Jesús Valdés

Circadian oscillations of RPCH gene expression in the eyestalk of the crayfish Cherax quadricarinatus

The RPCH and beta-actin cDNAs from the crayfish Cherax quadricarinatus were amplified, cloned and sequenced. The primary structure sequences of these cDNAs were compared to other members of the AKH/RPCH family. Fluctuations in the amount of the C. quadricarinatus RPCH and beta-actin mRNAs, as cDNAs, were quantified every 3h by RT-PCR. Single cosinor analysis supports the notion of beta-actin and RPCH mRNA circadian behavior in animals subjected to 12h:12h light/dark regimes. In constant darkness RPCH mRNA concentration changes to ultradian cycles.

A Possible Molecular Ancestor for Mollusk APGWamide, Insect Adipokinetic Hormone, and Crustacean Red Pigment Concentrating Hormone

Abstract. Precursor structures of various members of the neuropeptide family adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) of mandibular arthropods and the APGWamide family of mollusks were compared. Amino acid alignments showed a common overall architecture (signal peptide, active peptide, related peptide), with a similar α helix–random coil secondary structure. DNA sequence alignments revealed close similarities between the genes encoding for the peptides of the two families. The APGWamide genes are larger than the AKH/RPCH genes. The sequence environment occupied by introns is similar in AKH/RPCH and APGWamide genes. Such similarities suggest that these peptide families might have been originated by gene rearrangements from a common ancestor having either an AKH/RPCH/APGWamide-like structure or both an AKH/RPCH-like and an APGWamide-like structures. In the former model, DNA fragments could have been gained when the ancestor evolved to mollusks and it could have lost nucleotides when the progression to mandibular arthropods took place. In the second model, AKH/RPCH-like structures could have been fused during evolution toward mandibular arthropods, whereas in mollusks they could have been lost with the possible amplification of the APGWamide-like structure. Loss of domains in exon 1 may have originated the signal peptide and the first codon of the active RPCH. In exon 2, loss of domains possibly determined the junctions of codons 2 to 5 with the loss of a APGWamide copy; exon 3 underwent fewer variations. The similarity of the mollusk APGWamide precursors is closer to that of the RPCH family than the insect AKH family, indicating an earlier evolutionary departure.

Purification of bacterial genomic DNA in less than 20 min using chelex-100 microwave: examples from strains of lactic acid bacteria isolated from soil samples

We established a Chelex 100-Microwave method for the purification of bacterial genomic DNA (gDNA) in less than 20xa0min with high yield and good quality, useful for multiple purposes. It combines Chelex 100, proteinase K, RNase A and heating in a microwave oven. The resulting gDNA was used directly to identify bacterial species of the Order Lactobacillales by means of PCR amplification of their 16S rDNA gene, isolated from sediments on the Yucatan Peninsula, Mexico. This method produced gDNA free of phenolic and protein residual contaminants from 100 of these isolated bacteria. 16S rDNA amplification and sequencing showed Pediococcus acidilactici to prevail in inland lagoons, and Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sp., and Lactobacillus fermentum to be most abundant in the soils of livestock farms. The combination of Chelex 100, enzymes and microwave heating used in the Chelex 100-Microwave method produced large amounts of highly pure gDNA from Gram-positive and Gram-negative bacteria, in less than 20xa0min.

Expression of claudins -2 and -4 and cingulin is coordinated with the start of stratification and differentiation in corneal epithelial cells: retinoic acid reversibly disrupts epithelial barrier

Summary Although tight junctions (TJ) have been extensively studied in simple epithelial cells, it is still unknown whether their organization is coupled to cell differentiation in stratified epithelia. We studied the expression of TJ in RCE1(5T5) cells, an in vitro model which mimics the sequential steps of rabbit corneal epithelial differentiation. RCE1(5T5) cells expressed TJ components which were assembled once cells constituted differentiated epithelia, as suggested by the increase of transepithelial electrical resistance (TER) which followed a similar kinetic to the expression of the early differentiation marker Pax-6. TJ were functional as indicated by the establishment of an epithelial barrier nonpermeable to ruthenium red or a biotin tracer. In immunostaining experiments, TJ were located at the superficial cells from the suprabasal layers; Western blot and RT-PCR suggested that TJ were composed of claudins (cldn) -1, -2, -4, cingulin (cgn), occludin (ocln) and ZO-1. Semi-quantitative RT-PCR and TER measurements showed that TJ became organized when cells began to form a 3–5 layers stratified epithelium; TER increased once cells reached confluence, with a time course comparable to the raise in the expression of cgn, cldn-2 and -4. Nevertheless, cldn-1, -2, ZO-1 and ocln were present in the cells from the beginning of cultivation, suggesting that TER increases mainly depend on TJ assembly. While EGF increased epithelial barrier strength, retinoic acid disrupted it, increasing paracellular flux about 2-fold; this effect was concentration dependent and completely reversible. Our results suggest that TJ assembly is tightly linked to the expression of corneal epithelial terminal phenotype.

The genomic organization of the open reading frame of the red pigment concentrating hormone gene in the blue crab Callinectes sapidus

The open reading frame (ORF) of the gene for the precursor of the octapeptide Red Pigment Concentrating Hormone (RPCH) from the blue crab Callinectes sapidus was cloned by PCR with oligonucleotides targeted to the initiation and the end of the translation coding sequences. A 272 bp intron was characterized between nucleotides 343 and 344 of the reported cDNA, present in the region coding for the last amino acids of the precursor related peptide of RPCH. The intron genomic structure here described is similar to that reported for the gene coding for the Adipokinetic Hormone (AKH) of the grasshopper Schistocerca nitans.

Identification of repressive and active epigenetic marks and nuclear bodies in Entamoeba histolytica.

BackgroundIn human hosts, Entamoeba histolytica cysts can develop into trophozoites, suggesting that the life cycle of this parasite are regulated by changes in gene expression. To date, some evidence has suggested that epigenetic mechanisms such as DNA methylation and histone modification are involved in the regulation of gene expression in Entamoeba. Some post–translational modifications (PTMs) at the N-terminus of E. histolytica’s histones have been reported experimentally, including tri-methylation in the lysine 4 of histone H3 (H3K4me3) and dimethylation in the lysine 27 of histone H3 (H3K27me2), dimethylation of arginine 3 (H4R3me2) and the indirect acetylation of histone H4 in the N-terminal region. However, it is not known which residues of histone H4 are subject to acetylation and/or methylation or where in the nucleus these epigenetic marks are located.MethodsHistones from trophozoites of E. histolytica were obtained and analyzed by LC-MS/MS. WB assays were performed using antibodies against epigenetic marks (acetylated lysines and methylated arginines). Immunofluorescence assays (IFA) were carried out to determine the distribution of PTMs and the localization of DNA methylation as a heterochromatin marker. Nuclear bodies such as the nucleolus were identified by using antibodies against fibrillarin and nucleolin and speckles by using anti-PRP6 antibody.ResultsSome new PTMs in histone H4 of E. histolytica, such as the acetylation of lysines 5, 8, 12 and 16 and the monomethylation of arginine 3, were identified by WB. IFA demonstrated that some marks are associated with transcriptional activity (such as acetylation and/or methylation) and that these marks are distributed throughout the E. histolytica nucleus. Staining with antibodies against anti-pan-acetylated lysine H4 histone and 5-methyl cytosine showed that the activation and transcriptional repression marks converge. Additionally, two nuclear bodies, the nucleolus and speckles, were identified in this parasite.ConclusionsThis study provides the first evidence that the nucleus of E. histolytica is not compartmentalized and contains two nuclear bodies, the nucleolus and speckles, the latter of which was not identified previously. The challenge is now to understand how these epigenetic marks and nuclear bodies work together to regulate gene expression in E. histolytica.

The Entamoeba histolytica TBP and TRF1 transcription factors are GAAC-box binding proteins, which display differential gene expression under different stress stimuli and during the interaction with mammalian cells

BackgroundEntamoeba histolytica is the protozoan parasite responsible for human amebiasis. It causes up to 100,000 deaths worldwide each year. This parasite has two closely related basal transcription factors, the TATA-box binding protein (EhTBP) and the TBP-related factor 1 (EhTRF1). TBP binds to the canonical TATTTAAA-box, as well as to different TATA variants. TRF1 also binds to the TATTTAAA-box. However, their binding capacity to diverse core promoter elements, including the GAAC-element, and their role in gene regulation in this parasite remains unknown.MethodsEMSA experiments were performed to determine the binding capacity of recombinant TBP and TRF1 to TATA variants, GAAC and GAAC-like boxes. For the functional analysis under different stress stimuli (e.g. growth curve, serum depletion, heat-shock, and UV-irradiation) and during the interaction with mammalian cells (erythrocytes, MDCK cell monolayers, and hepatocytes of hamsters), RT-qPCR, and gene knockdown were performed.ResultsBoth transcription factors bound to the different TATA variants tested, as well as to the GAAC-boxes, suggesting that they are GAAC-box-binding proteins. The KD values determined for TBP and TRF1 for the different TATA variants and GAAC-box were in the range of 10-12xa0M to 10-11xa0M. During the death phase of growth or in serum depletion, Ehtbp mRNA levels significantly increased, whereas the mRNA level of Ehtrf1 did not change under these conditions. Ehtrf1 gene expression was negatively regulated by UV-irradiation and heat-shock stress, with no changes in Ehtbp gene expression. Moreover, Ehtrf1 gene also showed a negative regulation during erythrophagocytosis, liver abscess formation, and a transient expression level increase at the initial phase of MDCK cell destruction. Finally, the Ehtbp gene knockdown displayed a drastic decrease in the efficiency of erythrophagocytosis in G3 trophozoites.ConclusionsTo our knowledge, this study reveals that these basal transcription factors are able to bind multiple core promoter elements. However, their immediate change in gene expression level in response to different stimuli, as well as during the interaction with mammalian cells, and the diminishing of erythrophagocytosis by silencing the Ehtbp gene indicate the different physiological roles of these transcription factors in E. histolytica.

The Entamoeba histolytica Syf1 Homolog Is Involved in the Splicing of AG-Dependent and AG-Independent Transcripts

Syf1 is a tetratricopeptide repeat (TPR) protein implicated in transcription elongation, spliceosome conformation, mRNA nuclear-cytoplasmic export and transcription-coupled DNA repair. Recently, we identified the spliceosomal components of the human parasite Entamoeba histolytica, among them is EhSyf. Molecular predictions confirmed that EhSyf contains 15 type 1 TPR tandem α-antiparallel array motifs. Amoeba transformants carrying plasmids overexpressing HA-tagged or EhSyf silencing plasmids were established to monitor the impact of EhSyf on the splicing of several test Entamoeba transcripts. EhSyf Entamoeba transformants efficiently silenced or overexpressed the proteins in the nucleus. The overexpression or absence of EhSyf notably enhanced or blocked splicing of transcripts irrespective of the strength of their 3′ splice site. Finally, the absence of EhSyf negatively affected the transcription of an intron-less transcript. Altogether our data suggest that EhSyf is a bona fide Syf1 ortholog involved in transcription and splicing.

Postsplicing-Derived Full-Length Intron Circles in the Protozoan Parasite Entamoeba histolytica

Noncoding circular RNAs are widespread in the tree of life. Particularly, intron-containing circular RNAs which apparently upregulate their parental gene expression. Entamoeba histolytica, the causative agent of dysentery and liver abscesses in humans, codes for several noncoding RNAs, including circular ribosomal RNAs, but no intron containing circular RNAs have been described to date. Divergent RT-PCR and diverse molecular approaches, allowed us to detect bona fide full-length intronic circular RNA (flicRNA) molecules. Self-splicing reactions, RNA polymerase II inhibition with Actinomycin D, and second step of splicing-inhibition with boric acid showed that the production of flicRX13 (one of the flicRNAs found in this work, and our test model) depends on mRNA synthesis and pre-mRNA processing instead of self-splicing. To explore the cues and factors involved in flicRX13 biogenesis in vivo, splicing assays were carried out in amoeba transformants where splicing factors and Dbr1 (intron lariat debranching enzyme 1) were silenced or overexpressed, or where Rabx13 wild-type and mutant 5′ss (splice site) and branch site minigene constructs were overexpressed. Whereas SF1 (splicing factor 1) is not involved, the U2 auxiliary splicing factor, Dbr1, and the GU-rich 5′ss are involved in postsplicing flicRX13 biogenesis, probably by Dbr1 stalling, in a similar fashion to the formation of ciRNAs (circular intronic RNAs), but with distinctive 5′-3′ss ligation points. Different from the reported functions of ciRNAs, the 5′ss GU-rich element of flicRX13 possibly interacts with transcription machinery to silence its own gene in cis. Furthermore, introns of E. histolytica virulence-related genes are also processed as flicRNAs.

TvZNF1 is a C 2 H 2 zinc finger protein of Trichomonas vaginalis

The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental stimulus by biometals such as Zn2+. Many of these proteins have the classical C2H2 zinc finger motifs (C2H2-ZNFm) of approximately 30 amino acids, where a Zn2+ ion is coordinated by two cysteine and two histidine residues. Trichomonas vaginalis is a protozoan parasite than responds to environmental changes including Zn2+. Until now has not been described any ZNF that could be involved in the regulation of genic expression of T. vaginalis. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from T. vaginalis and isolated the gene, tvznf1 encoding it. TvZNF1 have eight C2H2-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn2+ upregulation expression of tvznf1 gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in T. vaginalis.