RRC ID 49571
Author Noji M, Murakoshi I, Saito K.
Title Molecular cloning of a cysteine synthase cDNA from Citrullus vulgaris (watermelon) by genetic complementation in an Escherichia coli Cys- auxotroph.
Journal Mol Gen Genet
Abstract We have isolated cDNA clones encoding cysteine synthase (CSase, EC 4.2.99.8), which catalyzes the terminal step in cysteine biosynthesis, by direct genetic complementation of a Cys- mutation in Escherichia coli with an expression library of Citrullus vulgaris (watermelon) cDNA. The library was constructed from 8-day-old etiolated seedlings of C. vulgaris in the lambda ZAPII vector, converted to a plasmid library by in vivo excision, and then used for transformation of cysteine auxotroph E. coli NK3, which lacks the cysK and cysM loci. The complementing cDNA containing a 560 bp 5'-untranslated region encodes a polypeptide of 325 amino acids of M(r) 34342. The translational product reacted with an antibody raised against CSase A of Spinacia oleracea. CSase and beta-pyrazolealanine synthase activities were demonstrated in vitro in extracts from E. coli cells expressing the cDNA. Genomic DNA blot analysis indicated the presence of a single copy of the gene, designated cysA, in the C. vulgaris genome. RNA blot hybridization indicated constitutive expression of cysA in cotyledons, hypocotyls and radicles of green and etiolated seedlings. These data suggested that this cDNA clone encodes CSase A the homolog of which in spinach is localized in the cytoplasm. The molecular phylogenetic tree of the amino acid sequences of CSases from plants and bacteria suggested that there are three families in the CSase superfamily; the plant CSase A family, the plant CSase B family and the bacterial CSase family. The proteins in the plant CSase A family are the most conserved relative to the ancestral CSase protein.
Volume 244(1)
Pages 57-66
Published 1994-7-8
DOI 10.1007/BF00280187
PMID 8041362
MeSH Amino Acid Sequence Base Sequence Blotting, Western Cloning, Molecular / methods Cysteine Synthase / genetics* Cysteine Synthase / metabolism DNA, Complementary Escherichia coli / genetics* Fruit / enzymology* Genetic Complementation Test Molecular Sequence Data Mutation Nucleic Acid Conformation Nucleic Acid Hybridization Phylogeny Sequence Homology, Amino Acid
Times Cited 43
Resource
DNA material PCCS7 (RDB01538)