Selective Disadvantage Research Articles

Changes in disease gene frequency over time with differential genotypic fitness and various control strategies

A spreadsheet model was constructed to describe the change in allelic frequency over time for a lethal recessive mutation in an animal population. The model allowed relative fitness to differ between genotypes, between sexes, and over time. Whereas a lethal recessive allele is naturally eliminated very slowly from a population, a small selective disadvantage of the heterozygote results in a large increase in the rate of elimination. With selective advantage of the heterozygote through linkage with a production trait or pleiotropy, the allele is never naturally eliminated but tends toward a stable equilibrium frequency. The model was used to investigate various alternative control programs based on the detection of heterozygotes by genotyping and their exclusion from breeding. The programs (genotyping males only, genotyping males and 50% of females, and genotyping all breeding animals) were modeled for various initial heterozygote frequencies, and the results were described in terms of the number of generations, number of tests, and number of culls required to reduce the heterozygote frequency to a predefined level. The model can be used to compare the feasibility and cost of various control strategies and to illustrate clearly to breeders the expected outcomes, as well as the danger of prematurely terminating a control program when there is a selective advantage of the heterozygote.

Influenza A Virus PB1-F2 Gene

To the Editor: Recently, Chen and co-workers described the expression of an 11th influenza A virus protein, designated PB1-F2 because this protein is encoded in the +1 open reading frame of the segment-2 RNA (1). Later, Chen et al. presented a preliminary analysis of 336 PB1 sequences from GenBank (2). We have extended the work on PB1-F2 and analyzed 1,864 partial and complete segment-2 sequences deposited in GenBank; these sequences belong to 79 influenza A virus subtypes. In summary, the following 8 observations should receive attention: First, the size of PB1-F2 polypeptides ranges from 79 to 101 amino acids (aa); most isolates encode versions of either 87 or 90 aa. Because polypeptides of 79 aa are located within mitochondria, their truncation has no effect on the protein function. The frequency of the 79-aa PB1-F2 is ≈5%. Second, a functional PB1-F2 is expressed by 92% of all segment-2 sequences, i.e., a polypeptide >78 aa. The proportion of intact PB1-F2 varies according to host (humans 90%, swine 76%, other mammals 100%, birds 95%). Third, the H1N1 subtype comprises 3 genetic lineages. One clade has 2 branches: 1 branch includes the human viruses, with the pandemic 1918 virus at its root; the other branch includes the classic swine viruses. The third clade represents the European porcine isolates. Although all classic swine sequences have a truncated PB1-F2 (in-frame stop codons after 11, 24, and 35 codons), the early human isolates (H1N1 sequences from 1918 through 1947) have an intact PB1-F2. After 1956, however, a mutation became prevalent such that the recent sequences starting from A/Beijing/1/56 terminate after 57 codons. An exception to this rule is A/Taiwan/3355/97. Two human H1N1 isolates with an intact PB1-F2 coding sequence cluster in the H3N2 clade (A/Kiev/59/79, A/Wisconsin/10/98). The PB1 sequences of European porcine influenza A virus isolates cluster with European porcine H3N2 and H1N2. Fourth, all H2N2 sequences are monophyletic and encode an intact PB1-F2. Fifth, the main sequence cluster of the H3N2 subtype comprises 3 branches: 1) porcine H3N2 and porcine H1N2 sequences from the United States, 2) porcine H3N2 isolates from Hong Kong and human H1N2, and 3) recent human H3N2 and some Japanese H3N2 isolates. Most of these sequences encode an intact PB1-F2. Sixth, the cluster of European porcine influenza A virus isolates comprises the subtypes H1N1, H1N2, and H3N2. The lack of distinct clades for each subtype indicates frequent reassortment in the evolution of these viruses. Of the segment-2 sequences, 56% encode an intact PB1-F2. Seventh, other porcine isolates of various subtypes represent transspecies infections or single reassortment events. And eighth, the segment-2 sequences of many avian influenza A virus isolates encode intact PB1-F2. Considerable proportions of truncated PB1-F2 genes were found in the H5N2, H6N6, H9N2, and H13N2 subtypes. However, because of the small number of sequences available, this observation may not be important. In conclusion, PB1-F2 is expressed in most avian and many porcine influenza A virus isolates. This finding contrasts with those in the initial publication, which stated that PB1-F2 is not expressed in many animal isolates, particularly those of porcine origin (1). Because PB1-F2 was described as a proapoptotic protein probably counteracting the host immune response, why numerous human and porcine isolates lack this protein without selective disadvantage remains unclear.

Correlation of TCE cometabolism with growth characteristics on aromatic substrates in toluene-degrading bacteria

We have constructed a bacterial library consisting of 97 strains of toluene-degrading bacteria from soil and activated sludge samples for examining their physiological properties in terms of cometabolism of TCE. Large variation of TCE degradation ability was observed in Gram-positive and Gram-negative strains, as well as diverse patterns of availability of aromatics as a growth substrate. No clear correlation was observed between the number of available substrates for growth and TCE degradation ability. However, the growth on some of the aromatics showed positive or negative correlations with TCE degradation ability. Kendall correlation constants (tau) for the growth on cumene, m-xylene, p-xylene, and m-cresol with TCE degradation ability were statistically significant ( P < 0.001): their values were −0.44, −0.31, −0.26, 0.37, respectively. Among 12 of aromatics, only m-cresol showed positive correlation with TCE degradation ability. These findings would be useful for enrichment and isolation of the microbes that have TCE-cometabolism ability, which is a selective disadvantage through the toxicity or competitive inhibition against growth substrates.

Partial trisomy 1p due to paternal t(1;9) translocation in a family with recurrent miscarriages

To report a new case with partial trisomy 1p due to paternal t(1;9) translocation in a family with recurrent miscarriages. Case report. Faculty of Medicine, Cukurova University. A couple with recurrent miscarriages, an abnormal fetus, a newborn infant, paternal grandfather and grandmother. Chorionic villi sampling (CVS), amniocentesis, lymphocytic karyotype, and genetic counseling. Chromosomal analysis of CVS, amniotic cells, and peripheral blood lymphocytes were performed according to standard cytogenetic methods using G-banding technique. We determined the reproductive risk in a couple who carried a balance and an unbalanced rearrangement of chromosomes 1 and 9 in two generations of a normal father with derivative 9 karyotype. The prenatal and postnatal karyotypes of the newborn infant were the same as the father [46,XY,der(9)t(1:9)(p34.2;q34.3)]. He was also phenotypically normal. The abnormal fetus that was miscarried also had a derivative 9 [46,XY,der(9)t(1:9)(p34.2;q34.3)fat]. The der(9) contained the partial short arm of chromosome 1. Both chromosome 1 showed normal. Trisomy 1p in the fetus was the result of familial derivative 9. Partial trisomy is associated with fetal wastage, and may play a role in the etiology of the other miscarriages in certain families. The apparent lack of increased reproductive failure may result from the selective disadvantage of aneusomic gamets at fertilization or very early spontaneous abortions of unbalanced conceptuses. The detection of couples with chromosomal anomalies can undoubtedly help prevent the births of malformed infants. These findings would be used widely in clinical genetics and as an effective tool for genetic counseling and reproductive guidance.

Conventional gene targeting protocols lead to loss of targeted cells when applied to a silent gene locus in primary fibroblasts

Gene targeting in livestock fibroblasts has proven difficult to achieve, particularly if the target gene is silent. We first tested whether efficient gene targeting at the transcriptionally active ovine alpha1(I) procollagen (COL1A1) locus required the use of a promoter trap vector. We compared gene targeting frequencies at the ovine COL1A1 locus using both a promoter trap and a non-promoter trap selection strategy. We demonstrated that targeted cells could be isolated regardless of whether an enrichment step (promoter trap) was used. Next, we used our optimised protocol to target a non-expressed gene, ovine beta-casein. We obtained clones that were scored positive by PCR for the targeting event, but were negative after cell expansion and Southern analysis. We propose that targeted cells were initially generated but that they were at a selective growth disadvantage during culture. We suggest modifications to the conventional targeting protocol that would prevent such loss of targeted cells.

Comparison of the reproductive biology between acaricide-resistant and acaricide-susceptible Rhipicephalus ( Boophilus) microplus (Acari: Ixodidae)

The reproductive fitness of Rhipicephalus ( Boophilus) microplus (Canestrini) strains resistant to organophosphate (OP), pyrethroid (P), or formamidine (F) acaricides was compared to an acaricide-susceptible (SUS) strain to determine whether the acquisition of resistance affected reproductive fitness in the resistant strains. The SUS strain females had a 3.0 days preoviposition period, a 12.1 days oviposition period, a 22.5 days egg incubation period, a mean of 3670 eggs per female, and a mean percentage egg hatch of 78.1%, which were all remarkably similar to these same parameters reported for this species throughout the world. The reproductive biology of the P-resistant strain (PYR) and the F-resistant strain (FOR) were, for the most part, similar to those of the SUS strain. In the few instances where statistical differences did occur there was little evidence that the variation had any biological basis that could be attributed to a reduction in fitness related to resistance to P or F acaricides. Although the comparison of reproductive parameters of the OP-resistant strain (OPR) and the SUS strain identified statistical differences between the mean egg incubation and oviposition periods, the magnitude of the differences was not sufficient to conclude that the OPR strain was biologically less fit than the SUS strain. However, the OPR strain produced 30% fewer eggs (2562 eggs per female) than the SUS strain (3670 eggs per female) indicating the acquisition of resistance placed the OPR at a selective disadvantage relative to the SUS strain. This coupled with a lower, though non-significant, egg hatch was used to predict there would be a reduction of at least 34.1% in larval numbers available to potentially re-infest subsequent cattle than were available from the SUS strain. These data may aid the development of management strategies that can be used to control OP-resistant ticks.

Infectious Molecular Clones from a Simian Immunodeficiency Virus-Infected Rapid-Progressor (RP) Macaque: Evidence of Differential Selection of RP-Specific Envelope Mutations In Vitro and In Vivo

A minor fraction of simian immunodeficiency virus (SIV)-infected macaques progress rapidly to AIDS in the absence of SIV-specific immune responses. Common mutations in conserved residues of env in three SIVsmE543-3-infected rapid-progressor (RP) macaques suggest the evolution of a common viral variant in RP macaques. The goal of the present study was to analyze the biological properties of these variants in vitro and in vivo through the derivation of infectious molecular clones. Virus isolated from a SIVsmE543-3-infected RP macaque, H445 was used to inoculate six naive rhesus macaques. Although RP-specific mutations dominated in H445 tissues, they represented only 10% of the population of the virus stock, suggesting a selective disadvantage in vitro. Only one of these macaques (H635) progressed rapidly to AIDS. Plasma virus during primary infection of H635 was similar to the inoculum. However, RP-specific mutations were apparently rapidly reselected by 4 to 9 weeks postinfection. Terminal plasma from H635 was used as a source of viral RNA to generate seven full-length, infectious molecular clones. With the exception of one clone, which was similar to SIVsmE543-3, clones with RP-specific mutations replicated with delayed kinetics in rhesus peripheral blood mononuclear cells and human T-cell lines. None of the clones replicated in monocyte-derived or alveolar macrophages, and all used CCR5 as their major coreceptor. RP variants appear to be well adapted to replicate in vivo in RP macaques but are at a disadvantage in tissue culture compared to their parent, SIVsmE543-3. Therefore, tissue culture may not provide a good surrogate for replication of RP variants in macaques. These infectious clones will provide a valuable reagent to study the roles of specific viral variants in rapid progression in vivo.

Abrupt Expression of TLR4 in TLR4-Deficient Macrophages Imposes a Selective Disadvantage: Genetic Evidence for TLR4-Dependent Responses to Endogenous, Nonmicrobial Stimuli

TLR4 is crucial for macrophage responses to LPS. It is less clear whether TLR4 may also transduce signals from host factors, and if so, with what consequences. Immortalized bone marrow-derived macrophage cell lines, termed T4Cr and T4ko, were established from TLR4null strains, C57BL/10ScNCr and TLR4 knockout mice, respectively. Multiple transfections and selections were conducted to stably introduce TLR4 into these cell lines. Among 196 individual clones isolated, 48 expressed TLR4 on the cell surface but did not respond to LPS due to a deletion in the MyD88 gene. The remaining clones integrated TLR4 DNA into the genome but expressed neither detectable TLR4 mRNA nor TLR4 protein. To test the possibility that TLR4null cells lack modulating factors to protect against a harmful effect of TLR4, 15 stably transfected clones were generated in the presence of conditioned media from wild-type macrophages. Some of these cells expressed a small amount of TLR4 and regained responsiveness to LPS. Because no microbial ligands were available to the cell lines during their generation, signaling via endogenous ligands is likely to have occurred in TLR4-expressing, signal-competent macrophages and imposed a proliferative or other selective disadvantage. These studies support the existence of constitutive signaling via TLR4 during in vitro culture of macrophages without microbial products, and help account for the lack of reports of restoration of TLR4 expression in normally TLR4-expressing types of cells in vitro whose TLR4 genes are deleted or disrupted.

The late Middle Eocene terrestrial vertebrate fauna from Seymour Island: the tails of the Eocene Patagonian size distribution

Abstract The Middle Eocene Antarctic terrestrial vertebrate palaeofauna from the La Meseta Formation on Seymour Island (= Isla Marambio), Antarctic Peninsula has a U-shaped, bimodal distribution of body sizes. This palaeofauna includes a wide range of body sizes from small insectivorous, omnivorous and granivorous marsupials, a rodent-like non-therian gondwanathere, large-sized ungulates, a sloth and cursorial birds (a ratite and a phororachoid). Medium-sized, homeothermic animals in the size range represented by rabbit to small ungulate-sized animals have not been found. For comparison, the Early Eocene Casamayoran (Vacan ‘subage’) mammalian palaeofauna from Patagonia has a reasonably normal distribution of body sizes, with the modal class represented by medium-sized mammals, a distribution that is the direct opposite of the Antarctic palaeofauna. A comparison of the Middle Eocene Antarctic palaeofauna from the La Meseta Formation to the early Eocene Vacan-aged mammal palaeofauna is appropriate, due to the taxonomic affinities of the Antarctic palaeofauna to Riochican (latest Palaeocene) and Vacan-aged palaeofaunas of Patagonia. If these Patagonian mammalian palaeofaunas (PMP) were the source for the La Meseta palaeofauna (LMP), then a similar normal distribution with less taxonomic diversity would be expected. However, the LMP does not meet this expectation or even a distribution where all size classes are equally represented. Thus, the pattern of size distribution is quite different from the PMPs. Floral data for the Early Eocene of Patagonia indicate subtropical conditions with mean annual temperatures (MAT) of 15.6 °C and equable winter temperatures (&gt;10 °C) generating high taxonomic diversity at the species level. Floral data from the La Meseta Formation of equivalent age to the mammalian fauna indicate a cooler MAT of 11–13 °C with a highly seasonal climate, where the mean winter temperature could have ranged from −3 to 2 °C. There is also a significant drop in floral taxonomic diversity, which is dominated by Nothofagus . A bimodal body size distribution pattern is not an unusual pattern for higher latitude mammalian faunas. Modern boreal mammalian faunas of North America have a low frequency of species in the medium body size range in response to cold winter temperatures in these higher latitudes. The smaller-sized mammals have adapted their physiology to the cold winter temperatures. The larger animals have adapted to the cold winter conditions by conserving heat through small surface-area-to-volume ratios as a result of their greater bulk. The low frequency of medium-sized animals is due to the fact that neither of these thermal strategies is available to them and thus they are at a selective disadvantage.

New Leontiniids (Class Mammalia, Order Notoungulata, Family Leontiniidae) from the Salla Beds of Bolivia (Deseadan, Late Oligocene)

A revised diagnosis of the Leontiniidae (Class Mammalia, Order Notoungulata) is provided and leontiniids of the Deseadan South American Land Mammal "age" (SALMA; late Oligocene) are summarized. Two new species of leontinids from the Deseadan Salla Beds of Bolivia are described and placed within the new genus, Anayatherium; the smaller A. ekecoa and a much larger A. fortis. A phylogenetic analysis suggests that these species are closely related to the derived Patagonian genera Ancylocoelus and Colpodon, with the loss of the canine serving as a putative synapomorphy uniting these taxa. Although leontiniids are the most frequently encountered taxa at many other Deseadan localities, they are exceedingly rare at Salla. This scarcity, the scarcity of other ungulates with low crowned cheek teeth and the heavy tooth wear of a relatively young individual of A. ekecoa suggest that ungulates with low crowned teeth were at a selective disadvantage at Salla.

The molecular pathology of new anti-cancer agents

Conventional anti-cancer drugs usually act against dividing cells and work on the premise that cancer cells divide more quickly than normal cells in the body, so that repeated doses will put the cancer cells at a selective disadvantage. In many tumours, however, it is not the rate of proliferation that causes tumour growth but the lack of tumour cell death by apoptosis, so these drugs will not be effective against such tumours. These agents also cause many side-effects because of their effect on normal rapidly dividing cells in the body, such as the bone marrow and the epithelial lining of the gut, and it is these side-effects which often limit the dose. There are now a whole range of new anti-cancer agents that target cancer cells more specifically because they are directed against unique protein targets in these cells. These targets often include specific growth factor receptors that are overexpressed in particular types of cancer. These new agents are often monoclonal antibodies directed against these proteins or small molecule inhibitors of their enzymatic intracytoplasmic domains. Examples include cetuximab, trastuzumab, bevacizumab, imatinib mesylate and gefitinib. This article reviews the molecular pathology of these agents and describes the role of histopathology testing in selecting these therapies for individual patients.

Reproductive Success Across The Black-Capped Chickadee (Poecile Atricapillus) and Carolina Chickadee (P. Carolinensis) Hybrid Zone in Ohio

AbstractBlack-capped Chickadees (Poecile atricapillus) and Carolina Chickadees (P. carolinensis) hybridize in an east-west band from New Jersey to Kansas. Within the past century, the Ohio portion of this hybrid zone and the Carolina Chickadee range to the south have been moving northward, whereas the Black-capped Chickadee range has retracted. In Ohio, we characterized the genetic composition of the hybrid zone using five diagnostic molecular loci. Although there was no evidence of assortative mating in the center of the hybrid zone, we found a relative paucity of genetically intermediate breeding females as compared with breeding males. That suggests viability selection against female hybrids, in line with Haldane’s rule. On the basis of reproductive variables (number of nestlings, reproductive success), we found a decrease in productivity of breeding pairs in the hybrid zone that is significantly and positively related to their probability of producing homozygous offspring at each autosomal or sex-linked locus. We also found that the decrease in productivity was significantly and positively related to the genetic composition of the male of the pair (i.e. pure male chickadees more productive). These data strongly suggest that hybrids are at a selective disadvantage. Because the zone of reduced reproductive success was considerably narrower than the zone of introgression, our results demonstrate that genetic introgression is occurring in the face of substantial selection against hybrids.Éxito Reproductivo a través de la Zona de Hibridación de Poecile atricapillus y P. carolinensis en Ohio

Diversity of thymidine analogue resistance genotypes among newly diagnosed HIV-1-infected persons

The introduction of highly active antiretroviral therapy (HAART) has resulted in a significant decrease in HIV and AIDS-related mortality and morbidity. However, these treatments can select for drug-resistant viruses which are associated with poor virological responses to the antiretroviral therapy and possible loss of clinical benefit. Drug-resistant viruses can also be transmitted between individuals. In the absence of drug pressure, transmitted drug-resistant viruses gradually lose resistance mutations that confer a selective disadvantage as they evolve to more fit viruses. As a result, unusual resistance-related genotypes not commonly seen in treated patients may arise in the population. Viruses with unique patterns of thymidine analogue-associated mutations (TAMs) have now been identified in a substantial proportion of treatment-naive recently diagnosed persons. In this leading article, I discuss these findings and the potential impact of these unique reverse transcriptase (RT) genotypes on evolution of resistance and treatment responses.

Conditional inactivation of the mouse Hus1 cell cycle checkpoint gene

The Hus1 cell cycle checkpoint protein plays a central role in genome maintenance by mediating cellular responses to DNA damage and replication stress. Targeted deletion of mouse Hus1 results in spontaneous chromosomal abnormalities and embryonic lethality. To study the physiological impact of Hus1 deficiency in adult mice, we generated a conditional Hus1 allele, Hus1 flox , in which exons two and three are flanked by loxP sites. Cre-mediated excision of the loxP-flanked region produces Hus1 Δ2,3 , which is capable of encoding only 19 of 281 Hus1 amino acids. Germline homozygosity for Hus1 Δ2,3 resulted in mid-gestational embryonic lethality that was indistinguishable from that caused by an established null allele, Hus1 Δ1n . Hus1 was inactivated in adult mice using a transgenic strain in which Cre is sporadically expressed in a variety of tissues from the Hsp70-1 promoter. Conditional Hus1 knockout mice were produced at unexpectedly low frequency and, unlike control animals, demonstrated limited inactivation of the conditional allele, suggesting that Hus1-deficient cells were at a strong selective disadvantage in adult animals. However, viable conditional Hus1 knockout mice consistently showed the greatest degree of Hus1 inactivation specifically in lung and mammary gland, highlighting varying requirements for Hus1 in different tissues. The novel tools described here hold promise for elucidating how the Hus1-dependent checkpoint mechanism contributes to chromosomal stability, DNA damage responses, and tumor suppression in adult mice.

PERSPECTIVE: THE EVOLUTION OF WARNING COLORATION IS NOT PARADOXICAL

Animals that are brightly colored have intrigued scientists since the time of Darwin, because it seems surprising that prey should have evolved to be clearly visible to predators. Often this self-advertisement is explained by the prey being unprofitable in some way, with the conspicuous warning coloration helping to protect the prey because it signals to potential predators that the prey is unprofitable. However, such signals only work in this way once predators have learned to associate the conspicuous color with the unprofitability of the prey. The evolution of warning coloration is still widely considered to be a paradox, because it has traditionally been assumed that the very first brightly colored individuals would be at an immediate selective disadvantage because of their greater conspicuousness to predators that are naive to the meaning of the signal. As a result, it has been difficult to understand how a novel conspicuous color morph could ever avoid extinction for long enough for predators to become educated about the signal. Thus, the traditional view that the evolution of warning coloration is difficult to explain rests entirely on assumptions about the foraging behavior of predators. However, we review recent evidence from a range of studies of predator foraging decisions, which refute these established assumptions. These studies show that: (1) Many predators are so conservative in their food preferences that even very conspicuous novel prey morphs are not necessarily at a selective disadvantage. (2) The survival and spread of novel color morphs can be simulated in field and aviary experiments using real predators (birds) foraging on successive generations of artificial prey populations. This work demonstrates that the foraging preferences of predators can regularly (though not always) result in the increase to fixation of a novel morph appearing in a population of familiar-colored prey. Such fixation events occur even if both novel and familiar prey are fully palatable and despite the novel food being much more conspicuous than the familiar prey. These studies therefore provide strong empirical evidence that conspicuous coloration can evolve readily, and repeatedly, as a result of the conservative foraging decisions of predators.

829. Design of a Safe and Efficient Vector for Bone Marrow Chemoprotection by O6-Methyl-Guanine-DNA-MethylTransferase

In order to achieve efficient and safe expression of the promising chemoselection marker, O6-Methylguanine-DNA-methyltransferase (MGMT) P140K, in hematopoietic cells, we designed and compared eight vectors, three gammaretroviral (murine leukemia virus, MLV) and five lentiviral (human immunodeficiency virus, HIV-1). All vectors had monocistronic cassettes as required for clinical studies of MGMT-mediated bone marrow chemoprotection. We compared either LTR-driven or safer SIN (self-inactivating LTR) vectors. In the SIN context, we chose the human phosphoglycerokinase (hPGK), elongation factor 1a (EF1a) or SFFV U3 (MLV-derived) as an internal promoter to reveal different expression strengths. All constructs generated high titers and showed correct processing in target cells, as demonstrated by Northern, Western Blot und immunofluorescence. FACS analysis allowed sensitive detection of MGMT expression in transduced cells and revealed significant differences in MGMT levels, depending on vector design. Expression differences correlated with results of a biochemical alkyltransferase assay. The highest expression levels of MGMT P140K could be achieved using the SFFV promoter, located either in the LTR or in internal position of a SIN backbone. Gammaretroviral and lentiviral SIN vectors with comparable internal cassettes had similar titers and expression levels. An intron did not improve MGMT P140K expression. All vectors were sufficiently strong to mediate polyclonal chemoresistance, which resulted in a chimerism of >95% transduced cells in vitro, following a single exposure to BCNU. However, abundant expression of MGMT P140K, in contrast to the wild-type protein, led to a selective disadvantage of transduced cells in the absence of drug. Use of a less potent SIN vector with an internal expression cassette driven by the human PGK promoter avoided this side effect, and still allowed polyclonal chemoprotection of transduced hematopoietic cells under conditions of a low multiplicity of infection. Thus, to achieve the desired properties of efficiency and safety, less potent expression vectors for MGMT P140K represent a reasonable choice for future in vivo studies. Moreover, our preliminary data suggest that SIN vectors reduce the incidence of insertional genotoxicity in primary murine hematopoietic cells.

Energy Constraints on the Evolution of Gene Expression

I here estimate the energy cost of changes in gene expression for several thousand genes in the yeast Saccharomyces cerevisiae. A doubling of gene expression, as it occurs in a gene duplication event, is significantly selected against for all genes for which expression data is available. It carries a median selective disadvantage of s > 10(-5), several times greater than the selection coefficient s = 1.47 x 10(-7) below which genetic drift dominates a mutant's fate. When considered separately, increases in messenger RNA expression or protein expression by more than a factor 2 also have significant energy costs for most genes. This means that the evolution of transcription and translation rates is not an evolutionarily neutral process. They are under active selection opposing them. My estimates are based on genome-scale information of gene expression in the yeast S. cerevisiae as well as information on the energy cost of biosynthesizing amino acids and nucleotides.

The Biased Distribution of Alus in Human Isochores Might Be Driven by Recombination

Alu retrotransposons do not show a homogeneous distribution over the human genome but have a higher density in GC-rich (H) than in AT-rich (L) isochores. However, since they preferentially insert into the L isochores, the question arises: What is the evolutionary mechanism that shifts the Alu density maximum from L to H isochores? To disclose the role played by each of the potential mechanisms involved in such biased distribution, we carried out a genome-wide analysis of the density of the Alus as a function of their evolutionary age, isochore membership, and intron vs. intergene location. Since Alus depend on the retrotransposase encoded by the LINE1 elements, we also studied the distribution of LINE1 to provide a complete evolutionary scenario. We consecutively check, and discard, the contributions of the Alu/LINE1 competition for retrotransposase, compositional matching pressure, and Alu overrepresentation in introns. In analyzing the role played by unequal recombination, we scan the genome for Alu trimers, a direct product of Alu-Alu recombination. Through computer simulations, we show that such trimers are much more frequent than expected, the observed/expected ratio being higher in L than in H isochores. This result, together with the known higher selective disadvantage of recombination products in H isochores, points to Alu-Alu recombination as the main agent provoking the density shift of Alus toward the GC-rich parts of the genome. Two independent pieces of evidence-the lower evolutionary divergence shown by recently inserted Alu subfamilies and the higher frequency of old stand-alone Alus in L isochores-support such a conclusion. Other evolutionary factors, such as population bottlenecks during primate speciation, may have accelerated the fast accumulation of Alus in GC-rich isochores.

Sequence analysis of murine leukemia virus envelope gene from inoculated mice

Introduction of amino acids substitutions in murine leukemia virus genome is a powerful method to determine the relative importance of various viral factors in pathogenesis. However, introduction of such amino acids substitution could result in viruses at a selective disadvantage, and eventual selection of revertants. It is thus essential to verify if the mutation is maintained stably in replicating virus and in infected tumor cells. In the present study, viral nucleic acid sequences from diseased animals were determined using different approaches. Small blood samples were found adequate for direct RNA extraction and reverse transcriptase-PCR amplification followed by automated DNA sequencing. Alternatively, replication-competent viruses were recovered specifically by applying blood samples onto permissive cells; viral RNA is then extracted from tissue culture medium and similarly sequenced. Tissue samples were also used to amplify viral sequences from tumors DNA while small pieces of tumors tissues were applied onto permissive cells to isolate replicating viruses. The combined experimental approach was used to show sequence conservation using a mutant altered in the intracytoplasmic region of viral envelope glycoprotein. No difference was observed between viruses recovered directly from the animal and those amplified onto cultured cells.

PERSPECTIVE: THE EVOLUTION OF WARNING COLORATION IS NOT PARADOXICAL

Animals that are brightly colored have intrigued scientists since the time of Darwin, because it seems surprising that prey should have evolved to be clearly visible to predators. Often this self-advertisement is explained by the prey being unprofitable in some way, with the conspicuous warning coloration helping to protect the prey because it signals to potential predators that the prey is unprofitable. However, such signals only work in this way once predators have learned to associate the conspicuous color with the unprofitability of the prey. The evolution of warning coloration is still widely considered to be a paradox, because it has traditionally been assumed that the very first brightly colored individuals would be at an immediate selective disadvantage because of their greater conspicuousness to predators that are naive to the meaning of the signal. As a result, it has been difficult to understand how a novel conspicuous color morph could ever avoid extinction for long enough for predators to become educated about the signal. Thus, the traditional view that the evolution of warning coloration is difficult to explain rests entirely on assumptions about the foraging behavior of predators. However, we review recent evidence from a range of studies of predator foraging decisions, which refute these established assumptions. These studies show that: (1) Many predators are so conservative in their food preferences that even very conspicuous novel prey morphs are not necessarily at a selective disadvantage. (2) The survival and spread of novel color morphs can be simulated in field and aviary experiments using real predators (birds) foraging on successive generations of artificial prey populations. This work demonstrates that the foraging preferences of predators can regularly (though not always) result in the increase to fixation of a novel morph appearing in a population of familiar-colored prey. Such fixation events occur even if both novel and familiar prey are fully palatable and despite the novel food being much more conspicuous than the familiar prey. These studies therefore provide strong empirical evidence that conspicuous coloration can evolve readily, and repeatedly, as a result of the conservative foraging decisions of predators.