Dmc1 of Schizosaccharomyces pombe plays a role in meiotic recombination

Abstract

We report here a Schizosaccharomyces pombe gene (dmc1(+)) that resembles budding yeast DMC1 in the region immediately upstream of the rad24(+) gene. We showed by northern and Southern blot analysis that dmc1(+) and rad24(+) are co-transcribed as a bicistronic mRNA of 2.8 kb with meiotic specificity, whereas rad24(+) itself is constitutively transcribed as a 1.0-kb mRNA species during meiosis. Induction of the bicistronic transcript is under the control of a meiosis-specific transcription factor, Ste11. Disruption of both dmc1(+) and rad24(+) had no effect on mitosis or spore formation, and dmc1Delta cells displayed no change in sensitivity to UV or gamma irradiation relative to the wild type. Tetrad analysis indicated that Dmc1 is involved in meiotic recombination. Analysis of gene conversion frequencies using single and double mutants of dmc1 and rhp51 indicated that both Dmc1 and Rhp51 function in meiotic gene conversion. These observations, together with a high level of sequence identity, indicate that the dmc1(+) gene of S. POMBE: is a structural homolog of budding yeast DMC1, sharing both similar and distinct functions in meiosis.

Figure 1

rad24⁺ but not rad25⁺ displayed a meiosis-specific transcript at 2.8 kb distinct from the 1.0 kb band representing the constitutively transcribed rad24⁺ transcript. (A and C) Northern blot analyses with rad24- and rad25-specific probes, showing that the 2.8-kb band was derived from the rad24⁺ but not from the rad25⁺ gene. CD16-1, a heterozygous (h⁺/h^–) diploid strain initiated meiosis upon nitrogen starvation, whereas CD16-5, a homozygous (h^–/h^–) diploid strain could not proceed to meiosis. Numbers above the lanes indicate hours after nitrogen starvation. In lane M, RNA size markers that were stained with ethidium bromide before northern blotting are presented. (B and D) Southern blot analyses performed with rad24- and rad25-specific probes (Materials and Methods), showing that these two probes did not cross-hybridize under the conditions used in these experiments.

Figure 2

Northern blot analyses showing that the dmc1⁺ and rad24⁺ genes are positioned in the order dmc1⁺ rad24⁺, and that the meiosis-specific eta1 mRNA is bicistronic, including both dmc1⁺ and rad24⁺ ORFs. (A) Structure of cloned eta1 and rad24 cDNA fragments. The rad24 cDNA was isolated from a subtracted cDNA library (CD16-1 minus CD16-5). Using this rad24 cDNA fragment as a probe, eta1-1, -2 and -3 cDNAs were isolated from a CD16-1 cDNA library by colony hybridization. (B) Schematic representation of the dmc1⁺ and rad24⁺ genes and the bicistronic eta1 mRNA. (C–F) Northern blot analyses of the dmc1⁺ and rad24⁺ genes; the locations of the five different DNA fragments used as probes are indicated by thick horizontal bars. The 1.0-kb band was observed only when the rad24⁺ probe was used, whereas the 2.8-kb band was observed only when the eta1 cDNA region was included in the probe. (G) The 2.8-kb band disappeared in northern analysis using an ste11⁺ null mutant (ste11Δ), indicating that meiosis-specific transcription of dmc1⁺ takes place downstream of the Ste11 transcriptional cascade.