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Dominant mutations in the Caenorhabditis elegans Myt1 ortholog wee-1.3 reveal a novel domain that controls M-phase entry during spermatogenesis

S. Todd Lamitina^* and Steven W. L'Hernault

Department of Biology, Emory University, Atlanta, GA 30322, USA, Program in Biochemistry, Cell and Developmental Biology, Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA 30322, USA
^* Present address: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37232, USA

View larger version (157K): [in a new window]   Fig. 1. Light and ultrastructural phenotypes of spe-37(gf)/+ mutant sperm. (A-D) Various stages of wild-type spermatogenesis at identical magnifications. (A) Primary spermatocyte; (B) dividing primary spermatocyte; (C) dividing secondary spermatocytes; (D) spermatids. (E-H) Terminal phenotype of various spe-37(gf)/+ mutant spermatocytes. The independently derived eb1947, q89 (not shown) and e104 mutants bear the identical spe-37 mutation. (I,J) Transmission EM of spe-37(e1947)/+ spermatocytes: (I) early mutant phenotype; (J) terminal mutant phenotype. n, nucleus; fb-mo, fibrous body-membranous organelle; mo, membranous organelle; ne, nuclear envelope. Scale bars: 5 µm in A-H; 1 µm in I,J.

View larger version (48K): [in a new window]   Fig. 2. Phospho-histone H3 staining of wild-type and spe-37(gf)/+ sperm. (A-D) Wild-type. (E-H) spe-37(q89)/+. (A,E) DIC; (B,F) FITC anti-phosphorylated histone H3; (C,G) DAPI staining of nuclei; (D,H) merge of DAPI and anti-phospho-H3. Arrow indicates a wild-type spermatid that does not stain for phospho-histone H3. Scale bars: 5 µm.

View larger version (70K): [in a new window]   Fig. 3. Light microscopic phenotypes of spe-37(q89) Class 1 semi-dominant suppressors. (A-C) DIC and (D-F) DAPI fluorescence light microscopy. (A,D) Sperm from spe-37(q89 eb61)/+ males; (B,E) spe-37(q89 eb62)/+ males; (C,F) spe-37(q89 eb94)/+ males. Arrowheads indicate abnormal sperm with multiple nuclei. Scale bars: 5 µm.

View larger version (28K): [in a new window]   Fig. 4. Genomic structure of C. elegans wee-1.3 and location of mutations. (A) The eight exons (including the trans spliced SL1 leader exon) of wee-1.3 RNA are indicated by differently shaded or filled rectangles. Introns are the lines between exons. The spe-37(gf) mutations are listed within a large box above exon 7. The missense suppressor mutations (eb94, eb62, eb90 and eb88) and the associated amino acid changes are indicated above exon 3. The two suppressor mutations that cause a premature stop (eb93 and eb60) are indicated below exon 7. The eb91 deletion that removes exon 2 and part of exon 3 (including the intron acceptor for trans spliced exon 1) is indicated below these exons. The eb61 suppressor mutation alters the intron 4 splice donor (shown in the box above intron 4), while the eb87 suppressor alters the intron 4 splice acceptor site mutation (shown in the box below intron 4). (B) Comparison of wee-1.3 mRNA produced by wild-type (top) and spe-37(q89 eb87) suppressor mutant homozygotes (bottom). Splicing is altered so that the exon 5 sequence encoding the predicted transmembrane domain is absent.

View larger version (104K): [in a new window]   Fig. 5. Alignment of C. elegans, C. briggsae, Xenopus, mouse and human wee-1.3 kinases. Red boxes indicate identical residues among all wee-1.3 orthologs. Black boxes indicate identity to the C. elegans sequence. Asterisks indicate the sites of mutation in C. elegans spe-37(gf), missense and nonsense suppressors. The phenotypic class of each missense or nonsense suppressor is indicated in parentheses (see Table 1). Alignment was performed using the ClustalX program (Thompson et al., 1997). The C. briggsae wee-1.3 sequence is publicly available unpublished data from the C. briggsae Genome Project (The Sanger Institute Cambridge, UK and The Genome Sequencing Center, Washington University, St Louis, MO). C. briggsae wee-1.3 cDNA was predicted using GeneMark hmm (Borodovsky, 1998) and sequence around the spe-37(gf) region was verified by examination of ABI sequence traces (see http://trace.ensembl.org). ATP, ATP-binding domain; kinase, Wee1p-like kinase domain; TM, predicted transmembrane domain. The GenBank Accession Number for C. elegans wee-1.3 is NP_496095, C. briggsae wee-1.3 is based on the GeneMark HMM prediction from contig C000100543, Xenopus Myt1 is A57247, Mouse Myt1 is NP_075545 and Human Myt1 is NP_004194.

View larger version (110K): [in a new window]   Fig. 6. Expression from a wee-1.3 promoter-GFP fusion. Paired GFP fluorescent (A,C,E) and DIC (B,D,F) images. Arrowheads indicate (A,B) sites of GFP expression during early embryogenesis; (C,D) larval distal germline proliferation; (E,F) larval neuronal and hypodermal cells. No fluorescence was observed in adult somatic tissues (not shown).