RESUMEN
Mariner-like elements (MLEs) are classII transposons with highly conserved sequence properties and are widespread in the genome of animal species living in continental environments. We describe here the first full-length MLE found in the genome of a marine crustacean species, the deep-sea hydrothermal crab Bythograea thermydron (Crustacea), named Bytmar1. A comparison of its sequence features with those of the MLEs contained in the genomes of continental species reveals several distinctive characteristics. First, Bytmar1 elements contains an ORF that may encode three transposase isoforms 349, 379, and 398 amino acids (aa) in long. The two biggest proteins are due to the presence of a 30- and 49-aa flag, respectively, at the N-terminal end of the 349-aa cardinal MLE transposase. Their GC contents are also significantly higher than those found in continental MLEs. This feature is mainly due to codon usage in the transposase ORF and directly interferes with the curvature propensities of the Bytmar1 nucleic acid sequence. Such an elevated GC content may interfere with the ability of Bytmar 1 to form an excision complex and, in consequence, with its efficiency to transpose. Finally, the origin of these characteristics and their possible consequences on transposition efficiency are discussed.
Asunto(s)
Braquiuros/genética , Elementos Transponibles de ADN/genética , Filogenia , Transposasas/genética , Secuencia de Aminoácidos , Animales , Composición de Base , Secuencia de Bases , Análisis por Conglomerados , Codón/genética , Cartilla de ADN , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Océano Pacífico , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The two inverted terminal repeats (ITRs) flanking the Mos-1 mariner element differ in sequence at four positions. Gel retardation experiments indicated that each of these differences has a significant impact on the quality of the interaction between the ITR and the Mos-1 transposase. We showed that the transposase binds to the 3' ITR better than to the 5' ITR. The results of transposition assays performed in Escherichia coli indicated that these differences have an influence on the rate of transposition and the stability of the transposition products. Finally, we find that the wild-type configuration of the Mos-1 element, with one 5' ITR and one 3' ITR, is less efficient for transposition in bacteria than that of an element having two 3' ITRs.
Asunto(s)
Elementos Transponibles de ADN , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Secuencias Repetidas Terminales , Secuencia de Bases , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Mutagénesis Insercional , Conformación de Ácido Nucleico , Unión Proteica , Transposasas/metabolismoRESUMEN
Mariner-like elements are widespread eukaryotic transposons, but Mos-1 is the only natural element that is known to be active. Little is known about the biochemistry of mariner transposition. The first step in the process is the binding of the transposase to the 5' and 3' inverted terminal repeats (ITRs) of the element. Using the 3' ITR of the element, we have determined the binding properties of a recombinant Mos-1 transposase produced in bacteria, and we have used deletion derivatives to localize the minimal ITR binding domain between amino acids 1 and 141. Its features and structure indicate that it differs from the ITR binding domain of the transposase encoded by Tc1-related elements.
Asunto(s)
Proteínas de Unión al ADN/química , Secuencias Repetidas Terminales , Transposasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Cationes Bivalentes/química , ADN Bacteriano/análisis , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Eliminación de SecuenciaRESUMEN
Mariner-like elements (MLE) belong to the Tc1/ mariner superfamily of class II transposons. We have analyzed the mariner related to the cecropia subfamily, and called mammal mar1, in four mammalian genomes, Bos taurus (Bovidae), Homo sapiens (Primata), Mus musculus (Rodentia), and Ovis aries (Ovidae). Three kinds of MLE sequences were found in all these species: full-length 1.3-kbp elements, shorter elements 80 bp-1.2 kbp, and single inverted terminal repeats (ITRs). All the 1.3-kbp genomic copies sequenced had an open reading frame encoding a transposase interrupted by stop codons or frame shifts. Phylogenetic analysis of the full-length elements suggested at least two distinct populations of mammal mar1 elements in each species. This was confirmed by using a statistical method that allows defining populations. Finally, the evolutionary origin of the mammal mar1 elements and the paradoxes are discussed.
Asunto(s)
Elementos Transponibles de ADN , Evolución Molecular , Genoma , Animales , Secuencia de Bases , Bovinos , Cartilla de ADN , Ratones , Datos de Secuencia Molecular , Filogenia , Ovinos , Especificidad de la EspecieRESUMEN
We show that oligodeoxyribonucleotides (oligos) composed of alpha- and beta-anomeric sections can be used as antisense compounds. An octamer has been chosen as an effector domain to form a substrate for RNaseH. This octamer is complementary to the translation start site of the pim-1 protooncogene mRNA. Chimeric alpha-beta oligos and their beta-analogs have a similar binding affinity for their target. These oligos also direct efficient RNaseH-mediated cleavage of target mRNA. Among all alpha-beta oligos studied, one with an alpha-fragment bound by its 3'-end to the 3'-end of the beta-octamer is the most resistant to nucleolytic digestion in biological media. The alpha-beta oligos have been found to inhibit in vitro translation of pim-1 RNA with specificity.
Asunto(s)
Oligonucleótidos Antisentido/farmacología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/genética , Animales , Secuencia de Bases , Sangre , Medios de Cultivo , Eritroblastos , Globinas/genética , Humanos , Hidrólisis , Ratones , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/metabolismo , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Ribonucleasa H/metabolismoRESUMEN
A new type of chimeric oligonucleotides composed of alpha- and beta-sections is described. The sequence of eight beta-nucleotides flanked at 3'- or/and 5'-ends by nuclease-resistant alpha-oligonucleotides has been chosen as an effector domain to form a substrate for RNase H. The synthesized oligonucleotides are complementary to the translation initiation site of the pim protooncogene mRNA. We used the chemical ligation method to prepare the chimeric oligonucleotides. The thermal stability of heteroduplexes formed by the alpha beta oligonucleotides with a complementary strand is not significantly altered compared to that of their beta-analogs. These oligonucleotides promote efficient RNase H-mediated cleavage of pim mRNA. Among the alpha beta oligonucleotides studied, one with an alpha-fragment bound by its 3'-end to the 3'-end of the beta-octanucleotide proved to be the most resistant to nucleolytic digestion in human plasma, calf serum, and murine fibroblast lysate. This alpha beta oligonucleotide directs more specific RNA cleavage by RNase H than its beta beta counterpart.