RESUMO
Beauveria bassiana has potential for Aedes aegypti biological control. However, its efficacy depends on the strain's geographic location, host susceptibility, and virulence. The present study aimed to evaluate the effectiveness of B. bassiana strain BBPTG4 conidia in controlling Ae. aegypti adults and its detection via introns profile on exposed mosquito corpses. Morphologic characteristics among strains were highly similar. Comprehensive testing of these strains demonstrated that BBPT4 exhibited the ideal biological activity for Ae. aegypti control, with a median lethal time (TL50) of 7.5 d compared to ~3 d and ~10 d for BB01 and BB37 strains, respectively. Infected mosquitoes died after GHA and BBPTG4 exposure, and corpses were analyzed for infecting strains detection. Differences among the seven evaluated strains were determined, assessing five different insertion group I intron profiles in BBTG4, BB01, GHA, BB37, and BB02 strains. Mosquitoes infected by BBPTG4 and non-exposed (negative control) intron profiles were obtained. We detected the presence of introns in the BBPTG4 strain, which were not present in non-exposed mosquitoes. In conclusion, B. bassiana strains showed similarities in terms of their cultural and microscopic morphological characteristics and biologicals virulence level, but different intron profiles. BBPTG4 strain-infected Ae. aegypti adult corpses, showing specific amplicons, enabled us to identify B. bassiana at the strain level among infected mosquitoes. However, monitoring and detection of field-infected insects is essential for further verification.
Assuntos
Aedes , Beauveria , Beauveria/genética , Beauveria/patogenicidade , Animais , Aedes/microbiologia , Íntrons/genética , Fenótipo , Genótipo , Variação Genética , Controle Biológico de Vetores , Controle de Mosquitos/métodos , Virulência/genética , Mosquitos Vetores/microbiologiaRESUMO
It has been the aim of this study to molecular-taxonomically identify 15 Beauveria isolates collected from different geographical regions and insect hosts in Argentina and to investigate the levels of inter- and intra-specific diversity across this set of isolates. Based on phylogenetic analyses of EF1A-RPB1-RPB2 concatenated genes and BLOC markers, all Beauveria strains were identify as Beauveria bassiana. Within the B. bassiana clades of both phylogenies, isolates from Argentina were not clustered according to geographic origin or host. The 15 fungal isolates were further analyzed by PCR amplification of the intron insertion hot spot region of the nuclear 28S rRNA encoding sequence. By intron sequence and position, seven different group-I intron combinations termed variants A, B1, B2, C, D, E and F were found in the 15 isolates under study. Variants B1/B2 consisting of a single 28Si2 intron were found in ten isolates, whereas variant A occurred twice and variants C through F were unique across the set of isolates under study. The determination of the different introns and intron combinations in the 28S rRNA gene is a powerful tool for achieving infraspecific differentiation of B. bassiana isolates from Argentina.
Assuntos
Beauveria , Variação Genética , Filogenia , RNA Ribossômico 28S , Beauveria/genética , Beauveria/classificação , Beauveria/isolamento & purificação , Argentina , RNA Ribossômico 28S/genética , Animais , DNA Fúngico/genética , Insetos/microbiologia , Análise de Sequência de DNA , Dados de Sequência Molecular , Íntrons , DNA Ribossômico/genética , Análise por ConglomeradosRESUMO
The species complexes Cryptococcus neoformans and Cryptococcus gattii are the causative agents of cryptococcosis. Virulence and susceptibility to antifungals may vary within each species according to the fungal genotype. Therefore, specific and easily accessible molecular markers are required to distinguish cryptic species and/or genotypes. Group I introns are potential markers for this purpose because they are polymorphic concerning their presence and sequence. Therefore, in this study, we evaluated the presence of group I introns in the mitochondrial genes cob and cox1 in different Cryptococcus isolates. Additionally, the origin, distribution, and evolution of these introns were investigated by phylogenetic analyses, including previously sequenced introns for the mtLSU gene. Approximately 80.5% of the 36 sequenced introns presented homing endonucleases, and phylogenetic analyses revealed that introns occupying the same insertion site form monophyletic clades. This suggests that they likely share a common ancestor that invaded the site prior to species divergence. There was only one case of heterologous invasion, probably through horizontal transfer to C. decagattii (VGIV genotype) from another fungal species. Our results showed that the C. neoformans complex has fewer introns compared to C. gattii. Additionally, there is significant polymorphism in the presence and size of these elements, both among and within genotypes. As a result, it is impossible to differentiate the cryptic species using a single intron. However, it was possible to differentiate among genotypes within each species complex, by combining PCRs of mtLSU and cox1 introns, for C. neoformans species, and mtLSU and cob introns for C. gattii species.
RESUMO
Beauveria bassiana (B. bassiana) is a significant entomopathogenic fungus (EPF) in agriculture as a sprayable biocontrol agent. It has the potential to be established as an endophyte (ENP) in various crops, resulting in beneficial effects for the host plants, including resistance to pest insects and increased growth and yield. However, it is not known whether a B. bassiana strain has such a favorable impact on the plant, since it is a common soil microorganism. Therefore, techniques that allow strain monitoring will be advantageous. To date, methods for detecting or monitoring a specific EPF strain after external application are scarce. In the present study, an in planta nested PCR technique was standardized to differentiate between three B. bassiana strains (GHA, PTG4, and BB37) established as endophytes in bean plants under laboratory conditions by detecting the insertion profile of four group I introns located in the 28S gene of B. bassiana ribosomal DNA. This technique recognized a distinct pattern of bands of different sizes for each strain, with a sensitivity of 1 pg per 10 ng of plant DNA. This molecular approach may be more effective monitoring B. bassiana strains after application to evaluate their significance on crops.
RESUMO
Bacterial genomes are composed of core and accessory genomes. The first is composed of housekeeping and essential genes, while the second is highly enriched in mobile genetic elements, including transposable elements (TEs). Insertion sequences (ISs), the smallest TEs, have an important role in genome evolution, and contribute to bacterial genome plasticity and adaptability. ISs can spread in a genome, presenting different locations in nearly related strains, and producing phenotypic variations. Few tools are available which can identify differentially located ISs (DLISs) on assembled genomes. Here, we introduce ISCompare, a new program to profile IS mobilization events in related bacterial strains using complete or draft genome assemblies. ISCompare was validated using artificial genomes with simulated random IS insertions and real sequences, achieving the same or better results than other available tools, with the advantage that ISCompare can analyze multiple ISs at the same time and outputs a list of candidate DLISs. ISCompare provides an easy and straightforward approach to look for differentially located ISs on bacterial genomes.
Assuntos
Elementos de DNA Transponíveis , Genoma Bacteriano , Bactérias/genética , Elementos de DNA Transponíveis/genéticaRESUMO
Snake venom thrombin-like enzymes (SVTLEs) are serine proteinases that clot fibrinogen. SVTLEs are distributed mainly in venoms from snakes of the Viperidae family, comprising venomous pit viper snakes. Bothrops snakes are distributed throughout Central and South American and are responsible for most venomous snakebites. Most Bothrops snakes display thrombin-like activity in their venoms, but it has been shown that some species do not present it. In this work, to understand SVTLE polymorphism in Bothrops snake venoms, we studied individual samples from two species of medical importance in Brazil: Bothrops jararaca, distributed in Southeastern Brazil, which displays coagulant activity on plasma and fibrinogen, and Bothrops erythromelas, found in Northeastern Brazil, which lacks direct fibrinogen coagulant activity but shows plasma coagulant activity. We tested the coagulant activity of venoms and the presence of SVTLE genes by a PCR approach. The SVTLE gene structure in B. jararaca is similar to the Bothrops atrox snake, comprising five exons. We could not amplify SVTLE sequences from B. erythromelas DNA, except for a partial pseudogene. These genes underwent a positive selection in some sites, leading to an amino acid sequence diversification, mostly in exon 2. The phylogenetic tree constructed using SVTLE coding sequences confirms that they are related to the chymotrypsin/kallikrein family. Interestingly, we found a B. jararaca specimen whose venom lacked thrombin-like activity, and its gene sequence was a pseudogene with SVTLE structure, presenting nonsense and frameshift mutations. Our results indicate an association of the lack of thrombin-like activity in B. jararaca and B. erythromelas venoms with mutations and deletions of snake venom thrombin-like enzyme genes.
RESUMO
The subfamily GH13_1 of alpha-amylases is typical of Fungi, but it is also found in some unicellular eukaryotes (e.g., Amoebozoa, choanoflagellates) and non-bilaterian Metazoa. Since a previous study in 2007, GH13_1 amylases were considered ancestral to the Unikonts, including animals, except Bilateria, such that it was thought to have been lost in the ancestor of this clade. The only alpha-amylases known to be present in Bilateria so far belong to the GH13_15 and 24 subfamilies (commonly called bilaterian alpha-amylases) and were likely acquired by horizontal transfer from a proteobacterium. The taxonomic scope of Eukaryota genomes in databases has been greatly increased ever since 2007. We have surveyed GH13_1 sequences in recent data from ca. 1600 bilaterian species, 60 non-bilaterian animals and also in unicellular eukaryotes. As expected, we found a number of those sequences in non-bilaterians: Anthozoa (Cnidaria) and in sponges, confirming the previous observations, but none in jellyfishes and in Ctenophora. Our main and unexpected finding is that such fungal (also called Dictyo-type) amylases were also consistently retrieved in several bilaterian phyla: hemichordates (deuterostomes), brachiopods and related phyla, some molluscs and some annelids (protostomes). We discuss evolutionary hypotheses possibly explaining the scattered distribution of GH13_1 across bilaterians, namely, the retention of the ancestral gene in those phyla only and/or horizontal transfers from non-bilaterian donors.
Assuntos
Basidiomycota/genética , Evolução Molecular , Transferência Genética Horizontal , Transformação Genética , alfa-Amilases/genética , Basidiomycota/metabolismo , Genes Fúngicos , Íntrons , FilogeniaRESUMO
Resumen: La hemofilia A (HA) es la coagulopatía ligada al cromosoma X más frecuente. Es causada por mutaciones en el gen del factor VIII (FVIII) de coagulación (F8). La HA puede ser severa cuando la actividad del FVIII es menor a 1% (FVIII: C<1IU/dL). Casi la mitad de las HA severas son producidas por inversiones del F8, como la del intrón 1 (Inv1) y del intrón 22 (Inv22). Los pacientes con HA severa experimentan sus primeros sangrados generales entre los 9,7 - 10,9 meses, ocurriendo principalmente en las articulaciones. Se investigó la presencia de la Inv1 e Inv22 en la región noreste de Uruguay (departamentos de Tacuarembó, Rivera y Cerro Largo) para estimar su frecuencia y detectar la presencia de portadoras. Fueron estudiados 14 individuos (ocho pacientes con HA severa, cuatro madres y dos hermanas de pacientes) de cinco familias. La investigación de las inversiones se realizó aplicando las pruebas de inverse shifting-PCR (IS-PCR). La Inv1 se encontró en dos pacientes (hermanos) de Tacuarembó, en su hermana y madre (portadoras), mientras que un paciente de Rivera y su madre (portadora) resultaron positivos para la Inv22. Preliminarmente, en conjunto, la Inv1 y la Inv22 representan la causa de la HA severa en el 40% de las familias del noreste de Uruguay, valor menor a lo esperado; sin embargo, debido a la reducida población estudiada, la Inv1 muestra una frecuencia preliminar (20%, 1/5 familias, 25%, 2/8 pacientes) considerablemente mayor a estudios previos. Estos datos permiten caracterizar la etiología genética de la hemofilia, la detección de las portadoras, conocer la distribución geográfica de las mutaciones y el asesoramiento genético.
Summary: Hemophilia A (HA) is the most common X-linked coagulopathy, it is caused by mutations in the coagulation factor VIII (FVIII) gene (F8). HA can be severe when the FVIII activity is less than 1% (FVIII: C <1IU / dL). Almost a half of the severe HAs are produced by inversions of F8, the intron 1 (Inv1) and intron 22 (Inv22). Patients with severe HA show their first general bleeding between 9.7 - 10.9 months, mainly in the joints. We researched the presence of Inv1 and Inv22 in the Northeast region of Uruguay (Departments: Tacuarembó, Rivera and Cerro Largo) to estimate their frequency and detect the presence of carriers. We studied 14 individuals in 5 different families (8 patients with severe HA, 4 mothers and 2 sisters of patients). The inversion study was carried out using inverse shifting-PCR (IS-PCR) tests. Inv1 was found in 2 patients (siblings) from Tacuarembó, in their sister and mother (carriers). A patient from Rivera and his mother (carrier) were positive for Inv22. Inv1 and Inv22 are the cause of severe HA in 40% of the patients in North East of Uruguay, less than expected; however, due to the reduced population studied, Inv1 shows a considerably higher frequency than previous studies. These data enable us to characterize the genetic etiology of hemophilia, to adequately monitor patients, detect carriers, the geographical distribution of mutations and the corresponding genetic counseling for families.
Resumo: A hemofilia A (HA) é a coagulopatia ligada ao cromossomo X mais frequente, causada por mutações no gene do fator VIII (FVIII) de coagulação (FVIII) (F8). A HA pode ser grave quando a atividade do FVIII é menor que 1% (FVIII: C <1IU / dL). Quase metade da HA grave é produzida por inversões de F8, como a do Íntron 1 (Inv1) e do Íntron 22 (Inv22). Pacientes com HA grave experimentam seu primeiro sangramento geral entre 9,7 e 10,9 meses, principalmente nas articulações. A presença de Inv1 e Inv22 na região nordeste do Uruguai (departamentos: Tacuarembó, Rivera e Cerro Largo) foi investigada para estimar a sua frequência e detectar a presença de portadora. Foram estudados 14 indivíduos (8 pacientes com HA grave, 4 mães e 2 irmãs de pacientes) de 5 famílias. A pesquisa das inversões foi realizada aplicando os testes de inverse shifting -PCR (IS-PCR). Encontramos Inv1 em 2 pacientes (irmãos) de Tacuarembó, na sua irmã e mãe (portadoras), enquanto 1 paciente de Rivera e sua mãe (transportadora) foram positivos para Inv22. Preliminarmente, Inv1 e Inv22 juntos representam a causa de HA grave em 40% das famílias do nordeste do Uruguai, valor inferior ao esperado, no entanto, devido à pequena população estudada, Inv1 mostra uma frequência preliminar (20%, 1/5 famílias, 25%, 2/8 pacientes) consideravelmente mais alta que os estudos anteriores. Esses dados permitem-nos caracterizar a etiologia genética da hemofilia, detectar aos portadores, conhecer a distribuição geográfica das mutações e realizar aconselhamento genético.
RESUMO
Fungi of the genus Colletotrichum are economically important and are used as models in plant-pathogen interaction studies. In this study, the complete mitochondrial genomes of two Colletotrichum lindemuthianum isolates were sequenced and compared with the mitochondrial genomes of seven species of Colletotrichum. The mitochondrial genome of C. lindemuthianum is a typical circular molecule 37,446 bp (isolate 89 A2 2-3) and 37,440 bp (isolate 83.501) in length. The difference of six nucleotides between the two genomes is the result of a deletion in the ribosomal protein S3 (rps3) gene in the 83.501 isolate. In addition, substitution of adenine for guanine within the rps3 gene in the mitochondrial genome of the 83.501 isolate was observed. Compared to the previously sequenced C. lindemuthianum mitochondrial genome, an exon no annotated in the cytochrome c oxidase I (cox1) gene and a non-conserved open reading frame (ncORF) were observed. The size of the mitochondrial genomes of the seven species of Colletotrichum was highly variable, being attributed mainly to the ncORF, ranging from one to 10 and also from introns ranging from one to 11 and which encode a total of up to nine homing endonucleases. This paper reports for the first time by means of transcriptome that then ncORFs are transcribed in Colletotrichum spp. Phylogeny data revealed that core mitochondrial genes could be used as an alternative in phylogenetic relationship studies in Colletotrichum spp. This work contributes to the genetic and biological knowledge of Colletotrichum spp., which is of great economic and scientific importance.
Assuntos
Colletotrichum/genética , Genoma Mitocondrial , Colletotrichum/isolamento & purificação , DNA Circular/química , DNA Circular/genética , DNA Mitocondrial/química , DNA Mitocondrial/genética , Éxons , Genes Mitocondriais , Tamanho do Genoma , Fases de Leitura Aberta , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Mutação Puntual , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
Cryptococcosis, one of the most important systemic mycosis in the world, is caused by different genotypes of Cryptococcus neoformans and Cryptococcus gattii, which differ in their ecology, epidemiology, and antifungal susceptibility. Therefore, the search for new molecular markers for genotyping, pathogenicity and drug susceptibility is necessary. Group I introns fulfill the requisites for such task because (i) they are polymorphic sequences; (ii) their self-splicing is inhibited by some drugs; and (iii) their correct splicing under parasitic conditions is indispensable for pathogen survival. Here, we investigated the presence of group I introns in the mitochondrial LSU rRNA gene in 77 Cryptococcus isolates and its possible relation to drug susceptibility. Sequencing revealed two new introns in the LSU rRNA gene. All the introns showed high sequence similarity to other mitochondrial introns from distinct fungi, supporting the hypothesis of an ancient non-allelic invasion. Intron presence was statistically associated with those genotypes reported to be less pathogenic (p < 0.001). Further virulence assays are needed to confirm this finding. In addition, in vitro antifungal tests indicated that the presence of LSU rRNA introns may influence the minimum inhibitory concentration (MIC) of amphotericin B and 5-fluorocytosine. These findings point to group I introns in the mitochondrial genome of Cryptococcus as potential molecular markers for antifungal resistance, as well as therapeutic targets.
RESUMO
Resumen Introducción. La hemofilia A es una enfermedad recesiva ligada al cromosoma X, con una incidencia de 1 en 5 000 a 10 000 varones y es el trastorno hemostático congénito más frecuente en varones. En pacientes con fenotipo severo, las inversiones de los intrones 22 y 1 son las mutaciones más comunes con una prevalencia del 45% a 50% y del 1% al 5% de los pacientes, respectivamente. Objetivo. Determinar la frecuencia de la inversión de los intrones 1 y 22 del gen del factor VIII de la coagulación en menores de 18 años con hemofilia A severa en Bogotá D.C. Materiales y métodos. Estudio descriptivo y transversal. La identificación de la inversión de los intrones 1 y 22 del gen del factor VIII se realizó mediante técnicas de reacción en cadena de polimerasa de larga distancia. Resultados. Se estudiaron 30 pacientes y se encontró inversión del intrón 22 en 12 pacientes (40%) e inversión 1 en 3 pacientes, cifras similares a las observadas en otros estudios. Conclusiones. Se encontraron las inversiones de los intrones 1 y 22 en la mitad de los pacientes evaluados. Los resultados son reproducibles, por lo que constituyen una herramienta útil para la identificación de las dos mutaciones más frecuentes en hemofilia A severa.
Abstract Introduction: Hemophilia A is an X-linked recessive disease with an incidence of 1 in 5 000 to 10 000 males. It is the most common congenital hemostatic disorder in men. The inversion of introns 1 and 22 in patients with a severe phenotype is considered the most frequent abnormality, with a prevalence of 1 to 5% and 45 to 50%, respectively. Objective: To determine the frequency of introns 1 and 22 inversions in factor VIII gene in children under 18 years with severe hemophilia A in Bogotá. Materials and methods: This is a non-experimental, descriptive, transverse study. The inversions of introns 1 and 22 for factor VIII gene were identified using long-distance polymerase chain reaction techniques in pediatric patients with severe Hemophilia A treated in different centers of Bogotá, Colombia. Results: Thirty patients were analyzed. Inversion of intron 22 was found in 12 patients (40%), while inversion of intron 1 was observed in 3 patients. These findings are similar to other studies. Conclusions: Inversions of intron 22 and 1 were found in half of this group of patients. These results are reproducible and useful to identify the two most frequent mutations in severe hemophilia A patients.
RESUMO
Isoprenoids belong to a large family of structurally and functionally different natural compounds found universally from prokaryotes to higher animals and plants. In Hevea brasiliensis, the commercially important cis-polyisoprene (rubber) is synthesised as part of its defence mechanism in addition to other common isoprenoids like phytosterols, growth hormones etc. Farnesyl diphosphate synthase (FDPS) is a key enzyme in this process which catalyses the conversion of isoprene units into polyisoprene. Although prior sequence information is available, the structural variants of the FDPS gene presently existing in Hevea population are largely unknown. Since gene structure has a major role in gene regulation, extensive sequence analysis of this gene from different genotypes was carried out to identify the prevailing structural variants. We identified several SNPs and large indels which were associated with a partial transposable element (TE). Modification of key regulatory motifs and splice sites induced by the retroelement was also identified in the first intron. Screening of popular rubber clones, wild germplasm accessions and Hevea species revealed that the retroelement is responsible for the generation of new alleles with varying degrees of sequence homology. Segregation analysis of a progeny population confirmed that the alleles are not paralogs and are inherited in a Mendelian mode. Our findings suggest that the first intron of the FDPS gene has been subjected to various chromosomal rearrangements due to the interaction of a retrotransposon, resulting in novel alleles which may substantially contribute towards the evolution of this major gene in rubber. Moreover, the results indicate the possible existence of a retrotransposon-mediated epigenetic gene regulatory mechanism in Hevea.