The components of the plastid ribosome are not accumulated synchronously during the early development of spinach plants.

The expression of components of the 70S plastid ribosome has been determined during the first 13 days of spinach plant development. Total cellular RNA and proteins were used to determine the relative steady-state levels of mRNA for ribosomal proteins (r-proteins) by dot blot hybridization and the relative amounts of proteins by immunodetection with specific antibodies. The 16S rRNA as well as mRNAs for 9 out of 11 proteins studied, including those for the 32 kDa polypeptide of photosystem II and the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco) show a marked increase at the beginning of the germination (day 5). At this time the plastid DNA content increases from 4% to 6% of total DNA content and so the plastome copy number can only in part account for the important increase in mRNA steady-state levels. Interestingly the transcripts of the rpl23 and rps19 genes show a different accumulation pattern, indicating either a differential gene transcription and/or an increased stability of the transcripts. In the western blot analysis a group of r-proteins can be detected in dry seeds or after 24 hours of imbibition while a second group of proteins accumulates after 3 to 5 days of development. The differential accumulation pattern of r-proteins and mRNA for r-proteins indicates that post-transcriptional control plays an important role in plastid r-protein synthesis.

Sequence organization of the chloroplast ribosomal spacer ofSpinacia oleracea including the 3' end of the 16S rRNA and the 5' end of the 23S rRNA.

The 2201-bp spacer between the chloroplast ribosomal 16S and 23S genes ofSpinacia oleracea was sequenced. It contains the genes of the tRNA(Ile) (GAU) and tRNA(Ala) (UGC) which are both interrupted by introns of respectively 728 and 816 bp. These introns belong to the class II according to the classfication of Michel and Dujon [17]. Comparison of the rDNA spacer sequence of maize, tobacco and spinach indicates that no conserved polypeptide is encoded within the introns of the two tRNA genes and that the two main insertions/deletions between the three plants are located within two loops of the class II introns secondary structure, which is therefore conserved. Based on the sequence complementarity observed between the upstream and downstream parts, of the 16S and 23S rRNA genes, RNase III-like secondary structures involved in the processing of the rRNA precursor are proposed.

Relations between the plastid gene dosage and the levels of 16S rRNA and rbcL gene transcripts during amyloplast to chloroplast change in mixotrophic spinach cell suspensions.

Spinach cell suspension cultures maintained in photomixotrophic conditions exhibit plastids which undergo cyclic morphological transformations along a growth cycle. Ultrastructural studies show that the green chloroplasts present at the initial stage differentiate into amyloplasts during the subsequent log phase and then return to chloroplasts in stationary phase. The changes of the levels of plastid DNA (pt DNA) per cell have been determined along the growth cycle, as a percentage of total DNA by hybridization of definite amounts of total DNA to a radioactive probe of cloned pt DNA. The number of pt DNA copies have been estimated to 1125 per cell at the maximum of amyloplast development and to 5940 copies per cell at the maximum of chloroplast differentiation. Hybridizations of defined amounts of total cellular RNA to labelled probes of the 16S rDNA and of the rbcL gene allowed estimations of the variations of the corresponding cellular RNA pools. These variations are well correlated with the changes of the ptDNA cellular levels. These results show that the ptDNA gene dosage plays a central role in the regulation of the plastid transcript levels in this system.

Cytochrome b-559 genes from Oenothera hookeri and Nicotiana tabacum show a remarkably high degree of conservation as compared to spinach. The enigma of cytochrome b-559: highly conserved genes and proteins but no known function.

Recent data suggest that cytochrome b-559, an intrinsic membrane protein of the oxygen-evolving photosystem II in chloroplasts, is a heme cross-linked heteromeric polypeptide unit (Herrmann et al. 1984, FEBS Lett 176:239-244). The genes for this cytochrome, designated psbE and psbF, have been located on the chloroplast chromosome of Oenothera hookeri and Nicotiana tabacum by hybridization with fragments of the corresponding spinach genes, and characterized. In both cases, the nucleotide sequence discloses 2 uninterrupted reading frames of 83 and 39 codons separated by a few nucleotides, as in spinach. The amber translation stop codon of psbE overlaps the putative ribosome-binding site for psbF in all cases. The predicted molecular weights of the proteins are 9.2 and 4.3 kd, respectively. In each of the three plant species, and the direction of transcription is opposite to that of the gene for cytochrome f which is located distal to the b-559 genes. Comparison of the deduced amino acid sequences with those from the corresponding spinach genes shows 97% homology. The ubiquitous presence and remarkably high degree of structural and functional conservation of this gene cluster supports the hypothesis of a heteromeric assembly and an important functional role for cytochrome b-559.

Mapping of transfer RNA genes on tobacco chloroplast DNA.

Tobacco chloroplast tRNAs have been purified by two-dimensional polyacrylamide gel electrophoresis, identified by aminoacylation, labelled at their 3'-end and hybridized to tobacco chloroplast DNA restriction fragments, in order to establish a tRNA gene map. These hybridization studies have revealed the localization of at least seven genes in each inverted repeat region, a minimum of 22 tRNA genes in the large single copy region and one tRNA gene in the small single copy region. Comparison of the tobacco chloroplast tRNA gene map to that of maize shows many similarities, but also some differences suggesting that DNA sequence rearrangements have occurred in the chloroplast genome during evolution.

A physical map of Nicotiana tabacum plastid DNA including the location of structural genes for ribosomal RNAs and the large subunit of ribulose bisphosphate carboxylase/oxygenase.

1) Tobacco plastids contain a homogeneous population of double-stranded circular DNA molecules, 101 Megadalton (160 kbp) in circumference. In neutral CsCl equilibrium gradients, this DNA displays a density of 1.697 g · cm(-3) which is equivalent to an average base composition of 37.7 mole-% G+C. 2) A restriction endonuclease fragment map of the tobacco plastid chromosome is presented for the enzymes Bgl I, Sal I, Xho I and Pvu II which together dissect the DNA molecule into about 60 fragments. The map was derived by sequential digestion employing the previously described Seaplaque technique. The tobacco plastid chromosome has an anatomy similar to that of many other higher plants; the circular DNA is segmentally organized into two unique sequence segments of approximately 24 and 95 kbp separated on each side by a large inverted duplication of at least 20.4 kbp. 3) Saturation and blot hybridization showed that the genes for the 16S and 23S pt-rRNAs are duplicated. Each copy of the inverted repeat contains one set of rRNA genes; about 26 kbp (short distance) separate the sets from each other. 4) Cloned fragments of spinach ptDNA nick translated to high specific activity in vitro were used to probe the location of the large subunit gene of ribulose bisphosphate carboxylase/oxygenase on tobacco ptDNA. A 3.5 kbp-long fragment of the large single-copy region of the tobacco chromosome is complementary to structural sequences of the spinach gene. 5) Mapping and hybridization data suggest that the tobacco and spinach ptDNAs share striking similarities in anatomy and sequence.