Molecular cloning and characterization of the DNA mismatch repair gene class 2 from the Trypanosoma cruzi.
Genes with homology to the bacterial mutS gene, which encodes a protein involved in post-replication DNA mismatch repair, are known in several organisms. Using a degenerate PCR strategy, we cloned a Trypanosoma cruzi genomic DNA fragment homologous to the mutS gene class two (MSH2). This fragment was used as a probe to select the corresponding cDNAs from a T. cruzi cDNA library. The complete sequence of the gene (3304 bp), denominated TcMSH2, was obtained. The sequence contained an open reading frame of 2889 bp coding for a putative protein of 962 amino acids. Computational analyses of the amino acid sequence showed 36% identity with MSH2 proteins from other eukaryotes and revealed the presence of all functional domains of MutS proteins. Hybridization analyses indicated that the TcMSH2 gene is present as a single copy gene that is expressed in all forms of the T. cruzi life cycle. The role of the product of the TcMSH2 gene in mismatch repair was investigated by negative dominance phenotype analyses in Escherichia coli. When eukaryotic muts genes are expressed in a prokaryotic system, they increase the bacterial mutation rate. The same phenomenon was observed with the TcMSH2 cDNA, indicating that T. cruzi MSH2 interferes with the bacterial mismatch system. Phylogenetic analyses showed that the T. cruzi gene grouped with the MSH2 clade confirming the nature of the gene isolated in this work.
Augusto-Pinto
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Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Bartholomeu
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Teixeira
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Pena
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Machado
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D008969:Molecular Sequence Data; D051718:MutS Homolog 2 Protein; D010802:Phylogeny; D015800:Protozoan Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000254:growth & development
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; C435509:MSH2 protein, Trypanosoma cruzi; D051718:MutS Homolog 2 Protein