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We have isolated and sequenced an anther-specific gene from sunflower which encodes an 800-nucleotide transcript detectable in the peripheral anther cells. It contains an intron of 2615 bp, which separates the first exon (77 bp) coding for a putative signal peptide of 21 amino acids, from the second exon (563 bp) coding for a 100 amino acid polypeptide. The 5' and 3' untranslated regions comprise respectively 13 and 264 bp. The SF2 gene is present in the sunflower genome in several copies, all or most of which contain a closely related intron.

Bean (Phaseolus vulgaris cv. Saxa) chloroplasts contain two tRNA(Phe) species, namely tRNA(Phe)1 and tRNA(Phe)2. By sequence determination, we show that tRNA(Phe)2 is identical to the previously sequenced tRNA(Phe)1 except for two undermodified nucleotides. By reversed-phase chromatography analyses, we demonstrate that the relative amounts of these two chloroplast tRNAs(Phe) vary during leaf development: in etiolated leaves the undermodified tRNA(Phe)2 only represents 15% of total chloroplast tRNA(Phe), during development and greening it increases to reach 60% in 8-day-old leaves, and it then decreases to 9% in senescing leaves.

In chloroplasts, all 61 sense codons are found in chloroplast (cp) DNA sequences coding for proteins. However among the sequenced cp tRNAs or tRNA genes, tRNAs with anticodons complementary to codons CUU/C (Leu), CCU/C (Pro), GCU/C (Ala) and CGC/A/G (Arg) [or CGC/A (Arg) in Marchantia] have not been found. In this paper we show that cp tRNA(Ala)(U*GC) cp tRNA(Pro)(U*GG) and cp tRNA(Arg)(ICG) are able to decode the corresponding four-codon family. In the case of leucine codons CUU/C, we show that 'U:U and U:C wobble' mechanisms can operate to allow the reading of these codons by cp tRNA(Leu)(UAm7G).

The nucleotide sequences of soybean chloroplast tRNAsLeu were determined using post-labeling techniques. Comparison of the primary structures of soybean chloroplast tRNAsLeu with their bean, maize and spinach counterparts only show few base differences. Contrary to previously published results (1) a re-examination of bean tRNALeu sequence shows that this tRNA resembles soybean and maize tRNA2Leu in structure.

Soybean chloroplast (cp) ribosomal RNA genes were located by hybridizing labeled homologous probes to filter bound cpDNA fragments. The soybean chloroplast chromosome, like those of most other plants, contains duplicate sets of ribosomal genes arranged in an inverted orientation. The inverted repeated regions are separated by two single copy regions of 21 and 116 kilobasepairs (kb). Cloned rDNA was mapped with numerous restriction endonucleases. This detailed study shows that the ribosomal genes are arranged in the sequence 5' -16S - Spacer - 23S - 3', and that both 16S and 23S rRNAs are transcribed from the same strand of DNA. Quantitative comparison of rDNA sequence divergence between soybean and tobacco indicates that the highest degree of divergence is in the spacer region, possibly due to an insertion/deletion.

Polysomes and their associated ribonucleic acids were extracted from ageing soybean cotyledons. Density gradient analysis showed that the ribosomes of older cotyledons were present largely as monosomes and light polysomes while younger cotyledons possessed a high proportion of heavy polysomes. Increased polysomal and mRNA activities were observed in 3- to 5-day-old and again in 10-day-old cotyledons. The presence of polyadenylated mRNA was determined by oligo (dT) cellulose chromatography and confirmed by hybridization with 3H-polyuridylic acid.

The nucleotide sequence of a spacer region between 16S and 23S rRNA genes from soybean chloroplasts has been determined. The spacer region is over 3000 bp long and contains two tRNA genes, coding for rRNA(Ile) and tRNA(Ala) which contain intervening sequences of 953 and 811 base pairs respectively. There is a strong homology between the two introns suggesting that they have a common origin. These spacer tRNAs are synthesized as part of a kb precursor molecule containing 16S and 23S rRNA sequences.

Seven major plastid protein encoding genes were positioned on the soybean chloroplast DNA by heterologous hybridization. These include the genes for the alpha, beta and epsilon subunits of the CF1 component of ATP synthase (atpA, atpB and atpE respectively), for subunit III of the CF0 component of ATP synthase (atpH), for the cytochrome f (cytF), for the '32 Kd' thylakoid protein (psbA), and for the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL), all of which map in the large single copy region. The atpB, atpE and rbcL genes are located in the region adjacent to one of the segments of the inverted repeat. The genetic organization of the soybean chloroplast DNA is compared to that of other plastid genomes.

Transfer RNAs and aminoacyl-tRNA synthetases were isolated from 5- and 20-day-old etiolated ageing soybean cotyledons. The relative concentrations of Mg2+ and ATP for optimal acylation (using homologous tRNAs and synthetases) for the 16 amino acids tested indicate that the amino acids can be grouped into 3 categories with a Mg:ATP ratio of 2:1, 5:1 and 10:1 (aspartic acid, isoleucine, leucine, serine, and valine). The total amino acid acceptance for 5-day-old cotyledons was 0.427 mumol tRNA and 0.349 mumol/mumol tRNA for 20-day-old cotyledons. This shows that as cotyledons age, tRNA preparations show an overall decrease of 16.6% amino acid acceptance ability compared to tRNA from younger cotyledons. Charged tRNA samples from 5- and 20-day cotyledons chromatographed on RPC-5 columns demonstrate pronounced qualitative and quantitative changes in tRNA species as cotyledons age, including displacements of peaks as in alanyl-tRNAs and glycyl-tRNAs; disappearance of some species as in valyl-tRNAs, glutamyl-tRNAs, leucyl-tRNAs, lysyl-tRNAs, arginyl-tRNAs, histidyl-tRNAs and tyrosyl-tRNAs and formation of new species as in phenylalanine-tRNA.

The sequence of the ribosomal spacer region of soybean chloroplast DNA including the 3' end of the 16S rRNA gene, the tRNA(Ala) and tRNA(Ile) genes (but not their introns), the three intergenic regions and the 5' end of the 23S rRNA gene, has been determined. This sequence has been compared to corresponding regions of other angiosperm chloroplast DNAs. Secondary structure models are proposed for the entirety of the intergenic regions a, b and c and for the flanking rRNA regions. A model for a common secondary structure of the ribosomal spacer intergenic regions from chloroplasts of higher plants is proposed, which is supported by comparative evidence.

The nucleotide sequence of elongator tRNA(Met) genes from soybean chloroplast and mitochondria and Arabidopsis thaliana mitochondria have been determined. The mitochondrial tRNA(Met) genes from soybean and A. thaliana are identical, and they differ from the soybean chloroplast tRNA(Met) gene by only four nucleotides. Analysis of the flanking regions indicates that the mitochondrial tRNA(Met) gene is not present on a large chloroplast DNA insertion in the mitochondrial genome, but it suggests that they have a common origin. Comparison of the three genes and the evolutionary implications are discussed.

Three tRNA genes have been isolated from a genomic library of Arabidopsis thaliana: a tRNASer (GCU), a tRNATyr (GUA) and a tRNAGlu (UUC) genes. These genes are located closely on the same DNA fragment. The tRNASer and the tRNAGlu genes have both 99% sequence similarity with their mitochondrial counterparts from higher plants indicating that these three tRNA genes are mitochondrial. The tRNATyr gene shows a particular high sequence similarity with the mitochondrial tRNAPhe pseudogene from maize, and both genes are flanked by a tRNASer gene in the upstream region. Extensive sequence comparisons of the Arabidopsis thaliana mitochondrial sequence containing the three tRNA genes and the corresponding region from maize and soybean mitochondria have shown evidence that the tRNA Tyr gene has been generated from a mitochondrial tRNAPhe gene. The conversion was accomplished by three genetic events: a 4 base-pair deletion, a mutation and a recombination, which led to the transformation of the acceptor stem and the anticodon.

A method is described for the preparation of a tRNA-dependent wheat germ protein-synthesizing system. This system can be supplied with exogenous tRNAs of eukaryotic or prokaryotic origin. In order to obtain maximal aminoacylation of the added tRNAs, the translation assays can be supplemented with homologous aminoacyl-tRNA synthetase preparations. Such a tRNA-dependent wheat germ protein-synthesizing system, which is easy to prepare, can be used not only to translate plant cytoplasmic mRNAs in the presence of added cytoplasmic tRNAs, but also to determine the translation activity of exogenous tRNAs from various sources in the presence of either natural or in vitro synthesized mRNAs.

Ribosomal subunits prepared by zonal centrifugation of 80 S ribosomes from 1-day-old soybean cotyledons were active in in vitro polyphenylalanine synthesis. 40 S and 60 S subunits contained 32 and 43 ribosomal proteins respectively, when analyzed by two-dimensional polyacrylamide gel electrophoresis. A cyclic AMP (cAMP) independent protein kinase associated with ribosomes was isolated from the ribosomal salt wash fraction and was found to be similar in chromatographic properties and substrate requirements to cAMP independent casein kinase II present in post-ribosomal supernatant. Soybean cotyledon ribosomal casein kinase in vitro phosphorylates 4 polypeptides of 40 S ribosomal subunit., (Copyright © 1980. The Japanese Society of Plant Physiologists.)

Three tRNAsLeu from soybean chloroplasts were isolated and hybridized to restriction fragments of soybean chloroplast DNA. Based on the hybridization pattern, the locations of four genes coding for tRNA1Leu, tRNA2Leu (two genes tRNA2aLeu and tRNA2bLeu, are present in the inverted repeat region) and tRNA3Leu were determined on the physical map of the soybean chloroplast genome.

In chloroplasts there is a correlation between the amounts of tRNAs specific for a given amino acid and the codons specifying this amino acid. Furthermore, for the amino acids coded for by more than one codon, the population of isoaccepting tRNAs is adjusted to the frequency of synonymous codons used in chloroplast protein genes. A comparison by two-dimensional gel electrophoresis of the tRNA populations extracted from chloroplasts and from chloroplast polysomes shows that all chloroplast tRNAs are involved in protein biosynthesis.

By using the spinach chloroplast atpE gene (epsilon-subunit coding gene) as a probe we have isolated, from a soybean mitochondrial DNA library, a sequence containing a 405 base-pairs (bp) open-reading frame (ORF). This ORF, which is unique in the soybean mitochondrial genome, is probably part of an exon of the gene coding for subunit IV of the NADH dehydrogenase complex. The predicted protein shows 42% sequence similarity with the C-terminal region the Aspergillus nidulans NAD4 protein. The gene is split by a class II intron which is larger than 1,950 bp. Transcription analysis revealed a single 2,200 nucleotide long transcript which does not contain the intron sequence.

The gene coding for subunit III of CF0-ATPase complex (atpH gene) has been localized on the soybean chloroplast genome by heterologous hybridization with the atpH gene of wheat chloroplast. The gene, 243 bp in length, is located in a 7.5-kb BamHI fragment generated from the 14.8-kb PvuII fragment in the large single-copy region of the chloroplast genome. The nucleotide sequence of the gene has been determined. The deduced amino acid (aa) sequence shows 2 aa changes relative to its homologue from wheat and spinach and 1 aa with respect to that found in tobacco chloroplast. The gene is apparently transcribed independently as an 850-nt transcript.