to 10 ng and 20 mg, respectively. The composition of buffer B was 1.2 M sorbitol and 10 mM KHPO4 at pH 7.5. After the second posthybridization wash in 40% formamide and 23 standard saline citrate (SSC), a 15min wash at room temperature in 23 SSC and 0.1% Triton X-100 was done. The oligonucleotide probes were as follows: 59-GCTT*GCCTTGTTGAATT*CTGGTGAATT*GCCTGGTGTT*AATGAGGAAATT*GG-39, 59-GATGCCT T*AGTGATGGT*AGGCT T TGT TGT*GGGCGCTCCGGT*CTCT TAGAT*A-39, 59-GGAACT T*GGACGACCTAGT*CGAT TCCAAT T*CCT TGCCGT*AAT TGAAACT*AT-39, 59-AT*GGT TCTAT T*GGT TGGTGGACT*CATCGGCGGTGT*GACGGGAGGAGTAAT*A-39, 59-AAGCT* T TGAAACTGT T*CGTCT T T TTGT*GACTGGCAT T T*GGCATGGGAAAT*G-39, and 59-GT*CGAGAGCAAATCTAT*GATAAT TGGG*GACCT TGGGCT* TGGAGTGTAT*GC-39. The probes were directly labeled with a Cy3 fluorochrome at amino-modified thymidine residues indicated by the asterisks (19). To detect poly(A)1 RNA, FISH was performed with T43 labeled with fluorescein isothiocyanate (20). In formamidecontaining solutions, the concentration was reduced to 10% for poly(A)1 RNA detection. Images were taken with an Olympus IX70 inverted epifluorescence microscope and Oncor (Gaithersburg, MD) imaging software, version 2.0.5. 24. Strain K5552, which encodes an epitope-tagged version of Ash1p (Ash1p-myc9), was grown to midlogarithmic phase, fixed, and processed for simultaneous FISH and immunofluorescence. After FISH, immunofluorescence was performed as described previously (21) with the following alterations. Antibody to myc was diluted 1:5 into a solution of 13 phosphate-buffered saline, 0.1% bovine serum albumin, 20 mM vanadyl ribonucleoside complex, and ribonuclease inhibitor (40 U/ml). The secondary antibody, goat antibody to mouse immunoglobulin G, conjugated to dichlorotrianzinyl amino fluorescein (Jackson Laboratories), was diluted 1:50 into the same solution. 25. Plasmid C3431 is a derivative of YEplac195 (17 ) carrying a Sal I–Sac I ASH1 fragment. 26. Plasmid pHZ18-poly(A) containing the ADHII 39-UTR has been described (5). Plasmid pXMRS25 was constructed from pHZ18 (22) by insertion of an ASH1 fragment generated by the polymerase chain reaction (PCR). The PCR product contained the last five amino acid codons of ASH1 and extended 250 nucleotides beyond the stop codon. The ASH1 fragment was subcloned into the Sac I site of pHZ18 by the inclusion of a Sac I restriction site in the PCR primers. The primers for PCR were 59-GGGCCCGAGCTCGAGACAGTAGAGAATTGATACATG39 and 59-GGGCCCGAGCTCATCAGGATGACCAATCTATTGCGC-39. To verify that no mutations were introduced by PCR, the ASH1 region of plasmid pXMRS25 was confirmed by DNA sequencing. 27. We thank M. Rosbash for initiating our collaboration and D. Amberg, S. Brown, A. Bretscher, B. Haarer, and P. Novick for providing yeast strains. Supported by NIH grant GM54887 (to R.H.S) and NIH–National Institute of Child Health and Human Development fellowship 7 F32 HD08088-02 (to R.M.L).