Non-selective transformation of Saccharomyces cerevisiae

Reipen, Gernot; Erhart, Elke; Breunig, Karin D.; Hollenberg, Cornelis P.

doi:10.1007/bf00390337

Cited by 24 publications

(9 citation statements)

References 15 publications

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“…This induces us to conclude that the major role of ars elements in S. pombe lies in the establishment of plasmids in the host cell. That the uptake of plasmids into the cell only rarely leads to their prolonged maintenance has been demonstrated for S. cerevisiae (30). Transformation under nonselective conditions and early monitoring for plasmid presence (transient expression of a marker gene) showed that up to 4% of regenerated protoplasts had taken up the plasmid.…”

Section: Discussionmentioning

confidence: 93%

Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element.

1986

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We characterized a number of widely used yeast-Escherichia coli shuttle vectors in the fission yeast Schizosaccharomyces pombe. The 2,um vectors pDB248 and YEp13 showed high frequency of transformation, intermediate mitotic and low meiotic stability, and a low copy number in S. pombe, analogous to their behavior in [ciro] strains of Saccharomyces cerevisiae. The S. cerevisiae integration vectors pLEU2 and pURA3 transformed S. pombe at very low frequencies but, surprisingly, in a nonintegrative fashion. Instead, they replicated autonomously, and they showed very high copy numbers (up to 150 copies per plasmid-containing cell). This could reflect a lack of sequence specificity for replication of plasmid DNA in S. pombe. pFL20, an S. pombe ars vector, and a series of plasmids derived from it were studied to analyze the unusually high stability of this plasmid. Mitotic stability and partitioning of the plasmids was measured by pedigree analysis of transformed S. pombe cells. An S. pombe DNA fragment (stb) was identified that stabilizes pFL20 by improvement of plasmid partitioning in mitosis and meiosis.Many of the molecular genetic techniques which have revolutionized the study of biological problems in Saccharomyces cerevisiae have now been applied to the fission yeast Schizosaccharomyces pombe. Although some of the S. cerevisiae cloning vectors will transform S. pombe and be maintained fairly well, several reports have suggested that the two yeasts differ in plasmid behavior (14,32,33). To better understand this we performed a comparative analysis of the transforming ability and mitotic and meiotic segregation characteristics of several types of plasmids which replicate autonomously in S. pombe.The first demonstration of high-frequency transformation of S. pombe was obtained with marker genes and plasmid vectors from S. cerevisiae (3). After this report, S. pombe gene banks were constructed employing different 2pum-based vectors, and genes were isolated by functional complementation of mutants (4-6, 31). In addition, S. poinbe DNA fragments were isolated which confer high frequency of transformation to plasmids (3,24,32,36) and thus resemble the ARS (autonomously replicating sequences) elements previously characterized in S. cerevisiae (34,35). Integrative transformation based on recombination between homologous sequences was successfully applied to map the rDNA locus of S. ponmbe (36) and was found to occur with cloned DNA from the mating-type gene region with high efficiency (4). Gaillardin et al. (14) found frequent rearrangements including deletions and integration of plasmids that contain the entire S. cerevisiae 2,um DNA. An extensive analysis of plasmids carrying the S. pombe oiralI gene and the associated arsl sequence has shown that these plasmids are established in S. pombe cells in a peculiar way. They assume a polymeric form by tandem amplification (32,33).Facing these disparate reports of varying plasmid behavior in S. pombe (14, 32, 33), we decided to characterize the most frequently used S. pombe v...

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Section: Discussionmentioning

confidence: 93%

Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element.

1986

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“…To demonstrate the presence of the plasmid pADH 040-2 all colonies were inoculated on the fl-lactamase indicator plates (0.67% YNB w/o, 0.2% starch, 2% glucose and 3% agar) and incubated at 30°C for 3 days. The plates were then covered with soft indicator agar (4 ml indicator agar with 1% agar, 1.5 m| IJKI solution, which contained 3 mg/ml .penicillin [9]). With one exception all colonies were fl-lactamasepositive.…”

Section: Resultsmentioning

confidence: 99%

“…0.67% yeast nitrogen base (Difco) without amino acids, 2% glucose, 30 btg/ml histidine, his-YNB is a minimal medium for the culture of strain AH22 transformed by pADH 040-2. The absence of leucine ensures that the maximum number of copies per cell of the plasmid is maintained [9].…”

Section: His-ynbmentioning

confidence: 99%

Large-scale production of yeast hybrids by electrofusion

Schnettler

Zimmermann

Emeis

1984

FEMS Microbiology Letters

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Large‐scale production of electrically fused yeast protoplasts from Saccharomyces cerevisiae AH 22[pADH 040‐2] and S. cerevisiae AH215 was achieved by the use of the so‐called helical fusion chamber. Both strains were of the same mating type a and carried the following auxotrophic markers: his4 in the case of AH22 and leu2, his3 in the case of AH215. AH22 also is a carrier of the plasmid pADH 040‐2. This plasmid confers the leu2 gene of yeast and the β‐lactamase gene from Escherichia coli, and this feature enables quick detection of plasmid‐positive cells. After dielectrophoresis (275 V/cm, 800 kHz) fusion was induced by two field pulses (10 kV/cm, 10 μs duration) applied at an interval of 0.5 s. 50 to 60 hybrids per run were isolated after regeneration on selection medium.

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“…Although apparently this process is rather specific, it does not play a significant role, if any, in vivo. This is demonstrated by the fact that the activity of 0-lactamase in pep4-3 transformants is as high as in other yeast transformants (Table I) [16]. Since, as shown above, the preprotein is almost inactive, a pep4 transformant should exhibit no or a very low enzyme activity if a PEP4-dependent protease would be responsible for in vivo processing.…”

Section: Processing Activities In Yeast Crude Extractsmentioning

confidence: 92%

“…Crude extracts of yeast transformants carrying the plasmid pADH040-2 exhibit high 0-lactamase activities [16] allowing the purification of the enzyme. The final preparation yielded homogeneous protein having the same specific activity and molecular mass as the purified P-lactamase from E coli (Fig.…”

Section: Properties Of Purified 0-lactamase From Yeast Transformantsmentioning

confidence: 99%

Specific processing of the bacterial β‐lactamase precursor in Saccharomyces cerevisiae

Roggenkamp

Hoppe²,

Hollenberg

1983

J of Cellular Biochemistry

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Synthesis and processing of the bacterial enzyme beta-lactamase (E.C. 3.5. 2.6) were studied in Saccharomyces cerevisiae. The 2-micron DNA vector pADH040-2 containing the yeast ADH1 promoter fused to the bacterial gene was used in order to obtain enhanced synthesis of the bacterial protein in yeast transformants. Both precursor and mature beta-lactamase were shown to be present in yeast cells, the precursor being the major product. The mature enzyme was purified about 500-fold over crude extracts to apparent homogeneity and thus represents nearly 0.2% of the total yeast protein. No difference in specific activity and molecular weight could be observed when compared with the authentic beta-lactamase from Escherichia coli. Specificity of the processing of beta-lactamase in yeast cells was verified by partial amino acid sequence analysis demonstrating the removal of the signal peptide at the correct position.

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Non-selective transformation of Saccharomyces cerevisiae

Cited by 24 publications

References 15 publications

Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element.

Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element.

Large-scale production of yeast hybrids by electrofusion

Specific processing of the bacterial β‐lactamase precursor in Saccharomyces cerevisiae

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