RESUMO
Spiny tailed iguanas are highly diverse clade of lizards in Mesoamerica, ranging from northern Mexico through Panama. Utilizing 2 regions of mitochondrial DNA (1948bp) and 4 nuclear loci (2232bp) we explored the relationships between these species and the phylogeographic history of the major clades. We discovered that the lineage endemic to the Yucatan Peninsula renders the genus Ctenosaura paraphyletic. To resolve this non-monophyly, we resurrect the taxon Cachryx Cope, 1866, and provide a new diagnosis for the genus. We also find that small body-size and highly spinose tails in the species previously referred to the subgenus Enyaliosaurus, have evolved independently 3 times. Cachryx were recovered as sister to the lineage of iguanines endemic to the Galapagos Islands, and we discuss biogeographic scenarios to explain this relationship as well as those among the primary clades of Ctenosaura in Mesoamerica.
Assuntos
Iguanas/classificação , Animais , Evolução Biológica , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Haplótipos , Iguanas/genética , México , Proteínas MutL/classificação , Proteínas MutL/genética , NADH Desidrogenase/classificação , NADH Desidrogenase/genética , Panamá , Filogenia , Filogeografia , Análise de Sequência de DNARESUMO
Mutator strains were identified by screening random Tn5 insertion clones of Caulobacter crescentus. We identified clones with robust increases in mutation rates with Tn5 insertions in the mutY, mutS, mutL and uvrD genes, known to act in mutation-preventing pathways in Escherichia coli. Analysis of mutations in the rpoB gene revealed that in both the parental strain and mismatch repair-deficient mutants, A:TâG:C transitions predominate by a large margin over C:GâT:A. We have also investigated the role of the error-prone polymerase encoded by imuC (dnaE2) in spontaneous mutagenesis, and found that a imuC mutant strain shows mutation rates and sequences comparable to the parental strain. Our study characterizes for the first time mutator strains in a member of the alphaproteobacteria group. In spite of the limitations of using a single marker, possible reasons for the observed mutational bias are discussed in the light of the repertoire of DNA repair genes in this bacterium.
Assuntos
Caulobacter crescentus/genética , Reparo de Erro de Pareamento de DNA , Mutagênese , DNA Helicases/genética , Proteínas MutL/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genéticaRESUMO
Lynch syndrome (LS) accounts for 3-5% of all colorectal cancers (CRC) and is inherited in an autosomal dominant fashion. This syndrome is characterized by early CRC onset, high incidence of tumors in the ascending colon, excess of synchronous/metachronous tumors and extra-colonic tumors. Nowadays, LS is regarded of patients who carry deleterious germline mutations in one of the five mismatch repair genes (MMR), mostly in MLH1 and MSH2, but also in MSH6, PMS1 and PMS2. To comprehensively characterize 116 Brazilian patients suspected for LS, we assessed the frequency of germline mutations in the three minor genes MSH6, PMS1 and PMS2 in 82 patients negative for point mutations in MLH1 and MSH2. We also assessed large genomic rearrangements by MLPA for detecting copy number variations (CNVs) in MLH1, MSH2 and MSH6 generating a broad characterization of MMR genes. The complete analysis of the five MMR genes revealed 45 carriers of pathogenic mutations, including 25 in MSH2, 15 in MLH1, four in MSH6 and one in PMS2. Eleven novel pathogenic mutations (6 in MSH2, 4 in MSH6 and one in PMS2), and 11 variants of unknown significance (VUS) were found. Mutations in the MLH1 and MSH2 genes represented 89% of all mutations (40/45), whereas the three MMR genes (MSH6, PMS1 and PMS2) accounted for 11% (5/45). We also investigated the MLH1 p.Leu676Pro VUS located in the PMS2 interaction domain and our results revealed that this variant displayed no defective function in terms of cellular location and heterodimer interaction. Additionally, we assessed the tumor phenotype of a subset of patients and also the frequency of CRC and extra-colonic tumors in 2,365 individuals of the 116 families, generating the first comprehensive portrait of the genetic and clinical aspects of patients suspected of LS in a Brazilian cohort.
Assuntos
Adenosina Trifosfatases/genética , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Proteínas de Neoplasias/genética , Adolescente , Adulto , Brasil , Neoplasias Colorretais Hereditárias sem Polipose/genética , Variações do Número de Cópias de DNA , Reparo de Erro de Pareamento de DNA , Feminino , Humanos , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteínas MutL , Adulto JovemRESUMO
The DNA mismatch repair (MMR) system is a major DNA repair pathway whose function is critical for the correction of DNA biosynthetic errors. MMR is initiated by the binding of MutS proteins to mismatches and unpaired nucleotides followed by the recruitment of MutL proteins. The major MutL activity in eukaryotes is performed by MutLα, the heterocomplex of MLH1-PMS1 in yeast and plants and MLH1-PMS2 in humans. We here report the effect the expression of Arabidopsis PMS1 protein exerts on Saccharomyces cerevisiae genomic stability. A strain carrying specific microsatellite instability reporter systems was chosen for the study. The plant protein failed to complement the hypermutator phenotype of a pms1 deficient strain but increased approximately 14-fold and 2,000-fold the mutation rates of his7-2 and lys2::InsE-A 14 loci of MMR proficient strains when compared to wild-type strains, respectively. Overexpressing AtMLH1 in the AtPMS1-overproducing strain generated an increase in mutation rate comparable to that of AtPMS1 expression alone. Deletion of the C-terminal residues implicated in protein-protein interaction and including the putative endonuclease sequence of AtPMS1 completely eliminated the mutator phenotype. Taken together, these results indicate that the plant proteins affect yeast genomic stability, very possibly altering protein-protein interactions that are necessary to complete repair.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Expressão Gênica , Instabilidade Genômica , Mutação , Fenótipo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Ordem dos Genes , Loci Gênicos , Vetores Genéticos , Dados de Sequência Molecular , Proteínas MutL , Taxa de Mutação , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/metabolismo , Alinhamento de SequênciaRESUMO
One of the major limitations when attempting to obtain detailed biochemical, biophysical and immunological characterization of plant DNA mismatch repair proteins is their extremely low abundance in vivo under normal growth conditions. An initial analysis of PMS1 transcript level in various Arabidopsis thaliana tissues was carried out by quantitative real-time RT-PCR. For calli, flowers and seedlings, the corresponding cDNA copies per ng RNA were 66.9, 3.1 and 2.7, respectively. This suggests an important role of this gene in rapidly dividing tissues. In order to obtain a high level of PMS1 from Arabidopsis thaliana, the protein production was successfully optimized in an Escherichia coli host. The corresponding coding sequence of PMS1 was inserted into pET28a downstream a hexa-histidyl leader sequence. The pET28a-AtPMS1 plasmid was efficiently expressed in JM109(DE3)-pRIL strain probably due to the genotype features of the cells (endA1, recA1, relA1, Δ(lac-proAB), laqIqZΔM15) and the presence of extra copies of argU, ileY, and leuW tRNA genes, which encode the RIL codons. This strategy has allowed us to obtain His-tagged PMS1 at about 7% of the total soluble E. coli cell protein. The protein was purified by standard Ni(+) affinity chromatography procedures and the electrophoretically homogeneous preparation was used as an antigen for antibody generation in rabbits. This approach provides effective tools for a further reconstitution of plant mismatch repair (MMR) system in vitro and for the analysis of protein expression and distribution of AtPMS1 in various tissues after different treatments (e.g. DNA mutagens).
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Cromatografia de Afinidade , Clonagem Molecular , Códon , Eletroforese em Gel de Poliacrilamida , Genes de Plantas , Genótipo , Proteínas MutL , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Escherichia colidam cells have an active but non-directed mismatch repair system; therefore, assembly of MutSLH complex at a mismatched base pair can result in MutH-mediated cleavage of GATC sites in both DNA strands. Unpaired double-strand breaks on a fraction of the replication errors occurring in dam cells presumably cause cell death, selectively eliminating these putative mutants from the population. We show that E. colidam cells transformed with plasmids containing either the mutS, mutL or mutH gene display a mutation frequency three to eight times lower than that of the parental dam strain, due to increased mismatch-stimulated cell killing. Transformed strains are also more susceptible to killing by the base analogue 2-aminopurine. However, dam and dam transformed cells have similar duplication time, proportion of live/dead cells and morphology.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Mutação , Plasmídeos , Transformação Bacteriana , 2-Aminopurina/farmacologia , Genes Bacterianos , Proteínas MutLRESUMO
This study searched for mutations in the MLH1 and MSH2 genes in 23 unrelated Colombian families with suspected hereditary nonpolyposis colorectal cancer (HNPCC). The families were grouped according to the fulfillment of the Amsterdam II criteria or the Bethesda guidelines. We screened all probands by single-strand conformational polymorphism (SSCP) and direct DNA sequencing. Eleven families fulfilled the Amsterdam criteria II and 12 families the Bethesda guidelines. Germline mutations were detected in 11 families, which corresponds to a mutation detection rate of 48%. When only families fulfilling the Amsterdam II criteria were analyzed, the mutation detection rate rose to 82%. Only 8% of the mutation detection rate was found in families following the Bethesda guidelines. Three mutations were shared by two different families, which corresponds to a total of eight different mutations, seven of them found in the MLH1 gene and one in the MSH2 gene. We have identified four mutations that have not been previously reported to the International Collaborative Group of HNPCC. Three of these are pathogenic, a single base substitution (C > T) at codon 640, exon 17, a G deletion at codon 619, exon 16 and in the MLH1 gene and a two-nucleotide deletion (TG) at codon 184, exon 3 in the MSH2. Also, an unclassified variant, a substitution (C > G) at the codon 141, exon 5 of the MLH1, was detected.
Assuntos
Proteínas de Transporte/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , DNA de Neoplasias/genética , Predisposição Genética para Doença , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Colômbia , Neoplasias Colorretais Hereditárias sem Polipose/epidemiologia , Análise Mutacional de DNA , Feminino , Mutação em Linhagem Germinativa , Humanos , Masculino , Proteína 1 Homóloga a MutL , Proteínas MutL , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
Escherichia coli MutS, MutL and MutH proteins act sequentially in the MMRS (mismatch repair system). MutH directs the repair system to the newly synthesized strand due to its transient lack of Dam (DNA-adenine methylase) methylation. Although Pseudomonas aeruginosa does not have the corresponding E. coli MutH and Dam homologues, and consequently the MMRS seems to work differently, we show that the mutL gene from P. aeruginosa is capable of complementing a MutL-deficient strain of E. coli. MutL from P. aeruginosa has conserved 21 out of the 22 amino acids known to affect functioning of E. coli MutL. We showed, using protein affinity chromatography, that the C-terminal regions of P. aeruginosa and E. coli MutL are capable of specifically interacting with E. coli MutH and retaining the E. coli MutH. Although, the amino acid sequences of the C-terminal regions of these two proteins are only 18% identical, they are 88% identical in the predicted secondary structure. Finally, by analysing (E. coli-P. aeruginosa) chimaeric MutL proteins, we show that the N-terminal regions of E. coli and P. aeruginosa MutL proteins function similarly, in vivo and in vitro. These new findings support the hypothesis that a large surface, rather than a single amino acid, constitutes the MutL surface for interaction with MutH, and that the N- and C-terminal regions of MutL are involved in such interactions.