Program R. For the analysis, T, C, G, and A bases were replaced by the numbers 1, 2, 3, and 4, respectively, and were converted into triplet codons. The proportion of genes affected by the presence of 59-UTRs was calculated by dividing the number of bases in the 59-UTRs by 3 and collecting those 59UTRs which had non-zero remainders. In order to calculate the number of 59-UTRs which contained stop codons, we combined the bases into triplets as described above (starting from the first base) and identified the 59-UTRs containing at least one stop codon.In-frame cDNA Expression LibraryFor the construction of an in-frame cDNA expression library, mRNA was isolated from human normal ureters using TRIzol reagent. (Life Technologies) The first-strand cDNA was synthesized using the polyT primer. 59-TAGATCCGGTGGATCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGC39 and SMARTScribe reverse transcriptase (BD Bioscience Clontech). The polyT primer contained the TAGATCCGGTGGATC ligation sequence to facilitate the subsequent homologous recombination of the 39 site of the amplified cDNAs. To synthesize double-stranded cDNAs without the 59-UTRs, we used a mixture of forward primers reflecting the combination of Kozak sequences in vertebrate genomes [8] (Table 1). All forward primers contained the same ligation sequence corresponding to positions 1?6, which facilitated the homologous recombination of the 59 site of the amplified cDNAs. Positions 17?32 in the forward primers corresponded to sequence variations around the translational start site (positions 212 to +4) of the reported Kozak sequences [8]. The most frequent consensus sequences for the initiation of translation are in primers 1 and 2, which 166518-60-1 correspond to approximately 40 of Kozak sequences in vertebrates. In order to complement mRNAs containing the remaining combinations of the Kozak sequences, we designed the primer mixes 3 and 4 as sequence combinations reflecting all reported variations at positions 17?5, 27, 28, and 32 of the primer design. At position 17, the sequence frequency was reported as A 23 , G 23 , C 35 , and T 19 . Thus, an equal mixture of A, G, and T at position 17 was used in primer mixesand 4 to approximate this frequency. The nucleotide C was excluded from the combinatorial design at position 17 AN-3199 web because it was already included in the design of primers 1 and 2. Then, in a similar manner, the sequence combinations for positions 18?4 and 32 were designed to reflect their approximate relative frequencies in vertebrate genomes. At position 25, the frequency of A was 25 , G 15 , and T 7 . Because the frequency of A was much higher than G or T at this position, we selected nucleotide A at position 25 for primer mix 3 and the mixture of G and T at position 25 for primer mix 4. Similar combinations were designed for positions 27 and 28 in primer mixes 3 and 4. In summary, in primer mixes 3 and 4, “D” was an equal mixture of A, G and T, “H” was an equal mixture of A, C and T, “K” was an equal mixture of G and T, and “W” was an equal mixture of A and T. There were 19,683 and 157,464 possible sequence combinations for primer mixes 3 and 4, respectively. We show nine sequence combinations for nucleotides at positions 17 and 18 in primer mix 4 as an example. Sequence combinations 1?3 at position 17 represent A, G, and T, respectively. All possible sequence combinations at position 17 are shown as combinations 4?, and the sequence combinations with an equal frequency of A, G, and T are shown fo.Program R. For the analysis, T, C, G, and A bases were replaced by the numbers 1, 2, 3, and 4, respectively, and were converted into triplet codons. The proportion of genes affected by the presence of 59-UTRs was calculated by dividing the number of bases in the 59-UTRs by 3 and collecting those 59UTRs which had non-zero remainders. In order to calculate the number of 59-UTRs which contained stop codons, we combined the bases into triplets as described above (starting from the first base) and identified the 59-UTRs containing at least one stop codon.In-frame cDNA Expression LibraryFor the construction of an in-frame cDNA expression library, mRNA was isolated from human normal ureters using TRIzol reagent. (Life Technologies) The first-strand cDNA was synthesized using the polyT primer. 59-TAGATCCGGTGGATCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGC39 and SMARTScribe reverse transcriptase (BD Bioscience Clontech). The polyT primer contained the TAGATCCGGTGGATC ligation sequence to facilitate the subsequent homologous recombination of the 39 site of the amplified cDNAs. To synthesize double-stranded cDNAs without the 59-UTRs, we used a mixture of forward primers reflecting the combination of Kozak sequences in vertebrate genomes [8] (Table 1). All forward primers contained the same ligation sequence corresponding to positions 1?6, which facilitated the homologous recombination of the 59 site of the amplified cDNAs. Positions 17?32 in the forward primers corresponded to sequence variations around the translational start site (positions 212 to +4) of the reported Kozak sequences [8]. The most frequent consensus sequences for the initiation of translation are in primers 1 and 2, which correspond to approximately 40 of Kozak sequences in vertebrates. In order to complement mRNAs containing the remaining combinations of the Kozak sequences, we designed the primer mixes 3 and 4 as sequence combinations reflecting all reported variations at positions 17?5, 27, 28, and 32 of the primer design. At position 17, the sequence frequency was reported as A 23 , G 23 , C 35 , and T 19 . Thus, an equal mixture of A, G, and T at position 17 was used in primer mixesand 4 to approximate this frequency. The nucleotide C was excluded from the combinatorial design at position 17 because it was already included in the design of primers 1 and 2. Then, in a similar manner, the sequence combinations for positions 18?4 and 32 were designed to reflect their approximate relative frequencies in vertebrate genomes. At position 25, the frequency of A was 25 , G 15 , and T 7 . Because the frequency of A was much higher than G or T at this position, we selected nucleotide A at position 25 for primer mix 3 and the mixture of G and T at position 25 for primer mix 4. Similar combinations were designed for positions 27 and 28 in primer mixes 3 and 4. In summary, in primer mixes 3 and 4, “D” was an equal mixture of A, G and T, “H” was an equal mixture of A, C and T, “K” was an equal mixture of G and T, and “W” was an equal mixture of A and T. There were 19,683 and 157,464 possible sequence combinations for primer mixes 3 and 4, respectively. We show nine sequence combinations for nucleotides at positions 17 and 18 in primer mix 4 as an example. Sequence combinations 1?3 at position 17 represent A, G, and T, respectively. All possible sequence combinations at position 17 are shown as combinations 4?, and the sequence combinations with an equal frequency of A, G, and T are shown fo.
