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(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) ,uCi/ml; 40 Ci/mmol) (both from New England Nuclear).Radioactive GAGs were isolated by anion-exchange chromatography and ethanol precipitation as described by Bame and Esko (20), except that samples bound to DEAE-Sephacel (Pharmacia LKB) were washed with 0.25 M NaCl in 20 mM sodium acetate (pH 6.0) and eluted with 2.0 M NaCl in acetate buffer. Samples were 3-eliminated with 0.5 M NaOH for 24 h at 40C and reduced with 1 M NaBH4 (21) to remove any covalently linked peptides. Chondroitin sulfate was depolymerized with chondroitinase ABC according to the manufacturer's directions (ICN), and the enzyme was inactivated by boiling.To measure GAGs chemically, preparations were first treated with 10 units of DNase I and RNase A (Sigma) at 10 ,ug/ml to degrade nucleic acids. The GAGs were repurified by anion-exchange chromatography, and uronic acid was

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The prp2 gene of fission yeast has previously been shown to encode the large subunit of the splicing factor spU2AF. SpU2AF59 is an evolutionarily conserved protein that has an arginine/serine‐rich region and three RNA recognition motifs (RRMs). We have sequenced three temperature‐sensitive alleles of prp2 and determined that the mutations result in single amino acid changes within one of the RRMs or between RRMs. All mutant alleles of prp2 have pre‐mRNA splicing defects at the non‐permissive temperature. Although the mutant strains are growth‐arrested at 37°C, they do not elongate like typical fission yeast cell cycle mutants. The DNA of the prp2− strains stains more intensely than a wild‐type strain, suggesting that the chromatin may be condensed. Ultrastructural studies show differences in the mutant nuclei including a prominent distinction between the chromatin‐ and non‐chromatin‐enriched regions compared to the more homogenous wild‐type nucleus. Two‐hybrid assays indicate that some of the wild‐type protein interactions are altered in the mutant strains. These results suggest that normal functioning of spU2AF59 may be essential not only for pre‐mRNA splicing but also for the maintenance of proper nuclear structure and normal cell cycle progression. Copyright © 2000 John Wiley & Sons, Ltd.

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