RESUMO
In Trypanosoma cruzi DNA is packaged into chromatin by octamers of histone proteins that form nucleosomes. Transcription of protein coding genes in trypanosomes is constitutive producing polycistronic units and gene expression is primarily regulated post-transcriptionally. However, chromatin organization influences DNA dependent processes. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities found in trypanosomes. To map nucleosomes genome-wide in several organisms, digestion of chromatin with micrococcal nuclease followed by deep sequencing has been applied. Nonetheless, the special requirements for cell manipulation and the uniqueness of the chromatin organization in trypanosomes entails a customized analytical approach. In this work, we adjusted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we implemented an exhaustive and thorough computational workflow to overcome the difficulties imposed by this complex genome. We tested the performance of two aligners, Bowtie2 and HISAT2, and discuss their advantages and caveats. Specifically, we highlight the relevance of using the whole genome as a reference instead of the commonly used Esmeraldo-like haplotype to avoid spurious alignments. Additionally, we show that using the whole genome refines the average nucleosome representation, but also the quality of mapping for every region represented. Moreover, we show that the average nucleosome organization around trans-splicing acceptor site described before, is not just an average since the same chromatin pattern is detected for most of the represented regions. In addition, we extended the study to a non-hybrid strain applying the experimental and analytical approach to Sylvio-X10 strain. Furthermore, we provide a source code for the construction of 2D plots and heatmaps which are easy to adapt to any T. cruzi strain.
Assuntos
Nucleossomos , Trypanosoma , Nucleossomos/genética , Cromatina/genética , Histonas/genética , Trypanosoma/genética , DNA , Nuclease do Micrococo/metabolismoRESUMO
Subtelomeres (ST) are chromosome regions that separate telomeres from euchromatin and play relevant roles in various biological processes of the cell. While their functions are conserved, ST structure and genetic compositions are unique to each species. This study aims to identify and characterize the subtelomeric regions of the 13 Toxoplasma gondii chromosomes of the Me49 strain. Here, STs were defined at chromosome ends based on poor gene density. The length of STs ranges from 8.1 to 232.4 kbp, with a gene density of 0.049 genes/kbp, lower than the Me49 genome (0.15 kbp). Chromatin organization showed that H3K9me3, H2A.X, and H3.3 are highly enriched near telomeres and the 5' end of silenced genes, decaying in intensity towards euchromatin. H3K4me3 and H2A.Z/H2B.Z are shown to be enriched in the 5' end of the ST genes. Satellite DNA was detected in almost all STs, mainly the sat350 family and a novel satellite named sat240. Beyond the STs, only short dispersed fragments of sat240 and sat350 were found. Within STs, there were 12 functional annotated genes, 59 with unknown functions (Hypothetical proteins), 15 from multigene FamB, and 13 from multigene family FamC. Some genes presented low interstrain synteny associated with the presence of satellite DNA. Orthologues of FamB and FamC were also detected in Neospora caninum and Hammondia hammondi. A re-analysis of previous transcriptomic data indicated that ST gene expression is strongly linked to the adaptation to different situations such as extracellular passage (evolve and resequencing study) and changes in metabolism (lack of acetyl-CoA cofactor). In conclusion, the ST region of the T. gondii chromosomes was defined, the STs genes were determined, and it was possible to associate them with high interstrain plasticity and a role in the adaptability of T. gondii to environmental changes.
RESUMO
Protein kinase A (PKA) activity is involved in dimorphism of the basal fungal lineage Mucor. From the recently sequenced genome of Mucor circinelloides we could predict ten catalytic subunits of PKA. From sequence alignment and structural prediction we conclude that the catalytic core of the isoforms is conserved, and the difference between them resides in their amino termini. This high number of isoforms is maintained in the subdivision Mucoromycotina. Each paralogue, when compared to the ones form other fungi is more homologous to one of its orthologs than to its paralogs. All of these fungal isoforms cannot be included in the class I or II in which fungal protein kinases have been classified. mRNA levels for each isoform were measured during aerobic and anaerobic growth. The expression of each isoform is differential and associated to a particular growth stage. We reanalyzed the sequence of PKAC (GI 20218944), the only cloned sequence available until now for a catalytic subunit of M. circinelloides. PKAC cannot be classified as a PKA because of its difference in the conserved C-tail; it shares with PKB a conserved C2 domain in the N-terminus. No catalytic activity could be measured for this protein nor predicted bioinformatically. It can thus be classified as a pseudokinase. Its importance can not be underestimated since it is expressed at the mRNA level in different stages of growth, and its deletion is lethal.
Assuntos
Domínio Catalítico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Mucor/enzimologia , Aerobiose , Anaerobiose , Perfilação da Expressão Gênica , Mucor/genética , Mucor/crescimento & desenvolvimento , Isoformas de Proteínas/genética , RNA Mensageiro/análise , Homologia de SequênciaRESUMO
The strength of the interaction between the catalytic and regulatory subunits in protein kinase A differs among species. The linker region from regulatory subunits is non-conserved. To evaluate the participation of this region in the interaction with the catalytic subunit, we have assayed its effect on the enzymatic properties of the catalytic subunit. Protein kinase A from three fungi, Mucor rouxii, Mucor circinelloides and Saccharomyces cerevisiae have been chosen as models. The R-C interaction is explored by using synthetic peptides of 8, 18 and 47 amino acids, corresponding to the R subunit autophosphorylation site plus a variable region toward the N terminus (0, 10, or 39 residues). The K(m) of the catalytic subunits decreased with the length of the peptide, while the V(max) increased. Viscosity studies identified product release as the rate limiting step for phosphorylation of the longer peptides. Pseudosubstrate derivatives of the 18 residue peptides did not display a competitive inhibition behavior toward either kemptide or a bona fide protein substrate since, at low relative pseudosubstrate/substrate concentration, stimulation of kemptide or protein substrate phosphorylation was observed. The behavior was mimicked by intact R. We conclude that in addition to its negative regulatory role, the R subunit stimulates C activity via distal interactions.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação Fúngica da Expressão Gênica , Mucor/enzimologia , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Aminoácidos/química , Catálise , Domínio Catalítico , Ativação Enzimática , Cinética , Dados de Sequência Molecular , Mucor/metabolismo , Peptídeos/química , Fosforilação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Protein kinase A from the fungus Mucor circinelloides shows high affinity interaction between regulatory (R) and catalytic (C) subunits. Its R subunit shows a differential presence of several acidic residues in linker I region, in the amino terminus. Mutants R1, lacking the N-terminal region, and R2, lacking the acidic cluster, were used to analyze its effect on the interaction with the C subunit, assessed through inhibition of catalytic activity and cAMP activation of reconstituted holoenzyme. A similar decrease in the interaction was obtained when using R1 and R2 with the homologous C subunit; however when using heterologous bovine C, only R1 had a decreased interaction. The results show the general importance of linker I region in the R-C interaction in protein kinases A and point to the importance of the acidic cluster present in the N-terminus of M. circinelloides R subunit in the high affinity interaction between R and C in this holoenzyme.