RESUMO
The Didymium iridis DiSSU1 intron is located in the nuclear SSU rDNA and has an unusual twin-ribozyme organization. One of the ribozymes (DiGIR2) catalyses intron excision and exon ligation. The other ribozyme (DiGIR1), which along with the endonuclease-encoding I-DirI open reading frame (ORF) is inserted in DiGIR2, carries out hydrolysis at internal processing sites (IPS1 and IPS2) located at its 3' end. Examination of the in vivo expression of DiSSU1 shows that after excision, DiSSU1 is matured further into the I-DirI mRNA by internal DiGIR1-catalysed cleavage upstream of the ORF 5' end, as well as truncation and polyadenylation downstream of the ORF 3' end. A spliceosomal intron, the first to be reported within a group I intron and the rDNA, is removed before the I-DirI mRNA associates with the polysomes. Taken together, our results imply that DiSSU1 uses a unique combination of intron-supplied ribozyme activity and adaptation to the general RNA polymerase II pathway of mRNA expression to allow a protein to be produced from the RNA polymerase I-transcribed rDNA.
Assuntos
Amoeba/genética , Íntrons/genética , Spliceossomos/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Ribossômico/genética , Regulação da Expressão Gênica/genética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Polirribossomos/genética , RNA Polimerase II/genética , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética , RNA Catalítico/genética , RNA Mensageiro/genética , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
DiSSU1 is an optional group I twintron present in the nuclear extrachromosomal ribosomal DNA of the myxomycete Didymium iridis. DiSSU1 appears to be complex both in structure and function. At the RNA level it has a twin-ribozyme organization composed of two group I ribozymes with different functions, separated by an open reading frame. Here, we show that DiSSU1 is mobile when haploid intron-containing and intron-less amoebae are mated. The mobility process is fast, being completed in 5-10 nuclear cycles after mating in the developing zygote and plasmodia. Analyses of progeny from genetic crosses confirm intron mobility. DiSSU1 is the first example of a mobile group I twintron. The intron-encoded protein was expressed in Escherichia coli and found to be an endonuclease, I-DirI, that cleaves an intron-less ribosomal DNA allele at the intron-insertion site, and is probably involved in intron homing. The endonuclease I-DirI seems to be a rare example of a protein that is expressed from a ribozyme-processed RNA polymerase I transcript in vivo.
Assuntos
DNA Ribossômico/genética , Mixomicetos/genética , Sequência de Aminoácidos , Sequência de Bases , Elementos de DNA Transponíveis , DNA Fúngico/genética , Endonucleases/genética , Genes Fúngicos , Íntrons , Dados de Sequência Molecular , Precursores de Ácido Nucleico/genética , Polimorfismo Genético , Processamento Pós-Transcricional do RNA , RNA Catalítico , RNA Fúngico/genéticaRESUMO
A novel gene designated cmr, which mapped to 18.8 min of the Escherichia coli K-12 genome, was shown to mediate resistance to chloramphenicol when it was expressed from a multicopy vector. The accumulation of chloramphenicol was significantly less in cells overexpressing cmr than in control cells harboring the vector without insert. After the addition of a proton motive force blocker, the level of accumulation of chloramphenicol in the resistant cells rapidly approached the levels found in sensitive cells carrying only the chromosomal cmr. Northern (RNA) blot analyses revealed that the cmr gene is expressed as a 1.3-kb transcript. This size corresponds very well with a predicted size of 1,293 nucleotides (nt) based on the mapping of the transcription initiation site to a G residue 24 nt upstream of the start codon and the presence of a putative rho-independent terminator sequence ending 36 nt downstream of the 1,233-nt open reading frame encoding the putative Cmr protein. The 411-residue-long derived amino acid sequence contains 12 putative transmembrane segments and displays significant sequence similarities to several known drug resistance protein sequences of the major facilitator family. We provide evidence strongly suggesting that the resistance mediated by Cmr involves active exclusion of chloramphenicol.
Assuntos
Resistência ao Cloranfenicol/genética , Escherichia coli/genética , Genes Bacterianos , Sequência de Aminoácidos , Sequência de Bases , Cloranfenicol/farmacocinética , Mapeamento Cromossômico , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Dados de Sequência Molecular , RNA Bacteriano/análiseRESUMO
Comparison of two group I intron sequences in the nucleolar genome of the myxomycete Physarum flavicomum to their homologs in the closely related Physarum polycephalum revealed insertion-like elements. One of the insertion-like elements consists of two repetitive sequence motifs of 11 and 101 bp in five and three copies, respectively. The smaller motif, which flanks the larger, resembles a target duplication and indicates a relationship to transposons or retroelements. The insertion-like elements are found in the peripheral loops of the RNA structure; the positions occupied by the ORFs of mobile nucleolar group I introns. The P. flavicomum introns are 1184 and 637 bp in size, located in the large subunit ribosomal RNA gene, and can be folded into group I intron structures at the RNA level. However, the intron 2s from both P. flavicomum and P. polycephalum contain an unusual core region that lacks the P8 segment. None of the introns are able to self-splice in vitro. Southern analysis of different isolates indicates that the introns are not optional in myxomycetes.