RESUMEN

Leptin is described as a pro-inflammatory signal in fat tissue, which is released from adipocytes and in turn activates immune cells. Also, leptin levels are known to be increased in pregnancies complicated with enhanced inflammatory processes in the placenta. Hence, we assumed that increased leptin amounts might contribute to inducing an inflammatory response in the placenta. To test this hypothesis, pregnant mice were continuously infused with recombinant murine leptin s. c. from day g13 to g16, resulting in a 3-fold increase of maternal circulating serum leptin levels. Dissected placentas were examined for the expression of pro-inflammatory cytokines IL-6 and TNF-alpha and the anti-inflammatory cytokine IL-10 using qPCR analysis. No changes were found except for TNF-alpha, which was slightly elevated upon leptin stimulation. However, TNF-alpha protein levels were not significantly higher in placentas from leptin treated mice. Also, leukocyte infiltration in the labyrinth section of placentas was not increased. In summary, our data demonstrate for the first time that elevated leptin levels alone do not induce an inflammatory response in the placenta.

Asunto(s)

Inflamación/patología , Leptina/metabolismo , Placenta/metabolismo , Placenta/patología , Animales , Citocinas/metabolismo , Conducta Alimentaria , Femenino , Inflamación/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Placenta/efectos de los fármacos , Placenta/enzimología , Embarazo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismo

RESUMEN

The placenta is a major barrier that prevents potentially infectious agents from causing fetal diseases or related complications during pregnancy. Therefore, we postulated that the placenta might express a broad repertoire of antimicrobial proteins as well as inflammatory chemokines and cytokines to combat invading microorganisms. Here we demonstrate that placental cells indeed express a wide range of AMPs (antimicrobial peptides and proteins) including bactericidal/permeability-increasing protein (BPI), secretory leukocyte protease inhibitor (SLPI), human ß-defensin 2 (hBD2), acyloxyacyl hydrolase (AOAH), and cathelicidin (CAP18). In addition, these cells also secrete pro-inflammatory cytokines and chemokines upon stimulation with bacterial ligands. Notably, we show that BPI expression by placental cells could be completely attributed to granulocytes while highly purified placental trophoblasts expressed only a subset of the AMPs like SLPI. Unexpectedly, trophoblast AMPs did not exhibit inducible secretion in response to various TLR ligands and further investigations showed that the unresponsiveness of trophoblasts to lipopolysaccharide (LPS) was due to a lack of TLR4 expression. In summary, we have shown that the expression of different AMPs can be allocated to various cells in the placenta and the repertoire of the AMPs expressed by placental cells is a result of a cooperation of leukocytes as well as cells from embryonic origin.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/genética , Placenta/citología , Placenta/fisiología , alfa-Defensinas/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Especificidad de Órganos , Placenta/inmunología , Placenta/metabolismo , Embarazo , Trofoblastos/citología , Trofoblastos/metabolismo , Trofoblastos/fisiología , Regulación hacia Arriba , alfa-Defensinas/metabolismo

RESUMEN

Mechanisms that control the activation of antigen-specific immune responses in vivo are poorly understood. It has been suggested that the initiation of adaptive immune responses is controlled by innate immune recognition. Mammalian Toll-like receptors play an essential role in innate immunity by recognizing conserved pathogen-associated molecular patterns and initiating the activation of NF-kappaB and other signaling pathways through the adapter protein, MyD88. Here we show that MyD88-deficient mice have a profound defect in the activation of antigen-specific T helper type 1 (TH1) but not TH2 immune responses. These results suggest that distinct pathways of the innate immune system control activation of the two effector arms of adaptive immunity.

Asunto(s)

Antígenos de Diferenciación/genética , Proteínas de Drosophila , Activación de Linfocitos , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Receptores Inmunológicos , Células TH1/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Caspasa 1/genética , Diferenciación Celular , Células Cultivadas , Células Dendríticas/inmunología , Inmunoglobulinas/biosíntesis , Interferón gamma/biosíntesis , Interleucina-13/biosíntesis , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide , Fenotipo , Células Th2/inmunología , Receptores Toll-Like

RESUMEN

We report here the sequence of the 1743 bp intergenic spacer (IGS) that separates the 3'-end of the large subunit ribosomal RNA (rRNA) gene from the 5'-end of the small subunit (SSU) rRNA gene in the circular, extrachromosomal ribosomal DNA (rDNA) of Euglena gracilis. The IGS contains a 277 nt stretch of sequence that is related to a sequence found in ITS 1, an internal transcribed spacer between the SSU and 5.8S rRNA genes. Primer extension analysis of IGS transcripts identified three abundant reverse transcriptase stops that may be analogous to the transcription initiation site (TIS) and two processing sites (A' and A0) that are found in this region in other eukaryotes. Features that could influence processing at these sites include an imperfect palindrome near site A0 and a sequence near site A' that could potentially base pair with U3 small nucleolar RNA. Our identification of the TIS (verified by mung bean nuclease analysis) is considered tentative because we also detected low-abundance transcripts upstream of this site throughout the entire IGS. This result suggests the possibility of 'read-around' transcription, i.e. transcription that proceeds multiple times around the rDNA circle without termination.

Asunto(s)

ADN Circular/genética , ADN Intergénico/genética , ADN Ribosómico/genética , Euglena/genética , ARN Ribosómico/biosíntesis , Transcripción Genética/genética , Animales , Emparejamiento Base , Secuencia de Bases , Secuencia Conservada/genética , Datos de Secuencia Molecular , Ensayos de Protección de Nucleasas , Procesamiento Postranscripcional del ARN , ARN Ribosómico/química , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Alineación de Secuencia , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo

RESUMEN

The common gamma-chain (gammac) is a component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 and is essential for their signal transduction. Western blotting and a newly established enzyme-linked immunosorbent assay detected substantial constitutive levels (50-250 ng/mL) of soluble gammac (sgammac) in sera of murine inbred strains. It was demonstrated that purified immune cells, such as T, B, and natural killer cells, and macrophages released this protein after activation. Transfection experiments with cDNA encoding the full-length gammac showed that shedding of the transmembrane receptor led to the release of sgammac. The shedding enzymes, however, appeared to be distinct from those cleaving other cytokine receptors because inhibitors of metalloproteases (eg, TAPI) did not influence sgammac release. In vivo, superantigen-induced stimulation of T cells enhanced sgammac serum concentrations up to 10-fold within 6 hours. Because these findings demonstrated regulated expression of a yet unknown molecule in the immune response, further experiments were performed to assess the possible function(s) of sgammac. A physiological role of sgammac was indicated by its capacity to specifically inhibit cell growth induced by gammac-dependent cytokines. Mutational analysis revealed that the C-terminus and the WSKWS motif are essential for the cytokine inhibitory effect of the sgammac and for binding of the molecule to cytokine receptor-expressing cells. Thus, competitive displacement of the transmembrane gammac by excess sgammac is the most likely mechanism of cell growth inhibition. It was implied that naturally produced sgammac is a negative modulator of gammac-dependent cytokines.

Asunto(s)

Citocinas/efectos de los fármacos , Citocinas/fisiología , Receptores de Interleucina-7/fisiología , Receptores de Interleucina/metabolismo , Transducción de Señal/efectos de los fármacos , Secuencias de Aminoácidos , Animales , Western Blotting , División Celular/efectos de los fármacos , Citocinas/farmacología , Depresión Química , Femenino , Subunidad gamma Común de Receptores de Interleucina , Activación de Linfocitos , Subgrupos Linfocitarios/metabolismo , Masculino , Metaloendopeptidasas , Ratones , Ratones Endogámicos/metabolismo , Ratones Mutantes/metabolismo , Ratones SCID/metabolismo , Mutagénesis Sitio-Dirigida , Péptido Hidrolasas/metabolismo , Estructura Terciaria de Proteína , Subunidades de Proteína , Receptores de Interleucina/sangre , Receptores de Interleucina/genética , Solubilidad , Células Tumorales Cultivadas

RESUMEN

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from approximately 11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an approximately 55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A' pre-rRNA processing sites within the 5' external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5' ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C. fasciculata and Trypanosoma brucei involves 3'-terminal addition of three A residues that are not present in the corresponding DNA sequences.

Asunto(s)

Crithidia fasciculata/genética , ADN Protozoario/genética , ADN Ribosómico/genética , ARN Nucleolar Pequeño/metabolismo , Animales , Emparejamiento Base , Secuencia de Bases , Secuencia Conservada , Crithidia fasciculata/metabolismo , ADN Protozoario/metabolismo , ADN Ribosómico/metabolismo , Heterogeneidad Genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Trypanosoma brucei brucei/genética

RESUMEN

The innate immune system evolved to recognize conserved microbial products, termed pathogen-associated molecular patterns (PAMPs), which are invariant among diverse groups of microorganisms. PAMPs are recognized by a set of germ-line encoded pattern recognition receptors (PRRs). Among the best characterized PAMPs are bacterial lipopolysaccharide (LPS), peptidoglycan (PGN), mannans, and other constituents of bacterial and fungal cell walls, as well as bacterial DNA. Recognition of bacterial DNA is the most enigmatic of these, as it depends on a particular sequence motif, called the CpG motif, in which an unmethylated CpG present in a particular sequence context accounts for a potent immunostimulatory activity of CpG DNA. Receptor(s) of the innate immune system that mediate recognition of CpG DNA are currently unknown. Here, we report that recognition of CpG DNA requires MyD88, an adaptor protein involved in signal transduction by the Toll-like receptors (TLRs), essential components of innate immune recognition in both Drosophila and mammals [1,2]. Signaling induced by CpG DNA was found to be unaffected in cells deficient in TLR2 or TLR4, suggesting that some other member of the Toll family mediates recognition of bacterial DNA.

Asunto(s)

Antígenos de Diferenciación/metabolismo , Islas de CpG/genética , ADN/metabolismo , Proteínas de Drosophila , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos de Diferenciación/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Islas de CpG/inmunología , ADN/genética , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Interleucina-6/metabolismo , Macrófagos Peritoneales/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide , Receptores de Superficie Celular/genética , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like

RESUMEN

Mollusks are an extraordinarily diverse group of animals with an estimated 200,000 species, second only to the phylum Arthropoda. We conducted a comparative analysis of complete mitochondrial ribosomal large subunit sequences (LSU) of a chiton, two bivalves, six gastropods, and a cephalopod. In addition, we determined secondary structure models for each of them. Comparative analyses of nucleotide variation revealed substantial length variation among the taxa, with stylommatophoran gastropods possessing the shortest lengths. Phylogenetic analyses of the nucleotide sequence data supported the monophyly of Albinaria, Euhadra herklotsi + Cepaea nemoralis, Stylommatophora, Cerithioidea, and when only transversions are included, the Bivalvia. The phylogenetic limits of the mitochondrial LSU rRNA gene within mollusks appear to be up to 400 million years, although this estimate will have to be tested further with additional taxa. Our most novel finding was the discovery of phylogenetic signal in the secondary structure of rRNA of mollusks. The absence of entire stem/loop structures in Domains II, III, and V can be viewed as three shared derived characters uniting the stylommatophoran gastropods. The absence of the aforementioned stem/loop structure explains much of the observed length variation of the mitochondrial LSU rRNA found within mollusks. The distribution of these unique secondary structure characters within mollusks should be examined.

Asunto(s)

ADN Mitocondrial/genética , ADN Ribosómico/genética , Moluscos/genética , Filogenia , Animales , Composición de Base , Secuencia de Bases , ADN/química , ADN/genética , ADN/aislamiento & purificación , ADN Mitocondrial/química , ADN Ribosómico/química , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Moluscos/clasificación , ARN Ribosómico/química , ARN Ribosómico/genética , Alineación de Secuencia , Análisis de Secuencia de ADN

RESUMEN

We report the complete nucleotide sequence of the Tetrahymena pyriformis mitochondrial genome and a comparison of its gene content and organization with that of Paramecium aurelia mtDNA. T. pyriformis mtDNA is a linear molecule of 47,172 bp (78.7 % A+T) excluding telomeric sequences (identical tandem repeats of 31 bp at each end of the genome). In addition to genes encoding the previously described bipartite small and large subunit rRNAs, the T. pyriformis mitochondrial genome contains 21 protein-coding genes that are clearly homologous to genes of defined function in other mtDNAs, including one (yejR) that specifies a component of a cytochrome c biogenesis pathway. As well, T. pyriformis mtDNA contains 22 open reading frames of unknown function larger than 60 codons, potentially specifying proteins ranging in size from 74 to 1386 amino acid residues. A total of 13 of these open reading frames ("ciliate-specific") are found in P. aurelia mtDNA, whereas the remaining nine appear to be unique to T. pyriformis; however, of the latter, five are positionally equivalent and of similar size in the two ciliate mitochondrial genomes, suggesting they may also be homologous, even though this is not evident from sequence comparisons. Only eight tRNA genes encoding seven distinct tRNAs are found in T. pyriformis mtDNA, formally confirming a long-standing proposal that most T. pyriformis mitochondrial tRNAs are nucleus-encoded species imported from the cytosol. Atypical features of mitochondrial gene organization and expression in T. pyriformis mtDNA include split and rearranged large subunit rRNA genes, as well as a split nad1 gene (encoding subunit 1 of NADH dehydrogenase of respiratory complex I) whose two segments are located on and transcribed from opposite strands, as is also the case in P. aurelia. Gene content and arrangement are very similar in T. pyriformis and P. aurelia mtDNAs, the two differing by a limited number of duplication, inversion and rearrangement events. Phylogenetic analyses using concatenated sequences of several mtDNA-encoded proteins provide high bootstrap support for the monophyly of alveolates (ciliates, dinoflagellates and apicomplexans) and slime molds.

Asunto(s)

ADN Mitocondrial/genética , ADN Protozoario/genética , Genoma , Paramecium/genética , Tetrahymena pyriformis/genética , Animales , Secuencia de Bases , Codón/genética , Evolución Molecular , Genes Duplicados/genética , Genes Protozoarios/genética , Genes de ARNr/genética , Código Genético/genética , Variación Genética/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Filogenia , Mapeo Físico de Cromosoma , Polimorfismo Genético/genética , Proteínas Protozoarias/genética , ARN de Transferencia/genética , Telómero/genética

RESUMEN

U5 snRNAs in trypanosomatid protozoa do not contain the trimethylguanosine cap structures that are often targeted in snRNA isolation procedures. As a result, the trypanosomatids are not well represented in the database of available U5 snRNA sequences. We have isolated and determined the sequence of the U5 snRNA from Crithidia fasciculata. Comparison with previously published trypanosomatid U5 snRNA sequences allows us to deduce the pattern of structural conservation and variation among these very divergent snRNA molecules.

Asunto(s)

ARN Nuclear Pequeño/genética , Trypanosoma/genética , Animales , Secuencia de Bases , Crithidia fasciculata/genética , Electroforesis en Gel de Poliacrilamida , Guanosina/análogos & derivados , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , ARN/química , ARN Nuclear Pequeño/química , Alineación de Secuencia

RESUMEN

In contrast to earlier proposals, recent evidence suggests that trans-spliceosomes in trypanosomatid protozoa may contain a homolog of U1 small nuclear (sn) RNA (Schnare, M.N. and Gray, M.W. (1999) J. Biol. Chem. 274, 23,691-23,694). However, the candidate trypanosomatid U1 snRNA is unconventional because it lacks the highly conserved stem/loop II present in all other U1 snRNAs. Trypanosomatids also possess a unique spliced leader-associated (SLA) RNA of unknown function. We present the complete sequence of the SLA RNA from Crithidia fasciculata and propose that it may contribute a U1 snRNA-like stem/loop II to the trans-spliceosome.

Asunto(s)

Crithidia fasciculata/química , ARN Protozoario/química , ARN Lider Empalmado/química , Animales , Secuencia de Bases , Crithidia fasciculata/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Protozoario/genética , ARN Nuclear Pequeño/química , ARN Lider Empalmado/genética , Homología de Secuencia de Ácido Nucleico

RESUMEN

In trypanosomatid protozoa, all mRNAs obtain identical 5'-ends by trans-splicing of the 5'-terminal 39 nucleotides of a small spliced leader RNA to appropriate acceptor sites in pre-mRNA. Although this process involves spliceosomal small nuclear (sn) RNAs, it is thought that trypanosomatids do not contain a homolog of the cis-spliceosomal U1 snRNA. We show here that a trypanosomatid protozoon, Crithidia fasciculata, contains a novel small RNA that displays several features characteristic of a U1 snRNA, including (i) a methylguanosine cap and additional 5'-terminal modifications, (ii) a potential binding site for common core proteins that are present in other trans-spliceosomal ribonucleoproteins, (iii) a U1-like 5'-terminal sequence, and (iv) a U1-like stem/loop I structure. Because trypanosomatid pre-mRNAs do not appear to contain cis-spliced introns, we argue that this previously unrecognized RNA species is a good candidate to be a trans-spliceosomal U1 snRNA.

Asunto(s)

Crithidia fasciculata/genética , ARN Protozoario/análisis , ARN Nuclear Pequeño/análisis , Animales , Secuencia de Bases , Datos de Secuencia Molecular , ARN Protozoario/química , ARN Nuclear Pequeño/química

RESUMEN

In the flagellated protozoon Euglena gracilis, characterized nuclear genes harbor atypical introns that usually are flanked by short repeats, adopt complex secondary structures in pre-mRNA, and do not obey the GT-AG rule of conventional cis-spliced introns. In the nuclear fibrillarin gene of E. gracilis, we have identified three spliceosomal-type introns that have GT-AG consensus borders. Furthermore, we have isolated a small RNA from E. gracilis and propose, on the basis of primary and secondary structure comparisons, that it is a homolog of U1 small nuclear RNA, an essential component of the cis-spliceosome in higher eukaryotes. Conserved sequences at the 5' splice sites of the fibrillarin introns can potentially base pair with Euglena U1 small nuclear RNA. Our observations demonstrate that spliceosomal GT-AG cis-splicing occurs in Euglena, in addition to the nonconventional cis-splicing and spliced leader trans-splicing previously recognized in this early diverging unicellular eukaryote.

Asunto(s)

Euglena gracilis/genética , ARN Nuclear Pequeño/genética , Empalmosomas/genética , Animales , Emparejamiento Base , Secuencia de Bases , Secuencia Conservada , ADN Complementario , Euglena gracilis/metabolismo , Intrones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , ARN Protozoario/química , ARN Protozoario/genética , ARN Nuclear Pequeño/química , Empalmosomas/metabolismo

RESUMEN

IL-4 is a pleiotropic cytokine that is essential for the differentiation of Th2 cells and is critically involved in the pathogenesis of certain infectious and allergic diseases. We have produced and functionally characterized a mutant of murine IL-4 (IL-4.Y119D) as a potential antagonist of IL-4. The analysis of IL-4R binding revealed no differences between wild-type and mutated IL-4. Despite this finding, IL-4.Y119D was unable to induce proliferation of several IL-4-responsive T cell lines mediated via the type I IL-4R (IL-4Ralpha/common gamma chain (gamma c chain)) and specifically inhibited the proliferative effect of wild-type IL-4. In contrast, with IL-4.Y119D we found induction of MHC class II and CD23 molecules on resting splenic B cells as well as proliferation of B9 plasmocytoma cells. In addition, IL-4.Y119D induced mRNA for soluble IL-4R, leading to the release of soluble IL-4R protein by spleen cells. In macrophages, mutated IL-4 in combination with IFN-gamma induced TNF-alpha-dependent killing of Leishmania major parasites such as wild-type IL-4. The agonistic effects of IL-4.Y119D were observed on cells expressing the IL-13R alpha-chain, including an IL-13R alpha-chain transfected T cell line, but were absent in T cells that lack this molecule, indicating that IL-4.Y119D conveys its activity via the type II IL-4R (IL-4Ralpha/IL-13Ralpha). The described IL-4 mutant, therefore, represents a new tool to use in dissecting different IL-4 functions that are mediated by either type I or type II IL-4R complexes.

Asunto(s)

Interleucina-4/genética , Interleucina-4/fisiología , Receptores de Interleucina-4/antagonistas & inhibidores , Sustitución de Aminoácidos/genética , Animales , Ácido Aspártico/genética , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , División Celular/genética , División Celular/inmunología , Línea Celular , Citotoxicidad Inmunológica , Femenino , Sustancias de Crecimiento/fisiología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Interleucina-13/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13 , Interleucina-4/metabolismo , Interfase/genética , Interfase/inmunología , Leishmania major/crecimiento & desarrollo , Leishmania major/inmunología , Activación de Linfocitos , Macrófagos/inmunología , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Unión Proteica/inmunología , Receptores de IgE/biosíntesis , Receptores de Interleucina/biosíntesis , Receptores de Interleucina/genética , Receptores de Interleucina-13 , Receptores de Interleucina-4/metabolismo , Solubilidad , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transfección/inmunología , Células Tumorales Cultivadas , Tirosina/genética

RESUMEN

U3 small nucleolar RNA (snoRNA) has been isolated from Euglena gracilis, an early diverging protist, and its primary sequence determined. Although this 180-nucleotide-long RNA is considerably smaller than its homolog in vertebrate animals, it contains the conserved sequence blocks (boxes A, Ao, B, C and D) characteristic of U3 snoRNAs from other organisms. A secondary structure can be modelled that displays many of the salient features found in published core structures of vertebrate, yeast and trypanosome U3 snoRNAs. The functional significance of this proposed secondary structure is discussed in relation to the role E. gracilis U3 snoRNA may have in pre-rRNA processing in this organism. Multiple expressed species of E. gracilis U3 snoRNA were found to differ in nucleotide sequence at a number of positions; some of these differences alter pairing in the proposed secondary structure. Analysis of E. gracilis genomic DNA revealed a complex pattern of U3-hybridizing sequences that parallels the multiplicity of expressed species of U3 snoRNA revealed by transcript analysis.

Asunto(s)

Euglena gracilis/genética , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Animales , Secuencia de Bases , Genoma Fúngico , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Nucleósidos/química , Precursores del ARN , ARN de Hongos , ARN Ribosómico

RESUMEN

Comparative modeling of secondary structure is a proven approach to predicting higher order structural elements in homologous RNA molecules. Here we present the results of a comprehensive comparison of newly modeled or refined secondary structures for the cytoplasmic large subunit (23 S-like) rRNA of eukaryotes. This analysis, which covers a broad phylogenetic spectrum within the eukaryotic lineage, has defined regions that differ widely in their degree of structural conservation, ranging from a core of primary sequence and secondary structure that is virtually invariant, to highly variable regions. New comparative information allows us to propose structures for many of the variable regions that had not been modeled before, and rigorously to confirm or refine variable region structures previously proposed by us or others. The present analysis also serves to identify phylogenetically informative features of primary and secondary structure that characterize these models of eukaryotic cytoplasmic 23 S-like rRNA. Finally, the work summarized here provides a basis for experimental studies designed both to test further the validity of the proposed secondary structures and to explore structure-function relationships.

Asunto(s)

Células Eucariotas/química , Conformación de Ácido Nucleico , ARN Ribosómico 23S/química , Secuencia de Bases , Redes de Comunicación de Computadores , Secuencia Conservada , Citoplasma/química , Bases de Datos Factuales , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia

RESUMEN

In Euglena gracilis, the cytoplasmic large subunit (LSU) rRNA is composed of 14 discrete small RNA species that must somehow interact in the functional ribosome. We have isolated native complexes of Euglena rRNA and show here that the largest of these complexes contains eight of the 14 LSU rRNA species. Several of these small rRNA species are able to associate in vitro to reform an isolated domain of LSU rRNA structure.

Asunto(s)

Euglena gracilis/metabolismo , ARN Protozoario/metabolismo , ARN Ribosómico/metabolismo , Animales , Secuencia de Bases , Citoplasma/metabolismo , Electroforesis en Gel de Poliacrilamida , Euglena gracilis/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , ARN Protozoario/química , ARN Protozoario/genética , ARN Ribosómico/química , ARN Ribosómico/genética , ARN Ribosómico 28S/química , ARN Ribosómico 28S/genética , ARN Ribosómico 28S/metabolismo , ARN Ribosómico 5.8S/química , ARN Ribosómico 5.8S/genética , ARN Ribosómico 5.8S/metabolismo , Ribosomas

RESUMEN

In a previous investigation of the rDNA region in Tetrahymena pyriformis mitochondrial DNA, we identified a putative tRNA(Met) gene [Heinonen et al. (1987) J. Biol. Chem. 262, 2879-2887]. On the basis of Northern hybridization analyses, we suggested that this gene is expressed, even though the resulting tRNA would be unusually small and have an atypical dihydrouridine stem-loop domain. We report here the complete nucleotide sequence and post-transcriptional modification pattern of this tRNA(Met), confirming its predicted primary structure and supporting the view that this structurally aberrant species functions in translation in T. pyriformis mitochondria.

Asunto(s)

Procesamiento Postranscripcional del ARN , ARN Protozoario/química , ARN de Transferencia de Metionina/química , ARN/química , Tetrahymena pyriformis/genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN/metabolismo , ARN Mitocondrial , ARN Protozoario/metabolismo , ARN de Transferencia de Metionina/metabolismo , Tetrahymena pyriformis/metabolismo

Asunto(s)

Bases de Datos Factuales , ARN Ribosómico 23S/genética , Animales , Secuencia de Bases , Bases de Datos Bibliográficas , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia

RESUMEN

We report the sequence of a 7.2 kilobase pair DNA fragment containing a copy of the wheat mitochondrial gene (rrn26) that encodes the mitochondrial large-subunit ribosomal RNA (26S rRNA). The mature 26S rRNA was determined by direct RNA sequencing to be 3467 nucleotides long, and to share a 5'-terminal pentanucleotide (5'-AUCAU), thought to be important in post-transcriptional processing, with the wheat mitochondrial small-subunit (18S) rRNA. Two other prominent features of the sequence were noted. First, upstream of rrn26 are located two tandem copies of a 70 base pair element containing a putative mitochondrial promoter motif (TCGTATAAAAA). Second, downstream of rrn26 is a sequence element that, if transcribed, would produce an RNA with a secondary structure resembling that of tRNAs but differing sufficiently from the latter structure to preclude any transcript from functioning normally in translation. These upstream and downstream sequence elements may play a role in the expression of rrn26 in wheat mitochondria.

Asunto(s)

ADN Mitocondrial/genética , ARN Ribosómico/genética , Triticum/genética , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Homología de Secuencia de Ácido Nucleico