Cloning and manipulation of the Schizosaccharomyces pombe his7+ gene as a new selectable marker for molecular genetic studies

Abstract

We have cloned the his7+ gene of the fission yeast Schizosaccharomyces pombe by complementation of the recessive mutant allele his7-366. The his7+ gene is able to complement a mutation of the Escherichia coli hisI gene, suggesting that his7+ encodes a phosphoribosyl-AMP cyclohydrase. Subcloning experiments localize the gene to a 1.9-kb XbaI-BglII fragment. We describe the construction of plasmids to facilitate the use of his7+ as a selectable marker in S. pombe studies. Plasmid pEA2 carries his7+ cloned into the pUC18 polylinker. From either pEA2 or the original his7+ clone, pMN1, fragments carrying his7+ can be isolated using a variety of restriction enzymes for the construction of gene disruptions. Plasmid pEA500 is a cloning vector that carries his7+ and ars1, yet retains the ability to use the blue/white color screen to identify recombinants.

Fig. 1

Plasmid maps for pEA2 and pEA500. Plasmid pEA2 was constructed by insertion of the 3.15-kb BglII fragment (indicated by vertical stripes) from pMN1 into the BamHI site of pUC18 (Norrander et al. 1983). See Fig. 2 for subcloning information with respect to sites useful for gene disruptions (shown in bold). Plasmid pEA500 was constructed by inserting the PstI-SmaI fragment from pEA2 into plasmid pSP2 (Cottarel et al. 1993) which was digested within the URA3 gene with NsiI and StuI. Unique sites in the polylinker are in bold. Sites in the polylinker that are also present elsewhere in the plasmid are underlined, and the additional sites are displayed

Fig. 2

Complementation analysis of his7 subclones. The polylinker and insert from pEA2 (Fig. 1) are shown. The sites in the polylinker are indicated in the boxed regions. The plasmids carrying various subclones, described in Materials and methods, were tested for their ability to transform E. coli strain I903 (hisI903) to His⁺. Sites that cut outside of the his7⁺ gene, and are therefore useful for constructing gene disruptions, are in bold (SphI, PstI, SalI, AccI, EcoRI and XbaI to the left of his7⁺; XmaI, SmaI, KpnI and EcoRI to the right of his7⁺). The sites generated by the ligation that created pEA2 (BglII/BamHI and BamHI/BglII) can be cut with BstYI; however, other subcloning experiments indicate that there is a BstYI site within his7⁺ (data not shown)