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1386–1391 Nucleic Acids Research, 1999, Vol. 27, No. 5

 1999 Oxford University Press

Mammalian polyadenylation sites: implications for differential display Michael R. Frost and Jeremy A. Guggenheim* Department of Optometry and Vision Sciences, University of Wales, Cardiff, King Edward VII Avenue, Cardiff CF1 3XF, UK Received October 19, 1998; Revised and Accepted January 7, 1999

ABSTRACT Differential display relies on a series of anchored primers to divide the total mRNA population into subsets of roughly equal size. However, this will only occur if the dinucleotide targeted by the anchor region of the anchored primers has a random frequency distribution [i.e. each of the 12 possible dinucleotides preceding the poly(A) tail occur with the same frequency]. Previous reports have suggested that this is not the case and that the frequency distribution of the targeted dinucleotide can vary as much as 10-fold. In an analysis of several hundred unrelated mammalian mRNA sequences, we confirmed that the frequency of this particular dinucleotide does vary, although C > G. This same order of site preference has also been described by Chen et al. (19) in their analysis of cleavage site determinants in the mammalian polyadenylation signal. Table 3. Theoretical and experimental evaluations of the proportion of the total mRNA population targeted by differential display anchored primers

We conclude that the similarity in the number of bands produced by each anchored primer is most likely due to promiscuous priming of the reverse transcriptase. Two types of mispriming event can be envisaged. First, mispriming of one or more bases in the anchor region of the primer and, second, priming at an internal site on the mRNA rather than to the poly(A) tail. There is experimental evidence that both types of mispriming event are common (see below). To explore the reverse transcriptase mispriming phenomenon in more detail, we tested the ability of a β-actin forward primer to amplify the 3′-region of tree shrew retinal β-actin mRNA when used in conjunction with each of the 12 differential display anchored primers. A series of 12 3′ RACE (rapid amplification of cDNA ends) reactions were performed, with each reverse transcription reaction being carried out exactly as for differential display, i.e. each reaction containing a different anchored primer (the RACE paradigm is analogous to differential display except that a specific forward primer is substituted for the non-specific arbitrary primer). Following reverse transcription, 19 cycles of high-stringency PCR were performed. (Note that the first, low-stringency cycle adopted in our differential display experiments was replaced in these RACE experiments by a high-stringency cycle to reduce mispriming by the β-actin primer. Otherwise, reaction conditions were identical to those for differential display.) A second set of PCR reactions was conducted as described above, but with an annealing temperature of 65 instead of 55C to increase the stringency of priming. Using the 3′ RACE paradigm, we expected not only to be able to identify reverse transcriptase mispriming, but also to be able to differentiate between the two types of mispriming event mentioned

Figure 3. Comparative 3′ RACE reactions, each employing one of the 12 different anchored primers (A–M) in combination with an upstream β-actin primer (5′-TGGAGAAGAGCTACGAGCTGCCTG-3′), using an annealing temperature of 55 (A) or 65C (B). The expected product of 1067 bp is evident in several lanes at both annealing temperatures.

above, namely mispriming in the anchor region adjacent to the poly(A) tail and internal priming. β-Actin was chosen as a target since it is abundantly expressed in most mammalian tissues and because its mRNA is likely to have only one possible 3′-end sequence. We based this assumption on the evidence that β-actin is usually transcribed from a single gene in mammals and without splice variants. Thus, although numerous β-actin pseudogenes exist in most mammalian genomes, they are not normally expressed (20,21). If reverse transcriptase mispriming were never to occur, we would expect only one of the 12 anchored primers to yield a β-actin 3′ RACE product. However, if mispriming did occur, we would expect several of the anchored primers to yield products. Whereas mispriming in the anchor region would generate products of equal length, internal priming would be expected to yield truncated products. Our results (Fig. 3) suggest that both types of reverse transcriptase mispriming have occurred. Full-length (∼1 kb) 3′ RACE products were produced by several anchored primers (subsequent DNA sequencing and high-stringency hybridisation with a β-actin probe showed that 11 of the 12 anchored primers gave rise to the full-length product (see Fig. S1 in supplementary material). Truncated products were also synthesised (a minority of which were homologous to β-actin).

1391 Nucleic Acids Acids Research, Research,1994, 1999,Vol. Vol.22, 27,No. No.15 Nucleic Even raising the annealing temperature of the subsequent PCR reaction above the optimal Tm of the primers in an attempt to increase stringency, and therefore further limit mispriming due to Taq polymerase rather than reverse transcriptase, did not prevent this effect. In conclusion, our results appear to substantiate one of the key theoretical principles of Liang and Pardee’s differential display paradigm (8), namely that anchored primers provide a mechanism for dividing the total mRNA population into 12 subsets of roughly equal size. In an analysis of several hundred unrelated mammalian mRNA sequences, we confirmed that although the frequency of the dinucleotide targeted by the anchor region of anchored primers does vary, the extent of this variation is