Do exons code for structural or functional units in proteins?

In considering the origin and evolution of proteins, the possibility that proteins evolved from exons coding for specific structure-function modules is attractive for its economy and simplicity but is not systematically supported by the available data. However, the number of correspondences between exons and units of protein structure-function that have so far been identified appears to be greater than expected by chance alone. The available data also show (i) that exons are fairly limited in size but are large enough to specify structure-function modules in proteins; (ii) that the position of introns for homologous domains in the same gene is reasonably stable, but there is also evidence for mechanisms that alter the position or existence of introns; and (iii) that it is possible that the observed relationship of exons to protein structure represents a degenerate state of an ancestral correspondence between exons and structure-function modules in proteins

Purine nucleoside phosphorylase. Allosteric regulation of a dissociating enzyme.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is a trimeric enzyme that readily dissociates to the monomer. Dilution of enzyme from 20 to 0.02 microgram of protein/ml is accompanied by a greater than 50-fold increase in the specific activity (vtrimer = 0.23 nmol/min/microgram; vmonomer = 12.5 nmol/min/micrograms). Gel permeation chromatography in the presence of the substrate phosphate shows the enzyme to be predominantly trimeric at 50 mM Pi and predominantly monomeric at 100 mM Pi, when experiments are done at 24 degrees C. No significant dissociation was observed at 4 degrees C with Pi or at either temperature with the substrate inosine. As measured by dissociation, the L0.5 for Pi is 88 mM and thus significantly higher than the Km of 3.1 mM for Pi. Enzyme activity as a function of phosphate concentration showed negative cooperativity, but the conformational response measured by the change in native Mr during dissociation showed positive cooperatively toward Pi. These data support a model for two separate phosphate binding sites on the enzyme. The activity and stability of purine nucleoside phosphorylase are quite sensitive to the concentration of the enzyme as well as appropriate substrates. Although the monomer is interpreted as being a fully active form of the enzyme, the data in general are most consistent with the enzyme functioning in vivo as a regulated trimer

The Chemistry of the Reaction Determines the Invariant Amino Acids during the Evolution and Divergence of Orotidine 5′-Monophosphate Decarboxylase

Orotidine 5'-phosphate (OMP) decarboxylase has the largest rate enhancement for any known enzyme. For an average protein of 270 amino acids from more than 80 species, only 8 amino acids are invariant, and 7 of these correspond to ligand-binding residues in the crystal structures of the enzyme from four species. It appears that the chemistry required for catalysis determines the invariant residues for this enzyme structure. A motif of three invariant amino acids at the catalytic site (DXKXXD) is also found in the enzyme hexulose-phosphate synthase. Although the core of OMP decarboxylase is conserved, it has undergone a variety of changes in subunit size or fusion to other protein domains, such as orotate phosphoribosyltransferase, during evolution in different kingdoms. The phylogeny of OMP decarboxylase shows a unique subgroup distinct from the three kingdoms of life. The enzyme subunit size almost doubles from Archaea (average mass of 24.5 kDa) to certain fungi (average mass of 41.7 kDa). These observed changes in subunit size are produced by insertions at 12 sites, largely in loops and on the exterior of the core protein. The consensus for all sequences has a minimal size of <20 kDa

Uridine kinase from Ehrlich ascites carcinoma. Purification and properties of homogeneous enzyme.

Uridine kinase from Ehrlich ascites tumor cells has been purified about 60,000-fold to apparent homogeneity and with an overall recovery of about 40%. This purification was achieved using phosphocellulose and adenosine 5'-triphosphate-agarose affinity chromatography. The subunit molecular mass as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31,000 daltons. With two-dimensional electrophoresis, only one spot was observed, indicating the absence of isoenzymes. Multiple peaks of activity are routinely observed on ion exchange chromatography or gel filtration, for both crude preparations or homogeneous uridine kinase, in agreement with our earlier results that this enzyme exists as multiple interconvertible oligomeric forms (Payne, R. C., and Traut, T. W. (1982) J. Biol. Chem. 257, 12485-12488). The purified enzyme has a specific activity of 283 mumol/min/mg of protein at 22 degrees C. Initial velocity studies using uridine and ATP are consistent with a sequential mechanism. Km values for uridine, cytidine, and ATP are 40, 57, and 450 microM, respectively. CTP and UTP are competitive inhibitors with respect to ATP, with Ki values for CTP and UTP of 10 and 61 microM, respectively. The enzyme was active with several nucleoside analogs, the Km values being 69 microM (5-fluorouridine), 200 microM (3-deazauridine), and 340 microM (6-azauridine). The pure enzyme is very sensitive to freezing, but can be maintained at O degrees C for 8 weeks with only 20% loss of activity. For long-term storage, enzyme in 50% glycerol can be maintained at -20 degrees C for many months with no detectable loss of activity

CO2 Targets, Trajectories and Trends for International Shipping

The Shipping in Changing Climates (SCC) project connects the latest climate change science with knowledge, understanding and models of the shipping sector in a whole systems approach. It seeks to explore the potential to cut CO2 through the use of technical and operational changes in shipping and to understand how the sector might transition to a more resilient and low-carbon future; it also seeks to explore different climate change scenarios and related food and fuel security issues to gain an understanding of the direct and indirect impacts of climate change on the shipping sector. These scenarios can be used to build evidence and understanding around the range of potential future directions that the shipping industry may take. The RCUK Energy funded project brings together researchers from UCL (Energy Institute, Mechanical Engineering and Laws), Manchester, Southampton, Newcastle and Strathclyde, in close collaboration with a core industry stakeholder group of Shell, Lloyd’s Register, Rolls Royce, BMT and Maritime Strategies International, but drawing on the expertise and connections of over 35 companies and organisations worldwide. This paper is non-peer- reviewed and represents the collective opinions of the authors and should not be assumed to represent the views of all the researchers across the project or the project’s industry partners and their organisations

About females and males: continuity and discontinuity in flies

Through the decades of relentless and dedicated studies in Drosophila melanogaster, the pathway that governs sexual development has been elucidated in great detail and has become a paradigm in understanding fundamental cell-fate decisions. However, recent phylogenetic studies show that the molecular strategy used in Drosophila deviates in some important aspects from those found in other dipteran flies and suggest that the Drosophila pathway is likely to be a derivative of a simpler and more common principle. In this essay, I will discuss the evolutionary plasticity of the sex-determining pathway based on studies in the common housefly, Musca domestica. Diversification appears to primarily arise from subtle differences in the regulation of the key switch gene transformer at the top of the pathway. On the basis of these findings I propose a new idea on how the Drosophila pathway may have evolved from a more archetypal system such as in M. domestica. In essence, the arrival of an X counting mechanism mediated by Sex-lethal to compensate for X linked gene dose differences set the stage for an intimate coupling of the two pathways. Its precedent recruitment to the dosage compensation pathway allowed for an intervention in the regulation of transformer where it gradually and eventually' completely substituted for a need of transformer autoregulation

UMP/CMPK Is Not the Critical Enzyme in the Metabolism of Pyrimidine Ribonucleotide and Activation of Deoxycytidine Analogs in Human RKO Cells

Human UMP/CMP kinase was identified based on its enzymatic activity in vitro. The role of this protein is considered critical for the maintenance of pyrimidine nucleotide pool profile and for the metabolism of pyrimidine analogs in cells, based on the in vitro study of partially purified enzyme and recombinant protein. However, no detailed study has yet addressed the role of this protein in nucleotide metabolism in cells.Two stable cell lines in which UMP/CMP kinase (mRNA: AF087865, EC 2.7.4.14) can be either up-regulated or down-regulated were developed using Tet-On Gene Expression Systems. The amount and enzymatic activity of UMP/CMP kinase extracted from these two cell lines can be induced up by 500% or down by 95-98%. The ribonucleotides of endogenous pyrimidine as well as the metabolism of exogenous natural pyrimidine nucleosides and their analogs were not susceptible to the altered amount of UMP/CMP kinase in these two stable RKO cell lines. The level of incorporation of pyrimidine nucleoside analogs, such as gemcitabine (dFdC) and troxacitabine (L-OddC), into cellular DNA and their potency in inhibiting cell growth were not significantly altered by up-regulation or down-regulation of UMP/CMP kinase expression in cells.The UMP/CMP kinase (EC 2.7.4.14) expressed in RKO cells is not critical for the phosphorylation of (d)CMP and the maintenance of natural nucleotide pools. It also does not play an important role in the activation of dFdC and L-OddC. The increase by 500% or decrease by 95-98% in the levels of UMP/CMP kinase do not affect steady state levels of dFdC and L-OddC in RKO cells. Overall, the activity and possible mechanisms of recombinant UMP/CMP kinase expressed in the in vitro system can not be extended to that of UMP/CMP kinase expressed in a cell system or an in vivo system

Sex-specific differences in the synaptonemal complex in the genus Oreochromis (Cichlidae)

Total synaptonemal complex (SC) lengths were estimated from Oreochromis aureus Steindachner (which has a WZ/ZZ sex determination system), O. mossambicus Peters and O. niloticus L. (both of which have XX/XY sex determination systems). The total SC length in oocytes was greater than that in spermatocytes in all three species (194±30 μm and 134±13 μm, 187±22 μm and 127±17 μm, 193±37 μm and 144±19 μm, respectively). These sex-specific differences did not appear to be influenced by the type of sex determination system (the female/male total SC length ratio was 1.45 in O. aureus, 1.47 in O. mossambicus and 1.34 in O. niloticus) and do not correlate with the lack of any overall sex-specific length differences in the current Oreochromis linkage map. Although based on data from relatively few species, there appears to be no consistent relationship between sex-specific SC lengths and linkage map lengths in fish. Neomale (hormonally masculinized genetic female) O. aureus and O. mossambicus had total SC lengths of 138±13 μm and 146±13 μm respectively, more similar to normal males than to normal females. These findings agree with data from other vertebrate species that suggest that phenotypic sex, rather than genotype, determines traits such as total SC length, chiasmata position and recombination pattern, at least for the autosomes

Mis-Spliced Transcripts of Nicotinic Acetylcholine Receptor α6 Are Associated with Field Evolved Spinosad Resistance in Plutella xylostella (L.)

The evolution of insecticide resistance is a global constraint to agricultural production. Spinosad is a new, low-environmental-risk insecticide that primarily targets nicotinic acetylcholine receptors (nAChR) and is effective against a wide range of pest species. However, after only a few years of application, field evolved resistance emerged in the diamondback moth, Plutella xylostella, an important pest of brassica crops worldwide. Spinosad resistance in a Hawaiian population results from a single incompletely recessive and autosomal gene, and here we use AFLP linkage mapping to identify the chromosome controlling resistance in a backcross family. Recombinational mapping with more than 700 backcross progeny positioned a putative spinosad target, nAChR alpha 6 (Pxα6), at the resistance locus, PxSpinR. A mutation within the ninth intron splice junction of Pxα6 results in mis-splicing of transcripts, which produce a predicted protein truncated between the third and fourth transmembrane domains. Additional resistance-associated Pxα6 transcripts that excluded the mutation containing exon were detected, and these were also predicted to produce truncated proteins. Identification of the locus of resistance in this important crop pest will facilitate field monitoring of the spread of resistance and offer insights into the genetic basis of spinosad resistance in other species