The parasitic protozoon produces multiple forms of cysteine proteinase (CP). The molecular basis for this has now been examined by cloning DNA fragments encoding CPs. Using generic degenerate oligonucleotide primers based on two well-conserved regions within the central region of all eukaryotic CPs, several polymerase chain reaction fragments were isolated from genomic DNA and shown to encode different CPs. One fragment with a well-represented sequence was used as a general probe to screen a cDNA library at moderate stringency and five different cDNA clones were isolated. Preliminary sequencing showed that they encoded similar but distinct CPs. In the process of confirming the 5′ end of one of these cDNA clones using RACE-PCR (rapid amplification of cDNA 5′ ends-polymerase chain reaction), an additional sequence encoding a different CP was identified. The corresponding clone (TvCP3) and the three longest clones from the library screen (TvCP1, TvCP2 and TvCP4) were characterized further. TvCP1 and TvCP2 were full-length and TvCP3 and TvCP4 were apparently slightly less than full-length. Comparison of the predicted amino acid sequences of the four clones showed that TvCP1 and TvCP4 are related (72% identity). TvCP2 is closer to TvCP1 (60%) and TvCP4 (65%) than is TvCP3, which has 53%, 59% and 56% identity to TvCP1, TvCP2 and TvCP4, respectively. Comparison with the sequences of other known CPs indicated that the gene products all belong to the cathepsin L/cathepsin H/papain branch of the papain superfamily. The TvCP1, TvCP2 and TvCP4 sequences are related (38-45% identity) to those of CP2 of , human cathepsin L, three CPs from lobster and CPs from black gram, oilseed rape and rice (oryzains α and β). TvCP3 shows less identity to the other eukaryotic CPs but is most similar to CP2 (38%). The four predicted amino acid sequences share some features distinct from the majority of CPs, which suggests they might have had a common evolutionary origin. The most striking feature of sequences TvCP1, TvCP2 and TvCP3 is the apparent lack of a pre-sequence (signal sequence) for TvCP1 and very short pre-sequences for TvCP2 and TvCP3. Southern analysis indicated that the organization of the genes corresponding to the TvCP cDNAs differs. The TvCP1, TvCP2 and TvCP3 genes are single-copy, whereas the TvCP4 gene appeared to be multiple-copy. Similarly sized, single abundant transcripts were present for all four sequences. Overall, the data show that we have identified a family of genes in which encode a number of CPs. In total, seven distinct sequences have been recognized. This suggests that the multiplicity of CP activities seen


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