Number Of Functional Sites Research Articles

Mutational Load and the Functional Fraction of the Human Genome.

The fraction of the human genome that is functional is a question of both evolutionary and practical importance. Studies of sequence divergence have suggested that the functional fraction of the human genome is likely to be no more than ∼15%. In contrast, the ENCODE project, a systematic effort to map regions of transcription, transcription factor association, chromatin structure, and histone modification, assigned function to 80% of the human genome. In this article, we examine whether and how an analysis based on mutational load might set a limit on the functional fraction. In order to do so, we characterize the distribution of fitness of a large, finite, diploid population at mutation-selection equilibrium. In particular, if mean fitness is ∼1, the fitness of the fittest individual likely to occur cannot be unreasonably high. We find that at equilibrium, the distribution of log fitness has variance nus, where u is the per-base deleterious mutation rate, n is the number of functional sites (and hence incorporates the functional fraction f), and s is the selection coefficient of deleterious mutations. In a large ( reproducing population, the fitness of the fittest individual likely to exist is . These results apply to both additive and recessive fitness schemes. Our approach is different from previous work that compared mean fitness at mutation-selection equilibrium with the fitness of an individual who has no deleterious mutations; we show that such an individual is exceedingly unlikely to exist. We find that the functional fraction is not very likely to be limited substantially by mutational load, and that any such limit, if it exists, depends strongly on the selection coefficients of new deleterious mutations.

Alternative oxidase: what information can protein sequence comparisons give us?

The finding that alternative oxidase (AOX) is present in most kingdoms of life has resulted in a large number of AOX sequences that are available for analyses. Multiple sequence alignments of AOX proteins from evolutionarily divergent organisms represent a valuable tool and can be used to identify amino acids and domains that may play a role in catalysis, membrane association and post-translational regulation, especially when these data are coupled with the structural model for the enzyme. I validate the use of this approach by demonstrating that it detects the conserved glutamate and histidine residues in AOX that initially led to its identification as a di-iron carboxylate protein and the generation of a structural model for the protein. A comparative analysis using a larger dataset identified 35 additional amino acids that are conserved in all AOXs examined, 30 of which have not been investigated to date. I hypothesize that these residues will be involved in the quinol terminal oxidase activity or membrane association of AOX. Major differences in AOX protein sequences between kingdoms are revealed, and it is hypothesized that two angiosperm-specific domains may be responsible for the non-covalent dimerization of AOX, whereas two indels in the aplastidic AOXs may play a role in their post-translational regulation. A scheme for predicting whether a particular AOX protein will be recognized by the alternative oxidase monoclonal antibody generated against the AOX of Sauromatum guttatum (Voodoo lily) is presented. The number of functional sites in AOX is greater than expected, and determining the structure of AOX will prove extremely valuable to future research.

Chromatin potentiation of the hsp70 promoter is linked to GAGA-factor recruitment

The events leading to transcription initiation of the Drosophila melanogaster heat-shock protein (hsp)70 gene have been demonstrated to be directly connected with nucleosome remodeling factor and GAGA-dependent chromatin remodeling on its promoter region. To investigate the relative importance of the multiple GAGA-factor binding sites in the process of chromatin remodeling and their effect on DNA conformation, the position of nucleosomes over the proximal region of the promoter was mapped. No real-positioned nucleosome was detected. By matching the relative position of the GAGA-factor binding sites with the distribution of nucleosomes over the hsp70 promoter, the GAGA site 2 appeared to be the most accessible, i.e., located close to a nucleosomal edge or within the linker DNA. This result, combined with previous observations, suggest a link between increased GAGA-factor accessibility and efficiency of transcription initiation. The effect of GAGA-binding-site mutations, both individually and in combination, on DNA structure and nucleosome remodeling was assessed using free DNA and fly embryo extract chromatin templates assembled in vitro. Results indicated that both the number of functional sites and their positions within the chromatin were important determinants for nucleosome-remodeling efficiency. Ultimately, the degree of accessibility of the GAGA factor to its cognate binding site(s) appears to be proportional to chromatin-remodeling competency of the hsp70 promoter.

Enzyme-induced coagulation of casein micelles: a number of different kinetic models

SummarySeveral mathematical models are presented in an attempt to describe the kinetics of the enzyme-induced coagulation of casein micelles. In each model the primary phase of the clotting reaction is assumed to follow first order kinetics. The only differences amongst the various models centre on the definition of the flocculation rate constant, which is defined in seven different ways. The rate constants are defined and discussed in terms of activation energy and functionality theory. The first model is such that the number of functional sites is two. The second is such that the number is much larger. The third and fourth are such that there is an exponential energy barrier, one which has a magnitude proportional to the extent of proteolysis caused by the clotting enzyme. These two definitions differ only in the pre-exponent. In one case the pre-exponent is a constant, whereas in the other it is dependent on the size of clotting particles. The fifth and sixth definitions are also energy barrier rate constants, but the energy barrier changes in an arbitrary fashion with respect to time during proteolysis. The seventh definition assumes a large number of functional sites, but such that the number increases with extent of proteolysis. In the Payens nomenclature (Payens, 1989), all models could be considered to be ‘source’ models, and all are derived using the Drake moment equation (Drake, 1972). Only the first model has a truly constant flocculation rate parameter, and only this model has a relatively simple analytical solution. All other models yield analytical solutions only by way of infinite series expansions. Thus, all models are presented in terms of power series expansions, and only through the first five time-dependent coefficients. This confines all models to the early stages of coagulation. In all cases the first three coefficients are virtually the same. The first two coefficients involve only proteolysis, and the third includes initial flocculation information. Time-dependent changes in the flocculation rate constant begin to take effect in the fourth coefficient. When the fourth coefficients of the third and seventh models are compared, a simple relationship is suggested between free energy barrier removal and functional site generation, but only assuming that the number of functionalities is large.

Coordinate binding of factor Va and factor Xa to the unstimulated platelet.

The interaction of Factor Xa and Factor Va at the platelet surface was investigated by direct, simultaneous binding measurements of both proteins to platelets and by immunochemical and kinetic techniques. Binding measurements of 125I-Factor Xa and 131I-Factor Va to unstimulated platelets indicate that the amount of Factor Xa bound is proportional to the amount of Factor Va bound. At saturating concentrations of Factor Xa, the ratio of platelet-bound Factor Xa to platelet-bound Factor Va was unity. At saturating levels of Factor Va (1.2 X 10(-8) M), 2300 molecules of Factor Xa are bound to the platelet, whereas at an equivalent concentration of unactivated Factor V, only 800 molecules of Factor Xa are bound. In the absence of exogenous Factor V or Va, thrombin-treated washed platelets bound only 200 Factor Xa molecules per platelet, suggesting that under these conditions, platelet Factor Va is the limiting component. Monovalent Fab fragments of burro antibovine Factor V inhibit, to the same degree, the rate of thrombin generation and the binding of Factor Va and Factor Xa to the platelet surface. Anti-Factor V Fab decreased the extent of Factor Va and Factor Xa binding equivalently. When the interaction of Factor Xa with platelets is modeled as Factor Xa binding to platelet-bound Factor Va, double reciprocal plots are linear, yielding a stoichiometry of 1.04 and a dissociation constant of 6 X 10(-10) M. Kinetic experiments indicate the presence of approximately 900 functional Factor Va platelet sites (Kd = 1.5 to 2.2 X 10(-10) M). This number of functional sites is equivalent to the number (837 +/- 48) of Factor Va high affinity binding sites (Kd = 4.0 X 10(-10) M). These sites most likely represent the Factor Xa binding sites involved in the function of the prothrombinase complex at the platelet surface.