Abstract:SummaryAn Arabidopsis thaliana cDNA bank has been constituted in a Saccharomyces cerevisiae expression vector based on the phosphoglycerate kinase (PGK) promoter and terminator. This bank was used to complement eight S. cerevisiae auxotrophic markers. All of them were corrected. These results confirm the quality of the bank and the feasibility of cloning plant genes by yeast mutant complementation. The cDNA complementing the ural yeast mutant was sequenced, analysed and shown to encode a dihydroorotic (DHO) de… Show more
“…Sequences encoding proteins with DHODH activity have been characterised in Arabidopsis and tobacco (Minet et al, 1992;Giermann et al, 2002). Plant DHODHs sequenced to date belong to the membrane-bound family 2 of dihydroorotate dehydrogenases, which are flavoproteins.…”
“…Sequences encoding proteins with DHODH activity have been characterised in Arabidopsis and tobacco (Minet et al, 1992;Giermann et al, 2002). Plant DHODHs sequenced to date belong to the membrane-bound family 2 of dihydroorotate dehydrogenases, which are flavoproteins.…”
“…The resultant plasmid gave a CCTA::LEU2 cassette by HindlII digestion. [16] c D N A library constructed from roots of Brassica napus L. cv. Jet Neuf.…”
Section: '-Aaacccgggttttttaatatatagttttatttttg-3' and A 3'-choi Fragmentioning
confidence: 99%
“…After 3-4 days roots were excised and RNA was purified [ 1 ]. cDNA was synthesized with a cDNA synthesis kit (Pharmacia) and purified on a SizeSep 400 span column (Pharmacia) before ligation into the plasmid pFL61 (URA3) [ 16], which can express foreign cDNAs in yeast under the control of a constitutive PGK promoter. A cDNA library represented by 5 x 105 independent clones was constructed, and was amplified in Escherichia coli DH5~ competent cells (Life Technologies, Tokyo).…”
Section: ( U R a 3 )mentioning
confidence: 99%
“…In our case, however, the B. napus CCT was recovered also in the soluble fraction. This could be explained if we assume that there is a certain limit of the capacity of yeast cell for binding CCT to membranes, because the pFL61-based expression system usually overexpresses a foreign c D N A in yeast [ 16]. Alternatively, it is also possible that targeting of the B. napus enzyme to membranes was less efficient than that of the native enzyme in yeast cells.…”
CTP:phosphocholine cytidylyltransferase is a rate-limiting enzyme in biosynthesis of phosphatidylcholine in plant cells. We have isolated four cDNAs for the cytidylyltransferase from a root cDNA library of Brassica napus by complementation in a yeast cct mutant. The deduced amino-acid sequences of the B. napus enzymes resembled rat and yeast enzymes in the central domain. Although all cytidylyltransferases ever cloned from B. napus and other organisms were predicted to be structurally hydrophilic, the yeast cct mutant transformed with one of the B. napus cDNA clones restored the cytidylyltransferase activity in the microsomal fraction as well as in the soluble fraction. These results are consistent with a recent view that yeast cells contained a machinery for targeting the yeast cytidylyltransferase to membranes, and may indicate that the B. napus enzyme was compatible with the machinery.
“…The successful use of yeast mutants to isolate plant cDNAs that complement the respective mutations has recently been reported [1,25,33]. To confirm that the isolated cDNA indeed codes for coprogen oxidase its ability to complement a yeast HEM13 mutant (S150-2B (heml3A::URA3)) was investigated.…”
Section: Complementation Of a Yeast (S Cerevisiae) Hem 13 Mutantmentioning
In plants the enzyme coproporphyrinogen oxidase catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX in the heme and chlorophyll biosynthesis pathway(s). We have isolated a soybean coproporphyrinogen oxidase cDNA from a cDNA library and determined the primary structure of the corresponding gene. The coproporphyrinogen oxidase gene encodes a polypeptide with a predicted molecular mass of 43 kDa. The derived amino acid sequence shows 50% similarity to the corresponding yeast amino acid sequence. The main difference is an extension of 67 amino acids at the N-terminus of the soybean polypeptide which may function as a transit peptide. A full-length coproporphyrinogen oxidase cDNA clone complements a yeast mutant deleted of the coproporphyrinogen oxidase gene, thus demonstrating the function of the soybean protein. The soybean coproporphyrinogen oxidase gene is highly expressed in nodules at the stage where several late nodulins including leghemoglobin appear. The coproporphyrinogen oxidase mRNA is also detectable in leaves but at a lower level than in nodules while no mRNA is detectable in roots. The high level of coproporphyrinogen oxidase mRNA in soybean nodules implies that the plant increases heme production in the nodules to meet the demand for additional heme required for hemoprotein formation.
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