Trypanosome mRNAs share a common 5' spliced leader sequence.

A 5'-terminal leader sequence of 35 nucleotides was found to be present on multiple trypanosome RNAs. Based on its representation in cDNA libraries, we estimate that many, if not all, trypanosome mRNAs contain this leader. This same leader was originally identified on mRNAs encoding the molecules responsible for antigenic variation, variant surface glycoproteins. Studies of selected cDNAs containing this leader sequence revealed that leader-containing transcripts can be stage-specific, stage-regulated, or constitutive. They can be abundant or rare, and transcribed from single or multigene families. No linkage between the genomic leader sequences and the structural gene exons was observed. Possible mechanisms by which the leader sequences are added to trypanosome mRNAs are discussed

Identification of a small RNA containing the trypanosome spliced leader: a donor of shared 5' sequences of trypanosomatid mRNAs?

The 35 nucleotide spliced leader (SL) sequence is found on the 5' end of numerous trypanosome mRNAs, yet the tandemly organized reiteration units encoding this leader are not detectably linked to any of these structural genes. Here we report the presence of a class of discrete small SL RNA molecules that are derived from the genomic SL reiteration units of Trypanosoma brucei, Trypanosoma cruzi, and Leptomonas collosoma. These small SL RNAs are 135, 105, and 95 nucleotides, respectively, and contain a 5'-terminal SL or SL-like sequence. S1 nuclease analyses demonstrate that these small SL RNAs are transcribed from continuous sequence within the respective SL reiteration units. With the exception of the SL sequence and a concensus donor splice site immediately following it, these small RNAs are not well conserved. We suggest that the small SL RNAs may function as a donor of the SL sequence in an intermolecular process that places the SL at the 5' terminus of many trypanosomatid mRNAs

Fungicidal activity of a phospholipase A2-derived synthetic peptide variant against Candida albicans

pEM-2, a 13-mer synthetic peptide variant derived from myotoxin II, a phospholipase A2 homologue present in Bothrops asper snake venom, has shown potent bactericidal activity in previous studies due to the combination of cationic and hydrophobic amino acids, including three tryptophan-substituted residues in its sequence. This study reports that pEM-2 also exerts potent fungicidal activity against a variety of clinically relevant Candida species, killing 100% of yeasts at concentrations near 10 mg/l (5 μM), as indicated by plate counting assays. Thus, this peptide displays a broad-spectrum antimicrobial activity, in the absence of hemolytic activity. The fungicidal action of pEM-2 against Candida can be partially inhibited by increasing concentrations of extracellular divalent cations (Ca+2 or Mg+2), in agreement with its proposed membrane- permeabilizing mechanism of action.pEM-2, una variante de péptido sintético derivada de la miotoxina II, un homólogo de la fosfolipasa A2 presente en el veneno de la serpiente Bothrops asper, ha mostrado en estudios previos una potente actividad bactericida debida a la combinación de aminoácidos catiónicos e hidrófobos en su secuencia, incluyendo tres residuos sustituidos por triptófano. Este estudio describe que el pEM-2 también ejerce una potente actividad fungicida contra una variedad de especies de Candida clínicamente relevantes, matando el 100% de las levaduras a concentraciones cercanas a 10 mg/l (5 μM), mediante ensayos de recuento en placas. De tal modo, este péptido muestra una acción antimicrobiana de amplio espectro, en ausencia de actividad hemolítica. La acción fungicida del pEM-2 sobre Candida es parcialmente inhibida por concentraciones crecientes de cationes divalentes extracelulares (Ca+2 o Mg+2), en concordancia con su mecanismo de acción propuesto, permeabilizante de membranas.Vicerrectoría de Investigación, University of Costa Rica/[]/UCR/Costa RicaNeTropica Sweden-Central America Research Network/[01-R-2003]/NeTropica/UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Alternative mechanism for bacteriophage adsorption to the motile bacterium Caulobacter crescentus

2D and 3D cryo-electron microscopy, together with adsorption kinetics assays of ϕCb13 and ϕCbK phage-infected Caulobacter crescentus, provides insight into the mechanisms of infection. ϕCb13 and ϕCbK actively interact with the flagellum and subsequently attach to receptors on the cell pole. We present evidence that the first interaction of the phage with the bacterial flagellum takes place through a filament on the phage head. This contact with the flagellum facilitates concentration of phage particles around the receptor (i.e., the pilus portals) on the bacterial cell surface, thereby increasing the likelihood of infection. Phage head filaments have not been well characterized and their function is described here. Phage head filaments may systematically underlie the initial interactions of phages with their hosts in other systems and possibly represent a widespread mechanism of efficient phage propagation

Ultraviolet mutagenesis and inducible DNA repair in Caulobacter crescentus

The ability to reactivate ultraviolet (UV) damaged phage ΦCbK (W-reactivation) is induced by UV irradiation of Caulobacter crescentus cells. Induction of W-reactivation potential is specific for phage ΦCbK, requires protein synthesis, and is greatly reduced in the presence of the rec-526 mutation. The induction signal generated by UV irradiation is transient, lasting about 1 1/2–2 h at 30°C; if chloramphenicol is present during early times after UV irradiation, induction of W-reactivation does not occur. Induction is maximal when cells are exposed to 5–10 J/m 2 of UV, a dose that also results in considerable mutagenesis of the cells. Taken together, these observations demonstrate the existence of a UV inducible, protein synthesis requiring, transiently signalled, rec -requiring DNA repair system analogous to W-reactivation in Escherichia coli . In addition, C. crescentus also has an efficient photoreactivation system that reverses UV damage in the presence of strong visible light.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47557/1/438_2004_Article_BF00329935.pd

A Human-Curated Annotation of the Candida albicans Genome

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications

Persistent ER Stress Induces the Spliced Leader RNA Silencing Pathway (SLS), Leading to Programmed Cell Death in Trypanosoma brucei

Trypanosomes are parasites that cycle between the insect host (procyclic form) and mammalian host (bloodstream form). These parasites lack conventional transcription regulation, including factors that induce the unfolded protein response (UPR). However, they possess a stress response mechanism, the spliced leader RNA silencing (SLS) pathway. SLS elicits shut-off of spliced leader RNA (SL RNA) transcription by perturbing the binding of the transcription factor tSNAP42 to its cognate promoter, thus eliminating trans-splicing of all mRNAs. Induction of endoplasmic reticulum (ER) stress in procyclic trypanosomes elicits changes in the transcriptome similar to those induced by conventional UPR found in other eukaryotes. The mechanism of up-regulation under ER stress is dependent on differential stabilization of mRNAs. The transcriptome changes are accompanied by ER dilation and elevation in the ER chaperone, BiP. Prolonged ER stress induces SLS pathway. RNAi silencing of SEC63, a factor that participates in protein translocation across the ER membrane, or SEC61, the translocation channel, also induces SLS. Silencing of these genes or prolonged ER stress led to programmed cell death (PCD), evident by exposure of phosphatidyl serine, DNA laddering, increase in reactive oxygen species (ROS) production, increase in cytoplasmic Ca2+, and decrease in mitochondrial membrane potential, as well as typical morphological changes observed by transmission electron microscopy (TEM). ER stress response is also induced in the bloodstream form and if the stress persists it leads to SLS. We propose that prolonged ER stress induces SLS, which serves as a unique death pathway, replacing the conventional caspase-mediated PCD observed in higher eukaryotes

A comparative genome-wide study of ncRNAs in trypanosomatids

<p>Abstract</p> <p>Background</p> <p>Recent studies have provided extensive evidence for multitudes of non-coding RNA (ncRNA) transcripts in a wide range of eukaryotic genomes. ncRNAs are emerging as key players in multiple layers of cellular regulation. With the availability of many whole genome sequences, comparative analysis has become a powerful tool to identify ncRNA molecules. In this study, we performed a systematic genome-wide in silico screen to search for novel small ncRNAs in the genome of <it>Trypanosoma brucei </it>using techniques of comparative genomics.</p> <p>Results</p> <p>In this study, we identified by comparative genomics, and validated by experimental analysis several novel ncRNAs that are conserved across multiple trypanosomatid genomes. When tested on known ncRNAs, our procedure was capable of finding almost half of the known repertoire through homology over six genomes, and about two-thirds of the known sequences were found in at least four genomes. After filtering, 72 conserved unannotated sequences in at least four genomes were found, 29 of which, ranging in size from 30 to 392 nts, were conserved in all six genomes. Fifty of the 72 candidates in the final set were chosen for experimental validation. Eighteen of the 50 (36%) were shown to be expressed, and for 11 of them a distinct expression product was detected, suggesting that they are short ncRNAs. Using functional experimental assays, five of the candidates were shown to be novel H/ACA and C/D snoRNAs; these included three sequences that appear as singletons in the genome, unlike previously identified snoRNA molecules that are found in clusters. The other candidates appear to be novel ncRNA molecules, and their function is, as yet, unknown.</p> <p>Conclusions</p> <p>Using comparative genomic techniques, we predicted 72 sequences as ncRNA candidates in <it>T. brucei</it>. The expression of 50 candidates was tested in laboratory experiments. This resulted in the discovery of 11 novel short ncRNAs in procyclic stage <it>T. brucei</it>, which have homologues in the other trypansomatids. A few of these molecules are snoRNAs, but most of them are novel ncRNA molecules. Based on this study, our analysis suggests that the total number of ncRNAs in trypanosomatids is in the range of several hundred.</p