New methods have been applied to the determination of single copy DNA sequence differences between the sea urchin species Strongylocentrotus purpuratus, S. franciscanus, S. drobachiensis, and Lytechinus pictus. The thermal stability of interspecies DNA duplexes was measured in a solvent (2.4 M tetraethylammonium chloride) that suppresses the effect of base composition on melting temperature. The lengths of duplexes were measured after digestion with S1 nuclease and correction made for the effect of length on thermal stability. The degree of base substitution that has occurred in the single copy DNA during sea urchin evolution is significantly larger than indicated by earlier measurements. We estimate that 19% of the nucleotides of the single copy DNA are different in the genomes of the two sea urchin congeners, S. purpuratus, and S. franciscanus, which apparently diverged only 15 to 20 million years ago.
We have isolated cDNA clones for the maize leaf enzymes phosphoenolpyruvate (P-ePrv) carboxylase [orthophosphate:oxaloacetate carboxy-lyase (phosphorylating) EC 4.1.1.311 and pyruvate,orthophosphate (Prv,Po) dikinase (ATP:pyruvate,orthophosphate phosphotransferase, EC 2.7.9.1) by exploiting the light-inducibility and large size of the mRNAs (3.5 kilobases) that encode the two enzymes. The clones were identified by hybrid-selection and immunoprecipitation assays. From a maize genomic library, two different types of genomic clones were screened with both the P-ePrv carboxylase and the Prv,Pi dikinase cDNA clones. Information from these genomic clones and genome blots indicates that the P-ePrv carboxylase gene family has at least three members and the Prv,P1 dikinase family at least two. Transcripts for both enzymes were detected in green leaves, etiolated leaves, and roots. The results show that the P-ePrv carboxylase mRNAs in green leaves and roots are encoded by different genes. Whereas the P-ePrv carboxylase mRNAs in all three tissues appear to be the same size, the Prv,P1 dikinase mRNA in green leaves is about 0.5 kilobases longer than the Prv,P1 dikinase mRNAs in etiolated leaves and roots. It Is possible that all these PrvPi dikinase transcripts are encoded by one gene, and the size differences may correspond to the presence or absence of a sequence encoding a chloroplast transit peptide.The enzymes phosphoenolpyruvate (P-ePrv) carboxylase [orthophosphateloxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.31] and pyruvate,orthophosphate (Prv,Pj) dikinase (ATP:pyruvate,orthophosphate phosphotransferase, EC 2.7.9.1) play important roles in C4 and crassulacean acid metabolism photosynthesis. P-ePrv carboxylase is responsible for the fixation of atmospheric CO2, while Prv,Pi dikinase produces the substrate phosphoenolpyruvate for P-ePrv carboxylase (1). In green leaves of the C4 plant maize (Zea mays), P-ePrv carboxylase is located in the cytoplasm of mesophyll cells (2). Prv,Pi dikinase is found primarily in the chloroplasts of mesophyll cells (3-5), although some is also detectable in bundle-sheath cells (6). The former enzyme has a subunit molecular mass of 100-103 kDa and has been estimated to comprise 8-15% of the total leaf soluble protein (7)(8)(9). The latter enzyme has a subunit molecular mass of 94-97 kDa and makes up 2-10o of the total leaf soluble protein (10-12). Maximal accumulation of both of these enzymes and their mRNAs is light-dependent (1,8,11,(13)(14)(15). In addition, P-ePrv carboxylase has been found in other maize tissues, including etiolated leaves and roots (8, 14.16), and Prv,Pi dikinase has been detected in maize seeds (17) and etiolated leaves (13). However, these forms of the enzymes have received much less study.Here we describe the isolation and partial characterization of P-ePrv carboxylase and Prv,Pi dikinase cDNA and genomic clones from maize. Our results indicate that each enzyme is encoded by a small number of genes that exhibit differential expression in leave...
The expression of maize (Zea mays) phophoenolpyruvate carboxylase (PPC) gene constructions was studied in transgenic tobacco plants (Nicotiana tabacum and fully coordinated C4 photosynthesis is a rare event. Thus, employing traditional breeding methods to incorporate C4 traits into C3 crops will be difficult if not impossible.With the development ofplant genetic engineering, transfer of foreign genes into plants has become commonplace, providing a new approach to altering plant traits. A genetic engineering approach to the transfer of C4 traits into C3 crops may bypass the problems that are encountered with traditional breeding.pPC2 performs an essential role in leaves of C4 plants, catalyzing the primary fixation of atmospheric carbon (for a review see ref. 7). The reaction catalyzed by PPC is the first step of the C4 pathway, a process that ultimately reduces the energy loss by photorespiration. In the C4 plant maize (Zea mays), carbon fixed by PPC is transported as malate from mesophyll cells to bundle sheath cells. The malate is decarboxylated in the chloroplasts of bundle sheath cells, elevating the ratio ofCO2 to 02. The elevated level ofCO2 in the bundle sheath chloroplast overcomes the wasteful process of photorespiration caused by the oxygenase activity of RuBPC (7, 15).Although PPC is best known for its role in C4 photosynthesis, isozymes of PPC serve nonphotosynthetic functions in both C3 and C4 plant species (25). Previously, we and others have characterized the maize PPC gene (Ppcl) encoding the isozyme involved in C4 photosynthesis (1 1, 21). In this report, we describe the expression of three different maize Ppcl gene constructions in transgenic tobacco and how this affects the biochemistry and physiology of these plants. MATERIALS AND METHODS Plasmid Constructions and Transformation of TobaccoThree different plasmids were constructed containing maize (Zea mays) Ppcl sequence. These constructions were designated BVI-9500, BVI-7000, and BVI-6400 (see Fig. IA
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