Suppression effect of Mnd1 overproduction and splicing of MND1 pre-mRNA. (A) The hop2-ts mutant (hop2-ts/hop2::ADE2) carrying the multicopy vector YEp24 containing either HOP2 or no insert was sporulated at permissive (23°C) and restrictive (33°C) temperatures and then replica plated to medium selecting for viable spores. OP, overproduction. (B) The hop2-ts mutant (hop2-ts/hop2::ADE2) and an isogenic hop2::ADE2 homozygote (referred to as hop2Δ) were transformed with YEp24 containing HOP2, MND1, or no insert. Transformants were sporulated at the restrictive temperature (33°C) for hop2-ts and then replica plated to medium selecting for viable spores. (C) Structure of the YGL183c and MND1 ORFs. The gray bar indicates the MND1 intron. The small arrows indicate the locations of the primers used to generate the PCR products shown in panel D. (D) Genomic DNA and cDNA derived from meiotic mRNA were amplified by PCR with primers flanking the putative intron (as shown in panel C). The positions of molecular size markers are indicated on the left.

DNA and amino acid sequences of MND1 and alignment of potential Mnd1 homologs. (A) DNA sequence and encoded amino acid sequence of MND1. Consensus splicing signals (37) are underlined. The start codon of the YGL183 ORF is boxed. (B) Mnd1 of budding yeast is aligned with potential homologs from other organisms: S. cerevisiae (Sc); Mus musculus (Mm; accession no. BAB27765); S. pombe (Sp; accession no. Q09739); and Arabidopsis thaliana (At; accession no. T08972). Identical amino acids are boxed in black; similar amino acids are boxed in gray.

Chromosome synapsis is aberrant in mnd1 mutants. (A) Spread nuclei from mnd1 mutants were stained with DAPI and anti-Zip1 antibodies. Examples of different patterns of Zip1 staining are shown. (B) Quantitative analysis of spread nuclei according to the pattern of Zip1 localization. Samples were collected after 28 h in sporulation medium. At least 100 nuclei were scored for each strain. Strains used were ndt80::LEU2, hop2::ADE2, and mnd1::kanMX; all are homozygous diploids isogenic with TBR001. Bar = 5 μm.

Homolog pairing is defective in mnd1 mutants. (A) Typical images of fluorescent in situ hybridization in wild-type (WT) and mnd1 strains are shown. (B) Quantitative analysis of homolog pairing. For each strain, 120 nuclei were scored after 24 h in sporulation medium. Strains used were ndt80::LEU2, hop2::ADE2, mnd1::kanMX, and hop2::ADE2 mnd1::kanMX; all are homozygous diploids isogenic with TBR001. Shown is the frequency of pairing for chromosomes III and V, as well as the frequency of nuclei in which both chromosomes III and V are paired. Chr, chromosome. Bar = 5 μm.

Meiotic recombination is impaired in mnd1. (A) Diploids containing one circular and one linear copy of chromosome III were introduced into sporulation medium, and samples were harvested at the time points indicated. The lane labeled 45* represents a shorter exposure of the 45-h sample from wild type (WT). (B) Cells were incubated in sporulation medium for 20 h and then subdivided. One subculture was further incubated in sporulation medium (SPM), and aliquots were harvested after 5 and 10 h. The other subculture was transferred to vegetative growth medium and incubated for an additional 5 h. Genomic DNA was subjected to pulsed-field gel electrophoresis followed by Southern blot analysis hybridizing with a probe containing the THR4 gene on chromosome III. Strains used were wild type, mnd1::TRP1/mnd1::kanMX, and hop2::ADE2/hop2::kanMX; all strains are isogenic with TBR266.

Rad51 foci accumulate on meiotic chromosomes in mnd1. Spread meiotic nuclei were stained with DAPI (blue) and antibodies to Rad51 (green) and Zip1 (red). Shown are spread nuclei from wild type (A) and the mnd1-null mutant (B). Arrowheads indicate the position of a polycomplex. (C) The fraction of nuclei with more than 10 Rad51 foci was scored at various time points after the introduction into sporulation medium. Note that cells do not proceed through meiosis with perfect synchrony; thus, a small fraction of wild-type cells are at the zygotene stage even at late time points. Strains analyzed were wild type, hop2::ADE2, and mnd1::TRP1; all strains are homozygous diploids isogenic with TBR001. WT, wild type. Bar = 5 μm.

Localization of Mnd1 to meiotic chromosomes and coimmunoprecipitation of Mnd1 and Hop2. (A) Meiotic chromosomes from cells producing Mnd1 tagged with GFP were stained with anti-Zip1, anti-Rad51, and anti-GFP antibodies. (B) Nuclei from spo11 cells producing Mnd1-GFP were stained with anti-GFP antibodies and DAPI. (C) Nuclei from cells producing myc-tagged Hop2 and GFP-tagged Mnd1 were stained with anti-myc and anti-GFP antibodies. Areas of overlap appear yellow in the merged image. (D) Shown in the top row is a nucleus from a hop2-null mutant producing GFP-tagged Mnd1 after staining with anti-GFP and anti-Rad51 antibodies and with DAPI. Shown on the bottom is a nucleus from a mnd1-null mutant producing myc-tagged Hop2 after staining with anti-myc and anti-Rad51 antibodies and with DAPI. (E) Extracts from meiotic cells containing untagged Hop2 and Mnd1, tagged Mnd1 only, or tagged versions of both Hop2 and Mnd1 were subjected to immunoprecipitation with anti-myc or anti-GFP antibodies. Antibodies used for precipitation are indicated under “IP”; antibodies used for Western blot analysis are indicated under “detection.” Strains used were wild type, spo11::ADE2 MND1-GFP, MND1-GFP, HOP2-myc MND1-GFP, hop2::ADE2 MND1-GFP, and mnd1::TRP1 HOP2-myc; all strains are homozygous diploids isogenic with TBR001. Bar = 5 μm.