Protein complexes in the archaeon Methanothermobacter thermautotrophicus analyzed by blue native/SDS-PAGE and mass spectrometry

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.     

Inducible expression of bacterio-opsin in transgenic tobacco and tomato plants

The development of new strategies to enhance resistance of plants to pathogens is instrumental in preventing agricultural losses. Lesion mimic, the spontaneous formation of lesions resembling hypersensitive response lesions in the absence of a pathogen, is a dramatic phenotype occasionally induced upon expression of certain transgenes in plants. These transgenes simulate the presence of a pathogen and, therefore, activate the plant anti-pathogen defense mechanisms and induce a state of systemic resistance. Lesion mimic genes have been successfully used to enhance the resistance of a number of different plants to pathogen attack. However, constitutive expression of these genes in plants is associated with the spontaneous formation of lesions on leaves and stems, reduced growth, and lower yield. We tested the possibility of using a wound-inducible promoter to control the expression of bacterio-opsin (bO), a transgene that confers a lesion mimic phenotype in tobacco and tomato plants when constitutively expressed. We found that plants with inducible expression of bO did not develop spontaneous lesions. Nevertheless, under controlled laboratory conditions, they were found to be resistant to infection by pathogens. The activation of defense mechanisms by the bO gene was not constitutive, and occurred in response to wounding or pathogen infection. Furthermore, wounding of transgenic tobacco plants resulted in the induction of systemic resistance to pathogen attack within 48 h. Our findings provide a promising initial assessment for the use of wound-inducible promoters as a new strategy to enhance pathogen resistance in transgenic crops by means of lesion mimic genes.     

Short-chain saturated fatty acids in the regulation of pollination-induced ethylene sensitivity of Phalaenopsis flowers

Pollination greatly accelerates petal senescence. The first observed event after pollination is an increase in the flower's sensitivity to ethylene, followed by an increase in ethylene biosynthesis. Our objectives were to study the mode of action of the increase in ethylene sensitivity and the possible involvement of short-chain saturated fatty acids (SCSFAs) in this process. Application of SCSFAs, ranging in chain length from 7 to 10 carbons onto stigmas of Phalaenopsis (Phalaenopsis hybrid, cv. Herbert Hager) flowers increased their sensitivity to ethylene in the same way as pollination. Following pollination, there was a significant increase in the endogenous content of these fatty acids in the flower's column and perianth, with octanoic acid (C₈) being the main SCSFA observed. The increase in SCSFA content was observed as early as 6 h after pollination and began to decline 6 h later. Incorporation of octanoic acid into liposomes or microsomal membranes isolated from Phalaenopsis petals resulted in a decrease in lipid order that was detected by fluorescence polarization of dansyl pyrrolidine (DNSP) but not of 1,6-diphenyl-1,3,5-hexatriene (DPH). At peak ethylene sensitivity, 10 h after pollination, there was a significant decrease in the lipid order of microsomal membranes isolated from Phalaenopsis columns and perianths, again as detected by DNSP but not by DPH. Stigmatic application of octanoic acid mimicked the effect of pollination on membrane lipid order. We suggest that SCSFAs may be the ethylene 'sensitivity factors' produced following pollination, and that their mode of action involves a decrease in the order of specific regions in the membrane lipid bilayer, consequently altering ethylene action.     

Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and beta1 integrin to promote cell migration

The WD repeat scaffolding protein RACK1 can mediate integration of the insulin-like growth factor I receptor (IGF-IR) and integrin signaling in transformed cells. To address the mechanism of RACK1 function, we searched for regulatory proteins that associate with RACK1 in an IGF-I-dependent manner. The serine threonine phosphatase protein phosphatase 2A (PP2A) was found associated with RACK1 in serum-starved cells, and it dissociated immediately upon stimulation with IGF-I. This dissociation of PP2A from RACK1 and an IGF-I-mediated decrease in cellular PP2A activity did not occur in cells expressing either the serine 1248 or tyrosine 1250/1251 mutants of the IGF-IR that do not interact with RACK1. Recombinant RACK1 could bind to PP2A in vitro and restore phosphatase activity to PP2A from IGF-I-stimulated cells. Ligation of integrins with fibronectin or Matrigel was sufficient to facilitate IGF-I-mediated dissociation of PP2A from RACK1 and also to recruit beta1 integrin as PP2A dissociated. By using TAT-fused N-terminal and C-terminal deletion mutants of RACK1, we determined that both PP2A and beta1 integrin interact in the C terminus of RACK1 within WD repeats 4 to 7. This suggests that integrin ligation displaces PP2A from RACK1. MCF-7 cells overexpressing RACK1 exhibited enhanced motility, which could be reversed by the PP2A inhibitor okadaic acid. Small interfering RNA-mediated suppression of RACK1 also decreased the migratory capacity of DU145 cells. Taken together, our findings indicate that RACK1 enhances IGF-I-mediated cell migration through its ability to exclusively associate with either beta1 integrin or PP2A in a complex at the IGF-IR.     

Quantitative trait loci for urinary albumin in crosses between C57BL/6J and A/J inbred mice in the presence and absence of Apoe

We investigated the effect of apolipoprotein E (Apoe) on albuminuria in the males of two independent F2 intercrosses between C57BL/6J and A/J mice, using wild-type inbred strains in the first cross and B6-Apoe(-/-) animals in the second cross. In the first cross, we identified three quantitative trait loci (QTL): chromosome (Chr) 2 [LOD 3.5, peak at 70 cM, confidence interval (C.I.) 28-88 cM]; Chr 9 (LOD 2.0, peak 5 cM, C.I. 5-25 cM); and Chr 19 (LOD 1.9, peak 49 cM, C.I. 23-54 cM). The Chr 2 and Chr 19 QTL were concordant with previously found QTL for renal damage in rat and human. The Chr 9 QTL was concordant with a locus found in rat. The second cross, testing only Apoe(-/-) progeny, did not identify any of these loci, but detected two other loci on Chr 4 (LOD 3.2, peak 54 cM, C.I. 29-73 cM) and Chr 6 (LOD 2.6, peak 33 cM, C.I. 11-61 cM), one of which was concordant with a QTL found in rat. The dependence of QTL detection on the presence of Apoe and the concordance of these QTL with rat and human kidney disease QTL suggest that Apoe plays a role in renal damage.     

From QTL to QTN identification in livestock--winning by points rather than knock-out: a review

Many quantitative trait loci (QTL) affecting economic traits in livestock have now been identified. However, the confidence interval (CI) of individual QTL as determined by linkage analysis often spans tens of map units, containing hundreds of genes. Linkage disequilibrium (LD) mapping can reduce the CI to individual map units, but this reduced interval will still contain tens of genes. Methods suitable for model animals to find and validate specific quantitative trait nucleotides (QTN) underlying the QTL cannot be easily applied to livestock species because of their long generation intervals, the cost of maintaining each animal and the difficulty of producing transgenics or 'knock-outs'. Considering these limitations, we review successful approaches for identifying QTN in livestock and outline a schematic strategy for QTN determination and verification. In addition to linkage and LD mapping, the methods include positional cloning, selection of candidate genes, DNA sequencing and statistical analyses. Concordance determination and functional assays are the critical tests for validation of a QTN; we provide a generalized formula for the probability of concordance by chance. Three genes that meet the burden of proof for QTN identification--DGAT1 in cattle, IGF2 in swine and GDF8 in sheep--are discussed in detail. The genetic and economic ramifications of identified QTN and the horizon for selection and introgression are also considered.