Murine Pkd1 introns 21 and 22 lack the extreme polypyrimidine bias present in human PKD1.

Autosomal dominant polycystic kidney disease (ADPKD) is estimated to affect between 1/200–1/1000 people, making it one of the most common genetic disorders in humans (Gabow 1993). Mutations of PKD1 on Chromosome (Chr) 16p13.3 account for 85–95% of all cases. PKD1 is a relatively compact gene, with its 46 exons embedded within an ∼50kb genomic segment. The gene has an unusual bipartite structure, with 70% of its 58 end duplicated in multiple copies elsewhere on Chr 16. The gene encodes a 14-kb mRNA that is translated into a protein product of >400kDa. It has not yet been determined whether the homologous loci, which also are transcribed, encode proteins or are pseudogenes. Another striking feature of the human PKD1 gene is a cluster of two lengthy polypyrimidine tracts in adjacent introns in the center of the gene. Intron 21 contains a 2.5-kb polypyrimidine tract which is comprised of 23 mirror repeats, while intron 22 has a much shorter segment of ∼550 bp. The CT-element of intron 21 is the longest polypyrimidine tract yet described in the human genome. While the biological significance of these structures in PKD1 is unknown, one can make some predictions based on the results of studies with much shorter polypyrimidine tracts. It has been previously shown that polypyrimidine tracts can form triple helices (H-DNA) that have diverse biological effects (Mirkin and Frank-Kamenetskii 1994). These alternative DNA structures have been shown to affect gene expression by inhibiting gene transcription and affecting spliceosome assembly and selection of splice sites. Triple helices also may play a role in the organization of the chromosomal environment by forming nuclear attachment regions (MARs) (Michel et al. 1993) or affecting nucleosome phasing (Westin et al. 1995). Finally, triplex structures may play a role in enhancing mutagenesis by a variety of mechanisms. In a bacterial system, investigators have shown that triplex formation between two homologous sequences promoted recombination when transcription of the downstream gene was activated (Kohwi and Panchenko 1993). Wang and associates (1996) found a tenfold increase in mutability in sequences adjacent to a site of triplex formation in a mammalian cell culture model. What is particularly noteworthy about the study by Wang and coworkers that the triplex segment was approximately 100-fold shorter than the one present in PKD1 intron 21. Given the unusual length of the polypyrimidine tracts in PKD1 and the large number of possible biological effects associated with their presence, we sought to determine whether the polypyrimidine tracts were present in the murine homolog (Lohning et al. 1997). We reasoned that if the structures were conserved, they might play an important role in regulating the gene’s activity. Conversely, their presence in humans but absence from the genome of mice might implicate the structures in the gene’s mutability in humans. In order to resolve this question, we isolated three P1 clones (A, B and C) containing murine genomic DNA (strain 129 Ola), using a PCR-based screening strategy for introns 1 and 2 of mPkd1. Further mapping determined that each clone was unique, but that only clone C contained exon 24 (data not shown). We next sought to determine whether the lengthy polypyrimidine tracts present in human introns 21 and 22 were conserved in the corresponding position in the mouse genome with hybridization methods. A Southern blot of restriction digests of clone C and cGGG10 (included as a positive control because it contains the corresponding segment of human PKD1) was probed with radiolabeled insert of pGGG13. The results are presented in Fig. 1. Only nonspecific hybridization is detected in lanes containing mPkd1, while a strong signal is present in the human counterpart. The hybridization data suggested that mPkd1 lacks the polypyrimidine tracts present in the human gene. We sought to confirm this result by determining the sequence of the region in question. Restriction analyses and Southern blot hybridization studies of clone C identified a 7.4-kb EcoRV fragment that contained both exons 20 and 24, and thus the introns of interest. We subcloned the fragment into pBluescript and determined the sequence of its ends, using vector-based primers. The sequence revealed that the fragment contained a genomic fragment that spanned from exon 15 to intron 26. A B1 element was identified in the segment immediately adjacent to the cloning site in intron 26. In order to sequence the region between exons 21 and 23, a XbaI/KpnI fragment was subcloned into pBlusescript and sequenced with T3, T7, and a gene-specific primer located in exon 21. A comparison of the murine and human PKD1 gene structures of this region, along with the respective CT content of each, is presented in Fig. 2. Murine intron 21, which is 760 bp in length, has no remarkable features and lacks an obvious polypyrimidine bias or high GC composition (CT content 48.4%, GC content 53.2%). Murine intron 22 is only 89 bp long but is pyrimidine-rich (75.3% CT with 46.1% C) and contains a potential short mirror repeat (stem length 8 nucleotides, loop length 10 nucleotides), which is formed by two direct repeats (Fig. 3). The direct repeats are present only once each in murine intron 22, but the smaller repeat is present 47 times in human intron 21, and 7 times in human intron 22. The larger repeat covers 18.8% of the polypyrimidine tract in human intron 21 (copy number 47) and is present once in human intron 22. Both introns contain consensus mammalian splice site and branch sequences (underlined in Fig. 3). Murine exon 21 is 150 bp in length, which is three nucleotides shorter than the corresponding human exon. The sequence data described in this study clearly show that mPkd1 does not contain the polypyrimidine tracts found in introns 21 and 22 of the human gene (American PKD1 Consortium 1995; Van Raay et al. 1996). The murine gene has no pyrimidine bias in intron 21 and only a modest enrichment for pyrimidines in its very short intron 22. This is particularly noteworthy given the otherwise conserved genomic structure such as the tail-to-tail organization with the neighboring TSC2 gene (Brook-Carter et al. 1994) and the B1 element found in intron 26 that corresponds to an Alu repeat in the human intron 26. Alu repeats and B1 elements share 70–80% Correspondence to: G.G. Germino Mammalian Genome 10, 194–196 (1999).