Coalescent estimates of HIV-1 generation time in vivo

The generation time of HIV Type 1 (HIV-1) in vivo has previously been estimated using a mathematical model of viral dynamics and was found to be on the order of one to two days per generation. Here, we describe a new method based on coalescence theory that allows the estimate of generation times to be derived by using nucleotide sequence data and a reconstructed genealogy of sequences obtained over time. The method is applied to sequences obtained from a long-term nonprogressing individual at five sampling occasions. The estimate of viral generation time using the coalescent method is 1.2 days per generation and is close to that obtained by mathematical modeling (1.8 days per generation), thus strengthening confidence in estimates of a short viral generation time. Apart from the estimation of relevant parameters relating to viral dynamics, coalescent modeling also allows us to simulate the evolutionary behavior of samples of sequences obtained over time.

Rapid decline of fitness in panmictic populations of Drosophila melanogaster maintained under relaxed natural selection

The parameters of the spontaneous deleterious mutation process remain poorly known, despite their importance. Here, we report the results of a mutation accumulation experiment performed on panmictic populations of Drosophila melanogaster without any genetic manipulations. Two experimental populations were kept for 30 generations under relaxed natural selection. Each generation, 100 pairs were formed randomly, and every fecund pair contributed a son and a daughter to the next generation. Comparison with two controls, one cryopreserved and the other kept as the experimental populations but with long generation time, showed that the number of surviving offspring per female declined by 0.2% and 2.0% per generation under benign and harsh, competitive conditions, respectively. Thus, the mutational pressure on fitness may be strong and depends critically on the conditions under which fitness is assayed.

Modulation of Life-span by Histone Deacetylase Genes in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has a limited life-span, which is measured by the number of divisions that individual cells complete. Among the many changes that occur as yeasts age are alterations in chromatin-dependent transcriptional silencing. We have genetically manipulated histone deacetylases to modify chromatin, and we have examined the effect on yeast longevity. Deletion of the histone deacetylase gene RPD3 extended life-span. Its effects on chromatin functional state were evidenced by enhanced silencing at the three known heterochromatic regions of the genome, the silent mating type (HM), subtelomeric, and rDNA loci, which occurred even in the absence of SIR3. Similarly, the effect of the rpd3Δ on life-span did not depend on an intact Sir silencing complex. In fact, deletion of SIR3 itself had little effect on life-span, although it markedly accelerated the increase in cell generation time that is observed during yeast aging. Deletion of HDA1, another histone deacetylase gene, did not result in life-span extension, unless it was combined with deletion of SIR3. The hda1Δ sir3Δ resulted in an increase in silencing, but only at the rDNA locus. Deletion of RPD3 suppressed the loss of silencing in rDNA in a sir2 mutant; however, the silencing did not reach the level found in the rpd3Δ single mutant, and RPD3 deletion did not overcome the life-span shortening seen in the sir2 mutant. Deletion of both RPD3 and HDA1 caused a decrease in life-span, which resulted from a substantial increase in initial mortality of the population. The expression of both of these genes declines with age, providing one possible explanation for the increase in mortality during the life-span. Our results are consistent with the loss of rDNA silencing leading to aging in yeast. The functions of RPD3 and HDA1 do not overlap entirely. RPD3 exerts its effect on chromatin at additional sites in the genome, raising the possibility that events at loci other than rDNA play a role in the aging process.

Facilitated diffusion of fructose via the phosphoenolpyruvate/glucose phosphotransferase system of Escherichia coli

From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate/glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (Km for growth ≈8 mM but with Vmax for generation time ≈1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak). The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme IIGlc of the PTS, which we designate ptsG-F. Exconjugants that had acquired ptsG+ from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (Vmax ≈7 h 20 min), even though they were rich in Mak activity. A mutant of E. coli also rich in Mak but unable to grow on glucose by virtue of transposon-mediated inactivations both of ptsG and of the genes specifying enzyme IIMan (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose. Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl α-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence.

Directionality principles in thermodynamics and evolution

Directionality in populations of replicating organisms can be parametrized in terms of a statistical concept: evolutionary entropy. This parameter, a measure of the variability in the age of reproducing individuals in a population, is isometric with the macroscopic variable body size. Evolutionary trends in entropy due to mutation and natural selection fall into patterns modulated by ecological and demographic constraints, which are delineated as follows: (i) density-dependent conditions (a unidirectional increase in evolutionary entropy), and (ii) density-independent conditions, (a) slow exponential growth (an increase in entropy); (b) rapid exponential growth, low degree of iteroparity (a decrease in entropy); and (c) rapid exponential growth, high degree of iteroparity (random, nondirectional change in entropy). Directionality in aggregates of inanimate matter can be parametrized in terms of the statistical concept, thermodynamic entropy, a measure of disorder. Directional trends in entropy in aggregates of matter fall into patterns determined by the nature of the adiabatic constraints, which are characterized as follows: (i) irreversible processes (an increase in thermodynamic entropy) and (ii) reversible processes (a constant value for entropy). This article analyzes the relation between the concepts that underlie the directionality principles in evolutionary biology and physical systems. For models of cellular populations, an analytic relation is derived between generation time, the average length of the cell cycle, and temperature. This correspondence between generation time, an evolutionary parameter, and temperature, a thermodynamic variable, is exploited to show that the increase in evolutionary entropy that characterizes population processes under density-dependent conditions represents a nonequilibrium analogue of the second law of thermodynamics.

Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution.

The internal transcribed spacers (ITS) of nuclear ribosomal DNA of 33 species of genus Paeonia (Paeoniaceae) were sequenced. In section Paeonia, different patterns of nucleotide additivity were detected in 14 diploid and tetraploid species at sites that are variable in the other 12 species of the section, suggesting that reticulate evolution has occurred. Phylogenetic relationships of species that do not show additivity, and thus ostensibly were not derived through hybridization, were reconstructed by parsimony analysis. The taxa presumably derived through reticulate evolution were then added to the phylogenetic tree according to additivity from putative parents. The study provides an example of successfully using ITS sequences to reconstruct reticulate evolution in plants and further demonstrates that the sequence data could be highly informative and accurate for detecting hybridization. Maintenance of parental sequences in the species of hybrid origin is likely due to slowing of concerted evolution caused by the long generation time of peonies. The partial and uneven homogenization of parental sequences displayed in nine species of putative hybrid origin may have resulted from gradients of gene conversion. The documented hybridizations may have occurred since the Pleistocene glaciations. The species of hybrid origin and their putative parents are now distantly allopatric. Reconstruction of reticulate evolution with sequence data, therefore, provides gene records for distributional histories of some of the parental species.

Genomic evolution during a 10,000-generation experiment with bacteria

Molecular methods are used widely to measure genetic diversity within populations and determine relationships among species. However, it is difficult to observe genomic evolution in action because these dynamics are too slow in most organisms. To overcome this limitation, we sampled genomes from populations of Escherichia coli evolving in the laboratory for 10,000 generations. We analyzed the genomes for restriction fragment length polymorphisms (RFLP) using seven insertion sequences (IS) as probes; most polymorphisms detected by this approach reflect rearrangements (including transpositions) rather than point mutations. The evolving genomes became increasingly different from their ancestor over time. Moreover, tremendous diversity accumulated within each population, such that almost every individual had a different genetic fingerprint after 10,000 generations. As has been often suggested, but not previously shown by experiment, the rates of phenotypic and genomic change were discordant, both across replicate populations and over time within a population. Certain pivotal mutations were shared by all descendants in a population, and these are candidates for beneficial mutations, which are rare and difficult to find. More generally, these data show that the genome is highly dynamic even over a time scale that is, from an evolutionary perspective, very brief.

Direct measurement of nitric oxide generation from nitric oxide synthase

Although nitric oxide synthase (NOS) is widely considered as the major source of NO in biological cells and tissues, direct evidence demonstrating NO formation from the purified enzyme has been lacking. It was recently reported that NOS does not synthesize NO, but rather generates nitroxyl anion (NO−) that is subsequently converted to NO by superoxide dismutase (SOD). To determine if NOS synthesizes NO, electron paramagnetic resonance (EPR) spectroscopy was applied to directly measure NO formation from purified neuronal NOS. In the presence of the NO trap Fe2+-N-methyl-d-glucamine dithiocarbamate, NO gives rise to characteristic EPR signals with g = 2.04 and aN = 12.7 G, whereas NO− is undetectable. In the presence of l-arginine (l-Arg) and cofactors, NOS generated prominent NO signals. This NO generation did not require SOD, and it was blocked by the specific NOS inhibitor N-nitro-l-arginine methyl ester. Isotope-labeling experiments with l-[15N]Arg further demonstrated that NOS-catalyzed NO arose from the guanidino nitrogen of l-Arg. Measurement of the time course of NO formation demonstrated that it paralleled that of l-citrulline. The conditions used in the prior study were shown to result in potent superoxide generation, and this may explain the failure to measure NO formation in the absence of SOD. These experiments provide unequivocal evidence that NOS does directly synthesize NO from l-Arg.

A new mechanism of sound generation in songbirds

Our current understanding of the sound-generating mechanism in the songbird vocal organ, the syrinx, is based on indirect evidence and theoretical treatments. The classical avian model of sound production postulates that the medial tympaniform membranes (MTM) are the principal sound generators. We tested the role of the MTM in sound generation and studied the songbird syrinx more directly by filming it endoscopically. After we surgically incapacitated the MTM as a vibratory source, zebra finches and cardinals were not only able to vocalize, but sang nearly normal song. This result shows clearly that the MTM are not the principal sound source. The endoscopic images of the intact songbird syrinx during spontaneous and brain stimulation-induced vocalizations illustrate the dynamics of syringeal reconfiguration before phonation and suggest a different model for sound production. Phonation is initiated by rostrad movement and stretching of the syrinx. At the same time, the syrinx is closed through movement of two soft tissue masses, the medial and lateral labia, into the bronchial lumen. Sound production always is accompanied by vibratory motions of both labia, indicating that these vibrations may be the sound source. However, because of the low temporal resolution of the imaging system, the frequency and phase of labial vibrations could not be assessed in relation to that of the generated sound. Nevertheless, in contrast to the previous model, these observations show that both labia contribute to aperture control and strongly suggest that they play an important role as principal sound generators.

Generation of in vivo activating factors in the ischemic intestine by pancreatic enzymes

One of the early events in physiological shock is the generation of activators for leukocytes, endothelial cells, and other cells in the cardiovascular system. The mechanism by which these activators are produced has remained unresolved. We examine here the hypothesis that pancreatic digestive enzymes in the ischemic intestine may be involved in the generation of activators during intestinal ischemia. The lumen of the small intestine of rats was continuously perfused with saline containing a broadly acting pancreatic enzyme inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, 0.37 mM) before and during ischemia of the small intestine by splanchnic artery occlusion. This procedure inhibited activation of circulating leukocytes during occlusion and reperfusion. It also prevented the appearance of activators in portal venous and systemic artery plasma and attenuated initiating symptoms of multiple organ injury in shock. Intestinal tissue produces only low levels of activators in the absence of pancreatic enzymes, whereas in the presence of enzymes, activators are produced in a concentration- and time-dependent fashion. The results indicate that pancreatic digestive enzymes in the ischemic intestine serve as an important source for cell activation and inflammation, as well as multiple organ failure.

A second-generation vaccine protects against the psychoactive effects of cocaine

The effects of immunization with the second-generation cocaine immunoconjugate GND-keyhole limpet hemocyanin (KLH) or with the anti-cocaine mAb GNC92H2 were assessed in a model of acute cocaine-induced locomotor activity. After i.p. administration of cocaine⋅HCl (15 mg/kg), rats were tested in photocell cages, and stereotypy was rated to determine preimmunization drug response (baseline). Experimental animals were subjected to an immunization protocol with GND-KLH or treated with the mAb GNC92H2. Rats were then challenged with systemic cocaine, and their locomotor responses were again measured. Active immunization with GND-KLH produced a 76% decrease in the ambulatory measure (crossovers) in the experimental group and a 12% increase in the control group compared with baseline values. Also, stereotypic behavior was significantly suppressed in the vaccinated animals. Decreases in both measures were seen in the experimental group on two subsequent challenges. The maximum effect was observed at the time of the second challenge with a dramatic 80% decrease in crossovers. Treatment with GNC92H2 resulted in a 69% decrease in crossovers compared with baseline. This effect persisted across two additional challenges over 11 days with decreases of 46–47%. In contrast, the control group showed increases of up to 28%. Significant differences between groups were observed in the stereotypic measure in all three challenges. The results indicate that these immunopharmacotherapeutic agents have significant cocaine-blockade potential and therefore may offer an effective strategy for the treatment of cocaine abuse.

Rapid generation of incremental truncation libraries for protein engineering using α-phosphothioate nucleotides

Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of α-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3′→5′ exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.

Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance

The recently cloned NPR1 gene of Arabidopsis thaliana is a key regulator of acquired resistance responses. Upon induction, NPR1 expression is elevated and the NPR1 protein is activated, in turn inducing expression of a battery of downstream pathogenesis-related genes. In this study, we found that NPR1 confers resistance to the pathogens Pseudomonas syringae and Peronospora parasitica in a dosage-dependent fashion. Overexpression of NPR1 leads to enhanced resistance with no obvious detrimental effect on the plants. Thus, for the first time, a single gene is shown to be a workable target for genetic engineering of nonspecific resistance in plants.

Role of cortical N-methyl-D-aspartate receptors in auditory sensory memory and mismatch negativity generation: implications for schizophrenia.

Working memory refers to the ability of the brain to store and manipulate information over brief time periods, ranging from seconds to minutes. As opposed to long-term memory, which is critically dependent upon hippocampal processing, critical substrates for working memory are distributed in a modality-specific fashion throughout cortex. N-methyl-D-aspartate (NMDA) receptors play a crucial role in the initiation of long-term memory. Neurochemical mechanisms underlying the transient memory storage required for working memory, however, remain obscure. Auditory sensory memory, which refers to the ability of the brain to retain transient representations of the physical features (e.g., pitch) of simple auditory stimuli for periods of up to approximately 30 sec, represents one of the simplest components of the brain working memory system. Functioning of the auditory sensory memory system is indexed by the generation of a well-defined event-related potential, termed mismatch negativity (MMN). MMN can thus be used as an objective index of auditory sensory memory functioning and a probe for investigating underlying neurochemical mechanisms. Monkeys generate cortical activity in response to deviant stimuli that closely resembles human MMN. This study uses a combination of intracortical recording and pharmacological micromanipulations in awake monkeys to demonstrate that both competitive and noncompetitive NMDA antagonists block the generation of MMN without affecting prior obligatory activity in primary auditory cortex. These findings suggest that, on a neurophysiological level, MMN represents selective current flow through open, unblocked NMDA channels. Furthermore, they suggest a crucial role of cortical NMDA receptors in the assessment of stimulus familiarity/unfamiliarity, which is a key process underlying working memory performance.

Modeling and optimization of populations subject to time-dependent mutation.

It has become clear that many organisms possess the ability to regulate their mutation rate in response to environmental conditions. So the question of finding an optimal mutation rate must be replaced by that of finding an optimal mutation schedule. We show that this task cannot be accomplished with standard population-dynamic models. We then develop a "hybrid" model for populations experiencing time-dependent mutation that treats population growth as deterministic but the time of first appearance of new variants as stochastic. We show that the hybrid model agrees well with a Monte Carlo simulation. From this model, we derive a deterministic approximation, a "threshold" model, that is similar to standard population dynamic models but differs in the initial rate of generation of new mutants. We use these techniques to model antibody affinity maturation by somatic hypermutation. We had previously shown that the optimal mutation schedule for the deterministic threshold model is phasic, with periods of mutation between intervals of mutation-free growth. To establish the validity of this schedule, we now show that the phasic schedule that optimizes the deterministic threshold model significantly improves upon the best constant-rate schedule for the hybrid and Monte Carlo models.