Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total soluble protein in mature potato tubers, and it has potent insecticidal activity against the corn rootworm. We determined the X-ray crystal structure of a His-tagged variant of an isozyme of patatin, Pat17, to 2.2 A resolution, employing SeMet multiwavelength anomalous dispersion (MAD) phasing methods. The patatin crystal structure has three molecules in the asymmetric unit, an R-factor of 22.0%, and an R(free) of 27.2% (for 10% of the data not included in the refinement) and includes 498 water molecules. The structure notably revealed that patatin has a Ser-Asp catalytic dyad and an active site like that of human cytosolic phospholipase A(2) (cPLA(2)) [Dessen, A., et al. (1999) Cell 97, 349-360]. In addition, patatin has a folding topology related to that of the catalytic domain of cPLA(2) and unlike the canonical alpha/beta-hydrolase fold. The structure confirms our site-directed mutagenesis and bioactivity data that initially suggested patatin possessed a Ser-Asp catalytic dyad. Alanine-scanning mutagenesis revealed that Ser77 and Asp215 were critical for both esterase and bioactivity, consistent with prior work implicating a Ser residue [Strickland, J. H., et al. (1995) Plant Physiol. 109, 667-674] and a Ser-Asp dyad [Hirschberg, H. J. H. B., et al. (2001) Eur. J. Biochem. 268, 5037-5044] in patatin's catalytic activity. The crystal structure aids the understanding of other structure-function relationships in patatin. Patatin does not display interfacial activation, a hallmark feature of lipases, and this is likely due to the fact that it lacks a flexible lid that can shield the active site.
Mixed lineage kinase domain-like pseudokinase (MLKL) mediates necroptosis by translocating to the plasma membrane and inducing its rupture. The activation of MLKL occurs in a multimolecular complex (the 'necrosome'), which is comprised of MLKL, receptor-interacting serine/threonine kinase (RIPK)-3 (RIPK3) and, in some cases, RIPK1. Within this complex, RIPK3 phosphorylates the activation loop of MLKL, promoting conformational changes and allowing the formation of MLKL oligomers, which migrate to the plasma membrane. Previous studies suggested that RIPK3 could phosphorylate the murine MLKL activation loop at Ser345, Ser347 and Thr349. Moreover, substitution of the Ser345 for an aspartic acid creates a constitutively active MLKL, independent of RIPK3 function. Here we examine the role of each of these residues and found that the phosphorylation of Ser345 is critical for RIPK3-mediated necroptosis, Ser347 has a minor accessory role and Thr349 seems to be irrelevant. We generated a specific monoclonal antibody to detect phospho-Ser345 in murine cells. Using this antibody, a series of MLKL mutants and a novel RIPK3 inhibitor, we demonstrate that the phosphorylation of Ser345 is not required for the interaction between RIPK3 and MLKL in the necrosome, but is essential for MLKL translocation, accumulation in the plasma membrane, and consequent necroptosis.
In the gibberellin (GA) biosynthesis pathway, 20-oxidase catalyzes the oxidation and elimination of carbon-20 to give rise to C 19 -GAs. All bioactive GAs are C 19 -GAs. We have overexpressed a cDNA encoding 20-oxidase isolated from Arabidopsis seedlings in transgenic Arabidopsis plants. These transgenic plants display a phenotype that may be attributed to the overproduction of GA. The phenotype includes a longer hypocotyl, lighter-green leaves, increased stem elongation, earlier flowering, and decreased seed dormancy. However, the fertility of the transgenic plants is not affected. Increased levels of endogenous GA 1 , GA 9 , and GA 20 were detected in seedlings of the transgenic line examined. GA 4 , which is thought to be the predominantly active GA in Arabidopsis, was not present at increased levels in this line. These results suggest that the overexpression of this 20-oxidase increases the levels of some endogenous GAs in transgenic seedlings, which causes the GAoverproduction phenotype.
Simple sequence repeats (SSRs), also known as microsatellites, are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. Three approaches were followed for the development of PCR primers for the amplification of DNA fragments containing SSRs from sorghum [Sorghum bicolor (L.) Moench]: a search for sorghum SSRs in public DNA databases; the use of SSR-specific primers developed in the Poaceae species maize (Zea mays L.) and seashore paspalum grass (Paspalum vaginatum Swartz); and the screening of sorghum genomic libraries by hybridization with SSR oligonucleotides. A total of 49 sorghum SSR-specific PCR primer pairs (two designed from GenBank SSR-containing sequences and 47 from the sequences of genomic clones) were screened on a panel of 17 sorghum and one maize accession. Ten primer pairs from paspalum and 90 from maize were also screened for polymorphism in sorghum. Length polymorphisms among amplification products were detected with 15 of these primer pairs, yielding diversity values ranging from 0.2 to 0.8 with an average diversity of 0.56. These primer pairs are now available for use as markers in crop improvement and conservation efforts.
ABSTRACTcDNA clones encoding proteins of -18 kDa in which 83% of the amino acids are conserved relative to the published sequences of mammalian cyclophilin/rotamase (CyP) have been isolated from tomato, maize, and Brassica napus. In correspondence with the mammalian genes, but in contrast with the Neurospora gene and one yeast CyP gene, the plant CyP genes encode only mature proteins lacking transit peptides. RNA blot analyses demonstrate that CyP genes are expressed in all plant organs tested. Southern blots of genomic DNA indicate that there are small families (two to eight members) of CyP-related genes in maize and B. napus. A vector was constructed for expression of the tomato cDNA in E. coil. SDS/polyacrylamide gels show that extracts of appropriately induced cells harboring this vector contain nearly 40% of the protein as a single "18-kDa band. While the majority of this protein is sequestered in insoluble inclusion bodies, the soluble extracts have higher levels of peptidyl-prolyl cis-trans isomerase (rotamase) activity than extracts of wild-type cells. This additional activity is sensitive to inhibition by the cyclic undecapeptide cyciosporin A.
We have isolated and characterized cDNA clones of a gene family (P2) expressed in Oenothera organensis pollen. This family contains approximately six to eight family members and is expressed at high levels only in pollen. The predicted protein sequence from a near full-length cDNA clone shows that the protein products of these genes are at least 38,000 daltons. We identified the protein encoded by one of the cDNAs in this family by using antibodies to beta-galactosidase/pollen cDNA fusion proteins. Immunoblot analysis using these antibodies identifies a family of proteins of approximately 40 kilodaltons that is present in mature pollen, indicating that these mRNAs are not stored solely for translation after pollen germination. These proteins accumulate late in pollen development and are not detectable in other parts of the plant. Although not present in unpollinated or self-pollinated styles, the 40-kilodalton to 45-kilodalton antigens are detectable in extracts from cross-pollinated styles, suggesting that the proteins are present in pollen tubes growing through the style during pollination. The proteins are also present in pollen tubes growing in vitro. Both nucleotide and amino acid sequences are similar to the published sequences for cDNAs encoding the enzyme polygalacturonase, which suggests that the P2 gene family may function in depolymerizing pectin during pollen development, germination, and tube growth. Cross-hybridizing RNAs and immunoreactive proteins were detected in pollen from a wide variety of plant species, which indicates that the P2 family of polygalacturonase-like genes are conserved and may be expressed in the pollen from many angiosperms.
The European corn borer [ECB; Ostrinia nubilalis (Hübner)] is an economically significant pest of corn (Zea mays L.). The ability to routinely transform corn has broadened the control options available to include the introduction of resistance genes from sexually incompatible species. In this study, microprojectile bombardment was used to introduce synthetic versions of cryIA insecticidal protein genes from Bacillus thuringiensis subsp, kurstaki (Btk) into embryogenitcis sue of the Hi‐II] (A188/B73 derivative) genotype of corn. Of 715 independent transgenic calli produced, 314 (44%) had insecticidal activity against tobacco hornworm (Manduca sexta L.) larvae. Plants were regenerated, self‐pollinated when possible, and crossed to B73. First‐generation progeny of 173 independent Btk‐protein expressing calli were evaluated under field conditions with artificial ECB infestations in 1992 or 1993. Approximately half (89/173) segregated in single‐gene manner for resistance to first‐generation ECB leaf‐feeding damage. All of the 89 lines evaluated in 1992 or 1993 for resistance to second‐generation ECB exhibited less stalk tunneling damage than the non‐transgenic controls. In 1993, 44% (34177) of the lines tested had ≤2.5 cm of tunneling, compared to severe damage (mean = 45.7 cm) in the B73 × Hi‐II controls. Experiments are in progress to evaluate the effect of the introduced genes on yield and other agronomic properties.
5‐Enol‐pyruvylshikimate‐3‐phosphate synthase from Agrobacterium sp. CP4 (CP4 EPSPS) confers tolerance to the nonselective herbicide glyphosate (marketed under the trade name Roundup1) when sufficiently expressed in transgenic plants. Dual CP4 EPSPS transgene cassettes were transformed into corn (Zea mays L.) under the transcriptional regulatory control of the rice (Oryza sativa L.) actin 1 (P‐Ract1) and the enhanced Cauliflower mosaic virus 35S (P‐e35S) promoters, respectively, to impart fully constitutive expression in corn. Resulting events were tested for lack of chlorosis and malformation injury after two sequential applications of 1.68 kg acid equivalents (a.e.) ha−1 glyphosate. Agronomic parameters, male fertility, appropriate Mendelian segregation of the trait, plus characteristics of the transgenic integration site were also evaluated. From this selection process, the NK603 event was chosen for commercialization as the event that embodied the most optimal profile of tolerance, agronomics, and molecular characteristics. The NK603 event exhibited high glyphosate tolerance from one transgenic locus bearing a single copy of the dual cassettes integrated into the corn genome with a minimum of target sequence disruption. Trait expression in the NK603 event has remained stable over more than eight generations as shown through tolerance testing, western blots of CP4 EPSPS accumulation, and Southern blot analysis of the transgene.
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