Slides were dried on a 50☌ hot plate for 30 s, then fixed in Mirsky's fixative for 30 min at room temperature and washed three times within 10 min with phosphate-buffered saline (PBS) and Tween 20 (PBS/Tween 20, 0.1%). They were then decapitated, and the heads were placed in OCT 4583 embedding medium (Tissue-Tek, Torrance, CA), frozen on dry ice, and sectioned. Whole flies were submerged in Mirsky's fixative (National Diagnostics, Atlanta, GA) for 1 to 2 min. These plasmids express polyglutamine tracts flanked by 8 amino acids on the NH 2-terminal side and 13 amino acids on the COOH-terminal side (MGGPPSTPQ nTSRTYPYDVPDYA) (37). The PCR fragment was digested with Eco RI and Spe I and, with a Pst I–Eco RI adapter, was inserted in-frame with an HA tag DNA sequence into the Pst I–Spe I fragment of the pINDY6 transgenic vector (36). To produce UAS-20Q and UAS-127Q, pol圜AG 20 and pol圜AG 127 and their flanking sequences were PCR-amplified by primers 5′ Gln2F (5′-CGG AAT TCG CCG CCA CCA TGG GAG GCC CAC CGT CAA CCC CCC AGC AG-3′) and 3′ GlnR (5′-ATT GCT GTT GCC GCC GTT ACT AGT CTG TTG CTG CTG CTG TTG-3′). To synthesize pol圜AG of 127 repeats, this procedure was repeated twice more. coli (Stratagene), the sequence between the two pol圜AG tracts was removed by digesting with Bst BI and Afl II (or Bfr I) and trimming the overhanging ends with mung bean nuclease (New England Biolabs), followed by ligation and transformation into XL1 Blue. After cloning and amplifying this construct in XL1 Blue strain of E. These fragments were digested with Bam HI (5′ fragment) or Bst XI (3′ fragment) and ligated with T4 DNA ligase (Gibco/BRL) into the Bam HI–Bst XI fragment of p139cAC1. Primers used to amplify two fragments containing pol圜AG tracts were as follows: (i) ProsBamHI3229F (5′-ATG CGC GGA TCC CAG CAG CTG GAG CAG AAC GAG GCC-3′) with 5′ phosphorylated–ProsAflIIR (5′-ATT GCT GTT GCC GCC GTT CTT AAG CTG TTG TTG TTG CTG TTG TTG-3′) and (ii) ProsBstBIF (5′-ACC GGA GGC CCA CCG TCA TTC GAA CAG CAG CAG CAA CAG-3′) with Pros3650R (5′-GCT GCG TGC GGA TTG AAG AAC GGC-3′). Because it has one of the longest known CAG tracts in the fly (20 repeats), the prospero gene in the p139cAC1 plasmid (35) was used as a template for PCR synthesis of expanded pol圜AG tracts.
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