ABSTRACT
An enzyme activity which converts dicamba (2-methoxy-3,6-dichlorobenzoic acid) to 3,6-dichlorosalicylic acid in vitro has been detected in cell lysates of Pseudomonas maltophilia DI-6. Phenyl-Sepharose column chromatography of a partially purified lysate resulted in the separation of this enzyme into three separate protein components tentatively identified as an oxygenase, a ferredoxin, and a reductase. The activity of dicamba O-demethylase was dependent on oxygen and required NADH and Mg(sup2+).
ABSTRACT
A method based on high-performance capillary electrophoresis (HPCE) was developed for the simultaneous analysis of dicamba and its metabolites in media containing the bacterium Pseudomonas maltophilia. Dicamba, 3,6-dichlorosalicylic acid (DCSA), and related products were extracted from media samples using ether under acidic conditions and injected onto an HPCE system containing a pH 10.0 running buffer. Baseline resolution between these compounds was obtained at 30 kV with a total run time of 6 min on a 50-microns i.d. x 50-cm capillary. The linear range for dicamba and DCSA detected at 274 nm extended from 0 to 100 mg/liter and the dynamic range for both compounds extended to 2000 mg/liter. The within-run precision was +/- 5% or less throughout the entire concentration range studied. The limits of detection for dicamba and DCSA in the media samples were 6 and 2 mg/liter. This corresponded to detection limits of 0.3 and 0.1 ng, respectively, in the injected extracts. With this method it was possible to conduct preliminary studies examining the kinetics of dicamba metabolism in P. maltophilia.
Subject(s)
Dicamba/analysis , Dicamba/metabolism , Electrophoresis/methods , Pseudomonas/metabolism , Capillary Action , Culture Media , Kinetics , Sensitivity and Specificity , Time FactorsABSTRACT
Hundreds of mutants with defects in a variety of physiologically important functions, such as photosynthesis, respiration, flagellar motility, phototaxis, circadian rhythms and the cell cycle, have been isolated from cultures of Chlamydomonas reinhardtii. In only a few cases have the genes responsible for these mutations been cloned and sequenced. The development of efficient methods for transformation with nuclear genes [7] has allowed the recent demonstration of gene isolation through genomic complementation with a pooled library of C. reinhardtii DNA [9]. To improve the efficiency with which genes complementing a particular mutation can be isolated, we have established an indexed (ordered) cosmid library of 11,280 individual clones contained in the separate wells of 120 microtiter plates. The average insert size is ca. 38 kb. PCR analysis of five sequenced nuclear genes present in the Chlamydomonas library revealed a range from two copies for the alpha 2- and beta 2-tubulin genes to at least seven copies for the argininosuccinate lyase gene. Overall, these five clones were represented an average of > or = 3.4 times in the library. Thus, the probability that any one particular nuclear gene of < 1000 bp will be found in the library is > or = 97%, and the probability that a gene of ca. 10,000 bp will be found in the library is ca. 92%. Rapid screening methods with cosmid DNAs pooled from individual microtiter dishes have been applied successfully. Bacteria containing clones of the argininosuccinate lyase gene have been identified through genomic complementation of a Chlamydomonas mutant bearing an inactive argininosuccinate lyase gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Chlamydomonas reinhardtii/genetics , Genes , Genetic Complementation Test , Genomic Library , Animals , Argininosuccinate Lyase/genetics , Cloning, Molecular/methods , Cosmids/genetics , Mutation/genetics , Polymerase Chain Reaction , Transformation, Genetic , Tubulin/geneticsABSTRACT
The GL1 gene is required for the initiation of differentiation of hair cells (trichomes) on the crucifer, Arabidopsis thaliana. This gene has been localized to a 4.5 kb DNA fragment by molecular complementation of gl1 mutants. DNA sequence analysis has shown that the protein encoded by GL1 contains a Myb DNA-binding motif. Southern analysis and subsequence analysis of isolated lambda clones has established that GL1 is a member of an extensive myb gene family in Arabidopsis. The putative GL1 promoter directs the expression of the GUS reporter gene in non-trichome-bearing structures that appear to be stipules. This pattern of expression suggests that GL1 may control the synthesis of a diffusible signal that activates the developmental pathway for trichome differentiation.
Subject(s)
Arabidopsis Proteins , DNA-Binding Proteins/genetics , Genes, Plant , Oncogenes , Plant Proteins/genetics , Plants/genetics , Amino Acid Sequence , Base Sequence , Cell Differentiation , DNA-Binding Proteins/chemistry , Gene Expression , Molecular Sequence Data , Oligonucleotides/chemistry , Polymerase Chain Reaction , Restriction Mapping , Sequence Alignment , Time FactorsABSTRACT
We are using the formation of trichomes in Arabidopsis thaliana as a model system to study gene expression during cellular differentiation. To initiate the molecular characterization of this system, we tagged and isolated a gene that is specifically required for the development of the specialized trichome cell. We confirmed the identity of this gene, GLABROUS1 (GL1), by complementation. These results demonstrate that a crucial gene in a plant developmental pathway can be successfully identified by complementation.
