The nucleotide sequence of the 3'-terminal portion of the tobacco etch virus (TEV) The TEV genome is considerably more complex than genomes of other plant viruses, such as brome mosaic virus and tobacco mosaic virus, which have been studied in detail. At least seven virus gene products are synthesized during infection (4). Five of these proteins have been identified and purified to homogeneity (5, 6). Therefore, TEV provides a model system to study plant virus gene expression and regulation.The genome of TEV is a plus-sense single-stranded RNA consisting of approximately 10,000 nucleotides (7). A small protein is covalently linked to the 5'-terminal nucleotide (7) and a polyadenylate tract is located at the 3' terminus (8).Cell-free translational analysis of TEV virion RNA or RNA extracted from TEV-infected tissue consistently results in the synthesis of a large molecular weight protein (-=85,000) that can be immunoprecipitated with monospecific antisera to either the TEV capsid or nuclear inclusion protein (4). Polyadenylylated subgenomic messenger RNAs are not detected when RNA extracted from TEV-infected tobacco leaf tissue is analyzed in gel hybridization experiments (9). These data suggest a strategy of genomic organization and expression for TEV in which the capsid protein is synthesized as part of a polyprotein that undergoes post-translational proteolytic processing.Nucleotide sequence determination studies were initiated to provide information on the organization of the TEV genome. Complementary DNA (cDNA) of TEV RNA was inserted into the plasmid vector pBR322, and the sequence of 2324 nucleotides adjacent to the 3' polyadenylate region of the viral genome was determined. A single open reading frame (ORF) of 2135 nucleotides was detected and the sequence coding for the capsid protein was identifed. These data support the hypothesis that the 30,000 molecular weight capsid protein is expressed via synthesis and processing of a polyprotein (4, 9). MATERIALS AND METHODSCloning and Sequencing. Double-stranded cDNA was prepared from the viral RNA of the not-aphid-transmitted (NAT) isolate of TEV (10) and inserted into the Sal I/EcoRI sites of the bacterial plasmid pBR322 (11). All experiments involving recombinant DNA were conducted under P1 containment conditions. Colonies resistant to ampicillin were screened on nitrocellulose by hybridization with 32P-labeled single-stranded cDNA of TEV RNA. The cDNA insert was removed by digestion with Sal I and EcoRI and separated from pBR322 DNA on an 0.8% agarose gel. The cloned cDNA fragment was isolated from the gel by using DEAE NA 45 membrane (Schleicher & Schuell), following the manufacturer's procedure. The following restriction enzymes were used either singly or in combination to digest the cDNA insert: Bal I, HindIII, Pst I, Rsa I, Sau3A, and Taq I. The resulting digestion products were inserted into the replicative form of an appropriate M13 bacteriophage cloning vector (12) and the nucleotide sequence was determined by the dideoxy nucleotide chain t...

Glutamate oxaloacetic transaminase (L-aspertate: 2-oxoglutarate aminotransferase, E.C. 2.6.1.1; GOT) was found to occur in five distinct electrophoretic forms in different tissue extracts from a number of highly inbred strains of Zea mays L. No major qualitative differences were detected in the various tissues examined, and the isozyme patterns did not undergo changes during temporal development of any given inbred strain. Cell fractionation studies showed one isozyme to be associated with the mitochondria (mGOT), another to be exclusively associated with the soluble fraction (sGOT), and a third to be associated with the glyoxysomes (gGOT). The glyoxysomal form occurs as two electrophoretically distinct variants which exist in different inbred strains of maize. The gGOT variants are under the control of two codiminant alleles (Got1A and Got1B) at the Got1 locus (isozyme5, gGOT). The genetic data and gene dosage effects suggest that GOT in maize is functionally a dimer.

Rates of catalase synthesis and degradation were measured in the scutellum of the germinating maize seedling by the technique of Price, Sterling, Tarantola, Hartley & Rechcigl [J. Biol. Chem. (1963) 237, 3468-3475] by using the porphyrinogenic drug 2-allyl-2-isopropylacetamide to inhibit catalase synthesis. Results indicate that developmental changes in catalase activity are determined by changes in the rate of enzyme degradation as well as by changes in the rates of its synthesis.

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A protein which has been shown to inhibit catalase in vitro appears to vary inversely with catalase activity in the maize scutellum during early sporophytic development when assayed using a catalase inhibition assay. This result suggested that the inhibitor protein may play a direct role in regulating catalase activity during this time period. Four experimental approaches were used to evaluate this putative regulatory role, including immunological quantitation of individual catalase isozymes during germination using rocket immunoelectrophoresis, perturbation of normal catalase expression with hydrogen peroxide or allylisopropylacetamide (AIA), examination of a mutant line with an altered catalase developmental program, and direct radioimmunoassay of the inhibitor protein during germination. The results of these experiments indicate that the quantitative changes in catalase activity during development are not mainly due to changes in the expression of the catalase inhibitor. Other possible roles of this protein in catalase regulation are discussed.