Preservation of a pseudogene by gene conversion and diversifying selection

Interlocus gene conversion is considered a crucial mechanism for generating novel combinations of polymorphisms in duplicated genes. The importance of gene conversion between duplicated genes has been recognized in the major histocompatibility complex and self-incompatibility genes, which are likely subject to diversifying selection. To theoretically understand the potential role of gene conversion in such situations, forward simulations are performed in various two-locus models. The results show that gene conversion could significantly increase the number of haplotypes when diversifying selection works on both loci. We find that the tract length of gene conversion is an important factor to determine the efficacy of gene conversion: shorter tract lengths can more effectively generate novel haplotypes given the gene conversion rate per site is the same. Similar results are also obtained when one of the duplicated genes is assumed to be a pseudogene. It is suggested that a duplicated gene, even after being silenced, will contribute to increasing the variability in the other locus through gene conversion. Consequently, the fixation probability and longevity of duplicated genes increase under the presence of gene conversion. On the basis of these findings, we propose a new scenario for the preservation of a duplicated gene: when the original donor gene is under diversifying selection, a duplicated copy can be preserved by gene conversion even after it is pseudogenized.     

Inhibition of Spirochaeta aurantia chemotaxis by neurotoxins.

The effects of neurotoxic compounds on the chemotactic response of Spirochaeta aurantia were investigated. In the presence of neurotoxins that affect action potential generation and transmission in excitable eucaryotic cells, D-xylose taxis was inhibited by 69 to 93%. Inhibition of chemotaxis was not due to decreased viability or motility. This study supports the hypothesis that the molecular basis for sensory signal transduction in S. aurantia involves ion fluxes across the cytoplasmic membrane.     

Norms of reaction and diversifying selection

The numbers of progeny produced by comparable numbers of female Drosophila melanogaster of 26 geographic strains on nine different culture media are examined in the context of norms of reaction. Having emphasized that diversifying selection is seldom discussed simultaneously with its seemingly related topic, norms of reaction, I present the following argument: diversifying selection has generally been viewed as involving sub-populations inhabiting separate localities and subject to different patterns of selection, norms of reaction as variation whose weighted average determines the relative fitnesses of different genotypes within individual sub-populations. Should environmental challenges frequently involve life or death (including sterility) outcomes, norms of reaction involving components of fitness engender diversifying selection within local populations (demes).     

Early evolution of histone mRNA 3' end processing

The replication-dependent histone mRNAs in metazoa are not polyadenylated, in contrast to the bulk of mRNA. Instead, they contain an RNA stem-loop (SL) structure close to the 3' end of the mature RNA, and this 3' end is generated by cleavage using a machinery involving the U7 snRNP and protein factors such as the stem-loop binding protein (SLBP). This machinery of 3' end processing is related to that of polyadenylation as protein components are shared between the systems. It is commonly believed that histone 3' end processing is restricted to metazoa and green algae. In contrast, polyadenylation is ubiquitous in Eukarya. However, using computational approaches, we have now identified components of histone 3' end processing in a number of protozoa. Thus, the histone mRNA stem-loop structure as well as the SLBP protein are present in many different protozoa, including Dictyostelium, alveolates, Trypanosoma, and Trichomonas. These results show that the histone 3' end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We also identified histone mRNAs from both metazoa and protozoa that are polyadenylated but also contain the signals characteristic of histone 3' end processing. These results provide further evidence that some histone genes are regulated at the level of 3' end processing to produce either polyadenylated RNAs or RNAs with the 3' end characteristic of replication-dependent histone mRNAs.     

Enhanced-rate biodegradation of organophosphate neurotoxins by immobilized nongrowing bacteria

Pesticide wastes generated from livestock dipping operations containing the organophosphate (OP) insecticide coumaphos (CP) are well suited for disposal by biodegradation since they are highly concentrated (approximately 1 g/L), generally contained, and lack additional toxic components. In this study, a significantly enhanced efficiency of degrading CP in cattle dip waste (CDW) is reported using a dense, nongrowing cell population that functions without the addition of nutrients required for growing cell cultures. A recombinant strain of Escherichia coli containing the opd gene for organophosphate hydrolase (OPH), which is capable of active hydrolysis of OP neurotoxins including CP, was cultivated in a rich medium containing all essential nutrients. Cells were harvested and utilized in lab scale experiments in the form of either freely suspended cells or cells immobilized within a macroporous gel matrix, poly(vinyl alcohol) (PVA) cryogel. Significantly higher degradation rates were achieved with either suspended or immobilized OPH(+) cells compared to rates with the microbial consortium naturally present in CDW. Of the two nongrowing cell systems, the detoxification rate with immobilized cells was approximately twice that of freely suspended cells, and kinetic studies demonstrated that a higher maximum reaction rate was achieved with the immobilized cell system. A comparative study using both the CDW and pure CP substrates with free cells indicated that the CDW contained one or more factors that reduced the bioavailability of CP. The immobilized cells retained their activity over a 4-month period of use and storage, demonstrating both sustained catalytic activity and long-term mechanical stability.     

Facilitation of transmitter release by neurotoxins from snake venoms

Toxins C13S1C3 and C13S2C3 from green mamba venom (Dendroaspis angusticeps) acted like dendrotoxin to increase acetylcholine release in response to nerve stimulation in the chick biventer cervicis preparation. Proteins B and E from black mamba venom (Dendroaspis polylepis) had no prejunctional facilitatory activity. All four proteins are trypsin inhibitor homologues. Binding of a prejunctional facilitatory toxin (Polylepis toxin I) to motor nerves was rapid and did not require the presence of Ca2+ or nerve stimulation. Binding was not prevented by protease inhibitors that lacked facilitatory actions. Prejunctional facilitatory toxins also augmented transmitter release in the chick oesophagus and the mouse vas deferens preparations. The effects were rapid in onset and could wane spontaneously. 125I-labelled dendrotoxin bound specifically to rat brain synaptosomes with a KD of about 3 nM. Binding was prevented by native dendrotoxin but not by beta-bungarotoxin or atropine. It is concluded that prejunctional facilitatory toxins affect transmitter release at many types of nerve endings in addition to motor nerve terminals. From consideration of the structures of active and inactive molecules, it is thought that binding of the active toxins may involve several exposed lysine residues.     

LG/LNS domains: multiple functions -- one business end?

The three-dimensional structures of LG/LNS domains from neurexin, the laminin alpha 2 chain and sex hormone-binding globulin reveal a close structural relationship to the carbohydrate-binding pentraxins and other lectins. However, these LG/LNS domains appear to have a preferential ligand-interaction site distinct from the carbohydrate-binding sites found in lectins, and this interaction site accommodates not only sugars but also steroids and proteins. In fact, the LG/LNS domain interaction site has features reminiscent of the antigen-combining sites in immunoglobulins. The LG/LNS domain presents an interesting case in which the fold has remained conserved but the functional sites have evolved; consequently, making predictions of structure-function relationships on the basis of the lectin fold alone is difficult.     

GAI homologues in the Hawaiian silversword alliance (Asteraceae-Madiinae): molecular evolution of growth regulators in a rapidly diversifying plant lineage

Accelerated evolution of regulatory genes has been proposed as an explanation for decoupled rates of morphological and molecular evolution. The Hawaiian silversword alliance (Asteraceae-Madiinae) has evolved drastic differences in growth form, including rosette plants, cushion plants, shrubs, and trees, since its origin approximately 6 MYA. We have isolated genes in the DELLA subfamily of putative growth regulators from 13 taxa of Hawaiian and North American Madiinae. The Hawaiian taxa contain two copies of DaGAI that form separate clades within the Madiinae, consistent with an allotetraploid origin for the silversword alliance. DaGAI retains conserved features that have previously been identified in DELLA genes. Selective constraint in the Hawaiian DaGAI copies remains strong in spite of rapid growth form divergence in the silversword alliance, although the constraint was somewhat relaxed in the Hawaiian copies relative to the North American lineages. We failed to detect evidence for positive selection on individual codons. Notably, selective constraint remained especially strong in the gibberellin-responsive DELLA region for which the gene subfamily is named, which is truncated or deleted in all identified dwarf mutants in GAI homologues in different angiosperm species. In contrast with the coding region, however, approximately 900 bp of the upstream flanking region shows variable rates and patterns of evolution, which might reflect positive selection on regulatory regions.     

Unique ganglioside binding by botulinum neurotoxins C and D-SA

The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G), based on a lack of cross-antiserum neutralization. The BoNT/C and BoNT/D serotypes include mosaic toxins that are organized as D-C and C-D toxins. One BoNT D-C mosaic toxin, BoNT/D-South Africa (BoNT/D-SA), was not fully neutralized by immunization with a vaccine composed of either prototype BoNT/C-Stockholm or BoNT/D-1873. Whereas several BoNT serotypes utilize dual receptors (gangliosides and proteins) to bind to and enter neurons, the basis for BoNT/C and BoNT/D entry into neurons is less well understood. Recent studies solved the crystal structures of the receptor-binding domains of BoNT/C, BoNT/D, and BoNT/D-SA. Comparative structural analysis showed that BoNT/C, BoNT/D and BoNT/D-SA lacked components of the ganglioside-binding pocket that exists within other BoNT serotypes. With the use of structure-based alignments, biochemical analyses, and cell-binding approaches, BoNT/C and BoNT/D-SA have been shown to possess a unique ganglioside-binding domain, the ganglioside-binding loop. Defining how BoNTs enter host cells provides insights towards understanding the evolution and extending the potential therapeutic and immunological values of the BoNT serotypes.     

Neuroprotective effect on brain injury by neurotoxins from the spider Phoneutria nigriventer

The role of calcium channels blockers in ischemic condition has been well documented. The PhTx3 neurotoxic fraction of the spider Phoneutria nigriventer venom is a broad-spectrum calcium channel blocker that inhibits glutamate release, calcium uptake and also glutamate uptake in synaptosomes. In the present study we describe the effect of PhTx3 (1.0 microg/mL), omega-conotoxin GVIA (1.0 micromol/L) and omega-conotoxin MVIIC (100 nmol/L) on neuroprotection of hippocampal slices and SN56 cells subjected to ischemia by oxygen deprivation and low glucose insult (ODLG). After the insult, cell viability in the slices and SN56 cells was assessed by confocal microscopy and epifluorescence, using live/dead kit containing calcein-AM and ethidium homodimer. Confocal images of CA1 region of the rat hippocampal slices subjected to ischemia insult and treated with omega-conotoxin GVIA, omega-conotoxin MVIIC and PhTx3 showed a percentage of dead cells of 68%, 54% and 18%, respectively. The SN56 cells subjected to ischemia were almost completely protected from damage by PhTx3 while with omega-conotoxin GVIA or omega-conotoxin MVIIC the cell protection was only partial. Thus, PhTx3 provided robust ischemic neuroprotection showing potential as a novel class of agents that targets multiple components and exerts neuroprotection in in vitro model of brain ischemia.     

r- and K-tactics in the evolution of protist developmental systems: cell and genome size, phenotype diversifying selection, and cell cycle patterns

I outline the significance for protist evolution of the r-, K-selection spectrum,, and of my earlier theory that the most fundamental way organisms adapt to this spectrum is by evolutionary variations in their cell volumes, cell growth rates and genome sizes. Then I introduce the concept of phenotype diversifying selection; this refers to those selective forces which favour an increase in the number of phenotypes produced during a single life cycle by an organism's genotype and epigenetic system. These ideas are then used to discuss the evolution of protist development, with special reference to modifications of the cell cycle whose evolutionary causes and consequences can be related to K-selection for large size and r-selection for rapid reproduction. The significance of multiple fission, syncytia, multicellularity, nuclear dimorphism plus polyploidy, and reversible polyploidy, is treated in detail. Predictions are made of the effects of these different developmental patterns on genome size and the distribution and amounts of nucleoskeletal RNA and heterochromatin. I suggest that heterochromatin exists primarily because of phenotype diversifying selection for differing nuclear volumes. The possibility of applying these ideas to other cell properties like mitotic or cytokinetic mechanisms is also briefly discussed.     

Ancient and recent adaptive evolution of primate non-homologous end joining genes

In human cells, DNA double-strand breaks are repaired primarily by the non-homologous end joining (NHEJ) pathway. Given their critical nature, we expected NHEJ proteins to be evolutionarily conserved, with relatively little sequence change over time. Here, we report that while critical domains of these proteins are conserved as expected, the sequence of NHEJ proteins has also been shaped by recurrent positive selection, leading to rapid sequence evolution in other protein domains. In order to characterize the molecular evolution of the human NHEJ pathway, we generated large simian primate sequence datasets for NHEJ genes. Codon-based models of gene evolution yielded statistical support for the recurrent positive selection of five NHEJ genes during primate evolution: XRCC4, NBS1, Artemis, POLλ, and CtIP. Analysis of human polymorphism data using the composite of multiple signals (CMS) test revealed that XRCC4 has also been subjected to positive selection in modern humans. Crystal structures are available for XRCC4, Nbs1, and Polλ; and residues under positive selection fall exclusively on the surfaces of these proteins. Despite the positive selection of such residues, biochemical experiments with variants of one positively selected site in Nbs1 confirm that functions necessary for DNA repair and checkpoint signaling have been conserved. However, many viruses interact with the proteins of the NHEJ pathway as part of their infectious lifecycle. We propose that an ongoing evolutionary arms race between viruses and NHEJ genes may be driving the surprisingly rapid evolution of these critical genes.     

Trophic interrelations at the neuromuscular junction as revealed by the use of botulinal neurotoxins

1. From denervation studies the trophic influence of the motor nerve on the muscle cell is well documented while little is known about the influence of the muscle on the nerve. Sectioning the axon invariably destroys the nerve terminals and produces nerve degeneration products which themselves may affect nerve and muscle properties. With regard to those difficulties we believe that the botulinal neurotoxins (BoTx) are valuable complements to denervation since they selectively interrupt impulse transmission across the synapse without damaging its morphology. 2. Paralysis of mouse or rat skeletal muscle in vivo with BoTx type A causes marked growth of motor nerve terminals. The sprouting terminals are rich in large dense-core synaptic vesicles containing various neuropeptides and they spontaneously release large quanta of ACh. Thus, it appears that paralysis by BoTx is a strong stimulus for motor nerve growth and the delivery of "trophic" substances to the nerve terminals. 3. Postsynaptically, in extrajunctional areas, paralysis by BoTx induces all the changes observed following denervation, i.e. atrophy, appearance of extra-junctional ACh receptors, TTX-resistant action potentials, a fall of resting membrane potential, fibrillation potentials and the disappearance of extrajunctional acetylcholinesterase activity. Endplate properties are, however, largely maintained. 4. BoTx blockade delays and prevents the retraction of polyneuronal innervation and motoneurone death during development. This supports the suggestion that the paralysed muscle secretes factors essential for growth and for the survival of motoneurones. 5. Like denervated muscle, BoTx paralysed ones, express a high endocytotic activity restricted to a segment in the endplate region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of chromosome loss by mixtures of organic solvents including neurotoxins

Twenty-three aprotic polar solvents - 3 nitriles, 8 organic esters, 10 ketones and 2 lactones - and LiCl were tested in combination with propionitrile alone or a mixture of ethyl acetate and propionitrile for the induction of mitotic chromosome loss in the D61.M strain of the yeast Saccharomyces cerevisiae. Propionitrile and ethyl acetate are very potent inducers of chromosome loss. Mixtures of propionitrile and ethyl acetate induced chromosome loss at much higher frequencies than was observed with the pure chemicals. To test the potentiating effects of propionitrile or mixtures of propionitrile with ethyl acetate on other chemicals, they were used in concentrations that were at or below the level for induction of chromosome loss. Twenty chemicals when tested in pure form were negative or only marginally active in the test for chromosome loss. Except for amyl propionate and benzyl acetate, the same chemicals showed strong induction in combination treatments with the potentiating chemicals. All the ketones including the neurotoxic methyl ethyl ketone, 2-hexanone and 2.5-hexanedione induced high frequencies of chromosome loss. Only methyl ethyl ketone is capable of inducing high levels of chromosome loss when tested in the pure form at much higher concentrations. 1-Methyl-2-pyrrolidinone and gamma-valerolactone had previously been shown to induce chromosome loss only when the treatment at a growth-supporting temperature was interrupted by a cold shock within a narrow range of low temperatures which prevented growth. Both gave very strong induction in combination treatment performed at a continuous growth-supporting temperature. LiCl is a weak inducer of chromosome loss: strong induction can be achieved in combination treatments.     

Secondary structural predictions for the clostridial neurotoxins

The primary structures of a family of ten clostridial. Neurotoxins have recently been deduced yet little information is presently available concerning their secondary or tertiary structures. Because the overall similarity percentage of multiply aligned sequences is high, the secondary structures of these metalloendopeptidases are also expected to be conserved. The neural net program, PHD (Rost and Sander, Proc. Natl. Acad. Sci. USA 90:7558–7562, 1993), predicted that the secondary structures of the neurotoxins were indeed conserved in both single and multiple sequence modes of analysis. Predictions for the amounts of helical, extended, and loop states from the single sequence analyses were consistent with previously published data from circular dichroism studies on some of these neurotoxins. In the single analysis mode, only the aligned regions were predicted to show conservation of the three-state structure. In contrast, the multiple sequence analysis predicted that a conserved state (variable loops) also exists in non-aligned regions. Alignments with the primary structure of the prototypic metalloendopeptidase thermolysin showed that about 25% of the residues within this enzyme are similar to those in the neurotoxins. A comparison of thermolysin's known secondary structure with the predictions from this study showed that about 80% of thermolysin's residues could be structurally aligned with those in the neurotoxins. These predictions provide the necessary. Framework to build a homologous low-resolution tertiary structure of the neurotoxin active site that will be essential in the development of synthetic inhibitors. © 1994 Wiley-Liss, Inc.     

Unique substrate recognition by botulinum neurotoxins serotypes A and E

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A-G. BoNT serotype A and serotype E cleave SNAP25 at residues 197-198 and 180-181, respectively. Unlike other zinc proteases, the BoNTs recognize extended regions of SNAP25 for cleavage. The basis for this extended substrate recognition and specificity is unclear. Saturation mutagenesis and deletion mapping identified residues 156-202 of SNAP25 as the optimal cleavage domain for BoNT/A, whereas the optimal cleavage domain for BoNT/E was shorter, comprising residues 167-186 of SNAP25. Two sub-sites were resolved within each optimal cleavage domain, which included a recognition or active site (AS) domain that contained the site of cleavage and a binding (B) domain, which contributed to substrate affinity. Within the AS domains, the P1', P3, and P5 sites of SNAP25 contributed to scissile bond cleavage by LC/A, whereas the P1' and P2 sites of SNAP25 contributed to scissile bond cleavage by LC/E. These studies provide insight into the development of strategies for small molecule inhibitors of the BoNTs.     

Comparative genomics and diversifying selection of the clustered vertebrate protocadherin genes

To explain the mechanism for specifying diverse neuronal connections in the brain, Sperry proposed that individual cells carry chemoaffinity tags on their surfaces. The enormous complexity of these connections requires a tremendous diversity of cell-surface proteins. A large number of neural transmembrane protocadherin (Pcdh) proteins is encoded by three closely linked human and mouse gene clusters (alpha, beta, and gamma). To gain insight into Pcdh evolution, I performed comprehensive comparative cDNA and genomic DNA analyses for the three clusters in the chimpanzee, rat, and zebrafish genomes. I found that there are species-specific duplications in vertebrate Pcdh genes and that additional diversity is generated through alternative splicing within the zebrafish "variable" and "constant" regions. Moreover, different codons (sites) in the mammalian Pcdh ectodomains (ECs) are under diversifying selection, with some under diversity-enhancing positive Darwinian selection and others, including calcium-binding sites, under strong purifying selection. Interestingly, almost all positively selected codon positions are located on the surface of ECs 2 and 3. These diversified residues likely play an important role in combinatorial interactions of Pcdh proteins, which could provide the staggering diversity required for neuronal connections in the brain. These results also suggest that adaptive selection is an additional evolutionary factor for increasing Pcdh diversity.     

Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa

Pyoverdine is the primary siderophore of the gram-negative bacterium Pseudomonas aeruginosa. The pyoverdine region was recently identified as the most divergent locus alignable between strains in the P. aeruginosa genome. Here we report the nucleotide sequence and analysis of more than 50 kb in the pyoverdine region from nine strains of P. aeruginosa. There are three divergent sequence types in the pyoverdine region, which correspond to the three structural types of pyoverdine. The pyoverdine outer membrane receptor fpvA may be driving diversity at the locus: it is the most divergent alignable gene in the region, is the only gene that showed substantial intratype variation that did not appear to be generated by recombination, and shows evidence of positive selection. The hypothetical membrane protein PA2403 also shows evidence of positive selection; residues on one side of the membrane after protein folding are under positive selection. R', previously identified as a type IV strain, is clearly derived from a type III strain via a 3.4-kb deletion which removes one amino acid from the pyoverdine side chain peptide. This deletion represents a natural modification of the product of a nonribosomal peptide synthetase enzyme, whose consequences are predictive from the DNA sequence. There is also linkage disequilibrium between the pyoverdine region and pvdY, a pyoverdine gene separated by 30 kb from the pyoverdine region. The pyoverdine region shows evidence of horizontal transfer; we propose that some alleles in the region were introduced from other soil bacteria and have been subsequently maintained by diversifying selection.