The rad3‐1 mutant is defective in axial core assembly and homologous chromosome pairing during meiosis in the basidiomycete Coprinus cinereus

Abstract

We have utilized spreading methods as well as serial sectioning three-dimensional reconstruction to examine meiotic chromosome behavior in cells homozygous for the rud3-1 mutation in Coprinus cinereus. Comparison of 42 wild-type nuclei that had been spread, stained with silver, and viewed by electron microscopy with 30 mutant nuclei treated in the same manner revealed several defects in the mutant. Axial core formation was defective in the mutant, although limited sideby-side association of axial cores was observed.To detect any differences in three-dimensional architecture between the wild-type and mutant nuclei, we reconstructed three of the former and six of the latter after serial sectioning. It was not possible to trace the expected number of axial cores from section to section in the mutant, although some tripartite synaptonemal complex was observed. Many axial core ends failed to terminate in the nuclear envelope in the mutant. This spectrum of defects (incomplete axial core assembly with some tripartite synaptonemal complex formation) had not been observed previously in either C. cinereus or other systems. We conclude that this combination of spreading and sectioning methods is very useful for analysis of meiotic mutants.0 1993 Wiley-Liss, lnc.

Coprinus cinereus rad50Mutants Reveal an Essential Structural Role for Rad50 in Axial Element and Synaptonemal Complex Formation, Homolog Pairing and Meiotic Recombination

“…Electron microscopy: Chromosome spreads were performed as described by Pukkila et al (Pukkila and Lu 1985;Pukkila et al 1992). Silver nitrate staining of meiotic nuclei rad50 Mutants of Coprinuswas performed as per Seitz et al (1996).…”

“…Silver nitrate staining of meiotic nuclei rad50 Mutants of Coprinuswas performed as per Seitz et al (1996). Grids were prepared as per Pukkila et al (1992) and viewed with a JEOL-1010 electron microscope. Images of stained nuclei were scanned into a computer and analyzed using Adobe Photoshop 7.0 (Adobe Systems), National Institutes of Health (NIH) Image 1.63 (Wayne Rasband, NIH), and Microsoft Excel '98.…”

“…Chromosome spreads were obtained as described (Pukkila et al 1992;Seitz et al 1996) except that we used uncharged slides (VWR), cleaned with Sparkle glass cleaner (A. J. Funk and Co.). Slides with chromosome spreads were wetted in PBS, pH 7.4, for 10 min.…”

Life History and Developmental Processes in the BasidiomyceteCoprinus cinereus

“…Two other genes act in sterigma formation following meiosis, 13 genes are involved in prespore formation, and 4 genes contribute to nuclear migration into the spores (214). Other studies report defects in premeiotic DNA replication and meiotic prophase I, but the majority of defects appear to be localized at the stage of meta-anaphase I (216,389,396,397,405,435,466,498,502,538,539,542). From ␥-radiation-sensitive mutants exhibiting defects in chromosome condensation and synapsis in meiosis, an epistasis group of rad3-9-11-12 has been defined (405,498,538,542).…”

Replication-dependent early meiotic requirement for Spo11 and Rad50

“…Microscopy. Chromosome spreads were silver-stained according to methods described in Pukkila et al (25). Spreads were viewed and photographed by using a JEOL transmission electron microscope.…”