ABSTRACT
The application of the polymerase chain reaction (PCR) method of DNA amplification for the isolation of full-length, infectious clones of geminiviruses is described. Non-overlapping, abutting 20-mer oligonucleotide primers were used to produce a linear product from the circular geminivirus genomic template. Clones of African cassava mosaic virus (ACMV) DNA A, obtained by this method, were infectious following mechanical inoculation (in the presence of ACMV DNA B) onto Nicotiana benthamiana. Normal ACMV symptoms resulted and typical geminate viral particles were detected by electron microscopy. The use of PCR for the detection and production of full-length, infectious geminivirus clones is discussed.
Subject(s)
Cloning, Molecular/methods , Mosaic Viruses/genetics , Polymerase Chain Reaction/methods , Amino Acid Sequence , Artifacts , Base Sequence , Capsid/genetics , DNA, Circular/genetics , DNA, Viral/genetics , DNA-Directed DNA Polymerase , Genetic Variation , Kenya , Molecular Sequence Data , Mosaic Viruses/classification , Mosaic Viruses/pathogenicity , Nigeria , Plant Diseases/microbiology , Plants, Toxic , Sequence Alignment , Sequence Homology , Taq Polymerase , Templates, Genetic , Nicotiana/microbiology , TransfectionABSTRACT
Evidence is presented for initial oxidation at the C-3 position of the flavonoid C-ring and for two bifurcating steps during catalysis by anthocyanidin synthase.
Subject(s)
Anthocyanins/biosynthesis , Anthocyanins/metabolism , Carbon/metabolism , Anthocyanins/chemistry , Catalysis , Isomerism , Magnetic Resonance Spectroscopy , Molecular Structure , Oxidation-Reduction , Oxygenases/metabolismABSTRACT
A system has been developed for the expression in Escherichia coli of 1-aminocyclopropane-1-carboxylate (ACC) oxidase from kiwi fruit. In this first report of site-directed mutagenesis of ACC oxidase, seven different mutants of the enzyme have been expressed, and their activities compared to that of the heterologoulsy expressed wild-type enzyme. No great loss of activity was observed when Lys172 was substituted by either Ala or Cys, or when Gly137 was substituted by Pro. However, the mutant proteins showed only 1% of the wild-type activity when substitutions were made of Asp179, His177, and Lys158. The results are discussed in terms of possible mechanisms by which ACC oxidase is activated by carbon dioxide, and in terms of structural motifs suggested by the known structure of isopenicillin N-synthase, an enzyme related by mechanism and sequence similarity to ACC oxidase. It is concluded that Lys172, a putative carbon dioxide binding site, has no role to play in the catalytic activity of the enzyme. The results support a previous suggestion that ACC oxidase shares important structural features with isopenicillin N-synthase.
Subject(s)
Amino Acid Oxidoreductases/biosynthesis , Amino Acid Oxidoreductases/chemistry , Fruit/enzymology , Amino Acid Oxidoreductases/isolation & purification , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA Primers , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Point Mutation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Homology, Amino AcidABSTRACT
Six genes that contain sequence encoding the DNA binding domain of the Myb oncoproteins have been isolated from a cDNA library prepared from Antirrhinum majus (snapdragon) flowers using oligonucleotide probes directed against part of this domain. The derived amino acid sequences of these genes reveal acidic domains in their carboxy termini, suggesting that they might act as transcriptional activators. Analysis of their expression patterns with respect to organ specificity, floral differentiation, and response to light suggests that these genes are not involved in controlling anthocyanin biosynthesis, unlike the characterized myb-related genes C1 and Pl from maize. One of the genes is expressed mainly in the nectary and the transmitting tract of the style, two major secretory tissues of the flower, suggesting that the function of this gene is related to active carbohydrate secretion. We conclude that plants contain a number of myb-related transcriptional activators involved in a diversity of gene regulation.
Subject(s)
DNA-Binding Proteins/genetics , Genes, Plant , Oncogenes , Plant Proteins/genetics , Plants/genetics , Proto-Oncogene Proteins c-myb , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Gene Expression , In Situ Hybridization , Light , Molecular Sequence Data , Sequence AlignmentABSTRACT
The use of broad-host-range plasmids derived from RP4 as intermediate vectors for the transfer of narrow-host-range recombinant plasmids from Escherichia coli to Agrobacterium tumefaciens as a preliminary to marker exchange is described. Recombinant plasmids having a ColE1 type origin were linked to the RP4 derivative. Cointegrate formation appeared to take place by RecA-independent, homologous recombination within a short piece of DNA derived from the beta-lactamase gene of Tn1/Tn3 carried by both vector components, so that it never disrupted the recombinant portion of the construction. pNJ5000 provides an unstable intermediate vector for use in marker exchange experiments, while its stable relative pNJ1020 provides a carrier for use in binary vector systems.