A cana-de-açúcar é uma monocotiledônea poliplóide, alógama que possui baixa taxa reprodutiva devido a dificuldade de florescimento. Devido estas características genéticas e fisiológicas os programas de melhoramento são longos e laboriosos. Alternativamente, modernas aplicações da biotecnologia visam contribuir com o desenvolvimento de novos cultivares. Neste trabalho estudou-se a metodologia de cultura de tecidos a partir de discos de folhas imaturas para o estabelecimento da cultura de calos embriogênicos e regeneração de plantas a partir dos calos embriogênicos e diretamente, a partir de folhas imaturas. O objetivo principal foi contribuir para o desenvolvimento de métodos eficientes para produção de plantas transgênicas a partir de calos e folhas imaturas, considerando-se a crescente necessidade de produção de novos cultivares com características agronômicas específicas. Diversas concentrações de 2,4-D e cinetina em meio MS foram testadas para o estabelecimento de calos altamente embriogênicos e para a indução da desdiferenciação celular nos discos foliares antecedendo a regeneração de plantas. Meios de cultura sem reguladores de crescimento (MS) e com a adição de BAP e ANA foram testados para a regeneração de plantas a partir de discos foliares. Calos embriogênicos com 12 a 20 semanas de cultivo produziram em média 3 a 5 plantas, em meio de regeneração MS. Folhas imaturas apresentaram elevado potencial de regeneração de plantas quando se utilizou 2,4-D em concentrações de 5 e 8 mg/L nos períodos de 5 e 8 dias no escuro. Houve indução a formação de embriões somáticos que resultaram em média 12 a 16 plantas por explante no período total de 7 a 10 semanas. Além disso, foi testado o pré-tratamento dos discos foliares em meio MS3K, contendo 2,4-D (3mg/L) e cinetina (0,1 mg/L), antes da transferência do discos para meio de regeneração MS. Os discos submetidos a este pré-tratamento durante 14, 21 e 28 dias apresentaram aumento significativo na eficiência de regeneração de plantas, variando em média de 41 a 50 plantas por disco foliar nas variedades RB835089 e RB855156. A redução no tempo para obtenção de plantas aliado ao aumento na média de plantas obtidas é a base para aumentar a eficiência de transformação genética de plantas. Experimentos de cotransformação dos genes neo e comt(AS), foram realizados por biolística. Em plantas regeneradas a partir de folhas imaturas da variedade RB835486, as análises de PCR confirmaram a incorporação do gene marcador neo em 57 e 90% das plantas em meio seletivo com geneticina (30 mg/L), sendo que a maior eficiência de regeneração de transgênicos (90%) foi obtida no pré-tratamento com o meio MS3K. Das plantas transgênicas para o gene neo, 7 e 38% também foram confirmadas para a incorporação do gene comt(AS). Nas plantas regeneradas a partir de calos embriogênicos em meio seletivo, as análises de PCR detectaram somente a incorporação do gene neo, o que ocorreu em 52% das plantas analisadas. Os resultados obtidos mostram que a cultura de discos de folhas imaturas para o processo de transformação genética por biolística é uma metodologia viável, rápida e menos onerosa, quando comparada com a cultura de calos embriogênicos. / Sugarcane is a polyploidy monocot and allogamous species that has low reproductive rate due to the difficulty of flowering. Because of these genetic and physiological characteristics breeding program takes long time and demand hard labor. Alternatively, modern biotechnology approaches contribute to the development of new cultivars. In this work we studied the methodology of plant tissue culture from immature leaf discs to establish callus culture and plant regeneration from those calli and from immature leaves, directly. The main objective was to contribute to the development of efficient methods to produce transgenic plants from callus and immature leaves, due to the growing need to produce new cultivars with specific agronomics traits. MS medium with different concentrations of 2,4-D and kinetin were tested to obtain highly embryogenic calli and to induce cellular dedifferentiation in the immature leaf discs prior to plant regeneration. Culture media without growth regulators (MS) and with the addition of BAP and NAA were tested for plant regeneration from leaf discs. Callus culture with 12 to 20 weeks resulted on average 3 to 5 plants on regeneration medium designed as MS. Immature leaves showed a high potential for plant regeneration when 2,4-D at concentrations of 5 and 8 mg/L in periods of 5 and 8 days in the dark. There were inducing of somatic embryos that resulted in average 12 to 16 plants per explant in the total period of 7 to 10 weeks. In addition, we tested the pre-treatment of leaf discs in MS3K medium which contain 2,4-D (3 mg/L) and kinetin (0.1 mg/L) before transfering to plant regeneration MS medium . The discs submitted to this pretreatment for 14, 21 and 28 days showed significant increase in the efficiency of plant regeneration, with on average of 41 to 50 plants per leaf disc in varieties RB835089 and RB855156. The reduction of time to obtain plants coupled with the increase of plants obtained is the basis for increasing the efficiency of plant genetic transformation. Co-transformation with genes neo and comt(AS), were performed by biolistics. Plants regenerated from immature leaves of the variety RB835486, PCR analysis confirmed the incorporation of the neo selection marker gene in 57 and 90% of the plants on selective medium with geneticin (30 mg/L), the higher efficiency of transgenic plants (90%) was obtained on pre-treatment in MS3K medium. Transgenic plants for the neo gene, 7 and 38% were also confirmed for the incorporation of comt (AS). PCR analysis of candidates transgenic plants from callus growing on selective medium, revelled only the insertion of the neo gene, which occurred in 52% of the analyzed plants. The results of this work showed that the approach of using immature leaf discs to obtain plant genetic transformation by biolistics methodology is a viable, cheaper and faster than using embryogenic callus.

During translation, the transfer RNAs (tRNAs) play the crucial role of adaptors between the messenger RNA and the amino acids. The tRNAs are first transcribed as pre-tRNAs which are then maturated. During this maturation, several nucleosides are modified by tRNA modification enzymes. These modifications are important for the functions of the tRNAs and for their correct folding. Many of the modifications are methylations of the bases or the ribose. Four families of tRNA methyltransferases are known, among which the SPOUT superfamily. Proteins of this superfamily are characterised by a C-terminal topological knot where the methyl donor is bound. With the exception of the monomeric Trm10, all known SPOUT proteins are dimeric and have an active site composed of residues of both protomers. Interestingly, depending on the organism, the same modification can be catalysed by completely unrelated enzymes. On the other hand, homologous enzymes can have different specificities or/and activities. These differences are well illustrated for the TrmJ and Trm10 enzymes.<p>In the first part of this work we have identified the TrmJ enzyme of Sulfolobus acidocaldarius (the model organism of hyperthermophilic Crenarchaeota) which 2’-O-methylates the nucleoside at position 32 of tRNAs. This protein belongs to the SPOUT superfamily and is homologous to TrmJ of the bacterium Escherichia coli. A comparative study shows that the two enzymes have different specificities for the nature of the nucleoside at position 32 as well as for their tRNA substrates. To try to understand these shifts of specificity at a molecular level we solved the crystal structure of the SPOUT domains of the two TrmJ proteins.<p>In the second part of this work, we have determined the crystal structure of the Trm10 protein of S. acidocaldarius. This is the first structure of a 1-methyladenosine (m1A) specific Trm10 and also the first structure of a full length Trm10 protein. The Trm10 protein of S. acidocaldarius is distantly related to its yeast homologues which are 1-methylguanosine (m1G) specific. To understand the difference of activity between the Trm10 enzymes, we compared the yeast and the S. acidocaldarius Trm10 structures. Remarkably several Trm10 proteins (such as Trm10 of Thermococcus kodakaraensis) are even able to form both m1A and m1G. To understand the capacity of the T. kodakaraensis protein to methylate A and G, a mutational study was initiated./Lors de la traduction, les ARN de transfert (ARNt) jouent le rôle crucial d’adaptateurs entre l’ARN messager et les acides aminés. Les ARNt sont transcrits sous forme de pré-ARNt qui doivent être maturés. Lors de cette maturation, plusieurs nucléosides sont modifiés. Un grand nombre de ces modifications sont des méthylations des bases ou du ribose. Quatre familles d’ARNt méthyltransferases sont actuellement connues, dont la superfamille des SPOUT. Les membres de cette superfamille sont caractérisés par un nœud dans la chaîne polypeptidique du côté C-terminal. C’est au niveau de ce nœud que se lie la S-adénosylméthionine qui est le donneur de groupement méthyle. A l’exception de Trm10 qui est monomérique, toutes les protéines SPOUT connues sont dimériques et leur site actif est formé de résidus provenant des deux protomères. Selon l’espèce, une même modification peut être formée à la même position dans la molécule d’ARNt par des enzymes qui appartiennent à des familles différentes. A l’opposé, des enzymes homologues peuvent présenter des spécificités ou des activités différentes.<p>Au cours de ce travail, nous avons identifié l’enzyme TrmJ de Sulfolobus acidocaldarius (l’organisme modèle des Crénarchées hyperthermophiles) qui méthyle le ribose du nucléoside en position 32 des ARNt. Cette protéine est un homologue de l’enzyme TrmJ de la bactérie Escherichia coli. L’étude comparative que nous avons réalisée a révélé que ces deux enzymes présentent une différence de spécificité pour la nature du nucléoside en position 32 ainsi que pour les ARNt substrats. Afin de comprendre ces différences de spécificité au niveau moléculaire, les structures des domaines SPOUT des deux TrmJ ont été déterminées et comparées.<p>En parallèle, nous avons résolu la structure cristalline de la protéine Trm10 de S. acidocaldarius. C’est la première structure disponible d’un enzyme Trm10 formant de la 1-méthyladénosine (m1A). C’est aussi la première structure complète d’une protéine Trm10. Les enzymes homologues des levures Saccharomyces cerevisiae et Schizosaccharomyces pombe qui n’ont que peu d’identité de séquence avec l’enzyme de S. acidocaldarius, forment de la 1-méthylguanosine (m1G). Dans le but de comprendre comment ces enzymes homologues peuvent présenter des activités différentes, leurs structures ont été comparées. De manière surprenante, certains homologues de Trm10 (comme l’enzyme de l’Euryarchée Thermococcus kodakaraensis) sont capables de former du m1A et du m1G. Afin de mieux comprendre comment ces protéines sont capables de méthyler deux types de bases, nous avons initié l’étude de l’enzyme Trm10 de T. kodakaraensis par mutagenèse dirigée.<p><p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

No description available.

Helicobacter pylori is one of the most genetically diverse bacterial species that successfully colonizes at least 50% of the world population. It has been associated with humans for thousands of years and most probably evolved from ancestral gastric Helicobacter species in early mammals. One of the important characteristics of this pathogen is the degree of allelic diversity and genetic variability which helps it to adapt and colonize. Phase variation is one of the mechanisms used by H. pylori to generate variation. The presence of homopolymeric nucleotide or dinucleotide repeats in an ORF make it prone to frequent length changes as a consequence of slipped strand mispairing mediated mutagenesis. Interestingly, R-M genes comprise a significant percentage of H. pylori strain-specific genes and are more prevalent in H. pylori than in other bacterial species whose genomes have been fully sequenced. R-M systems in H. pylori have been identified on the basis of sequence similarity to known restriction endonucleases and methyltransferases, genetic organization, and specific enzyme isolation and characterization. Analysis of genome sequences of H. pylori strains 26695, J99, HPAGI and 26 others has revealed the presence of more than 20 R-M systems in each stain, which are far more than detected in any other bacterial genome sequence till date. hp1369 and hp1370 are two ORFs in stain 26695 coding for hypothetical proteins. hp 1369 has a stretch of poly-G repeats, thus making hp1369-hp1370, a candidate of phase variation. hpag1_1313 is homolog of hp1369-hp1370 which got up-regulated, in a person suffering from acute gastritis, thus making these genes an interesting subject of investigation. This study was therefore initiated with the following objectives: 1. Cloning, over-expression and purification of Type III MTase (ORF- hp1369- hp1370) and its cognate restriction enzyme (hp1371). 2. Biochemical characterization of MTase (HP1369-HP1370): Determination of oligomeric status, kinetic properties, binding affinities for AdoMet and DNA. Sequence analysis shows the presence of a poly-G track (10 Gs) at 3’-end of hp1369 which is a signature sequence for phase variation. Addition of a single nucleotide can place both hp1369 and hp1370 in-frame, which could code for a single polypeptide. hp1369 and hp1370 in H. pylori strain 26695 alone do not code for any functional protein but with the fusion of hp1369 and hp1370 can code for a protein with all the nine motifs of a DNA MTase. Interestingly, on the basis of arrangement of Motifs, it is probably the first example of ε type of methyltransferase. By site-directed mutagenesis a single G nucleotide was inserted in the poly-G track and both the ORFs (hp1369 and hp1370 ) became in-frame, coding for fully functional HP1369-HP1370 MTase. Kinetic parameters for functional HP1369-HP1370 MTase were determined, and has shown that there was substrate inhibition in methylation reaction at higher concentrations of AdoMets. When preincubation studies were done, enzyme-DNA complex was found to be more competent than enzyme-AdoMet complex. HP1369-HP1370 MTase exists as dimer in solution, having affinity for duplex DNA and does not bind to single-stranded DNA. Binding affinity for ligand (AdoMet) was determined by Isothermal Titration Calorimetry method. H. pylori has evolving restriction-modification systems. It is capable of taking new R-M systems from the environment in the form of DNA released from other bacteria or other Helicobacter strains. H. pylori genome is dynamic with high mutation rates. Random mutations in R-M genes can result in a non-functional R-M systems or R-M systems with new properties. The dynamics of R-M system plays a vital role in shaping up the genome.

<div>Protein N-terminal methyltransferases (NTMTs) are a family of enzymes that methylate the α-N-terminus of a variety of protein substrates. Both NTMT1 and NTMT2 recognize a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) to install 1-3 methyl group(s) on the substrates. NTMT1 plays important roles in mitosis regulation, chromatin interactions, and DNA damage repair. Another member NTMT2 shares ~50% sequence similarity and the same substrate recognition motif although NTMT2 was initially characterized as a mono-methyltransferase. To understand the molecular mechanism of NTMT2, we obtained the first co-crystal structure of NTMT2 in complex with its peptide substrate. After an extensive investigation of substrate recognition and methylated products of NTMT1/2, we found out that NTMT2 can fully methylate G/P-PKRIA peptides despite a predominant mono-methyltransferase. Moreover, we identified a gatekeeper N89 in NTMT2 that controls the substrate entry and the product specificity of NTMT2.</div><div>To elucidate the biological functions of NTMT1/2-catalyzed N-terminal methylation, we applied two different strategies to discover cell-potent inhibitors. Guided by the co-crystal structures of NTMT1 in complex with previously reported inhibitors, we designed and synthesized a series of new peptidomimetic inhibitors. By introducing more hydrophobic groups, the most cell-potent peptidomimetic inhibitor GD562 (IC50 = 0.93 ± 0.04 µM) exhibited over 2-fold increased inhibition on cellular N-terminal methylation levels with an IC50 value of ~50 µM compared to previously reported peptidomimetic inhibitor DC541. Meanwhile, we also discovered the first potent small molecule inhibitor Genz-682452 (IC50 = 0.5 ± 0.04 µM) after screening ~58,000 compounds. Subsequent structural modifications led to the discovery of GD433 (IC50 = 27 ± 0.5 nM) with a 20-fold increased potency compared to the initial hit Genz-682452. Inhibition mechanism indicated both inhibitors bind to peptide-binding pocket and co-crystal structures of both Genz-682452 and GD433 with NTMT1 confirmed their binding modes. Furthermore, GD433 shows over 7-fold selectivity over other major 40 protein methyltransferases and DNA methyltransferase and exhibits improved selectivity for NTMT1 over glucosylceramide synthase (GCS). GD433 significantly decreases the cellular N-terminal methylation level of NTMT1 substrates RCC1 and SET at 10 nM in both HEK293 and HCT116 cells, providing a valuable probe for cell-based studies in the future.<br></div><p><br></p>

Skeletal muscle differentiation requires synergy between tissue-specific transcription factors, chromatin remodeling enzymes and the general transcription machinery. Here we demonstrate that two distinct protein arginine methyltransferases are required to complete the differentiation program. Prmt5 is a type II methyltransferase, symmetrically dimethylates histones H3 and H4 and has been shown to play a role in transcriptional repression. An additional member of the Prmt family, Carm1 is a type I methyltransferase, and asymmetrically methylates histone H3 and its substrate proteins. MyoD regulates the activation of the early class of skeletal muscle genes, which includes myogenin. Prmt5 was bound to and dimethylates H3R8 at the myogenin promoter in a differentiation-dependent fashion. When proteins levels of Prmt5 were reduced by antisense, disappearance of H3R8 dimethylation and Prmt5 binding was observed. Furthermore, binding of Brg1 to regulatory sequences of the myogenin promoter was abolished. All subsequent events relying on Brg1 function, such as chromatin remodeling and stable binding by muscle specific transcription factors such as MyoD, were eliminated. Robust association of Prmt5 and dimethylation of H3R8 at myogenin promoter sequences was observed in mouse satellite cells, the precursors of mature myofibers. Prmt5 binding and histone modification were observed to a lesser degree in mature myofibers. Therefore, these results indicate that Prmt5 is required for dimethylating histone at the myogenin locus during skeletal muscle differentiation in order to facilitate the binding of Brg1, the ATPase subunit of the chromatin remodeling complex SWI/SNF. Further exploration of the role of Prmt5 during the activation of the late class of muscle genes revealed that though Prmt5 is associated with and dimethylates histones at the regulatory elements of late muscle genes in tissue and in culture, it was dispensable for late gene activation. Previous reports had indicated that Carm1 was involved during late gene activation. We observed that Carm1 was bound to and responsible for dimethylating histones at late muscle gene promoters in tissue and in culture. In contrast to Prmt5, a complete knockout of Carm1 resulted in abrogation of late muscle gene activation. Furthermore, loss of Carm1 binding and dimethylated histones resulted in a disappearance of Brg1 binding and chromatin remodeling at late muscle gene loci. Time course chromatin immunoprecipitations revealed that Carm1 binding and histone dimethylation occurred concurrently with the onset of late gene activation. In vitro binding assays revealed that an interaction between Carm1, myogenin and Mef2D exists. These results demonstrate that Carm1 is recruited to the regulatory sequences of late muscle genes via its interaction with either myogenin or Mef2D and is responsible for dimethylates histones in order to facilitate the binding of Brg1. Therefore, these results indicate that during skeletal muscle differentiation, distinct roles exist for these Prmts such that Prmt5 is required for activation of early genes while Carm1 is essential for late gene induction.

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first protocol tackles this problem by developing a way to obtain active untagged recombinant PRMT proteins. The second protocol describes a fast and efficient method for quantitative measurement of AdoMet-dependent methyltranseferase activity with protein substrates. In addition to being very sensitive, this method decreases the processing time for the analysis of PRMT activity to a few minutes compared to weeks by traditional methods, and generates 3000-fold less radioactive waste. We then used these methods to investigate the effect of truncating the NT of human PRMT1 variant 1 (hPRMT1-V1) on enzyme activity, protein-protein interactions, and substrate specificity. Our studies show that the NT of hPRMT1-V1 influences enzymatic activity and protein-protein interactions. In particular, methylation of a variety of protein substrates was more efficient when the first 10 amino acids of hPRMT1v1 were removed, suggesting an autoinhibitory role for this small section of the N-terminus. Likewise, as portions of the NT were removed, the altered hPRMT1v1 constructs were able to interact with more proteins. Overall, my studies suggest the the sequence and length of the NT of hPRMT1v1 is capable of enforcing specific protein interactions.

No description available.

Background: The impact of genetic polymorphisms on cognition is assumed to increase with age as losses of brain resources have to be compensated for. We investigate the relation of catechol-O-methyltransferase (COMT) p.Val158Met polymorphism and cognitive capacity in the course of adult development, healthy aging and the development of mild cognitive impairment (MCI) in two birth cohorts of subjects born between 1930 and 1932 or between 1950 and 1952. Methods: Thorough neuropsychological assessment was conducted in a total of 587 participants across three examination waves between 1993 and 2008. The COMT genotype was determined as a restriction fragment length polymorphism after PCR amplification and digestion with Nla III. Results: Significant effects of the COMT p.Val158Met polymorphism were identified for attention and cognitive flexibility in the younger but not the older cohort. Conclusion: These results confirm the importance of the COMT p.Val158Met genotype on tasks assessing attention and cognitive flexibility in midlife but not in healthy aging and the development of MCI. Our findings suggest that the influence of COMT changes as a function of age, decreasing from midlife to aging.

No description available.