The complete set of predicted genes from Saccharomyces cerevisiae in a readily usable form - PubMed (original) (raw)

The complete set of predicted genes from Saccharomyces cerevisiae in a readily usable form

J R Hudson Jr et al. Genome Res. 1997 Dec.

Abstract

Nearly all of the open reading frames (ORFs) of the yeast Saccharomyces cerevisiae have been synthesized by PCR using a set of approximately 6000 primer pairs. Each of the forward primers has a common 22-base sequence at its 5' end, and each of the back primers has a common 20-base sequence at its 5' end. These common termini allow reamplification of the entire set of original PCR products using a single pair of longer primers-in our case, 70 bases. The resulting 70-base elements that flank each ORF can be used for rapid and efficient cloning into a linearized yeast vector that contains these same elements at its termini. This cloning by genetic recombination obviates the need for ligations or bacterial manipulations and should permit convenient global approaches to gene function that require the assay of each putative yeast gene.

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Figures

Figure 1

Figure 1

Strategy for cloning each of the yeast ORFs. A segment of the yeast genome containing illustrative ORFs 1 and 2, both of which would be transcribed left to right, is shown at the top. Each ORF is flanked by two bent arrows representing the unique pairs of primers, with the solid black fill (_rightward_-pointing arrows) indicating the common 5′ termini of the forward primers and the horizontal-lined fill (leftward pointing arrows) indicating the common 5′ termini of the back primers. The sequence of each forward primer is 5′-GGAATTCCAGCTGACCACCATGN17–29-3′ corresponding to 19 bases of non-yeast sequence, the initiator ATG of the ORF, and 17–29 subsequent bases of the ORF. The sequence of each back primer is 5′-GATCCCCGGGAATTGCCATGENDN17–29-3′ corresponding to 20 bases of non-yeast sequence, followed by the reverse complement of a stop codon (noted above as “END”), followed by the reverse complement of 17–29 bases found at the end of the ORF. The product of the PCR of ORF1, shown as a box with diagonal-lined fill flanked by the 19 and 20 bp of non-yeast sequences, is template for the rePCR. The 70-base sequences of the rePCR primers, shown as bent arrows with checkered and “brick” 5′ termini, and 3′ termini matching the sequences in the first set of PCR primers, are provided in Methods. The product of the rePCR is ORF1 flanked by 70-base elements that are identical to those in the two-hybrid vector pOAD. The positions of the translation initiation codon (ATG) and termination codon (term.) of the ORF1 rePCR product are shown. Digestion of the vector with _Nco_I and _Pvu_II and cotransformation with the rePCR product results in two recombination events that precisely insert the ORF in-frame with the activation domain of Gal4p.

Figure 2

Figure 2

Products of a sample of rePCRs. Fifteen ORFs were reamplified using the 70-base activation domain rePCR primers. The sizes of these ORFs from the genome sequence are YBR139W (1527 bp), YBR186w (1611 bp), YCL038c (1587 bp), YCR028c (1539 bp), YDL238c (1470 bp), YDL207w (1617 bp), YDL197c (1578 bp), YDL189w (1612 bp), YDL178w (1593 bp), YDL170w (1587 bp), YDL160c (1521 bp), YDL159w (1548 bp), YDL156w (1569 bp), YDL146w (1476 bp), and YDL143w (1587 bp). The DNA size marker in the right lane has fragments of 23, 9.4, 6.6, 4.4, 2.3, 2.0, 1.4, 1.1, 0.9, and 0.6 kb. The rePCR products all migrate slightly slower than the predicted sizes of their ORFs because of the 70 bp of extra sequence at each end.

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