A crude extract of commercial wheat germ is capable of translating mRNAs from widely different sources with high efficiencies. Of six wheat germs analyzed only one was found capable of a high level or incorporation with natural mRNAs. Under optimum conditions at a saturating level of Tobacco Mosaic Virus (TMV) RNA (4.5 mug) and labeled amino acid, 68% of all the available (14)C leucine is incorporated in 70 min. at 30 degrees C with a stimulation of 425 fold above background (with an efficiency of 252 moles leucine/mole TMV RNA). Thus this system which is 30 fold more efficient for TMV translation than previous reported wheat germ cell free systems is capable of yielding 568 pmoles of (14)C leucine incorporated into protein in a 50 mul assay. 80% of the proteins produced have a molecular weight greater than TMV coat protein (17,400). This level of incorporation requires optimization of extract concentration, pH, Mg(+2), K(+) and spermine concentration as well as the method of extract preparation. Samples of crude polysomal RNA from hen oviducts (3% mRNA) and chorionating moth follicular cells (1% mRNA) are also translated in the wheat germ cell free system with high efficiency.

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An unusual minor species of bovine liver serine tRNA has previously been isolated, sequenced, and found to suppress the UGA termination codon in protein synthesis in vitro [Diamond, A., Dudock, B. & Hatfield, D. (1981) CeU25, 497-506]. We have now found that this tRNA can be a substrate in a specific phosphorylation reaction in which phosphoseryl-tRNA is formed. Moreover, bovine liver contains a second UGA suppressor serine tRNA (tRNASerA; N is a modified nucleoside) which also forms phosphoseryl-tRNA. The nucleotide sequence and coding properties of tRNASeA are presented.The suppression of termination codons by naturally occurring suppressor tRNAs has been best studied in prokaryotic systems. The genomes ofboth bacteriophage Q(3 (1, 2) and A (3) code for proteins that arise as a consequence of partial readthrough of UGA termination signals by the normal tryptophan tRNA ofthe host. In eukaryotes, tRNAs that suppress nonsense codons have been observed in yeast and Drosophila (4) as well as in mammalian cells (5, 6). A tyrosine tRNA from Drosophila that lacks the highly modified Q base in its anticodon is responsible for suppressing the termination codon UAG, whereas the corresponding tyrosine tRNA with a fully modified Q base does not read UAG (4). In mammalian cells a naturally occurring suppressor tryptophan tRNA has been shown to read the UGA stop codon of -hemoglobin mRNA both in rabbit reticulocyte lysates and in intact reticulocytes (5). In addition, we have reported the characterization and nucleotide sequence of an unusual bovine liver UGA suppressor serine tRNA (tRNACrA) (6).In this paper we report that tRNASCA can be a substrate in a specific phosphorylation reaction in which phosphoseryltRNA is formed. In addition, there exists another bovine liver suppressor serine tRNA (tRNASerA in which N is a modified nucleoside whose structure has not yet been determined) which also forms phosphoseryl-tRNA. The nucleotide sequence and coding properties of tRNAeA are presented here. This tRNA, like tRNACerA is aminoacylated with serine and suppresses the UGA termination codon in vitro. The nucleotide sequences of tRNAASer and tRNACerA differ in only six positions, all located in the 5' 40% of the molecule.MATERIALS AND METHODS tRNA was isolated from bovine liver (7), estrogen-treated rooster liver (8), and rabbit reticulocytes (7). Aminoacyl-tRNA synthetases were prepared from rabbit reticulocytes as described (7). Conditions of aminoacylation were the same as those described (6, 7) except that [3H]serine (14-28 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) was used. Bovine liver tRNAeCA was purified as described (6). tRNASA was isolated from fresh bovine liver (9) and subsequently purified by three successive chromatographic runs on RPC-5 columns, first in a linear 0.525-0.725 M NaCl gradient as described (7), followed by the same NaCl gradient run in the absence of Mg2", and finally in a 0.45 M NaCl (150 ml) to 1.0 M NaCl (75 ml) concave gradient. The final purification was by preparative electrophoresis on a ...

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Summary.A complete physical map of the Codium fragile chloroplast genome was constructed and the locations of a number of chloroplast genes were determined. Several features of this circular genome are unusual. At 89 kb in size, it is the smallest chloroplast genome known. Unlike most chloroplast genomes it lacks any large repeat elements. The 8 kb spacer region between the 16S and 23S rRNA genes is the largest such spacer characterized to date in chloroplast DNA. This spacer region is also unusual in that it contains the rps12 gene or at least a portion thereof. Three regions polymorphic for size are present in the Codium chloroplast genome. The psbA and psbC genes map closely to one of these regions, another region is in the spacer between the 16 S and 23 S rRNA genes and the third is very close to or possibly within the 16S rRNA gene.The gene order in the Codium genome bears no marked resemblance to either the "consensus" vascular plant order or to that of any green algal or bryophyte genome.

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