RESUMO
The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.
RESUMO
Transcription of the 2.6 kbp psbK-psbI-trnG cluster in tobacco chloroplasts has been studied. This cluster contains, in linear sequence, the genes encoding two low-molecular-mass polypeptides, K and I, of photosystem II (psbK and psbI, respectively), and tRNA(Gly) (UCC) (trnG). Northern blot hybridization revealed that the largest transcript (2.6 kb) hybridizes to psbK, psbI and trnG, but not to the following trnR-UCU. Ten other transcripts ranging from 0.1 to 1.3 kb were also detected. Three of these transcripts overlap the divergent transcript arising from trnS-GCU located on the opposite DNA strand. S1 mapping and primer extension experiments showed that these multiple transcripts comprise eight distinct 5' ends. By in vitro capping assays two of them were determined to be transcriptional initiation sites; one is located 163 bp upstream of psbK and the other is 6 bp upstream of trnG. The 3' ends of transcripts were determined by S1 mapping; one lies between psbI and trnG and the other is at the end of trnG. The presence of dual promoters of trnG is discussed.
Assuntos
Cloroplastos , DNA/genética , Família Multigênica , Nicotiana/genética , Plantas Tóxicas , Regiões Promotoras Genéticas , Autorradiografia , Sequência de Bases , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmídeos , RNA/genética , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
A 6.5 kb region from the genome of the cyanobacterium, Anacystis nidulans 6301 was cloned using the tobacco chloroplast gene for ribosomal protein S12 as a probe. Sequence analysis revealed the presence of genes for ribosomal proteins S12 and S7 and elongation factors EF-G and EF-Tu in this DNA region. The arrangement is rps12 (124 codons) - 167 bp spacer - rps7 (156 codons) - 77 bp spacer - fus (694 codons) - 26 bp spacer - tufA (409 codons), which is similar to that of the Escherichia coli str operon. The deduced amino acid sequences of the A. nidulans S12 and EF-Tu show high homology (72%-82%) with the E. coli and chloroplast counterparts while those of the A. nidulans S7 and EF-G give low homology (51%-59%). Striking structural homology was found between the potential S7 binding region of 16S rRNA and the beginning of S7 mRNA, suggesting that feedback regulation of rps7 expression operates in A. nidulans.
Assuntos
Cianobactérias/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA/genética , Escherichia coli/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos , Fator Tu de Elongação de Peptídeos/genética , Fatores de Alongamento de Peptídeos/genética , RNA Mensageiro/genética , RNA Ribossômico 16S/genética , Proteínas Ribossômicas/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Transcription of the psbD-psbC gene cluster in tobacco chloroplasts has been studied. This cluster contains in linear sequence the overlapping genes encoding the D2 and 43 kDa proteins of Photosystem II (psbD and psbC, respectively), and ORF62. Eight major transcripts ranging from 1.5 to 4.4 kb were detected by northern blot analysis. S1 mapping experiments revealed that these multiple transcripts comprise five distinct 5' ends whose precise positions were further determined by primer extension analysis. Two of the five 5' ends were determined to be the transcriptional initiation sites by in vitro capping assays: the main site is located 905 bp upstream from the ATG codon of psbD and the additional site is 194 bp upstream from the first ATG codon of psbC. The latter site and the preceding prokaryotic promoter motif are within the protein-coding region of psbD. The 3' ends of transcripts were determined by S1 mapping.
Assuntos
Clorofila/genética , DNA/genética , Genes de Plantas , Família Multigênica , Nicotiana/genética , Proteínas de Plantas/genética , Plantas Tóxicas , Plantas/genética , Regiões Promotoras Genéticas , Sequência de Bases , Complexos de Proteínas Captadores de Luz , Dados de Sequência Molecular , Mapeamento de Nucleotídeos , Complexo de Proteínas do Centro de Reação Fotossintética , Complexo de Proteína do Fotossistema II , Plantas/metabolismo , Plasmídeos , RNA/genética , RNA/isolamento & purificação , Mapeamento por Restrição , Nicotiana/metabolismo , Transcrição GênicaRESUMO
Transcription of the psaA operon in tobacco chloroplasts has been studied. This operon contains in linear sequence the genes encoding the P700 chlorophyll a A1 and A2 apoproteins (psaA and psaB) and the gene encoding the ribosomal CS14 protein (rps14). Northern blot hybridization revealed that a 5.2 kb transcript hybridizes to psaA, psaB, and rps14, but not to the fMet-tRNA (trnfM) gene which follows. Primer extension and in vitro capping assays indicated that the transcriptional initiation site is 194 bp upstream of psaA. The 3' end of the transcript was determined by S1 mapping to be 105 bp downstream of rps14. The transcript is calculated to be 5,207 nucleotides long.
Assuntos
Clorofila/genética , Nicotiana/genética , Proteínas de Plantas/genética , Plantas Tóxicas , Capuzes de RNA , Proteínas Ribossômicas/genética , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Cloroplastos/ultraestrutura , DNA , Endonucleases , Complexos de Proteínas Captadores de Luz , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Óperon , Complexo de Proteínas do Centro de Reação Fotossintética , Mapeamento por Restrição , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
Sequences (ndhA-F) homologous to human mitochondrial genes for components of the respiratory chain NADH dehydrogenase have been found in the chloroplast DNA of tobacco. The ndhA, D, E and F sequences corresponding to the mitochondrial URF1, 4, 4L and 5 are located in the small single copy region, the ndhB sequence corresponding to URF2 in the inverted repeat and the ndhC sequence corresponding to URF3 in the large single copy region of the chloroplast DNA. Northern blot hybridization revealed that all six ndh sequences are actively expressed in the chloroplasts. The ndhA and ndhB sequences contain single introns and the splice sites of their pre-mRNAs were determined by reverse transcription analysis. These findings suggest that potential components of an NADH dehydrogenase are synthesized in the chloroplasts.
Assuntos
Cloroplastos/fisiologia , Redutases do Citocromo/genética , DNA Mitocondrial/genética , Mitocôndrias/fisiologia , NADH Desidrogenase/genética , Sequência de Aminoácidos , Sequência de Bases , Regulação da Expressão Gênica , Genes , Íntrons , Dados de Sequência Molecular , Plantas Tóxicas , Homologia de Sequência do Ácido Nucleico , NicotianaRESUMO
The entire chloroplast genome of the monocot rice (Oryza sativa) has been sequenced and comprises 134525 bp. Predicted genes have been identified along with open reading frames (ORFs) conserved between rice and the previously sequenced chloroplast genomes, a dicot, tobacco (Nicotiana tabacum), and a liverwort (Marchantia polymorpha). The same complement of 30 tRNA and 4 rRNA genes has been conserved between rice and tobacco. Most ORFs extensively conserved between N. tabacum and M. polymorpha are also conserved intact in rice. However, several such ORFs are entirely absent in rice, or present only in severely truncated form. Structural changes are also apparent in the genome relative to tobacco. The inverted repeats, characteristic of chloroplast genome structure, have expanded outward to include several genes present only once per genome in tobacco and liverwort and the large single copy region has undergone a series of inversions which predate the divergence of the cereals. A chimeric tRNA pseudogene overlaps an apparent endpoint of the largest inversion, and a model invoking illegitimate recombination between tRNA genes is proposed which accounts simultaneously for the origin of this pseudogene, the large inversion and the creation of repeated sequences near the inversion endpoints.