Fig. 1. A 12% SDS-polyacrylamide gel of eluants from the ssDNA column. Lanes 1 and 5, molecular weight standards; lane 2, 0.3 M KCl eluant; lane 3, 0.5 M KCl eluant; lane 4, 1.0 M KCl eluant. Molecular weight standards include: phosphorylase B, 97 400, serum albumin, 66 200, ovalbumin, 45 000, carbonic anhydrase, 31 000, trypsin inhibitor, 21 500, and lysozyme, 14 400.

Fig. 2. Schematic map representing the p34 and p37 genes. Panel A, major RT-PCR products from cDNA. Panel B, major genomic DNA fragment amplified from genomic DNA. The thin bars represent cDNA or genomic DNA templates used for PCR. AAA indicates the polyA tail of the original mRNA. Primers annealing to specific positions are shown below by short arrows pointing in the 3′ direction (F for forward primers and R for reverse primers). ME indicates the miniexon primer. The thick bars represent major RT-PCR and PCR products. The unique 54 nt insertion is marked by a gray square. Panel C, genomic restriction map of the two gene loci based on sequence information and Southern blot analysis. The two black boxes represent the two respective gene coding regions as indicated. Southern blot probes are shown below by thick bars. Abbreviations for restriction sites: B, BamHI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SacI; X, XhoI.

Fig. 3. Alignment and comparison of nucleotide and deduced amino acid sequences of cDNA1 and cDNA2 [encoding p34 and p37 respectively]. The dots indicate positions with identical nucleotides or amino acid residues. The dashed lines indicate gaps introduced to allow optimal sequence alignment. Nonidentical sequence is indicated with letters. The internal peptide sequences obtained previously are denoted by underlines. The primers used in this study are indicated by arrows. In the 3′-UTRs, all sequences are indicated by letters due to the lack of obvious homology.

Fig. 4. Alignment of p34/p37 RBD homology regions with RBD consensus and homologous sequences. The RBD consensus sequences are indicated with single-letter amino acid codes. Alternative consensus residues at the same position are shown on the second or third line. Non-consensus sequences in the RBD are indicated with (x). Asterisks (*) above the p34/37 sequences indicate amino acids identical to the consensus sequence. Gaps (-) are introduced to allow optimum alignment. Chicken nucleolin, Xenopus laevis (Xl) nucleolin, human inducible poly A-binding protein (Hs iPABP), Schizosaccharomyces pombe (Sp) PABP, Trypanosoma cruzi (Tc) PABP, and Drosophila melanogaster (Dm) lark protein homology region sequences are aligned with the p34/37 amino acids 172– 209/190–217. Residues in this region similar or identical to at least four aligned sequences are indicated by pluses.

Fig. 5. Summary of the sequence motifs in p34 and p37. The p34 and p37 amino acid sequences are represented by blocks. Amino acids different between the two proteins are indicated between the blocks. Sequence motifs indicated are as described in the text. The asterisk indicates the lack of the carboxy-terminal residue of p37 as compared with p34.

Fig. 6. Competition assays to determine the binding specificity of p34/37. Competitors used are as indicated. Tb RNA, T. brucei procyclic form total RNA, Tb ssDNA/dsDNA, T. brucei procyclic form denatured genomic DNA/native genomic DNA. The ratio (w/w) of each competitor versus the amount of ssDNA is indicated above each lane. Proteins bound to the ssDNA beads were detected by Western blot analysis. The upper panel represents competition assays with recombinant p37, the middle panel with recombinant p34, and the lower panel with native p34 and p37 from the ssDNA agarose column.

Fig. 7. Analysis of purified recombinant proteins. Panel A. 12% SDS-polyacrylamide gel. Panel B, Western blot analysis. Lane 1, native p34/37 in 1.0 M fraction from a ssDNA agarose column. Lane 2, recombinant p34, lane 3, recombinant p37.