No abstract

A simple and efficient method is described to introduce structurally pre-determined mutations into recombinant genomes of filamentous phage M13. The method rests on gapped duplex DNA (gdDNA) molecules of the phage M13 genome as the key intermediate. In this gdDNA, the (+) and the (shorter) (-) strand carry different genetic markers in such a way, that a rigorous selection can be applied for phage carrying the markers of the (-) strand. For introduction of the mutation, a synthetic oligonucleotide with partial homology to a target site within the single stranded DNA region is annealed to the gdDNA. The oligonucleotide subsequently becomes part of the (-) strand by enzymatic DNA gap filling and sealing. This physical linkage is preserved at the genetic level after transfection of a recipient E.coli strain deficient in DNA mismatch correction, so that the synthetic marker can be selected from the phage progeny independent from its potential phenotype. It is demonstrated that by this method mutants can be constructed with marker yields in excess of 70%.

a high to intermediate efficiency. The mismatch C/C and a 38-nucleotide loop were corrected with low efficiency. This substrate specificity pattern resembles that found in Escherichia coli and Streptococcus pneumoniae, suggesting an evolutionary relationship of DNA mismatch repair in pro-and eucaryotes. Repair of the listed mismatches was severely impaired in the putative S. cerevisiae DNA mismatch repair mutants pmsl and pms2. Low-efficiency repair also characterized pms3 strains, except that correction of single-nucleotide loops occurred with an efficiency close to that of PMS wild-type strains. A close correlation was found between the repair efficiencies determined in this study and the observed postmeiotic segregation frequencies of alleles with known DNA sequence. This suggests an involvement of DNA mismatch repair in recombination and gene conversion in S. cerevisiae.

The PMS1 gene from Saccharomyces cerevisiae, implicated in DNA mismatch repair in yeast cells (M. S. Williamson, J. C. Game, and S. Fogel, Genetics 110:609446, 1985), was cloned, and the nucleotide sequence was determined. The MATERIALS AND METHODS Yeast strains. The following haploid yeast strains were used: MW3069-5A (trpl ura3-52 hom3-10 his3-KpnI leu2-3, 112 MATa pmsl-J), MW3069-15A (trpl ura3-52 ade2-1 hom3-10 lys2-20 his3-KpnI MATa), MW3071-8A (Atrpl ura3-52 ade2-1 ade8-14 hom3-10 leu2-3,112 MATa pmsl -1), MW3157-63C (ura3-52 ade2-1 hom3-10 his4-519 met4 MATa pmsl-1), MW3070-8B (trpl ura3-52 ade2-1 ade8-14 hom3-10 his3-HindIII leu2-3,112 MATa), MW3070-8BpmslA (same as MW3070-8B but Apmsl), MW3271-1OC (ura3-52 ade2 Aade8 hom3-10 lysJ-l lys2-20 his4-519 leu2-3,112, thrl, MATa). MW3271-lOCpmslA (same as MW3271-1OC but Apmsl), MW3317-21A (Atrpl ura3-52 ade2 Aade8 hom3-10 his3-KpnI met4 metl3 MATo), and MW3317-2lApmslA (same as MW3317-21A but Apmsl). The alleles his3-HindIII and his3-KpnI were constructed in vitro by cleavage with the respective enzymes and fill-in (his3-HindIII) or removal (his3-KpnI) of the protruding ends with subsequent religation (unpublished data). Haploids MW 3271-10C and MW3317-21A are the parents of diploid WC149, and haploids MW3271-lOCpmslA and MW3317-2lApmslA are the parents of diploid WC150.Media. Yeast media were as previously described (7). Regeneration agar was a modified synthetic medium lacking uracil and containing 1 M sorbitol and 3% agar.Genetic techniques. Mass mating, isolation of zygotes by micromanipulation, and plate dissection of unseleted tetrads were carried out as described previously (14,29). Postmeiotic segregations were scored as sectored prints on the appropriate selective medium. For some markers, e.g., his3 versus his4, postmeiotic segregations were scored by complementation as described elsewhere (44).

Localization of soluble endoplasmic reticulum (ER) resident proteins is likely achieved by the complementary action of retrieval and retention mechanisms. Whereas the machinery involving the H/KDEL and related retrieval signals in targeting escapees back to the ER is well characterized, other mechanisms including retention are still poorly understood. We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal normally found at the C terminus of ER residents in Dictyostelium discoideum. Here we demonstrate that its 57 residue C-terminal domain is necessary for intracellular retention of Dd-PDI and sufficient to localize a green fluorescent protein (GFP) chimera to the ER, especially to the nuclear envelope. Dd-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes. The overexpression of GFP-PDI57 leads to disruption of endogenous PDI complexes and induces the secretion of PDI, whereas overexpression of a GFP-HDEL chimera induces the secretion of endogenous calreticulin, revealing the presence of two independent and saturable mechanisms. Finally, low-level expression of Dd-PDI but not of PDI truncated of its 57 C-terminal residues complements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating functional disulfide isomerase activity and ER localization. Altogether, these results indicate that the PDI57 peptide contains ER localization determinants recognized by a conserved machinery present in D. discoideum and Saccharomyces cerevisiae.