The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression.

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.

Overlap and cotranscription of the genes for the beta and epsilon subunits of tobacco chloroplast ATPase.

The nucleotide sequences of the genes for the beta and epsilon subunits of tobacco chloroplast ATPase have been determined. The coding regions for the beta and epsilon subunits contain 1494 bp (498 codons) and 399 bp (133 codons), respectively. The 3' end of the beta-coding region overlaps by one nucleotide with the 5' end of the epsilon-coding region. The overlapping termination and initiation codons are ATGA. The beta and epsilon genes are cotranscribed as a 2.7-kb polycistronic mRNA. The amount of the beta and epsilon mRNA in the chloroplast is about one-twentieth that of the LS mRNA.

The nucleotide sequences of tRNASer (GCU) and tRNAGln (UUG) genes from tobacco chloroplasts.

The nucleotide sequence of tobacco chloroplast genes for tRNASer (GCU) and tRNAGln (UUG) have been determined. These tRNA genes are encoded on the same DNA strand and separated by 1144 bp. Two open reading frames of 52 codons and 98 codons have been found in this spacer region. The tRNASer (GCU) and tRNAGln (UUG) deduced from the DNA sequences show 67% and 76% sequence homologies with E. coli tRNASer (GCU) and tRNAGln (UUG), respectively.

Chloroplast tRNA gene contains a long intron in the D stem: Nucleotide sequences of tobacco chloroplast genes for tRNA (UCC) and tRNA (UCU).

The nucleotide sequences of tobacco chloroplast genes for tRNA(Gly) (UCC) and tRNA(Arg) (UCU) have been determined. The tRNA(Gly) gene has a 691-base-pair intron located in the D stem while the tRNA(Arg) gene does not have any intron. The tRNA(Gly) and tRNA(Arg) genes are encoded on the same strand and separated by a 169-base-pair spacer. The tRNA(Gly) gene is transcribed as a 900-base precursor RNA molecule. The tRNA(Gly) and tRNA(Arg) deduced from the DNA sequences show 84% and 55% sequence homologies with Escherichia coli tRNA(Gly) (UCC) and phage T4 tRNA(Arg) (UCU), respectively.

Nucleotide sequence of tobacco chloroplast gene for the alpha subunit of proton-translocating ATPase.

The tobacco chloroplast gene for the alpha subunit of proton-translocating ATPase has been cloned and sequenced. The coding region contains 1521 bp (507 codons). The nucleotide sequence and the deduced amino acid sequence show 55% and 54% homologies with those of the E. coli alpha subunit, respectively. The deduced amino acid composition is quite similar to that estimated for the spinach alpha subunit.

Tobacco chloroplast gene coding for subunit I of proton-translocating ATPase: comparison with the wheat subunit I and E. coli subunit b.

The tobacco chloroplast gene for subunit I of proton-translocating ATPase is located 405 bp down-stream from the gene for subunit III and 58 bp upstream from the gene for subunit alpha in the same DNA strand. This gene is interrupted by a 695 bp intron. The coding region contains 552 bp (184 codons) and its deduced amino acid sequence shows 78% homology with that of the wheat gene for subunit I.

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon : VI. Production of shikonin derivatives by a two-layer culture containing an organic solvent.

A two-layer culture method was established that uses an organic solvent to remove shikonin derivatives produced on cell surfaces during the culture of suspension cells of Lithospermum erythrorhizon. Some paraffins and a fatty acid ester made suitable solvents, whereas olefins and aromatic solvents extensively inhibited the production of shikonin derivatives. The yield of derivatives increased with an increase in the carbon chain length of the n-paraffin used as the solvent and when the oxygen supply was sufficient it reached the value found for the ordinary culture method.

An atpE-specific promoter within the coding region of the atpB gene in tobacco chloroplast DNA.

The atpB and atpE genes encode beta and epsilon subunits, respectively, of chloroplast ATP synthase and are co-transcribed in the plant species so far studied. In tobacco, an atpB gene-specific probe hybridizes to 2.7- and 2.3-kb transcripts. In addition to these, a probe from the atpE coding region hybridizes also to a 1.0-kb transcript. The 5' end of the atpE-specific transcript has been mapped 430/431 nt upstream of the atpE translation initiation site, within the coding region of the atpB gene. In-vitro capping revealed that this transcript results from a primary transcriptional event and is also characterized by -10 and -35 canonical sequences in the 5' region. It has been found to share a common 3' end with the bi-cistronic transcripts that has been mapped within the coding region of the divergently transcribed trnM gene, approximately 236 nt downstream from the atpE termination codon. Interestingly, this transcript accumulates only in leaves and not in proplastid-containing cultured (BY-2) cells, indicating that, unless it is preferentially degraded in BY-2 cells, its expression might be transcriptionally controlled.

Nucleotide sequences of tobacco chloroplast genes for elongator tRNAMet and tRNAVal (UAC): the tRNAVal (UAC) gene contains a long intron.

The nucleotide sequences of tobacco chloroplast genes for elongator tRNAMet and tRNAVal (UAC) have been determined. The tRNAVal gene contains a 571 base pairs intron located in the anticodon loop. The tRNAVal gene is transcribed as a 750 bases precursor RNA molecule. Both tRNAs deduced from the DNA sequences show 97% sequence homologies with those of spinach chloroplasts.

Effects of Exogenous Polyamines on Tropane Alkaloid Production by a Root Culture of Duboisia myoporoides.

The addition of putrescine, a precursor of tropane alkaloids, to the culture medium increased scopolamine production of DUBOISIA MYOPOROIDES R. Br. (Solanaceae) roots (RB-1), whereas, other precursors (ornithine, arginine, and tropine) increased the content of scopolamine only slightly or not at all. Spermidine, a natural polyamine and a direct metabolite of putrescine, increased the scopolamine content of the cultured roots to the same degree as putrescine increased it. High scopolamine root contents were obtained in media supplemented with 3 mM putrescine (0.96%) and 3 mM spermidine (0.94%), whereas spermine decreased scopolamine production. The putrescine analogues diaminomethane, diaminoethane, diaminopropane, and cadaverine also increased the scopolamine content of the DUBOISIA root culture from 0.56% to the respective values of 0.86%, 0.90%, 1.06%, and 1.04%. Other diamines with long alkyl chains and agmatine did not increase scopolamine production.