Dissection of Arp2/3 complex actin nucleation mechanism and distinct roles for its nucleation-promoting factors in Saccharomyces cerevisiae

Actin nucleation by the Arp2/3 complex is under tight control, remaining inactive until stimulation by nucleation-promoting factors (NPFs). Although multiple NPFs are expressed in most cell types, little is known about how they are coordinated and whether they perform similar or distinct functions. We examined genetic relationships among the four S. cerevisiae NPFs. Combining las17delta with pan1-101 or myo3delta myo5delta was lethal at all temperatures, whereas combining pan1-101 with myo3delta myo5delta showed no genetic interaction and abp1delta partially suppressed las17delta. These data suggest that NPFs have distinct and overlapping functions in vivo. We also tested genetic interactions between each NPF mutant and seven different temperature-sensitive arp2 alleles and purified mutant Arp2/3 complexes to compare their activities. Two arp2 alleles with mutations at the barbed end were severely impaired in nucleation, providing the first experimental evidence that Arp2 nucleates actin at its barbed end in vitro and in vivo. Another arp2 allele caused partially unregulated ("leaky") nucleation in the absence of NPFs. Combining this mutant with a partially unregulated allele in a different subunit of Arp2/3 complex was lethal, suggesting that cells cannot tolerate high levels of unregulated activity. Genetic interactions between arp2 alleles and NPF mutants point to Abp1 having an antagonistic role with respect to other NPFs, possibly serving to attenuate their stronger activities. In support of this model, Abp1 binds strongly to Arp2/3 complex, yet has notably weak nucleation-promoting activity and inhibits Las17 activity on Arp2/3 complex in a dose-responsive manner.     

Anion binding and controlled aggregation of human interleukin-1 receptor antagonist

Highly concentrated human recombinant interleukin-1 receptor antagonist (IL-1ra) aggregates at elevated temperature without perturbation in its secondary structure. The protein aggregation can be suppressed depending on the buffer ionic strength and the type of anion present in the sample solution. Phosphate is an approximately 4-fold weaker suppressant than either citrate or pyrophosphate on the basis of the measured protein aggregation rates. This is in agreement with the strength of protein-anion interactions at the IL-1ra single anion-binding site as judged by the estimated dissociation constant values of 2.9 mM, 3.8 mM, and 13.7 mM for pyrophosphate, citrate, and phosphate, respectively. The strength of binding also correlates with the anion size and with the number of ionized groups available per molecule at a given pH. Affinity probing of IL-1ra with methyl acetyl phosphate (MAP) in combination with proteolytic digestion and mass spectral analysis show that an anion-binding site location on the IL-1ra surface is contributed by lysine-93 and lysine-96 of the loop 84-98 as well as by lysine-6 of the unstructured N-terminal region 1-7. The replacement of lysine-93 with alanine by site-directed mutagenesis results in dramatically suppressed IL-1ra aggregation. Furthermore, when the unstructured N-terminal region of IL-1ra is removed by limited proteolysis, a 2-fold increase in the time course of the aggregation lag phase is observed for the truncated protein. An anion-controlled mechanism of IL-1ra aggregation is proposed by which the anion competition for the protein cationic site prevents formation of intermolecular cation-pi interactions and, thus, interferes with the protein asymmetric self-association pathway.     

Base composition analysis of human mitochondrial DNA using electrospray ionization mass spectrometry: a novel tool for the identification and differentiation of humans

In traditional approaches, mitochondrial DNA (mtDNA) variation is exploited for forensic identity testing by sequencing the two hypervariable regions of the human mtDNA control region. To reduce time and labor, single nucleotide polymorphism (SNP) assays are being sought to possibly replace sequencing. However, most SNP assays capture only a portion of the total variation within the desired regions, require a priori knowledge of the position of the SNP in the genome, and are generally not quantitative. Furthermore, with mtDNA, the clustering of SNPs complicates the design of SNP extension primers or hybridization probes. This article describes an automated electrospray ionization mass spectrometry method that can detect a number of clustered SNPs within an amplicon without a priori knowledge of specific SNP positions and can do so quantitatively. With this technique, the base composition of a PCR amplicon, less than 140 nucleotides in length, can be calculated. The difference in base composition between two samples indicates the presence of an SNP. Therefore, no post-PCR analytical construct needs to be developed to assess variation within a fragment. Of the 2754 different mtDNA sequences in the public forensic mtDNA database, nearly 90% could be resolved by the assay. The mass spectrometer is well suited to characterize and quantitate heteroplasmic samples or those containing mixtures. This makes possible the interpretation of mtDNA mixtures (as well as mixtures when assaying other SNPs). This assay can be expanded to assess genetic variation in the coding region of the mtDNA genome and can be automated to facilitate analysis of a large number of samples such as those encountered after a mass disaster.     

Physical and hydrodynamic properties of flocs produced during biological hydrogen production

Dense flocs readily form in continuous culture bioreactors used for hydrogen production, but the fractal and hydrodynamic properties of these flocs have not been previously analyzed. We therefore examined the size distribution, fractal dimension, and hydrodynamic properties of flocs formed in a continuous flow, well-mixed reactor treating synthetic wastewater at a fixed condition of a 4.5 h hydraulic detention time (23 degrees C, pH 5.5). The reactor was operated for a total of 3 months at three different organic loading rates (27, 53, and 80 g-COD/L-d) with influent glucose concentrations of 5, 10, and 15 g-COD/L. At all three loading rates the removal of glucose was nearly complete (98.6-99.4%) and biomass was produced in proportion to the organic loading rate (0.86 +/- 0.11, 2.40 +/- 0.26, and 4.59 +/- 1.55 g/L of MLVSS in the reactor). Overall conversion efficiencies of glucose to hydrogen, evaluated on the basis of a maximum of 4 mol-H2/mol-glucose, increased with organic loading rates in the order 17.7%, 23.1%, and 25.6%. The gas contained 56.1 +/- 4.9% hydrogen, with the balance as carbon dioxide. No methane gas was detected. Under these conditions, flocs were produced with mean sizes that increased with organic loading, in the order 0.12 cm (5 g-COD/L), 0.35 cm (10 g-COD/L), and 0.58 cm (15 g-COD/L). As the average floc size increased, the flocs became on average denser and less fractal, with fractal dimensions increasing from 2.11 +/- 0.17 to 2.48 +/- 0.13. Floc porosities ranged from 0.75-0.96, and resulted in aggregate densities that allowed little intra-aggregate flow through the floc. As a result, average settling velocities were not appreciably larger than those predicted by Stokes' law for spherical, impermeable flocs. Our results demonstrate that dense, relatively impermeable flocs are produced in biohydrogen reactors that have settling properties in reasonable agreement with Stokes' law.     

Isolation of a strain of Agrobacterium tumefaciens (Rhizobium radiobacter) utilizing methylene urea (ureaformaldehyde) as nitrogen source

Methylene ureas (MU) are slow-release nitrogen fertilizers degraded in soil by microbial enzymatic activity. Improved utilization of MU in agricultural production requires more knowledge about the organisms and enzymes responsible for its degradation. A Gram-negative, MU-degrading organism was isolated from a soil in Sacramento Valley, California. The bacterium was identified as Agrobacterium tumefaciens (recently also known as Rhizobium radiobacter) using both genotypic and phenotypic characterization. The pathogenic nature of the organism was confirmed by a bioassay on carrot disks. The MU-hydrolyzing enzyme (MUase) was intracellular and was induced by using MU as a sole source of nitrogen. The bacterial growth was optimized in NH4Cl, urea, or peptone, whereas the production and specific activity of MUase were maximized with either NH4Cl or urea as a nitrogen source. The result has a practical significance, demonstrating a potential to select for this plant pathogen in soils fertilized with MU.     

Expression and biochemical analysis of the entire HIV-2 gp41 ectodomain: determinants of stability map to N- and C-terminal sequences outside the 6-helix bundle core

The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2(ST) gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591-Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529-Trp-589 and Val-604-Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides indicated that the N-terminal polar segment, 521-528, and C-terminal membrane-proximal segment, 658-666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion.     

Regulation of the Drosophila epidermal growth factor-ligand vein is mediated by multiple domains

Vein (Vn), a ligand for the Drosophila epidermal growth factor receptor (Egfr), has a complex structure including a PEST, Ig, and EGF domain. We analyzed the structure-function relationships of Vn by assaying deletion mutants. The results show that each conserved domain influences Vn activity. A PEST deletion increases Vn potency and genetic evidence suggests that Vn is regulated by proteasomal degradation. The Ig deletion causes toxic effects not seen following expression of native Vn, but the Ig domain is not required for Vn localization or for the activation of Egfr signaling in wing vein patterning. Remarkably, when the EGF domain is deleted, Vn functions as a dominant negative ligand, implying that Vn normally physically interacts with another factor to promote its activity. We identified additional highly conserved sequences and found several regions that affect Vn potency and one that may mediate the effect of dominant negative Vn molecules. Together the results show that the activity of Vn is controlled both positively and negatively, demonstrating the existence of additional levels at which Egfr signaling can be regulated.     

A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F(2) population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map.