Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA

Science

Volumn 277, Issue 5324, 1997, Pages 383-387

  Long, Roy M ^a   Singer, Robert H ^a   Meng, Xiuhua ^a   Gonzalez, Isabel ^a   Nasmyth, Kim ^a   Jansen, Ralf Peter ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

ACTIN; CHIMERIC PROTEIN; ENDONUCLEASE; MESSENGER RNA; MYOSIN; REPRESSOR PROTEIN;

ANIMAL CELL; ARTICLE; CELL DIVISION; CONTROLLED STUDY; MESSENGER RNA SYNTHESIS; NONHUMAN; PRIORITY JOURNAL; PROGENY; SACCHAROMYCES CEREVISIAE;

ACTINS; CELL CYCLE; CELL NUCLEUS; DEOXYRIBONUCLEASES, TYPE II SITE-SPECIFIC; DNA-BINDING PROTEINS; FUNGAL PROTEINS; GENES, FUNGAL; GENES, MATING TYPE, FUNGAL; IN SITU HYBRIDIZATION, FLUORESCENCE; MICROTUBULES; MUTATION; MYOSIN HEAVY CHAINS; MYOSIN TYPE V; MYOSINS; REPRESSOR PROTEINS; RNA, FUNGAL; RNA, MESSENGER; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; TRANSCRIPTION FACTORS; TRANSFORMATION, GENETIC; TROPOMYOSIN; ZINC FINGERS;

EID: 0030875775     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5324.383     Document Type: Article

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41. + RNA detection. Images were taken with an Olympus IX70 inverted epifluorescence microscope and Oncor (Gaithersburg, MD) imaging software, version 2.0.5.
42. 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 1X phosphate-buffered saline, 0.1% bovine serum albumin, 20 mM vanadyl ribonucleoside complex, and ribonuclease inhibitor (40U/μl). The secondary antibody, goat antibody to mouse immunoglobulin G, conjugated to dichlorotrianzinyl amino fluorescein (Jackson Laboratories), was diluted 1:50 into the same solution.
43. Plasmid C3431 is a derivative of YEplac195 (17) carrying a Sal I-Sac I ASH1 fragment.
44. Plasmid pHZ18-poly(A) containing the ADHII 3′-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 5′-GGGCCCGAGCTCGAGACAGTAGAGAATTGATACATG-3′ and 5′-GGGCCCGAGCTCATCAGGATGACCAATCTATTGCGC-3′. To verify that no mutations were introduced by PCR, the ASH1 region of plasmid pXMRS25 was confirmed by DNA sequencing.
45. 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).