PrP-like gene
a technology of prp and gene, applied in the field of prplike gene, can solve the problem that dpl may also be toxic to other non-neuronal cell types
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example 1
Identification of the Prnd Locus
[0197] Previous large-scale sequencing studies of phage and cosmid molecular clones encompassing human, sheep, and the "a" allele of the mouse PrP gene, Prnp.sup.a, failed to reveal any evidence of additional coding regions, either immediately adjacent to PrP or within intronic sequences (Lee et al., Genome Res. 8:1022-1037 (1998)). However, sequencing of the mouse Prnp.sup.b cosmid clone I / LnJ-4 (Westaway et al., Neuron 7:59-68 (1991)), which extends further in a 3' direction and analysis with the "XGrail 1.3 c" program (Uberbacher et al, ORNL / TM 11741 (1991)) revealed a candidate coding region at nt 36119 to 36,751 (FIG. 1A).
[0198] Cosmids and YAC clones were propagated by standard procedures and have been described previously (D. Westaway, Cell 76:117-29 (1994)). A 129Sv / J BAC clone 321L17 was identified from a library kindly provided by Millenium Pharmaceuticals.
[0199] The predicted amino acid sequence of this ORF (FIG. 2) exhibits homology to mam...
example 2
Structure of the Prnd Locus
[0201] Prnd cDNAs were isolated to demonstrate the splicing of mRNAs transcribed from this region and to define appropriate coding exons. The complimentary approaches of cDNA library screening and rapid amplification of cDNA ends (RACE) were undertaken using wild type adult mouse brain cDNA as starting material.
[0202] RACE cloning of PrnD RNAs
[0203] For 5' RACE analysis, "Marathon" mouse brain cDNA (Clontech, Palo Alto, Calif.) was amplified with adapter primer AP1 and the Prnd anti-sense strand primer DW112, 5'-CGGTTGGTCCACGGCGACCCGAA-3'(0.2 FM), using a Perkin-Elmer 2400 thermocycler and "touchdown" PCR conditions of 94 EC 20 seconds, 5 cycles of 94 EC, 5 sec and 72 EC, 360 seconds; 5 cycles of 94 EC, 5 seconds and 70 EC 360 seconds; and 30 cycles of 94 EC, 5 seconds and 68 EC, 360 seconds. Taq polymerase was used in conjunction with "TaqStart" antibody (Clontech, Palo Alto, Calif.). Size-fractionated reaction products were resuspended in Tricine buffer ...
example 3
Expression of Transcripts from the Prnd Locus
[0225] Northern blot analyses were performed using the entire 570 bp PrnD ORF fragment derived from the ILn / J-4 cosmid using total (50 g loading) or oligo-dT selected RNA (7 g loading). Total RNA was made using Trizol reagent (Gibco) following the manufacturers instructions. Poly A+ RNA was isolated using a Oligotex mRNA kit (Qiagen). RNA samples were heated for 30 minutes to 50 deg C. in glyoxal sample buffer and electrophoresed on 1.2% agarose gels as recommended by the manufacturer (Ambion Inc., Woodland, Tex.). RNA was transferred onto Hybond N+ (Amersham) in 5 XSSC 10 mM NaOH for 2 hours using a turboblotter (Schleicher and Schuell), rinsed in 5 XSSC, UV fixed (Statagene, La Jolla, Calif.) then prehybridized (6 xSSC, 1% SDS, 3% (w / v) dextran sulfate, 10 ug / ml sonicated herring sperm DNA) for 1 hour at 65 EC prior to the addition of a 540 bp Dpl ORF PCR a-dCTP.sup.32 random-primed probe (Feinberg et al., Anal. Biochem. 132:6-13 (1983)...
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