Trypanosoma cruzi: genetic group with peculiar biochemical and biological behavior

Thirty-two Trypanosoma cruzi strains, isolated from chronic chagasic patients in the northwest of the state of Paraná (Brazil), were analyzed using molecular, biochemical and biological characteristics. Genotypic analysis using randomly amplified polymorphic DNA and simple sequence repeat-anchored polymerase chain reaction amplified profiles showed a large, genetically well-correlated group that contained the majority of the strains and a divergent group that included the PR-150 strain. For glycoconjugate composition, the PR-150 strain was different from the other strains considering the absence or presence of specific bands in aqueous or detergent phases. This strain was also totally different from the others in one out of the six parameters related to in vitro and in vivo biological behavior. We highlight the fact that the PR-150 was totally resistant to benznidazole. For the other biological parameters this strain was not totally distinct from the others, but it showed a peculiar behavior.

Evolutionary history of almond tree domestication in the Mediterranean basin.

Genetic diversity of contemporary domesticated species is shaped by both natural and human-driven processes. However, until now, little is known about how domestication has imprinted the variation of fruit tree species. In this study, we reconstruct the recent evolutionary history of the domesticated almond tree, Prunus dulcis, around the Mediterranean basin, using a combination of nuclear and chloroplast microsatellites [i.e. simple sequence repeat (SSRs)] to investigate patterns of genetic diversity. Whereas conservative chloroplast SSRs show a widespread haplotype and rare locally distributed variants, nuclear SSRs show a pattern of isolation by distance with clines of diversity from the East to the West of the Mediterranean basin, while Bayesian genetic clustering reveals a substantial longitudinal genetic structure. Both kinds of markers thus support a single domestication event, in the eastern side of the Mediterranean basin. In addition, model-based estimation of the timing of genetic divergence among those clusters is estimated sometime during the Holocene, a result that is compatible with human-mediated dispersal of almond tree out of its centre of origin. Still, the detection of region-specific alleles suggests that gene flow from relictual wild preglacial populations (in North Africa) or from wild counterparts (in the Near East) could account for a fraction of the diversity observed.

Distinct genetic structure in populations of Chrysoperla externa (Hagen) (Neuroptera, Chrysopidae) shown by genetic markers ISSR and COI gene

Distinct genetic structure in populations of Chrysoperla externa (Hagen) (Neuroptera, Chrysopidae) shown by genetic markers ISSR and COI gene. Green lacewings are generalist predators, and the species Chrysoperla externa presents a great potential for use in biological control of agricultural pests due to its high predation and reproduction capacities, as well as its easy mass rearing in the laboratory. The adaptive success of a species is related to genetic variability, so that population genetic studies are extremely important in order to maximize success of the biological control. Thus, the present study used nuclear (Inter Simple Sequence Repeat - ISSR) and mitochondrial (Cytochrome Oxidase I - COI) molecular markers to estimate the genetic variability of 12 populations in the São Paulo State, Brazil, as well as the genetic relationships between populations. High levels of genetic diversity were observed for both markers, and the highest values of genetic diversity appear associated with municipalities that have the greatest areas of native vegetation. There was high haplotype sharing, and there was no correlation between the markers and the geographic distribution of the populations. The AMOVA indicated absence of genetic structure for the COI gene, suggesting that the sampled areas formed a single population unit. However, the great genetic differentiation among populations showed by ISSR demonstrates that these have been under differentiation after their expansion or may also reflect distinct dispersal behavior between males and females.

Genetic differentiation for size at first reproduction through male versus female functions in the widespread Mediterranean tree Pinus pinaster.

BACKGROUND AND AIMS: The study of local adaptation in plant reproductive traits has received substantial attention in short-lived species, but studies conducted on forest trees are scarce. This lack of research on long-lived species represents an important gap in our knowledge, because inferences about selection on the reproduction and life history of short-lived species cannot necessarily be extrapolated to trees. This study considers whether the size for first reproduction is locally adapted across a broad geographical range of the Mediterranean conifer species Pinus pinaster. In particular, the study investigates whether this monoecious species varies genetically among populations in terms of whether individuals start to reproduce through their male function, their female function or both sexual functions simultaneously. Whether differences among populations could be attributed to local adaptation across a climatic gradient is then considered. METHODS: Male and female reproduction and growth were measured during early stages of sexual maturity of a P. pinaster common garden comprising 23 populations sampled across the species range. Generalized linear mixed models were used to assess genetic variability of early reproductive life-history traits. Environmental correlations with reproductive life-history traits were tested after controlling for neutral genetic structure provided by 12 nuclear simple sequence repeat markers. KEY RESULTS: Trees tended to reproduce first through their male function, at a size (height) that varied little among source populations. The transition to female reproduction was slower, showed higher levels of variability and was negatively correlated with vegetative growth traits. Several female reproductive traits were correlated with a gradient of growth conditions, even after accounting for neutral genetic structure, with populations from more unfavourable sites tending to commence female reproduction at a lower individual size. CONCLUSIONS: The study represents the first report of genetic variability among populations for differences in the threshold size for first reproduction between male and female sexual functions in a tree species. The relatively uniform size at which individuals begin reproducing through their male function probably represents the fact that pollen dispersal is also relatively invariant among sites. However, the genetic variability in the timing of female reproduction probably reflects environment-dependent costs of cone production. The results also suggest that early sex allocation in this species might evolve under constraints that do not apply to other conifers.

Species selectivity of mixed-lineage leukemia/trithorax and HCF proteolytic maturation pathways.

Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1(PRO) repeats. We demonstrate here that the Drosophila homologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms.

Mechanisms of HCF protein proteolytic maturation

Abstract : Post-translational modifications such as proteolytic processing, phosphorylation, and glycosylation, add extra layers of complexity to proteomes and allow a finely tuned regulation of the activity of many proteins. The evolutionarily conserved cell-cycle and transcriptional regulator HCP-] is regulated by proteolytic maturation via which a stable heterodirneric complex of two cleaved subunits is formed from a single precursor protein. The human HCF-1 precursor is cleaved at six nearly identical 26 amino acid sequence repeats, called HCF-1pro repeats, which represent uncommon protease recognition sites dedicated to human HCF-1 proteolysis. This proteolytic maturation process is conserved in vertebrate HCF-1 homologues and is essential for the functions of the human protein in cell-cycle regulation; the mechanisms that execute and control HCF-1 proteolysis, however, remain poorly understood. In this dissertation I investigate the mechanisms of proteolytic maturation of HCF-1 proteins in different species. I show that the Drosophila homolog of human HCF-1, called dHCP, is proteolytically cleaved via a different mechanism than human HCF-1. dHCP is processed by the same protease, called Taspase], which cleaves one of the key developmental regulators in flies, the Trithorax protein. Maturation of HCP proteins via Taspase] cleavage is probably not particular to dHCP as many invertebrate HCP proteins, particularly insects and flatworms, possess Taspase] recognition sites. In contrast, the vertebrate HCF-1 proteins lack Taspase] recognition sites and the HCF-1pro repeats are not Taspase1 substrates, suggesting that multiple mechanisms for HCF-1 proteolytic maturation have appeared during evolution. I also show that the proteolytic activity responsible for the cleavage of the HCP- 1pro repeats is very difficult to characterize, being resistant to most protease inhibitors and very sensitive to biochemical fractionation. Moreover, the HCF-1pro repeats represent complex protease recognition sites and I demonstrate that, in addition to be the HCF-1 cleavage sites, these repeated sequences, also recruit the OG1cNAc transferase OGT. The OGT protein and the OG1cNAc modification of HCF-1 are both important for HCF-1pro repeat proteolysis. Interestingly, a human recombinant OGT purified from insect cells is able to induce cleavage of a HCF-1pro-repeat precursor in vitro, indicating that OGT either (i) induces HCF-1 autoproteolysis,(ii) is the HCF-1pro- repeat proteolytic activity itself, or (iii) physically associates with a proteolytic activity that is conserved in insect cells. In any case, OGT plays an important role in HCF-1 proteolytic maturation and perhaps a broader role in HCF-1 biological function. Résumé : Les modifications post-traductionelles pomme le clivage protéolytique, la phosphorylation, et la glycosylation, augmentent significativement la complexité des protéomes et permettent une régulation fine de l'activité de beaucoup de protéines. La protéine HCF-1, qui est un régulateur du cycle cellulaire et de la transcription, est elle- même régulée par clivage protéolytique. La protéine HCF-1 est en effet coupée en deux sous-unités qui s'associent l'une a l'autre pour former la protéine mature. Le précurseur de la protéine HCF-1 humaine est clivé à six sites correspondant à six séquences répétées nommées les HCF-1pro repeats, chacune composée de 26 acide aminés. Les HCF-1pro- repeats ne ressemblent ai aucune séquence de clivage protéolytique connue et sont présentes seulement dans les protéines HCF-1 chez les vertébrés. Bien que la maturation protéolytique d'HCF-1 soit essentielle pour les activités de cette protéine pendant le cycle cellulaire, les mécanismes qui la contrôlent restent inconnus. Au cours de mon travail de thèse, j'ai analysé les mécanismes de clivage protéolytique des protéines HCF dans différentes espèces. J'ai montré que la protéine de Drosophile homologue d'HCF-1 humaine nommée dHCF est clivée par une protéase nommée Taspase1. Ainsi, dHCF est clivé par la même protéase que celle qui induit la maturation protéolytique d'un des principaux facteurs du développement chez la mouche, la protéine Trithorax. La maturation de dHCF via le clivage par la Taspase1 n'est pas spécifique à la mouche, mais est probablement étendu à plusieurs protéines HCF chez les invertébrés, surtout dans les familles des insectes et des plathehninthes, car ces protéines HCF présentent des sites de reconnaissance pour la Taspasel. Par contre, les protéines HCF-1 chez les vertébrés n'ont pas de sites de reconnaissance pour la Taspasel et cela suggère que différents mécanismes de maturation des protéines HCF- ls ont apparu au cours de l'évolution. J'ai montré aussi que les HCF-1pro-repeats sont clivés par une activité protéolytique très difficile a identifier, car elle est résistante à la plupart des inhibiteurs de protéases, mais elle est très sensible au fractionnement biochimique. En plus, les HCF-1pro-repeats sont un site de protéolyse complexe qui ne sert pas seulement au clivage des protéines HCF- chez les vertébrés mais aussi à recruter l'enzyme responsable de la O- GlcNAcylation nommée OGT. La protéine OGT et la O-GlcNAcylatio d'HCF-1 sont toutes les deux importantes pour le clivage protéolytique des HCF1pro-repeats. Curieusement, la protéine OGT humaine produite dans des cellules d'insectes est capable de cliver les HCF-1pro repeats in vitro et cela suggère que OGT soit (i) induit le clivage autocatalytique cl'HCF-1, soit (ii) est elle-même l'activité protéolytique qui clive HCF4, soit (iii) est associée à une activité protéolytique conservée dans les cellules d'insectes qui a été co-purifiée avec OGT. En conclusion, OGT joue un rôle important dans la maturation protéolytique d'HCF-1 et peut-être aussi un rôle plus large dans les fonctions biologiques de la protéine HCF-1.

Dissecting the role of low-complexity regions in the evolution of vertebrate proteins

Low-complexity regions (LCRs) in proteins are tracts that are highly enriched in one or a few aminoacids. Given their high abundance, and their capacity to expand in relatively short periods of time through replication slippage, they can greatly contribute to increase protein sequence space and generate novel protein functions. However, little is known about the global impact of LCRs on protein evolution. We have traced back the evolutionary history of 2,802 LCRs from a large set of homologous protein families from H.sapiens, M.musculus, G.gallus, D.rerio and C.intestinalis. Transcriptional factors and other regulatory functions are overrepresented in proteins containing LCRs. We have found that the gain of novel LCRs is frequently associated with repeat expansion whereas the loss of LCRs is more often due to accumulation of amino acid substitutions as opposed to deletions. This dichotomy results in net protein sequence gain over time. We have detected a significant increase in the rate of accumulation of novel LCRs in the ancestral Amniota and mammalian branches, and a reduction in the chicken branch. Alanine and/or glycine-rich LCRs are overrepresented in recently emerged LCR sets from all branches, suggesting that their expansion is better tolerated than for other LCR types. LCRs enriched in positively charged amino acids show the contrary pattern, indicating an important effect of purifying selection in their maintenance. We have performed the first large-scale study on the evolutionary dynamics of LCRs in protein families. The study has shown that the composition of an LCR is an important determinant of its evolutionary pattern.

Seleção tradicional e associada a marcadores moleculares na avaliação genética animal

O objetivo deste trabalho foi comparar a seleção, utilizando valores genéticos preditos pelo BLUP clássico (BLUP), BLUP marcadores (BLUPM) e pela seleção individual (SI), usando simulação com o programa Genesys. Para obter a matriz de similaridade genética utilizada no BLUPM, foram simulados cem marcadores moleculares do tipo microssatélite (SSR - Simple Sequence Repeat), por meio de um coeficiente de similaridade correspondente à distância euclideana média para dados quantitativos. A fim de comparar os diferentes métodos, utilizaram-se populações com tamanho efetivo de 66,66 e média de 30 repetições, avaliando-se os valores fenotípicos médios. Os ganhos ao longo das 20 gerações de seleção foram maiores para o BLUP em relação ao BLUPM, e este foi superior à SI. Quanto ao ganho obtido nas cinco primeiras gerações, o BLUPM apresentou ganhos semelhantes ao BLUP e superiores à SI. Diferentes sistemas de acasalamento dos reprodutores selecionados não revelaram diferenças em ganho genético nos métodos baseados no BLUP.

Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis.

Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168-166 system were explained by postulating the existence of intrachromosomal homology regions. This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains. Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPbeta established the existence of one of the postulated homology sequences. Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPbeta, plays a key role in the formation of so-called trpE30, trpE30+, as well as of new class I merodiploids. Analysis of junctions involved in the transfer of the trpE26 mutation, i.e. simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination. We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome. The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of 'transstamping'. In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X-ray-induced, rearrangement.

Genetic variability of Brazilian rice landraces determined by SSR markers

The objective of this study was to evaluate the genetic variability of rice (Oryza sativa) landraces collected in Brazilian small farms. Twelve simple sequence repeat (SSR) markers characterized 417 landraces collected in 1986, 1987 and 2003, in the state of Goiás, Brazil. The number of landraces with long and thin grain type increased in the evaluated period, probably due to market demand. Based on the molecular data, the genetic variability increased during this period and, as per to the factorial correspondence analysis, most of the accessions were grouped according to the year of collection. The incorporation of modern rice cultivars in landrace cultivation areas and the selection carried out by small farmers are the most probable factors responsible for increasing landrace genetic variability, during the evaluated period. Genotype exchange between farmers, selection practice and local environmental adaptation are able to generate novel adapted allele combinations, which can be used by breeding programs, to reinitiate the process.

In situ and genetic characterization of Gossypium barbadense populations from the states of Pará and Amapá, Brazil

The objective of this work was to characterize the populations of Gossypium barbadense in the states of Amapá and Pará, Brazil. In situ characterization was conducted through interviews with the owners of the plants and environmental observations. Leaf or petal tissue as well as seed samples were collected for genetic characterization by single sequence repeats markers and for storage in germplasm banks, respectively. The plants were maintained in dooryards and used mainly for medical purposes. The genetic analysis showed no heterozygous plants at the loci tested (f = 1), indicating that reproduction occurs mainly through selfing. The total genetic diversity was high (He = 0.39); and a high level of differentiation was observed between cotton plants from the two states (F ST = 0.36). Conventional methods of in situ maintenance of G. barbadense populations are not applicable. The conservation of the genetic variability of populations present in the two states could be achieved through germplasm collection and establishing of ex situ seed banks.

Genetic diversity in soybean germplasm identified by SSR and EST-SSR markers

The objectives of this work were to investigate the genetic variation in 79 soybean (Glycine max) accessions from different regions of the world, to cluster the accessions based on their similarity, and to test the correlation between the two types of markers used. Simple sequence repeat markers present in genomic (SSR) and in expressed regions (EST-SSR) were used. Thirty SSR primer-pairs were selected (20 genomic and 10 EST-SSR) based on their distribution on the 20 genetic linkage groups of soybean, on their trinucleotide repetition unit and on their polymorphism information content. All analyzed loci were polymorphic, and 259 alleles were found. The number of alleles per locus varied from 2-21, with an average of 8.63. The accessions exhibit a significant number of rare alleles, with genotypes 19, 35, 63 and 65 carrying the greater number of exclusive alleles. Accessions 75 and 79 were the most similar and accessions 31 and 35, and 40 and 78, were the most divergent ones. A low correlation between SSR and EST-SSR data was observed, thus genomic and expressed microsatellite markers are required for an appropriate analysis of genetic diversity in soybean. The genetic diversity observed was high and allowed the formation of five groups and several subgroups. A moderate relationship between genetic divergence and geographic origin of accessions was observed.

Microsatellite markers in analysis of resistance to coffee leaf miner in Arabica coffee

The objective of this work was to analyze coffee (Coffea arabica) genotypes resistant to the coffee leaf miner (Leucoptera coffeella) using microsatellite markers. Sixty-six loci were evaluated, of which 63 were obtained from the Brazilian Coffee Expressed Sequence Tag (EST) database. These loci were amplified in bulks of individuals from F5 progenies of 'Siriema' (C. arabica x C. racemosa) resistant and susceptible to the insect, in eight samples of C. racemosa, and in a F6 population of 'Siriema' with 91 individuals segregating for resistance to the leaf miner. Polymorphisms were verified for two simple sequence repeat (SSR) loci in bulks of the susceptible progenies. The two polymorphic alleles were present in around 70% of the susceptible genotypes in F5 and in approximately 90% of the susceptible individuals in F6. However, the polymorphic EST-SSR markers among populations contrasting for resistance to leaf miner were not correlated to the evaluated characteristics. SSR markers show inter- and intraspecific polymorphism in C. arabica and C. racemosa.

Genetic variability of rice recurrent selection populations as affected by male sterility or manual recombination

The objective of this work was to determine the effect of male sterility or manual recombination on genetic variability of rice recurrent selection populations. The populations CNA-IRAT 4, with a gene for male sterility, and CNA 12, which was manually recombined, were evaluated. Genetic variability among selection cycles was estimated using14 simple sequence repeat (SSR) markers. A total of 926 plants were analyzed, including ten genitors and 180 individuals from each of the evaluated cycles (1, 2 and 5) of the population CNA-IRAT 4, and 16 genitors and 180 individuals from each of the cycles (1 and 2) of CNA 12. The analysis allowed the identification of alleles not present among the genitors for both populations, in all cycles, especially for the CNA-IRAT 4 population. These alleles resulted from unwanted fertilization with genotypes that were not originally part of the populations. The parameters of Wright's F-statistic (F IS and F IT) indicated that the manual recombination expands the genetic variability of the CNA 12 population, whereas male sterility reduces the one of CNA-IRAT 4.

Optimal use of SSR markers for varietal identification of upland cotton

Abstract: The objective of this work was to identify polymorphic simple sequence repeat (SSR) markers for varietal identification of cotton and evaluation of the genetic distance among the varieties. Initially, 92 SSR markers were genotyped in 20 Brazilian cotton cultivars. Of this total, 38 loci were polymorphic, two of which were amplified by one primer pair; the mean number of alleles per locus was 2.2. The values of polymorphic information content (PIC) and discrimination power (DP) were, on average, 0.374 and 0.433, respectively. The mean genetic distance was 0.397 (minimum of 0.092 and maximum of 0.641). A panel of 96 varieties originating from different regions of the world was assessed by 21 polymorphic loci derived from 17 selected primer pairs. Among these varieties, the mean genetic distance was 0.387 (minimum of 0 and maximum of 0.786). The dendrograms generated by the unweighted pair group method with arithmetic average (UPGMA) did not reflect the regions of Brazil (20 genotypes) or around the world (96 genotypes), where the varieties or lines were selected. Bootstrap resampling shows that genotype identification is viable with 19 loci. The polymorphic markers evaluated are useful to perform varietal identification in a large panel of cotton varieties and may be applied in studies of the species diversity.