Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs

Abstract

An Arabidopsis thaliana cDNA bank has been constituted in a Saccharomyces cerevisiae expression vector based on the phosphoglycerate kinase (PGK) promoter and terminator. This bank was used to complement eight S. cerevisiae auxotrophic markers. All of them were corrected. These results confirm the quality of the bank and the feasibility of cloning plant genes by yeast mutant complementation. The cDNA complementing the ura1 yeast mutant was sequenced, analysed and shown to encode a dihydroorotic (DHO) dehydrogenase sequence.

Hexokinase as a sugar sensor in higher plants.

“…Mutated yeast cells transformed with either the AtHXKI or AtHXK2 cDNA restored growth on the selection medium. The vector pFL61 (Minet et al, 1992) alone was not able to complement the mutant (Figure 1 respectively (Figure 2). AtHXKI and AIHXK2 share 82% nucleotide identity, and AtHXKI and AtHXK2 share 85% amino acid identity.…”

“…We first identified the Arabidopsis HXK genes from an expression library (Minet et al, 1992) by functional complementation of the yeast Saccharomyces cerevisiae hxkl hxk2 double mutant (DBY2219) that completely lacks HXK activity. Two cDNAs, AtHXKI and AtHXK2 (2.0 and 1.9 kb, respectively), were able to complement the yeast mutant.…”

“…The Saccharomyces cerevisiae hxkl hxk2 double mutant (DBY2219) (Ma and Botstein, 1986) was transformed with an Arabidopsis thaliana expression library (Minet et al, 1992), as described by Gietz et al (1 992). The glucose repression assay was performed using aYP plate(1 % Bacto-yeast extract, 2% Bacto-peptone, and 2% Bacto-agar) with 2% raffinose as the carbon source in the presence of 0.02% 2-deoxyglucose (2-dGlc).…”

Functional expression of the plant K+ channel KAT1 in insect cells

“…KATI cDNA was introduced in pGmAc 34T transfer vector [6]. KATI cDNA was provided in the yeast/E, coli shuttle vector pFL61 between two NotI sites [17,23]. In pGmAc 34T plasmid, the initiator ATG of polyhedrin was removed by changing G to T, and a linker containing a NotI site was introduced at the SmaI cloning site present at a deletion between nucleotides +44 and +462.…”

OmZnT1 and OmFET, two metal transporters from the metal-tolerant strain Zn of the ericoid mycorrhizal fungus Oidiodendron maius, confer zinc tolerance in yeast

“…The cDNAs were cloned into the yeast expression plasmid pFL61 ( Minet et al, 1992). Transformation of Δzrc1 yeast cells with the cDNA library followed the same procedure described above except that, after the 42 °C shock, cells were washed with 1 ml of sterile water and incubated in liquid YPD medium for 4 h. Cells were plated on SD-ura medium containing 17.5 mM ZnCl 2 .…”