Restriction digests of the clone give the following sizes (kb): PstI-5.4, 3.1; PvuII--4.8, 3.9; BamHI--6.6, 1.9; EcoRI--4.6, 4.2. Insert contains these sites in kb from 5' EcoRI site: SstI/0.2; XbaI/?; BglII/0.57; PstI/2.3; XbaI/2.4; BamHI/2.7; AvaI/?; XbaI/3.6; SstI/3.7; EcoRI/4.0. The insert includes exon 2 to exon 6 of the intronless gene in a 0.57 EcoRI/BglII fragment at the 5' end, and a poly(A) tail. The 5' portion of the insert between EcoRI and AvaI (about 2.9 kb) contains sequences homologous to the 3' end of the normal DHFR gene. The sequence is unique, but also cross-hybridizes to pseudogenes. Recorded as not polymorphic by Collaborative Research, meaning either not polymorphic in 0.8% agarose gels, or difficult to work with. PstI detects a deletion polymorphism with the A2 allele resulting from the absence of the pseudogene. EcoRI (-/4.0), BamHI (-/5.0), and BglII (6.7/3.85) also detect the polymorphism. The polymorphism can be detected with a specific 0.42 kb XbaI/EcoRI fragment of the insert. |
