Vibrationally enhanced hydrogen tunneling in the Escherichia coli thymidylate synthase catalyzed reaction

The enzyme thymidylate synthase (TS) catalyzes a complex reaction that involves forming and breaking at least six covalent bonds. The physical nature of the hydride transfer step in this complex reaction cascade has been studied by means of isotope effects and their temperature dependence. Competitive kinetic isotope effects (KIEs) on the second-order rate constant (V/K) were measured over a temperature range of 5-45 degrees C. The observed H/T ((T)V/K(H)) and D/T ((T)V/K(D)) KIEs were used to calculate the intrinsic KIEs throughout the temperature range. The Swain-Schaad relationships between the H/T and D/T V/K KIEs revealed that the hydride transfer step is the rate-determining step at the physiological temperature of Escherichia coli (20-30 degrees C) but is only partly rate-determining at elevated and reduced temperatures. H/D KIE on the first-order rate constant k(cat) ((D)k = 3.72) has been previously reported [Spencer et al. (1997) Biochemistry 36, 4212-4222]. Additionally, the Swain-Schaad relationships between that (D)k and the V/K KIEs reported here suggested that at 20 degrees C the hydride transfer step is the rate-determining step for both rate constants. Intrinsic KIEs were calculated here and were found to be virtually temperature independent (DeltaE(a) = 0 within experimental error). The isotope effects on the preexponential Arrhenius factors for the intrinsic KIEs were A(H)/A(T) = 6.8 +/- 2.8 and A(D)/A(T) = 1.9 +/- 0.25. Both effects are significantly above the semiclassical (no-tunneling) predicted values and indicate a contribution of quantum mechanical tunneling to this hydride transfer reaction. Tunneling correction to transition state theory would predict that these isotope effects on activation parameters result from no energy of activation for all isotopes. Yet, initial velocity measurements over the same temperature range indicate cofactor inhibition and result in significant activation energy on k(cat) (4.0 +/- 0.1 kcal/mol). Taken together, the temperature-independent KIEs, the large isotope effects on the preexponential Arrhenius factors, and a significant energy of activation all suggest vibrationally enhanced hydride tunneling in the TS-catalyzed reaction.     

Canonical labeling of proteome maps

We propose a canonical labeling of proteome maps, which enables one to sort and catalog the maps in a simple way. The canonical label of a proteome map is based on the canonical labeling of vertexes of Hasse diagram embedded in the map resulting in the adjacency matrix, the rows of which when viewed as binary numbers are the smallest possible such numbers. The use of the approach in documentation is illustrated with the proteome maps of liver cells of healthy male Fisher F344 rats and the rats treated with different peroxisome proliferators.     

Sequential ATP-induced allosteric transitions of the cytoplasmic chaperonin containing TCP-1 revealed by EM analysis

The eukaryotic cytoplasmic chaperonin containing TCP-1 (CCT) is a hetero-oligomeric complex that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. To understand the allosteric transitions that occur during the functional cycle of CCT, we imaged the chaperonin complex in the presence of different ATP concentrations. Labeling by monoclonal antibodies that bind specifically to the CCTalpha and CCTdelta subunits enabled alignment of all the CCT subunits of a given type in different particles. The analysis shows that the apo state of CCT has considerable apparent conformational heterogeneity that decreases with increasing ATP concentration. In contrast with the concerted allosteric switch of GroEL, ATP-induced conformational changes in CCT are found to spread around the ring in a sequential fashion that may facilitate domain-by-domain substrate folding. The approach described here can be used to unravel the allosteric mechanisms of other ring-shaped molecular machines.     

Photoinduction of Massive beta-Carotene Accumulation by the Alga Dunaliella bardawil: Kinetics and Dependence on Gene Activation

The massive accumulation of β-carotene by the halotolerant micro alga Dunaliella bardawil, in response to high light intensity and several other environmental factors, has been studied so far under different sets of fixed conditions. To determine the kinetics and characteristics of the induction of β-carotene accumulation, cells continuously grown under white light of approximately 27 microeinsteins per square meter per second were exposed to light of approximately 1650 microeinsteins per square meter per second. The exposed cells accumulate β-carotene in two stages: the first stage, lasting for 24 hours, starts shortly after exposure, whereas the second stage starts concomitantly with the onset of the stationary phase and persists until the cells collapse. Actinomycin D, chloramphenicol, or cycloheximide added to low-illuminated cultures abolish the subsequent induction of β-carotene accumulation by high light intensity. These results, together with the early exponential kinetics of accumulation, point to the role of gene activation in the process. In vivo labeling of proteins and in vitro translation of poly(A)⁺ mRNA revealed several pronounced differences between low-illuminated and high-illuminated cells. A strongly light-induced protein of approximately 55 kilodaltons, as well as other light-induced proteins could possibly fulfill a carotenogenic function.     

Single nucleotide polymorphisms generated by genotyping by sequencing to characterize genome-wide diversity,linkage disequilibrium,and selective sweeps in cultivated watermelon

Background

A large single nucleotide polymorphism (SNP) dataset was used to analyze genome-wide diversity in a diverse collection of watermelon cultivars representing globally cultivated, watermelon genetic diversity. The marker density required for conducting successful association mapping depends on the extent of linkage disequilibrium (LD) within a population. Use of genotyping by sequencing reveals large numbers of SNPs that in turn generate opportunities in genome-wide association mapping and marker-assisted selection, even in crops such as watermelon for which few genomic resources are available. In this paper, we used genome-wide genetic diversity to study LD, selective sweeps, and pairwise F_ST distributions among worldwide cultivated watermelons to track signals of domestication.

Results

We examined 183 Citrullus lanatus var. lanatus accessions representing domesticated watermelon and generated a set of 11,485 SNP markers using genotyping by sequencing. With a diverse panel of worldwide cultivated watermelons, we identified a set of 5,254 SNPs with a minor allele frequency of ≥ 0.05, distributed across the genome. All ancestries were traced to Africa and an admixture of various ancestries constituted secondary gene pools across various continents. A sliding window analysis using pairwise F_ST values was used to resolve selective sweeps. We identified strong selection on chromosomes 3 and 9 that might have contributed to the domestication process. Pairwise analysis of adjacent SNPs within a chromosome as well as within a haplotype allowed us to estimate genome-wide LD decay. LD was also detected within individual genes on various chromosomes. Principal component and ancestry analyses were used to account for population structure in a genome-wide association study. We further mapped important genes for soluble solid content using a mixed linear model.

Conclusions

Information concerning the SNP resources, population structure, and LD developed in this study will help in identifying agronomically important candidate genes from the genomic regions underlying selection and for mapping quantitative trait loci using a genome-wide association study in sweet watermelon.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-767) contains supplementary material, which is available to authorized users.     

Mycorrhized Ri-transformed roots facilitate in vitro inoculation of Cistus incanus with Tuber melanosporum

Progress was made towards a reliable in vitro system for mycorrhizing Cistus incanus seedlings with Tuber melanosporum. A rich growth medium favored extensive growth of mycorrhized Ri-transformed roots (MTR) but inhibited mycelial outgrowth into the medium. A minimal medium, on the other hand, inhibited MTR growth but supported considerable mycelial outgrowth into the medium. While the presence of a C.␣incanus propagule clearly enhanced mycelial growth into the minimal medium, a highly significant factor appeared to be the use of MTR inoculant, which supported mycorrhizal development to the Hartig net stage. The advantages of MTR for in vitro mycorrhization of host plant seedlings are discussed.     

Gamma-irradiated RecD overproducers become permanent recB-/C- phenocopies for extrachromosomal DNA processing due to prolonged titration of RecBCD enzyme on damaged Escherichia coli chromosome

The RecBCD enzyme of Escherichia coli consists of three subunits RecB, RecC and RecD. RecBCD enzyme activities are regulated by its interaction with recombination hotspot Chi. Biochemical and genetic evidence suggest that interaction with Chi affects RecD subunit, and that RecD polypeptide overproduction antagonizes this interaction, suggesting that intact RecD replaces a Chi-modified one. We used bacteria with fragmented chromosomes due to double-strand breaks inflicted by UV and gamma-irradiation to explore in which way increased concentrations of RecBCD's individual subunits affect DNA metabolism. We confirmed that RecD overproduction alters RecBCD-dependent DNA repair and degradation in E. coli. Also, we found that RecB and RecC overproduction did not affect these processes. To determine the basis for the effects of RecD polypeptide overproduction, we monitored activities of RecBCD enzyme on gamma-damaged chromosomal DNA and, in parallel, on lambda and T4 2 phage DNA duplexes provided at intervals. We found that gamma-irradiated wild-type bacteria became transient, and RecD overproducers permanent recB(-)/C(-) phenocopies for processing phage DNA that is provided in parallel. Since this inability of irradiated bacteria to process extrachromosomal DNA substrates coincided in both cases with ongoing degradation of chromosomal DNA, which lasted much longer in RecD overproducers, we were led to conclude that the RecB(-)/C(-) phenotype is acquired as a consequence of RecBCD enzyme titration on damaged chromosomal DNA. This conclusion was corroborated by our observation that no inhibition of RecBCD activity occurs in gamma-irradiated RecBCD overproducers. Together, these results strongly indicate that RecD overproduction prevents dissociation of RecBCD enzyme from DNA substrate and thus increases its processivity.