T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis

Bundock, Paul; Mroczek, K; Winkler, Aaron A.; Steensma, H. Yde; Hooykaas, Paul J. J.

doi:10.1007/s004380050948

Cited by 65 publications

(19 citation statements)

References 35 publications

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“…None of these contained the correct Klacs2 disruption as tested by Southern analysis or PCR. Even the use of Agrobacterium tumefaciens for conjugation with K. lactis GG1916, which reportedly increases homologous recombination in K. lactis (Bundock et al , 1999), was unsuccessful. Eventually, transformation with the entire subcloned 7 kb Sph I fragment resulted in proper integration, although it took 3 weeks for the primary transformants to form sizeable colonies.…”

Section: Resultsmentioning

confidence: 99%

The acetyl co‐enzyme A synthetase genes of Kluyveromyces lactis

Zeeman

Steensma

2002

Yeast

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Two Kluyveromyces lactis genes encoding acetyl co-enzyme A synthetase isoenzymes were isolated. One we named KlACS1, as it has high similarity to the ACS1 gene of Saccharomyces cerevisiae. The other gene, KlACS2, showed more similarity to S. cerevisiae ACS2 than to KlACS1 or ScACS1. This suggests that divergence of the two isogenes occurred before the evolutionary separation of the species and that the different functions have been conserved. In line with this idea is the regulation of transcription of the genes. The mode of regulation appeared to be maintained between ScACS1 and KlACS1 and between ScACS2 and KlACS2. The KlACS1 transcript was absent in glucose-grown cells, whereas transcription levels in ethanol-and acetategrown cells were high. Disruption of the KlACS1 gene did not result in growth defects on glucose or ethanol. The growth rate on acetate, however, was reduced by a factor of two. KlACS2 was expressed at similar levels during growth on glucose and acetate, whereas expression on ethanol was slightly higher. A null mutant in this gene showed a reduced growth rate on all three carbon sources. Taken together, these data suggest that KlACS2 is used during growth on glucose and that KlACS1 is most dominant during growth on acetate. Strains in which both ACS genes are deleted could only be retrieved when a plasmid containing the ACS2 gene was present, suggesting that the double mutant is lethal. Tetrad analysis confirmed that non-viable spores with a deduced Klacs1Klacs2 genotype germinated but could not divide further. It therefore appears that, as in S. cerevisiae, the pyruvate dehydrogenase bypass formed by the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl co-enzyme A synthetase is essential for growth. These results are in apparent contradiction with the growth on glucose of a strain with a disruption in the only structural pyruvate decarboxylase gene of K. lactis. Residual enzyme activity might, however, account for this discrepancy, or Acs fulfils an additional as yet unknown function, separate from its enzymatic activity. The sequences of KlACS1 and KlACS2 have been deposited in the EMBL database under Accession Nos AF061265 and AF134491, respectively.

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Section: Resultsmentioning

confidence: 99%

The acetyl co‐enzyme A synthetase genes of Kluyveromyces lactis

Zeeman

Steensma

2002

Yeast

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“…Nevertheless, the T‐DNA derived plasmids that could be recovered provide ample evidence to support the proposed model. Besides that, the model is corroborated by the fact that integrated T‐DNA multimers have been recovered from the yeast strain Kluyveromyces lactis after AMT (Bundock et al ., ). Furthermore, earlier research on conjugal plasmids provided evidence for site‐specific recombination at the oriT sequence both within and between plasmids (Warren and Clark, ; Llosa et al ., ).…”

Section: Discussionmentioning

confidence: 97%

Involvement of Rad52 in T‐DNA circle formation during Agrobacterium tumefaciens‐mediated transformation of Saccharomyces cerevisiae

Rolloos

Dohmen

Hooykaas

et al. 2014

Molecular Microbiology

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SummaryAgrobacterium tumefaciens cells carrying a tumour inducing plasmid (Ti-plasmid) can transfer a defined region of transfer DNA (T-DNA) to plant cells as well as to yeast. This process of Agrobacterium-mediated transformation (AMT) eventually results in the incorporation of the T-DNA in the genomic DNA of the recipient cells. All available evidence indicates that T-strand transfer closely resembles conjugal DNA transfer as found between Gram-negative bacteria. However, where conjugal plasmid DNA transfer starts via relaxase-mediated processing of a single origin of transfer (oriT), the T-DNA is flanked by two imperfect direct border repeats which are both substrates for the Ti-plasmid encoded relaxase VirD2. Yeast was used as a model system to investigate the requirements of the recipient cell for the formation of T-DNA circles after AMT. It was found that, despite the absence of selfhomology on the T-DNA, the homologous repair proteins Rad52 and Rad51 are involved in T-DNA circle formation. A model is presented involving the formation of T-DNA concatemers derived from T-strands by a process of strand-transfer catalysed by VirD2. These concatemers are then resolved into T-DNA circles by homologous recombination in the recipient cell.

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“…Co‐cultivations between A. tumefaciens strain LBA1126 (pRAL7207) and RSY12 (Schiestl et al ., 1991) were carried out as previously described (Bundock et al ., 1999). The co‐cultivations between LBA1119 (pSDM8000) and YPH250 (Sikorski and Hieter, 1989) were performed in a slightly different way.…”

Section: Methodsmentioning

confidence: 99%

“…T‐DNA can therefore be used as a gene vector for these organisms, which is most relevant for species that cannot be otherwise efficiently transformed. In the absence of homology the T‐DNA integrates into the yeast chromosomal DNA by non‐homologous recombination (NHR) (Bundock et al ., 1995, 1999; Bundock and Hooykaas, 1996; De Groot et al ., 1999). This suggested that the T‐DNA could be used as an effective insertional mutagen for yeasts and fungi.…”

Section: Introductionmentioning

confidence: 99%

Insertional mutagenesis in yeasts using T‐DNA from Agrobacterium tumefaciens

Bundock¹,

Attikum²,

Dulk‐Ras³

et al. 2002

Yeast

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Insertional mutagenesis is a powerful tool for the isolation of novel mutations. The gene delivery system of the bacterium Agrobacterium tumefaciens, which mediates transfer not only to plants but also to yeasts and fungi, could be exploited to generate collections of yeasts containing insertional mutations if there were no bias towards particular integration sites, as is the case in plants. To test this, we have analysed a small collection of Saccharomyces cerevisiae strains with T-DNA copies integrated in the S. cerevisiae genome. The position of 54 of these T-DNAs was determined. The T-DNA showed no clear preference for certain DNA sequences or genomic regions. We have isolated insertions in the coding regions of the genes YGR125w, YDR250c, YGR141w, YGR045c, YPL017c, YGR040w, YDL052c, YJL148w, YCL033c, YFL061w, YJR033c, YDR175c and YLR309c confirming that these genes are non-essential for S. cerevisiae haploid growth on minimal medium. Given the advantages of T-DNA, we propose its use as an ideal mobile DNA element for insertional mutagenesis in yeasts.

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T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis

Cited by 65 publications

References 35 publications

The acetyl co‐enzyme A synthetase genes of Kluyveromyces lactis

The acetyl co‐enzyme A synthetase genes of Kluyveromyces lactis

Involvement of Rad52 in T‐DNA circle formation during Agrobacterium tumefaciens‐mediated transformation of Saccharomyces cerevisiae

Insertional mutagenesis in yeasts using T‐DNA from Agrobacterium tumefaciens

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