References 29 publications

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“…For yeast transformations, competent cells were prepared as described in Thomson et al (96), mixed with ∼500 ng of plasmid DNA, and pulsed at 1.5 kV in an Eppendorf electroporator. S. cerevisiae strain 31019b was transformed with pAG416 GPD vAmt or pAG416 GPD empty vector, as described above.…”

Section: Methodsmentioning

confidence: 99%

Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

Monier

Chambouvet

Milner

et al. 2017

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

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Phytoplankton community structure is shaped by both bottomup factors, such as nutrient availability, and top-down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer.Phycodnaviridae | NCLDV | prasinophytes | Mamiellophyceae | lateral gene transfer

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Section: Methodsmentioning

confidence: 99%

Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

Monier

Chambouvet

Milner

et al. 2017

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

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“…Yeast cells (EGY48L) were transformed with the NTT cDNA libraries following the method of Thompson et al (1998). Transformants were incubated on a synthetic dropout (SD) medium plate lacking Leu and His (Leu-/His-) at 308C for 3 to 7 d to detect LEU2 reporter gene expression.…”

Section: Ntt Assay and Sequencing Of Inserted Cdnasmentioning

confidence: 99%

Functional Isolation of Novel Nuclear Proteins Showing a Variety of Subnuclear Localizations

et al. 2005

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Nuclear proteins play key roles in the fundamental regulation of genome instability, the phases of organ development, and physiological responsiveness through gene expression. Although nuclear proteins have been shown to account for approximately one-fourth of total proteins in yeast, no efficient method to identify novel nuclear proteins has been applied to plants. In this study, a trial to isolate nuclear proteins in rice was attempted, and several novel nuclear proteins showing a variety of subnuclear localizations were identified. The nuclear transportation trap (NTT) system, which is a modified twohybrid system, isolated many nuclear proteins from rice (Oryza sativa) NTT cDNA libraries. Nuclear localization of the isolated proteins was confirmed by transient introduction of green fluorescent protein fusion constructs for a subset of protein genes into onion (Allium cepa) cells. The majority of these proteins, including novel proteins and proteins initially categorized as cytoplasmic proteins, were revealed to be localized in the nucleus. Detailed characterization of unknown proteins revealed various subnuclear localizations, indicating their possible association with chromatin and the nuclear matrix with a foci or speckle-like distribution. Some also showed dual distribution in the nucleus and cytoplasm. In the novel protein fraction, a protein was further identified for its chromatin-associated localization in a specific organ of rice by immunostaining. Thus, a variety of novel nuclear architectural proteins with chromatin or matrix associating abilities, which are important in nuclear organization by influencing certain organ developments or cell responsiveness, can be isolated using the NTT method. Because nuclear proteins other than transcription regulators have rarely been characterized in plants, such as matrix proteins and development-specific chromatin proteins, their identification and subsequent characterization could provide important information for genome-wide regulatory mechanisms controlled by nuclear organization.

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“…Yeasts were transformed by electroporation (Thompson et al, 1998). Selection and maintenance of the transformants were carried out on MSD medium.…”

Section: Yeast Transformationmentioning

confidence: 99%

A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

Guo

Zhang

et al. 2010

J. Zhejiang Univ. Sci. B

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Abstract:The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and expressed under the control of the GAL1 promoter. α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.

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An improved protocol for the preparation of yeast cells for transformation by electroporation

Cited by 187 publications

References 29 publications

Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

Functional Isolation of Novel Nuclear Proteins Showing a Variety of Subnuclear Localizations

A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

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