Conservation of matrix attachment region-binding filament-like protein 1 among higher plants.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher-order chromatin organization and the regulation of gene expression. We have previously isolated a novel nuclear matrix-localized protein (MFP1) from tomato (Lycopersicon esculentum) that preferentially binds to MAR DNA. Tomato MFP1 has a predicted filament-protein-like structure and is associated with the nuclear envelope via an N-terminal targeting domain. Based on the antigenic relationship, we report here that MFP1 is conserved in a large number of dicot and monocot species. Several cDNAs were cloned from tobacco (Nicotiana tabacum) and shown to correspond to two tobacco MFP1 genes. Comparison of the primary and predicted secondary structures of MFP1 from tomato, tobacco, and Arabidopsis indicates a high degree of conservation of the N-terminal targeting domain, the overall putative coiled-coil structure of the protein, and the C-terminal DNA-binding domain. In addition, we show that tobacco MFP1 is regulated in an organ-specific and developmental fashion, and that this regulation occurs at the level of transcription or RNA stability.

Cytochrome P-450-catalysed monoterpenoid oxidation in catmint (Nepeta racemosa) and avocado (Persea americana); evidence for related enzymes with different activities.

A cytochrome P-450 present in ripening avocado (Persea americana) fruit mesocarp (CYP71A1) had previously been shown to metabolize the monoterpenoids nerol and geraniol (Hallahan et al. (1992) Plant Physiol. 98, 1290-1297). Using DNA encoding CYP71A1 as a hybridization probe, we have shown by Southern analysis that a related gene is present in the catmint, Nepeta racemosa. RNA blot analysis, together with Western analysis of catmint leaf polypeptides using avocado cyt P-450 antiserum, showed that a closely related gene is expressed in catmint leaves. Cytochrome P-450 in catmint microsomes catalysed the specific hydroxylation of nerol and geraniol at C-10, whereas avocado CYP71A1, in either avocado microsomes or heterologously expressed in yeast, catalysed 2,3- or 6,7-epoxidation of these substrates. These results suggest that orthologous genes of the CYP71 family are expressed in these two plant species, but catalyse dissimilar reactions with monoterpenoid substrates.

Low carbon monoxide affinity allene oxide synthase is the predominant cytochrome P450 in many plant tissues.

A cytochrome P450 with low affinity (2.9 x 10(3) M-1) for CO appears to be the major microsomal P450 in some plant tissues. The presence of low CO affinity cytochrome P450 correlates with its lack of NADPH reducibility and with the presence of high levels of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate peroxidase activity. This activity and low CO affinity are retained by purified tulip cytochrome P450, which appears to be catalytically identical to a flaxseed-derived fatty acid allene oxide synthase P450 described previously [Song, W.-C., & Brash, A.R. (1991) Science 253, 781-784]. Other heme-binding ligands, such as CN- and imidazoles, bind weakly to the allene oxide synthase P450s, suggesting that axial coordination in the heme distal pocket may be hindered. We conclude that low CO affinity is characteristic of the allene oxide synthase P450s and that these P450s constitute a major portion of the microsomal P450 in a variety of plant tissues, particularly from monocot species.

Occurrence and biological function of cytochrome P450 monooxygenases in the actinomycetes.

Many species within the order Actinomycetales contain one or more soluble cytochrome P450 monooxygenases, often substrate-inducible and responsible for a variety of xenobiotic transformations. The individual cytochromes exhibit a relatively broad substrate specificity, and some strains have the capacity to synthesize large amounts of the protein(s) to compensate for low catalytic turnover with some substrates. All three of the Streptomyces cytochromes sequenced to date are exclusive members of one P450 family, CYP105. In several instances, monooxygenase activity arises from induction of a P450 and associated ferredoxin, or of a P450 only, suggesting that some essential electron donor proteins (reductase and ferredoxin) are not co-ordinately regulated with the cytochrome. The overall properties of these systems suggest an adaptive strategy whose twofold purpose is to maintain a competitive advantage via the production of secondary metabolites, and, whenever possible, to utilize unusual growth substrates by introducing metabolites from these reactions into the more substrate-specific primary metabolic pathways.

Isolation and characterization of Streptomyces griseolus deletion mutants affected in cytochrome P-450-mediated herbicide metabolism.

Metabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450SU1 and P-450SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of greater than 15 kb of DNA, including the structural genes for cytochrome P-450SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the subC,B gene products (cytochrome P-450SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450SU2 in the absence of P-450SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.

Characterization of the OCT plasmid encoding alkane oxidation and mercury resistance in Pseudomonas putida.

Transformation of Pseudomonas putida and analysis for plasmid DNA revealed that both n-alkane oxidation and mercury resistance are encoded on a single 220-megadalton OCT plasmid molecule. Derivatives of OCT having lost the mercury resistance function could be readily isolated and contained a smaller plasmid estimated to be 170 megadaltons. The results show that segregation of the mercury resistance property occurs not by loss of a separate MER plasmid as previously thought but by a deletion in the OCT plasmid.