Figure 6

C3H/He and 129/SV mice lack an intracisternal A-particle (IAP) insert in the first intron of the nocturnin/CCR4 gene. A. Diagram showing the results of partial sequencing of a nocturnin genomic clone derived from a 129/SV bacterial artificial chromosome (BAC) library. Green boxes identify regions of medium repetitive sequences in the mouse genome. The blue arrows represent mNoc coding regions corresponding to Xenopus exons II and III separated by an intron. The IAP element identified in BALB/c mice (bar above) was expected between bp 6583 and 6584 in our BAC sequence based on published data [39] but was lacking in this BAC sequence. F1, F2 and R1 are the positions of forward and reverse primers used for genomic PCR. Note that there are two potential polyadenylation sites in the 3' UTR 650 base pairs apart. The 3' UTR probe used in our northern analysis lies between the two potential polyadenylation sites, and hybridizes to the same mRNA band as the probe derived from Exon II. This suggests that only the most 3' site is used. B. Genomic PCR with primers from the BAC clone flanking the IAP site (labeled as F1 and R1 in A) resulted in a 107 bp band in C3H/He and 129SV mice that was lacking in BALB/c mice. Pairing the R1 primer from the BAC clone with primer F2 derived from the IAP sequence (see A) resulted in a 1500 bp band in BALB/c mice but not 129SV or C3H/He. DNA sequencing revealed the expected sequence from the BAC clone for the 107 bp band and the expected sequence from the IAP insert for the 1500 bp band. The lanes labeled 412D6 are control PCR reactions using the 129/SV BAC as template. Note that the absence of ≅ 5 Kb band for BALB/c mice with primers F1 and R1 is due to the inefficiency of Taq polymerase for large products; we have separately obtained the full IAP insert in BALB/c mice using a long PCR procedure. DNA size markers are included in the two outer lanes.