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.

No abstract

No abstract

We have isolated and characterized the genomic clone 2CHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on 2CHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN 17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspensioncultured cells, the chimeric gene consisting of 1.1 kb CHN50 5' upstream region fused to the coding sequence of fl-glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5' deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region ( -68 to -4 7 ) proximal to the TATA box functions as a putative silencer.

The location and nucleotide sequences of tobacco chloroplast genes for tRNAGlu(UUC), tRNATyr(GUA) and tRNAAsp(GUC) have been determined. These genes lie midway between the genes for alpha and beta/epsilon subunits of H+-ATPase on the large single-copy region of the chloroplast DNA. The gene organization is tRNAGlu - 59bp spacer - tRNATyr - 108bp spacer - tRNAAsp on the same DNA strand. Northern blot hybridization studies revealed that these three tRNA genes are cotranscribed. The transcription initiation site was localized at 24 bp upstream from the tRNAGlu coding region and its termination site at 90 bp downstream from the tRNAAsp coding region by S1 mapping. The tricistronic tRNA precursor is thus calculated to be 512 bases long. Its processing was also studied by S1 mapping.

The location and nucleotide sequences of tobacco chloroplast genes for tRNA(Ile) (CAU), tRNA(Leu) (CAA), tRNA(Cys) (GCA), tRNA(Ser) (UGA) and tRNA(Thr) (GGU) (trnI-CAU, trnL-CAA, trnC-GCA, trnS-UGA and trnT-GGU, respectively) have been determined. The trnI and trnL are located in the inverted repeat region. The trnC, trnS and trnT are present in the large single copy region. These five tRNA genes together with the 25 different tRNA genes previously published have been compiled and compared. These 30 tRNA genes corresponding to 20 amino acids are most likely to be all of the tRNA genes encoded in tobacco chloroplast genome.

No abstract

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.