Article Figures & Data
Figures
- Fig. 1.
Schematic representation of the yeast chromosomal regions contained in the YEp24 genomic DNA clones obtained by direct selection with MQ for clone 1 (MQR2c) or QD for clone 2 (QDR7.3) and clone 3 (QDR9.13). Vector arms are shown as open boxes, while the genomic insert is represented by a black slashed line. ORFs are indicated by black arrows pointing in the direction of transcription, with the name of the ORF given above or below each arrow. Partial ORFs are indicated by slashed lines under the genomic insert line, with the corresponding name given under each ORF. The insert in clone 1 carries two partial ORFs. YBR231c is an ORF of 912 bp, of which 613 bp is contained within the insert, while ORF YBR235wrepresents a 3.3 kb gene, of which only 726 bp is contained within the insert of clone 1. Likewise, in clone 2 only 210 bp of the 1,025-bpBUB3 gene is contained within the insert. The chromosomal nucleotide positions (nt pos) of the flanking nucleotides for each of the three inserts are indicated above the genomic insert line, with a thin arrow pointing to the insert end.
- Fig. 2.
MQ resistance in S. cerevisiae can be conferred by overexpression of yeast and human members of the hnRNPK family. (A) MQ resistance of clone 1 (MQR2c) is caused by the ORFYBR233w. The plate on the left shows growth of yeast transformed with the three ORFs YBR232c, YBR233w, and YBR234w, the YEp24 vector controls, and primary transformant clone 1 (MQR2c) in the absence of MQ. The middle plate shows the growth of the same yeast transformants in the presence of 225 μg of MQ per ml. The right plate depicts the plating scheme used. (B) Multiple amino acid sequence alignment of PCBP-1, hnRNPK, and YBR233w proteins. The extensive sequence homology in the region of the acidic and the KH domains identifies YBR233w as a yeast member of the hnRNPK family. (C) Overexpression of human hnRNPK genes can confer MQ resistance in yeast. The left plate shows the growth of a yeast transformed with the expression vector pVT-U containing ORFYBR233w in the sense and the antisense directions and cDNAs for the human genes PCBP-1, hnRNPK, andhnRNPK-A as an example of a splice variant plated on MQ-free agar. The middle plate shows the growth of the same yeast transformants in the left plate plated in the presence of 225 μg of MQ per ml. The right plate depicts the plating scheme used.
- Fig. 3.
Identification of three genes that confer resistance to QD when they are overexpressed in yeast. (A) CIN5 andSTI1 are two genes that confer QD resistance and that are contained within the same QD-resistant primary transformant clone 2 (QDR7.3). The right plate depicts the plating scheme and the identification of transformants. The left plate shows equal growth of all yeast transformants in the absence of QD. The middle plate shows only growth of the yeast transformed with primary library clone 2 (QDR7.3) or with CIN5 and STI1 expressed under the control of the ADH promoter in pVT-U. (B) The ORFYOR273c contained in the QD-resistant primary transformant clone 3 (QDR9.13) confers QD resistance when it is overexpressed in yeast. The right plate depicts the plating scheme and the identities of the transformants. The left plate shows equal growth of all yeast transformants in the absence of QD. The middle plate shows only the growth of yeast transformed with primary library clone 3 (QDR9.3) and with YOR273c cloned in YEp24 or pVT-U (YOR273c) in the presence of QD.
Tables
- Table 1.
Quantitation of resistance to QD, MQ, QN, and CQ
Yeast clone Fold resistancea QD MQ QN CQ Clone 2b 6.5 ± 1.0 5.6 ± 0.8 2.5 ± 0.5 5.6 ± 3.8 STI1c 7.0 ± 0.4d 3.4 ± 0.5e 2.0 ± 0.8f 2.7 ± 1.7f Anti-STI1c 1.0 ± 0.1 1.3 ± 0.6 1.3 ± 0.6 1.3 ± 0.6 CIN5c 3.9 ± 0.5d 9.6 ± 1.4d 1.2 ± 0.2f 5.4 ± 0.7d Anti-CIN5c 1.0 ± 0.1 1.5 ± 0.5 1.0 ± 0.1 1.3 ± 0.5 Clone 3b 4.8 ± 1.3 1.5 ± 0.6 1.3 ± 0.5 2.7 ± 0.6 YOR273cc 4.3 ± 1.5g 1.0 ± 0.0f 1.4 ± 0.5f 1.3 ± 0.6f Anti-YOR273cc 1.5 ± 0.5 1.0 ± 0.0 1.3 ± 0.6 1.5 ± 0.5 Clone 1b 2.5 ± 1.3 22.4 ± 12.4 1.6 ± 0.6 1.8 ± 1.3 YBR233wc 1.3 ± 0.6f 13.5 ± 3.6d 1.2 ± 0.3f 2.9 ± 1.7f Anti-YBR233wc 1.2 ± 0.3 1.3 ± 0.6 1.2 ± 0.3 1.3 ± 0.6 ↵a Significance of resistance for individual ORFs was assessed by comparison with drug resistance conferred by the same ORF cloned in the antisense orientation in the pVT-U expression vector.
↵b Fold resistance over that of transformants containing insertless vector YEp24.
↵c Fold resistance over that of transformants containing insertless vector pVT-U.
↵d P < 0.001.
↵e P < 0.01.
↵f Not significant.
↵g P < 0.05.
