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Five Arabidopsis mutants have been isolated on the basis of hypersensitivity of leaf tissue to UV light. For each mutant, the UV-hypersensitive phenotype (uvh) was inherited as a single recessive Mendelian trait. In addition, each uvh mutant represented a separate complementation group. Three of the mutations producing the UV hypersensitive phenotype have been mapped relative to either genetic markers or physical microsatellite polymorphisms. Locus UVH1 is linked to nga76 on chromosome 5, UVH3 to GL1 on chromosome three, and UVH6 to nga59 on chromosome 1. Each uvh mutant has a characteristic pattern of sensitivity based on UV sensitivity of leaf tissue, UV sensitivity of root tissue, and ionizing radiation sensitivity of seeds. On the basis of these patterns, possible molecular defects in these mutants are discussed.

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We employed a rapid fractionation method coupled with a sensitive enzyme-linked immunosorbent assay to quantify the globulins and avenins in developing and mature oat seeds. On a molar basis, there is approximately 10-11 times as much globulin as avenin. Pulse labeling of endosperm proteins indicated that the rate of globulin synthesis is approximately nine times that of avenin. In addition, neither protein class showed any signs of degradation during this experiment. Analysis of the storage protein mRNAs indicates that both globulin and avenin transcripts are associated with membrane-bound polysomes and are found in similar concentrations within the membrane-bound polysome fraction. We found that avenin and globulin mRNAs are fully loaded with ribosomes, suggesting that initiation is not rate-limiting for translation of either protein. Rates of globulin and avenin synthesis were similar when synthetic storage protein mRNAs were translated in vitro. Translation of equimolar amounts of globulin and avenin mRNAs in the same reaction showed equivalent amounts of protein synthesized when compared with globulin and avenin mRNAs translated in separate reaction mixes. We propose that translation elongation or termination reactions are likely regulatory steps for controlling storage protein synthesis in oat endosperm.

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We have isolated genomic clones encoding the two major classes of seed storage proteins in oats, the 12 S globulins and the avenins. The globulin genes encode glutamine-rich, sulfur-poor storage proteins that are highly conserved in sequence and structure. The globulin genes contain three short introns whose positions in the coding sequence are the same as in storage globulin genes in legumes and other dicots. The avenin genomic clone contains four tightly linked genes that belong to both of the two avenin gene subfamilies. The avenin genes encode glutamine-rich, lysine-poor proteins that vary in length due to differences in the number of peptide repeats. Although globulin and avenin genes are expressed coordinately during oat seed development, their promoter regions do not contain any conserved sequence elements that might determine developmental timing. Previous studies showed that there are roughly equal amounts of globulin and avenin mRNAs in developing oat seed, despite there being much more globulin than avenin in mature seed. Storage protein synthesis in oats must therefore be controlled partially by post-transcriptional mechanisms. Sequence analysis of globulin and avenin genes has provided several clues as to why globulin mRNAs may be translated more efficiently than avenin mRNAs.

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We have isolated and characterized cDNA clones encoding avenins, the prolamine storage proteins of oat seeds. Sequence analysis shows that avenins are a related group of polypeptides and that their mRNAs differ from each other by point mutations and small insertions and deletions. Avenin proteins have structural homology to the alpha/beta-gliadins and gamma-gliadins of wheat, the B-hordeins of barley, and the gamma-secalins of rye. Hybridization analysis of DNA from various diploid, tetraploid, and hexaploid oat species shows that the oat genome contains more globulin storage protein genes than avenin genes and that some restriction fragments containing these genes are conserved between species with common genomes. We estimate that there are 25 avenin genes and 50 globulin genes per haploid genome in Avena sativa and similar ratios of globulin to avenin genes in other Avena species. Avenin and globulin polypeptides begin to accumulate between 4 days and 6 days after anthesis. Messenger RNAs encoding avenin and globulin proteins become abundant 4 days after anthesis and reach peak concentrations at 8 days after anthesis. Avenin mRNAs are present in somewhat greater molar amounts than globulin mRNAs beginning at 4 days after anthesis. Because there is considerably more globulin than avenin in the mature oat seed, the expression of globulin and avenin genes may be regulated both transcriptionally and post-transcriptionally.

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We have isolated full-length cDNA clones that encode oat (Avena sativa) seed storage globulin mRNAs from a cDNA library in the expression vector lambda gtll. The longest of these clones, pOG2, has an 1840-base pair insert that encodes a complete precursor subunit with a signal peptide of 24 amino acids followed by an acidic polypeptide of 293 amino acids and a basic polypeptide of 201 amino acids. Near the C terminus of the acidic polypeptide are four repeats of a highly conserved, glutamine-rich octapeptide. Other oat globulin cDNA clones contain five of these repeats. Nucleotide sequence comparisons between these clones indicate that the genes encoding these proteins are highly conserved. We estimate there to be 7 to 10 genes for the oat globulin per haploid genome. Comparisons of amino acid sequences show that the oat globulin is 30 to 40% homologous with storage globulins of legumes and about 70% homologous with the rice seed storage globulin (glutelin).

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Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and tryptophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.

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We have taken two approaches to the study of the genetics of leucine transport in mammalian cells. First, from a mutant Chinese hamster ovary cell line that has a temperature-sensitive leucyl-tRNA synthetase, we isolated temperature-resistant revertants with increased leucine transport activity. This transport elevation is reflected by increased Vmax values of leucine uptake and unchanged Km values of uptake. The temperature resistance in each revertant appears to result from the increased transport and not from any change in the leucyl-tRNA synthetase. We conclude that in each revertant there is a stable derepression of amino acid transport system L. In a second approach, we started with a Chinese hamster-human hybrid strain formed by the fusion of a temperature-sensitive leucyl-tRNA synthetase mutant hamster cell line and normal human leukocytes. From this temperature-sensitive hybrid strain we selected temperature-resistant hybrids, one class of which we found to have greatly elevated leucine transport activity. We have allowed human chromosomes to segregate from these high-transport hybrids, promoted by the presence of low concentrations of colcemid. The loss of the high-transport phenotype coincides with the loss of a single small human chromosome, which we are attempting to identify by using G-11 and G-banding staining techniques.

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We have isolated mutants defective in the regulation of leucine transport by selecting temperature-resistant revertants from the CHO-tsH1 strain, a temperature-sensitive leucyl-tRNA synthetase mutant of the Chinese hamster ovary cell line. In each revertant, there is a stable 2- to 3-fold enhancement in the activity of transport System L, which serves for the uptake of branched-chain amino acids. This increased transport is reflected by an increase in the Vmax for System L transport without a significant change in the Km value and results in increased intracellular pools of leucine. The thermal stability of the leucyl-tRNA synthetase activity in each of these revertants is not changed significantly from that of the starting strain, CHO-tsH1. We conclude from these studies that the temperature resistance in the revertants arises from alterations in the regulation of transport System L, leading to constitutively high System L transport and increased intracellular pools of leucine, complementing the leucyl-tRNA synthetase defect.

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The regulation of the activity of transport System L for neutral amino acids has been investigated in Chinese hamster ovary cells. Incubation of the temperature-sensitive leucyl-tRNA synthetase mutant CHO-tsH1 at marginally permissive temperatures results in leucine-limited growth and increased transport of branched chain and aromatic amino acids. This temperature-dependent transport enhancement is restricted to transport System L and results in increased Vmax values of uptake of System L substrates with unchanged Km values of uptake. Several lines of evidence suggest that trans-stimulation by intracellular amino acids cannot account for the increased System L activity in CHO-tsH1. Other temperature-sensitive aminoacyl-tRNA synthetase mutants of the Chinese hamster ovary line show increased transport of the amino acid corresponding to the synthetase defect after incubation at elevated temperatures. Increased transport by System L occurs in CHO-S, the parental line of CHO-tsH1, after growth in limiting levels of leucine. The System L enhancement can be prevented by cycloheximide but not, at early times, by actinomycin D. We conclude that the activity of transport System L in Chinese hamster ovary cells is regulated by a mechanism which appears to act at the level of translation.

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The neutral amino acid transport systems A, ASC, and L have been characterized in the Chinese hamster ovary cell. System A, defined by its sodium ion dependency and inhibition by 2-methylaminoisobutyric acid, was found to be extremely sensitive to the pH of the external medium and to increase in response to starvation for amino acids. System ASC, identified by its sodium ion dependency and intolerance of N-methylation of substrates, was found to be relatively insensitive to external pH and nutrient limitation. System ASC in Chinese hamster ovary cells has been shown to be the major mode of entry of neutral amino acids. A much broader substrate specificity was observed for System ASC than has been reported for other mammalian cell types, with nearly every amino acid tested showing significant uptake by this system. In addition, the highest observed velocities of uptake were for System ASC. System L, defined by its sodium ion independency and inhibition by 2-aminobicyclo-[2,2,2]-heptane-2-carboxylic acid, was enhanced in activity by lowered pH. The starvation-induced enhancement in System A activity was prevented by the presence of a single substrate of this system, the nonmetabolizable analog 2-methylaminoisobutyric, or by the presence of the protein synthesis inhibitor cycloheximide.

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