Mechanistic Constraints Research Articles

Evolution of dominance in metabolic pathways.

Dominance is a form of phenotypic robustness to mutations. Understanding how such robustness can evolve provides a window into how the relation between genotype and phenotype can evolve. As such, the issue of dominance evolution is a question about the evolution of inheritance systems. Attempts at explaining the evolution of dominance have run into two problems. One is that selection for dominance is sensitive to the frequency of heterozygotes. Accordingly, dominance cannot evolve unless special conditions lead to the presence of a high frequency of mutant alleles in the population. Second, on the basis of theoretical results in metabolic control analysis, it has been proposed that metabolic systems possess inherent constraints. These hypothetical constraints imply the default manifestation of dominance of the wild type with respect to the effects of mutations at most loci. Hence, some biologists have maintained that an evolutionary explanation is not relevant to dominance. In this article, we put into question the hypothetical assumption of default metabolic constraints. We show that this assumption is based on an exclusion of important nonlinear interactions that can occur between enzymes in a pathway. With an a priori exclusion of such interactions, the possibility of epistasis and hence dominance modification is eliminated. We present a theoretical model that integrates enzyme kinetics and population genetics to address dominance evolution in metabolic pathways. In the case of mutations that decrease enzyme concentrations, and given the mechanistic constraints of Michaelis-Menten-type catalysis, it is shown that dominance of the wild type can be extensively modified in a two-enzyme pathway. Moreover, we discuss analytical results indicating that the conclusions from the two-enzyme case can be generalized to any number of enzymes. Dominance modification is achieved chiefly through changes in enzyme concentrations or kinetic parameters such as k(cat), both of which can alter saturation levels. Low saturation translates into higher levels of dominance with respect to mutations that decrease enzyme concentrations. Furthermore, it is shown that in the two-enzyme example, dominance evolves as a by-product of selection in a manner that is insensitive to the frequency of heterozygotes. Using variation in k(cat) as an example of modifier mutations, it is shown that the latter can have direct fitness effects in addition to dominance modification effects. Dominance evolution can occur in a frequency-insensitive manner as a result of selection for such dual-effects alleles. This type of selection may prove to be a common pattern for the evolution of phenotypic robustness to mutations.

Substrate water interactions within the Photosystem II oxygen evolving complex

The 18O exchange rates for the substrate water in photosystem II can be obtained by time-resolved mass spectrometric approaches and provide insight into the chemical nature of the substrate binding sites in the oxygen evolving complex (OEC). In this communication, we report additional data on the resolvable S-state dependence of the substrate exchange rates in untreated spinach thylakoids. The data reveals that the exchange for one substrate water molecule is resolvable in all S states, i.e. S0 (k1 ≈ 10 s−1), S1 (k1 ≈ 0.02 s−1), S2 (k1 ≈ 2.0 s−1) and S3 (k1 ≈ 2.0 s−1), and that the second substrate water molecule is resolvable in S2 (k2 ≈ 120 s−1) and S3 (k2 ≈ 40 s−1) states, and faster than 120 s−1 during the S0 and S1 states. The temperature dependencies of the exchange rates are also reported. The corresponding activation energies for the slow exchange substrate water in S1, S2 and S3 are 83 ± 4, 71 ± 9, and 78 ± 9 kJ mol−1, respectively. In contrast, the activation energy for the fast exchange substrate water in S3, is 40 ± 5 kJ mol−1. Finally we also report the secondary isotope effect of deuterium on S3 state measurements, where the slow exchange substrate water remains unaffected while the rate for the fast exchange substrate water increases by a factor of ∼35%, resulting in an inverse H/D isotope effect. The exchange rates, activation energies and deuterium effects provide further mechanistic constraints on the water oxidation reaction in photosystem II and provide strong evidence that the slow exchange substrate water does not undergo any significant changes in its binding properties during the S2–S3 transition.

On the evolution of redundancy in genetic codes.

We simulate a deterministic population genetic model for the coevolution of genetic codes and protein-coding genes. We use very simple assumptions about translation, mutation, and protein fitness to calculate mutation-selection equilibria of codon frequencies and fitness in a large asexual population with a given genetic code. We then compute the fitnesses of altered genetic codes that compete to invade the population by translating its genes with higher fitness. Codes and genes coevolve in a succession of stages, alternating between genetic equilibration and code invasion, from an initial wholly ambiguous coding state to a diversified frozen coding state. Our simulations almost always resulted in partially redundant frozen genetic codes. Also, the range of simulated physicochemical properties among encoded amino acids in frozen codes was always less than maximal. These results did not require the assumption of historical constraints on the number and type of amino acids available to codes nor on the complexity of proteins, stereochemical constraints on the translational apparatus, nor mechanistic constraints on genetic code change. Both the extent and timing of amino-acid diversification in genetic codes were strongly affected by the message mutation rate and strength of missense selection. Our results suggest that various omnipresent phenomena that distribute codons over sites with different selective requirements--such as the persistence of nonsynonymous mutations at equilibrium, the positive selection of the same codon in different types of sites, and translational ambiguity--predispose the evolution of redundancy and of reduced amino acid diversity in genetic codes.

Designing Lymphocyte Functional Structure for Optimal Signal Detection: Voilà, T cells

One basic task of immune systems is to detect signals from unknown “intruders” amidst a noisy background of harmless signals. To clarify the functional importance of many observed lymphocyte properties, I ask: What properties would a cell have if one designed it according to the theory of optimal detection, with minimal regard for biological constraints? Sparse and reasonable assumptions about the statistics of available signals prove sufficient for deriving many features of the optimal functional structure, in an incremental and modular design. The use of one common formalism guarantees that all parts of the design collaborate to solve the detection task. Detection performance is computed at several stages of the design. Comparison between design variants reveals e.g. the importance of controlling the signal integration time. This predicts that an appropriate control mechanism should exist. Comparing the design to reality, I find a striking similarity with many features of T cells. For example, the formalism dictates clonal specificity, serial receptor triggering, (grades of) anergy, negative and positive selection, co-stimulation, high-zone tolerance, and clonal production of cytokines. Serious mismatches should be found if T cells were hindered by mechanistic constraints or vestiges of their (co-)evolutionary history, but I have not found clear examples. By contrast, fundamental mismatches abound when comparing the design to immune systems of e.g. invertebrates. The wide-ranging differences seem to hinge on the (in)ability to generate a large diversity of receptors.

Multiple independent origins of mitochondrial gene order in birds.

Mitochondrial genomes of all vertebrate animals analyzed to date have the same 37 genes, whose arrangement in the circular DNA molecule varies only in the relative position of a few genes. This relative conservation suggests that mitochondrial gene order characters have potential utility as phylogenetic markers for higher-level vertebrate taxa. We report discovery of a mitochondrial gene order that has had multiple independent originations within birds, based on sampling of 137 species representing 13 traditionally recognized orders. This provides evidence of parallel evolution in mitochondrial gene order for animals. Our results indicate operation of physical constraints on mitochondrial gene order changes and support models for gene order change based on replication error. Bird mitochondria have a displaced OL (origin of light-strand replication site) as do various other Reptilia taxa prone to gene order changes. Our findings point to the need for broad taxonomic sampling in using mitochondrial gene order for phylogenetic analyses. We found, however, that the alternative mitochondrial gene orders distinguish the two primary groups of songbirds (order Passeriformes), oscines and suboscines, in agreement with other molecular as well as morphological data sets. Thus, although mitochondrial gene order characters appear susceptible to some parallel evolution because of mechanistic constraints, they do hold promise for phylogenetic studies.

The Limits to Reproductive Output: Offspring Size Versus Number in the Sand Lizard (Lacerta agilis)

Natural selection does not always (or indeed, usually) produce phenotypes, because of a variety of phenomena including trade-offs (conflicting optimal phenotypes for different functions) and mechanistic constraints (e.g., physical limitations, lack of additive genetic variance, genetic correlations between the sexes). The importance of trade-offs and constraints has been widely appreciated (e.g., Gould and Lewinton 1979), but the ways in which such factors influence life-history traits remain obscure. One vigorous debate in reptilian biology concerns the determinants of reproductive output. In many reptile species, clutch sizes and offspring sizes are related to each other and to maternal body size. The causal basis for these relationships remains controversial, with two main approaches evident in the literature. One view stresses energy allocation, such that reproductive output (e.g., relative clutch mass [RCM] or reproductive effort [RE]) is determined by the optimal allocation of resources among maintenance, storage, growth, and reproduction (Tinkle and Hadley 1973, 1975; Congdon et al. 1982). Under this approach, the upper limit to reproductive investment is set by energy availability and, hence, is likely to vary in response to proximate factors (e.g., Ballinger 1983; James and Whitford 1994). An alternative approach suggests that physical constraints, such as the space available to hold the clutch in the female's abdominal cavity, determine the upper limit for reproductive investment (Vitt and Congdon 1978; Shine 1988, 1992). The central disagreement between the two approaches centers on which factor determines the upper limit to reproductive output: resources or physical constraints. The best evidence that such an upper limit does exist is the widespread occurrence of trade-offs between offspring size and clutch size: that is, larger clutches consist of smaller offspring (Elgar and Heaphy 1989; Madsen and Shine 1992, 1996; King 1993). The existence of such a trade-off implies that there is a fixed upper limit to investment. Interspecific comparisons suggest that RCMs are correlated with body shape, in keeping with the notion of physical constraints (if all females fill themselves with eggs, body shape should constrain reproductive output) (Shine 1992). However, the volume constraint hypothesis is difficult to reconcile with the high intrapopulational variance often observed in RCMs or the

Doing the Conjugative Two-step: Evidence of Recipient Autonomy in Retrotransfer

Bidirectional exchange of genetic information, called retrotransfer, during bouts of bacterial conjugation has drawn the interest of those concerned with the risk of releasing genetically engineered microbes, the fluidity of genes among species, and the mechanism of DNA transport between cells. The phenomenon has generated two models in explanation, both of which yield highly testable predictions. The first model, called the one-step, predicts that the flow of genes from recipient bacteria to donor bacteria is mechanistically distinct from, but dependent on, conjugation between donors and recipients. The second model, called the two-step, predicts that the same genetic requirements and mechanistic constraints apply to the process of gene flow from recipients to donors as for gene flow from donors to recipients. The requirement for expression of at least 10 plasmid-encoded genes in recipients, sensitivity of the reverse flow (recipient to donor) to restriction of DNA transferring from the donor, and the requirement of an additional 30-90 min for DNA to flow from recipients back to donors are predictions of the two-step model and directly refute the one-step model. Retrotransfer of genes to donors during conjugation remains genetically and physically indistinguishable from two successive rounds of conjugation between neighbors.

Mechanistic Constraints on Diversity in Human V(D)J Recombination

We have analyzed a large collection of coding junctions generated in human cells. From this analysis, we infer the following about nucleotide processing at coding joints in human cells. First, the pattern of nucleotide loss from coding ends is influenced by the base composition of the coding end sequences. AT-rich sequences suffer greater loss than do GC-rich sequences. Second, inverted repeats can occur at ends that have undergone nucleolytic processing. Previously, inverted repeats (P nucleotides) have been noted only at coding ends that have not undergone nucleolytic processing, this observation being the basis for a model in which a hairpin intermediate is formed at the coding ends early in the reaction. Here, inverted repeats at processed coding ends were present at approximately twice the number of junctions as P nucleotide additions. Terminal deoxynucleotidyl transferase (TdT) is required for the appearance of the inverted repeats at processed ends (but not full-length coding ends), yet statistical analysis shows that it is virtually impossible for the inverted repeats to be polymerized by TdT. Third, TdT additions are not random. It has long been noted that TdT has a G utilization preference. In addition to the G preference, we find that TdT adds strings of purines or strings of pyrimidines at a highly significant frequency. This tendency suggests that nucleotide-stacking interactions affect TdT polymerization. All three of these features place constraints on the extent of junctional diversity in human V(D)J recombination.

Inversions Produced during V(D)J Rearrangement at IgH, the Immunoglobulin Heavy-Chain Locus

Diversity in immunoglobulin antigen receptors is generated in part by V(D)J recombination. In this process, different combinations of gene elements are joined in various configurations. Products of V(D)J recombination are coding joints, signal joints, and hybrid junctions, which are generated by deletion or inversion. To determine their role in the generation of diversity, we have examined two sorts of recombination products, coding joints and hybrid junctions, that have formed by inversion at the mouse immunoglobulin heavy-chain locus. We developed a PCR assay for quantification and characterization of inverted rearrangements of DH and JH gene elements. In primary cells from adult mice, inverted DJH rearrangements are detectable but they are rare. There were approximately 1,100 to 2,200 inverted DJH coding joints and inverted DJH hybrid junctions in the marrow of one adult mouse femur. On day 16 of gestation, inverted DJH rearrangements are more abundant. There are approximately 20,000 inverted DJH coding joints and inverted DJH hybrid junctions per day 16 fetal liver. In fetal liver cells, the number of inverted DJH rearrangements remains relatively constant from day 14 to day 16 of gestation. Inverted DJH rearrangements to JH4, the most 3' JH element, are more frequently detected than inverted DJH rearrangements to other JH elements. We compare the frequencies of inverted DJH rearrangements to previously determined frequencies of uninverted DJH rearrangements (DJH rearrangements formed by deletion). We suggest that inverted DJH rearrangements are influenced by V(D)J recombination mechanistic constraints and cellular selection.

The oxidation of tetrasubstituted alkenes by cytochrome P450.

Hexamethyl(Dewar benzene) (HMDB) was selected to probe the intermediacy of an alkene radical cation during cytochrome P450 (P450) catalyzed epoxidation. P450 catalyzed the allylic oxidation of HMDB exclusively; no rearranged products (indicative of a radical cation intermediate) nor epoxide was detected. In addition, P450 exclusively catalyzed the allylic oxidation of 1,2-dimethylcyclohexene (DMCH) and 1,2,4,5-tetramethyl-1,4- cyclohexadiene. The double bonds of HMDB and DMCH were readily epoxidized by (tetraphenylporphyrinato)iron-(III) chloride with minor production of allylic alcohols. The rates of alkene epoxidation by model systems tend to increase with increasing alkene substitution, but it is apparent that P450 has additional mechanistic options or constraints that depress the rate of epoxidation with increasing alkene substitution. The failure of P450 to epoxidize tetrasubstituted alkenes illustrates a failure of P450 model systems to truly mimic the chemistry of the P450 iron-oxo species.

Mechanistic and selective constraints act on the establishment of V lambda J lambda junctions in the B cell repertoire.

Only four different subtypes of lambda Ig chains have been described in the mouse: lambda 1, lambda 2(V2), lambda 2(Vx), and lambda 3. These chains are encoded by gene segments all sequenced and well localized in chromosome 16. Although the lambda Ig system is both simple and well characterized, no exhaustive analysis has been done concerning V lambda J lambda junctions in nonintentionally stimulated B cells. To get an insight into the lambda B cell repertoire, we analyzed a large number of V lambda J lambda rearrangements isolated from spleen mRNA or genomic DNA of unimmunized adult BALB/c mice. By PCR amplification, more than 160 clones were obtained covering all V lambda J lambda recombinations. Simple recombinations of trimmed gene segments explain most sequences. Certain junctions have been found to be prevalent in each subtype, and an analysis of V lambda J lambda recombination sites shows that the splenic lambda repertoire can result from both differential efficiencies of rearrangement and selective processes.

Homologous DNA Targeting with RecA Protein-coated Short DNA Probes and Electron Microscope Mapping on Linear Duplex Molecules

We demonstrate that RecA protein-coated, short single-stranded DNA probes paired with a specific homologous DNA sequence in a linear duplex target molecule and accurately targeted the selected DNA sequence. RecA protein-coated complementary ssDNA probes were reacted with linear duplexes, and the homologously paired molecules were observed by electron microscopy. The sites of interaction between the RecA protein-coated DNA probes and the uncoated duplex DNA targets were directly visible on individual target DNA molecules by high-resolution darkfield electron microscopy, without chemical fixation or sample shadowing. The efficiency and specificity of pairing were verified with 446 and 222 base single-stranded DNA probes that shared no homology with one another, and several linear duplex target DNAs with their respective probe homology sites at different locations with respect to the ends of the double-stranded DNA molecules. Measurements of the position of RecA protein-coated probes paired to individual target molecules, observed at high magnification, showed that DNA probes specifically paired at their corresponding homologous target sequences. This RecA protein-mediated DNA mapping method allows homologous sequence positioning and gene mapping on individual double stranded DNA molecules. Targeting reactions in which two different probe/target sites were 900 bases apart on a single duplex target molecule allowed both sites to be mapped in the same targeting reaction; although targets displaying both probes simultaneously were seen much less frequently than expected. The possible torsional or mechanistic constraints related to these reactions are briefly discussed.

Purification and properties of a recombinant sulfur analog of murine selenium‐glutathione peroxidase

We previously constructed plasmids for synthesis of glutathione-peroxidase (GPx) mutants in an Escherichia coli expression system. In these recombinant proteins either cysteine ([Cys]GPx mutant) or serine ([Ser]GPx mutant) were present in place of the active-site selenocysteine (SeCys) of the natural enzyme. We have now investigated GPx activity of [Cys]GPx and [Ser]GPx mutants. Enzyme assays performed on preparations of these partially purified proteins demonstrated that the [Cys]GPx mutant exhibited a significant GPx activity, unlike the [Ser]GPx mutant. Purification of [Cys]GPx was performed in two steps of ion-exchange chromatography giving a 98% homogenous protein in 50% yield. The purified [Cys]GPx protein was shown to be a symmetrical tetramer by the means of gel-filtration HPLC and SDS/PAGE. Two isoelectric points were found (6.8 and 7.2) which may reflect two different oxidation states of the mutant protein. The GPx activity of the [Cys]GPx mutant was optimal at pH 8.5. The [Cys]GPx mutant had a specific activity approximately 1000-fold smaller than that of the natural enzyme, and was very easily inactivated by hydroperoxides. Inhibition of the activity with iodoacetate determined a pKa of 8.3, presumably that of the active-site cysteine. Unlike that of SeGPx, the GPx activity of [Cys]GPx was only slightly inhibited by mercaptosuccinate. We discuss hypothetical mechanistic constraints of either catalytic cycle, which may explain such results.

Length polymorphism in the threonine-glycine-encoding repeat region of theperiod gene inDrosophila

Single-fly polymerase chain reaction amplification and direct DNA sequencing revealed high levels of length polymorphism in the threonine-glycine encoding repeat region of the period (per) gene in natural populations of Drosophila melanogaster. DNA comparison of two alleles of identical lengths gave a high number of synonymous substitutions suggesting an ancient time of separation. However detailed examination of the sequences of different Thr-Gly length variants indicated that this divergence could be understood in terms of four deletion/insertion events. In Drosophila pseudoobscura a length polymorphism is observed in a five-amino acid degenerate repeat, which corresponds to melanogaster's Thr-Gly domain. In spite of the differences between D. melanogaster and D. pseudoobscura in the amino acid sequence of the repeats, the predicted secondary structures suggest evolutionary and mechanistic constraints on the per protein of these two species.

Primitive subunits: Models for cooperative motion in glass-forming liquids. Application to an idealized polymer melt

A family of mechanistic models are proposed to describe cooperative molecular displacements in glass-forming liquids. These models assume that within each cooperative domain motion occurs by sequences of discrete, localized events, but that each of these events involves synchronous displacement of a smaller cluster of neighboring components. The size and properties of this cluster, described as a ‘‘primitive subunit,’’ are assumed to reflect intrinsic details of local structure, not sensitive to external degrees of freedom. However, the length of the sequence of events by which each subunit moves is assumed to be a statistical function of some communal degree of freedom such as the free volume. Two examples are explored for the case of a polymer melt in which idealized conformational rearrangements are constrained by steric interferences. The distribution of lengths for cooperative sequences of events required to remove these interferences is derived as a function of a parameter β related to free volume. It is shown that the mean length of such sequences diverge to infinity for some nonzero (critical) value of β, and that the divergence obeys a scaling law. The divergence is a time-invariant feature of the model, similar in physical significance to the equilibrium phase transition which has been proposed as the underlying basis for the glass transition. However, in the present models it follows from mechanistic constraints, independent of any thermodynamic considerations.

The Emerging Paradigm of Computational Vision

Vision is the process by which structure in the world is inferred from images. But as such it is underconstrained; somehow additional constraints must be used to guide the inferences from two-dimensional (2-D) image data to three-dimensional (3-D) scene descriptions. The discovery of such constraints, and of techniques for using them, has led to the emergence of a new discipline called computational vision. It lies somewhere between the more classical attempts to solve engineering problems involving vision (as they arise, say, in robotics) and attempts to understand biological vision systems. Engineering applications suggest what should be solved; its scien­ tific counterparts suggest why; and biology suggests particular mechanisms that might be employed. But computational theories suggest how solutions can be obtained for different classes of visual tasks in the abstract, thereby filling the gap between particular problems and particular solutions. In this chapter I try to characterize the emergence of computational vision as its own discipline, contributing fundamental ideas to the study of both biological and engineered vision systems, rather than just supporting them with programming tools for testing existing ideas (cf Barlow et al 1972; Beck et al 1983; Braddick & Sleigh 1983). To illustrate the idea behind computational vision, consider an analogy with timekeeping. Mechanistic constraints come from clocks (the material from which they are composed, how they store energy, the mechanics of

Mechanistic Constraints and Optimality Models: Thermoregulatory Strategies in Colias Butterflies

To explore mechanistic constraints on the evolution of optimal phenotypes, we develop an optimality model for thermoregulation in Colias butterflies. The model identifies the thermoregulatory characteristics of butterflies that maximize time available for flight activity subject to an overheating constraint, and was tested in three Colias populations along an elevational gradient. Model simulations and field results showed that environmental variation limits available flight time in higher elevation populations, even for optimally designed butterflies. Optimal predicted thermoregulatory characteristics were within two standard deviations of the observed characteristics in each population. Model simulations and field transplant experiments showed that large departures from optimality would have deleterious consequences for flight and overheating. This represents the first test of a strategy model for optimal thermoregulatory characteristics in animals. Our results illustrate how the constraints imposed by environmental variation and the mechanisms of adaptation affect the evolution of locally optimal phenotypes. We argue that, used in this mechanistic context, optimality modeling remains a powerful tool in studying adaptation.

Multiphoton dissociation: A maximum entropy formalism with mechanistic constraints and applications to alkyl iodides

The relative importance of the different possible fragmentation pathways in multiphoton ionization—fragmentation is determined by a maximum entropy formalism. Absorption by fragment ions is allowed for, and leads to significantly lower temperatures. Differences between electron impact and laser-induced mass spectra are possibly due to the absence of secondary absorption in the former.

Methods and conclusions in functional analysis: a reply

This paper is a reply to criticism presented by C. R. C. Paul and R. Cowen in immediately preceding articles in Lethaia. Contrary to the assertion by Paul, the present author did not reject the paradigmatic method but (1) criticized it for its limitations and seemingly erroneous results, (2) applied it to the richthofeniacean and lyttoniacean brachiopods by use of the pump as paradigm, and (3) extended it to include anatomic, metabolic, and phylogenetic considerations that go beyond the structural and mechanistic constraints of the original formulation. The rhythmic flow mechanism was rejected primarily on the basis of evidence for a ptycholophous lophophore in the Productidina. and the relative inefficiency of ‘valve flapping’ in contrast to ciliary action in pumping nutrient-bearing fluid into the shell. A Chinese blast furnace was claimed by Cowen to employ a single oscillating panel as a pump, but instead this panel must act as a valve that admits air in surges while the draft in the furnace maintains unidirectional flow through the system. This is analogous to ciliary feeding in brachiopods, where undirectional flow is maintained by ciliary pumping, and the valve opens to admit water. A richthofeniid with a Composita entrapped beneath the apertural meshwork is introduced as additional evidence against the likelihood of ‘valve flapping’.