Evidence for a three-nucleon-force effect in proton-deuteron elastic scattering.

Developments in spin-polarized internal targets for storage rings have permitted measurements of 197 MeV polarized protons scattering from vector polarized deuterons. This work presents measurements of the polarization observables A(y), iT11, and C(y,y) in proton-deuteron elastic scattering. When compared to calculations with and without three-nucleon forces, the measurements provide further evidence that three-nucleon forces make a contribution to the observables. This work indicates that three-body forces derived from static nuclear properties appear to be crucial to the description of dynamical properties.

Replication-dependent early meiotic requirement for Spo11 and Rad50.

Spo11 and the Rad50-Mre11 complex have been indirectly implicated in processes associated with DNA replication. These proteins also have been shown to have early meiotic roles essential for the formation of a programmed DNA double-strand break known in Saccharomyces cerevisiae to initiate meiotic recombination. In both S. cerevisiae and the basidiomycete Coprinus cinereus, spo11 and rad50 mutants are defective in chromosome synapsis during meiosis. Here we demonstrate that a partial restoration of synapsis occurs in C. cinereus spo11 and rad50 mutants if premeiotic DNA replication is prevented. Double mutants were constructed with spo11-1 or rad50-4 and another mutant, spo22-1, which does not undergo premeiotic DNA replication. In both cases, we observed an increase in the percentage of nuclei containing synaptonemal complex (SC) structures, with concomitant decreases in the percentage of nuclei containing axial elements (AE) only or no structures. Both types of double mutants demonstrated significant increases in the average numbers of AE and SC, although SC-containing nuclei did not on average contain more AE than did nuclei showing no synapsis. Our results show that Spo11-induced recombination is not absolutely required for synapsis in C. cinereus, and that the early meiotic role of both Spo11 and Rad50 in SC formation partially depends on premeiotic S phase. This dependency likely reflects either a requirement for these proteins imposed by the premeiotic replication process itself or a requirement for these proteins in synapsis when a sister chromatid (the outcome of DNA replication) is present.

Insertional mutagenesis in Coprinus cinereus: use of a dominant selectable marker to generate tagged, sporulation-defective mutants.

We have constructed a dominant selectable marker, PHT1, for transformation of the basidiomycete Coprinus cinereus. PHT1 consists of a bacterial hygromycin B resistance gene fused to the promoter and terminator regions of the C. cinereus beta-tubulin gene. We found in transformation experiments that PHT1 confers hygromycin B resistance to all strains of C. cinereus tested, that it integrates without apparent bias into the genome, and that it is stable through meiotic crosses. We used a plasmid containing this marker, pPHT1, for restriction enzyme-mediated integration (REMI) and found that this technique could increase transformation efficiencies more than seven-fold. In REMI experiments using KpnI, the integrated DNA was flanked by intact KpnI sites in 53% of the cases examined, single-copy insertions represented 60% of the integration events, and most multicopy insertions were oriented head-to-tail. A screen of REMI-generated transformants yielded sporulation-defective mutants at a frequency of 1.2%. Genetic analysis showed that in six of nine mutants examined, the defect in spore formation is most likely a direct result of the pPHT1 insertion, and in three of these mutants a single pPHT1 locus was shown to cosegregate with the sporulation defect. We used semi-random PCR to isolate the genomic DNA adjacent to one pPHT1 insertion in a sporulation-defective mutant and found that we had disrupted the C. cinereus spo11 gene. Thus, REMI, in combination with pPHT1, is a powerful tool for the dissection of the meiotic process in C. cinereus.

Functions of DNA repair genes during meiosis.

One of the most basic functions in any organism is DNA repair. In addition, programmed DNA "damage," in the form of DNA double-strand breaks (DSBs), is a regular part of the physiology of most organisms. There are three main types of DSB repair: homologous recombination; single-strand annealing; and nonhomologous end joining. The gene products known to be required for these repair processes are conserved in evolution, but the relative dependence on different pathways for DSB repair is different when systems are compared. In the yeast Saccharomyces cerevisiae, the formation and repair of DNA double-strand breaks (DSBs) is apparently an essential feature of meiotic recombination. However, it is not clear whether DSBs are a conserved feature of meiotic recombination in eukaryotes. The basidiomycete Coprinus cinereus presents an experimental system which is amenable to genetic analysis, processes DSBs in a manner similar to complex eukaryotes, and has a naturally synchronous meiosis. An understanding of the functions of conserved genes in DSB repair in C. cinereus and other similar systems will help to determine whether DSB repair is a unifying theme in meiotic recombination or whether conserved gene products have other essential functions that tie together DNA repair and meiosis.

The rad9 gene of Coprinus cinereus encodes a proline-rich protein required for meiotic chromosome condensation and synapsis.

The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stage of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus.