Accumulation Of Random Mutations Research Articles

Abstract GS3-09: Loss of ASXL1 tumor suppressor promotes resistance to CDK4/6 inhibitors in ER+ breast cancer

Abstract Background: CDK4/6 inhibitors (CDK4/6i) in combination with antiestrogens have prolonged survival of patients with ER+ metastatic breast cancer. However, this combination is not curative mainly due to acquired drug resistance. Knowledge about mechanisms of such resistance remains quite incomplete. We report herein a forward-genetics screen to discover a broad spectrum of novel somatic mutations causal to CDK4/6i resistance. Methods: We used CRISPR/Cas9 to delete the DNA mismatch repair (MMR) gene MSH2 in MCF7 and T47D ER+ breast cancer cells. Deficiency of DNA MMR proteins such as MSH2 results in a high nucleotide substitution rate which, in turn, predisposes cells to acquire drug resistance-associated mutations. MSH2-/- MCF7 and T47D cells were infected with a lentiviral barcode library containing ~1000 unique DNA barcodes. MSH2-/- barcoded cells were expanded for ~25 doublings to allow the accumulation of random mutations. Clones resistant to CDK4/6i were selected in the presence of IC90 of palbociclib (200 nM) or abemaciclib (500 nM) for 4-6 weeks. CDK4/6i resistant clones with unique barcode IDs were subjected to whole exome sequencing (WES). Results: Following drug selection, ~73 uniquely barcoded resistant colonies emerged from MCF7 and T47D MSH2-/- clonal lines. As expected, MCF7 and T47D MSH2-/- clones harbored a high mutation burden compared to parental cells. Candidate variants were distilled based on (a) functionality prediction and (b) mutation frequency in Project GENIE. We observed RB1 (5/73 clones; 6.8%) mutations in CDK4/6i resistant clones, providing proof-of-principle that this approach can identify clinically-relevant drug resistant alterations. Overall, we identified non-synonymous alterations in 2,206 genes in T47D palbociclib-resistant, 2,195 genes in T47D abemaciclib-resistant, and 1,312 genes in MCF7 palbociclib-resistant lines. A secondary screen of the 10 genes recurrently mutated in all three CDK4/6i resistant groups identified loss of ASXL1 as top hit. ASXL1 encodes a polycomb repressive complex protein that regulates chromatin accessibility. Loss of ASXL1 has been implicated in myeloid transformation through epigenetic reprogramming. WES of 76 CDK4/6i resistant tumor biopsies (DFCI/MBCproject cohort) identified ASXL1 alterations in two and four patients with acquired and primary resistance, respectively (6/76=7.9%). One of the tumors that progressed after an initial response to palbociclib had acquired the same ASXL1 R549C mutation that was identified in our screen. Among 1,769 tumors from patients treated with CDK4/6i (TEMPUS database), 37 exhibited ASXL1 alterations (4 frameshift, 6 truncating, 3 in-frame del, 24 missense mutations). DNAseq of patient-derived organoids established from post-CDK4/6i metastases identified ASXL1 mutations in 2/7 organoids (29%). ASXL1-/- MCF7 and T47D cells were cross-resistant to fulvestrant. GSEA analysis of RNA-seq data showed upregulation of E2F targets in palbociclib-treated cells stably transduced with ASXL1 shRNA but not control shRNA (Enrichment score=0.75, q=1.00E-09). This was associated with maintenance of RB phosphorylation in the presence of CDK4/6i, markedly higher levels of CDK2, CDK6, cyclins E and A, and downregulation of p21 and p27. Finally, siRNAs targeting CDK2 or cyclin A reduced the viability of ASXL1-deficient T47D cells by 50% and 90%, respectively. Conclusions: An accelerated mutagenesis approach using MMR-deficient ER+ breast cancer cells identified loss of ASXL1 as a novel mechanism of resistance to CDK4/6i. ASXL1 alterations were found in ~8% of tumors from patients with de novo or acquired resistance to CDK4/6i. Knockdown of CDK2 and cyclin A restored sensitivity to CDK4/6i and reduced viability of ASXL1 deficient cells, suggesting CDK2 inhibitors are a treatment approach against these drug-resistant tumors. Citation Format: Dhivya R. Sudhan, Sumanta Chatterjee, Jiwoong Kim, Yunguan Wang, Vishal Kandagatla, Dan Ye, Chang-Ching Lin, Jorge Gomez Tejeda Zanudo, Esha Jain, Arnaldo Marin, Alberto Servetto, Kyung-min Lee, Juan Manuel Povedano, David McFadden, Alex Barrett, Nikhil Wagle, Ariella B. Hanker, Carlos L. Arteaga. Loss of ASXL1 tumor suppressor promotes resistance to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS3-09.

Abstract 1113: Synergistic drug combinations prevent drug resistance in anaplastic large cell lymphoma preclinical models

Abstract Neoplastic cells are characterized by genetic instability, leading to the random accumulation of mutations in different tumor subpopulations, which therefore respond differently to treatments. Given the average mutational rate of tumors, the probability to acquire resistance to a single drug usually exceeds the number of cells present at diagnosis, making drug resistance a fact in most cases. Statistically, the chance of a cell being resistant to two simultaneous treatments is much lower, suggesting that drug combinations may help prevent resistance. Anaplastic large cell lymphoma (ALCL) is an aggressive type of Non-Hodgkin Lymphoma, characterized by expression of the NPM/ALK oncogenic fusion tyrosine kinase. ALK+ ALCL patients resistant or relapsed (R/R) to front-line chemotherapy have a very poor prognosis. The therapeutic activity of the ALK inhibitor crizotinib has been shown in patients with R/R ALK+ ALCL. However, 30-40% of patients develop resistance to crizotinib monotherapy and relapse within short time, leaving no further therapeutic option than bone marrow transplant, when feasible. This highlights the need to identify new first-line therapies for high-risk patients.We explored the potential of upfront drug combinations to prevent the rise of resistant disease, in vitro and in vivo. Crizotinib was combined with CHOP chemotherapy, an epigenetic drug (decitabine), another targeted inhibitor (trametinib- MEK inhibitor), or a second-generation ALK inhibitor. ALK+ ALCL cells were kept in culture for ~100 days in the presence of different single or combined drugs. We found that in most cases, combined treatments were more effective than single agents in the long-term control of cells expansion. Upon further functional characterization using quantitative PCR and western blot, we found that combination treatments upregulated cell cycle inhibitors and pro-apoptotic genes as well as modulated survival factors to induce deeper inhibition of oncogenic signalling and higher rates of apoptosis. Crizotinib+decitabine combination treatment promoted downregulation of DNMT1 and increase of TGFB1, both of which indirectly affect cell cycle progression. In murine model, crizotinib+decitabine significantly delayed tumour growth, compared to single treatments. Our results indicate that combining a selective drug with a less specific anticancer agent, which allows unspecific killing of resistant clones that might potentially arise, would be beneficial.In conclusion, our data suggest that a polypharmacology approach combining ALK inhibition with other agents could be a valuable therapeutic option for ALK+ ALCL patients, to prevent relapses and improve outcomes. Citation Format: Geeta G. Sharma, Giulia Arosio, Matteo Villa, Mario Mauri, Marina Zappa, Vera Magistroni, Monica Ceccon, Sara Redaelli, Ilaria Crespiatico, Diletta Fontana, Cristina Mastini, Anna Garbin, Federica Lovisa, Lara Mussolin, Rocco Piazza, Carlo Gambacorrti-Passerini, Luca Mologni. Synergistic drug combinations prevent drug resistance in anaplastic large cell lymphoma preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1113.

Idiosyncratic epistasis creates universals in mutational effects and evolutionary trajectories.

Patterns of epistasis and shapes of fitness landscapes are of wide interest because of their bearings on a number of evolutionary theories. The common phenomena of slowing fitness increases during adaptations and diminishing returns from beneficial mutations are believed to reflect a concave fitness landscape and a preponderance of negative epistasis. Paradoxically, fitness drops tend to decelerate and harm from deleterious mutations shrinks during accumulation of random mutations, patterns thought to indicate a convex fitness landscape and a predominance of positive epistasis. Current theories cannot resolve this apparent contradiction. Here we show that the phenotypic effect of a mutation varies substantially depending on the specific genetic background and that this idiosyncrasy in epistasis creates all of the above trends without requiring a biased distribution of epistasis. The idiosyncratic epistasis theory explains the universalities in mutational effects and evolutionary trajectories as emerging from randomness due to biological complexity.

Adaptation of turnip mosaic potyvirus to a specific niche reduces its genetic and environmental robustness.

Robustness is the preservation of the phenotype in the face of genetic and environmental perturbations. It has been argued that robustness must be an essential fitness component of RNA viruses owed to their small and compacted genomes, high mutation rates and living in ever-changing environmental conditions. Given that genetic robustness might hamper possible beneficial mutations, it has been suggested that genetic robustness can only evolve as a side-effect of the evolution of robustness mechanisms specific to cope with environmental perturbations, a theory known as plastogenetic congruence. However, empirical evidences from different viral systems are contradictory. To test how adaptation to a particular environment affects both environmental and genetic robustness, we have used two strains of turnip mosaic potyvirus (TuMV) that differ in their degree of adaptation to Arabidopsis thaliana at a permissive temperature. We show that the highly adapted strain is strongly sensitive to the effect of random mutations and to changes in temperature conditions. In contrast, the non-adapted strain shows more robustness against both the accumulation of random mutations and drastic changes in temperature conditions. Together, these results are consistent with the predictions of the plastogenetic congruence theory, suggesting that genetic and environmental robustnesses may be two sides of the same coin for TuMV.

Intra and Interspecific Variations of Gene Expression Levels in Yeast Are Largely Neutral: (Nei Lecture, SMBE 2016, Gold Coast).

It is commonly, although not universally, accepted that most intra and interspecific genome sequence variations are more or less neutral, whereas a large fraction of organism-level phenotypic variations are adaptive. Gene expression levels are molecular phenotypes that bridge the gap between genotypes and corresponding organism-level phenotypes. Yet, it is unknown whether natural variations in gene expression levels are mostly neutral or adaptive. Here we address this fundamental question by genome-wide profiling and comparison of gene expression levels in nine yeast strains belonging to three closely related Saccharomyces species and originating from five different ecological environments. We find that the transcriptome-based clustering of the nine strains approximates the genome sequence-based phylogeny irrespective of their ecological environments. Remarkably, only ∼0.5% of genes exhibit similar expression levels among strains from a common ecological environment, no greater than that among strains with comparable phylogenetic relationships but different environments. These and other observations strongly suggest that most intra and interspecific variations in yeast gene expression levels result from the accumulation of random mutations rather than environmental adaptations. This finding has profound implications for understanding the driving force of gene expression evolution, genetic basis of phenotypic adaptation, and general role of stochasticity in evolution.

Dynamics of evolutionary radiation under ecological neutrality

The most spectacular phenomenon of evolutionary biota is the explosive radiation that occurs in depauperate environments in which there are fewer competitors and predators, such as oceanic islands and crater lakes. Adaptation to divergent niches has been proposed as a major cause for this accelerated speciation. Here, we show that neutral mutation, genetic drift, and neutral community dynamics are sufficient to lead to radiation. In addition, these processes yield overshooting dynamics with a decline in species richness in the later stages of radiation. We constructed an ecologically neutral model for a community on an island with a uniform environment. For the speciation process, we introduced a null model with minimal assumptions in which the incompatibilities between alleles in different lineages evolve by a random accumulation of mutations via genetic drift. Our simulations showed that the speciation rate, extinction rate and genetic variation of the species colonizing the island rapidly increased to a sharp peak followed by a decrease that approached zero. Because the extinction rate reached a peak later than the speciation rate, the species richness initially increased, but declined in the later stage, exhibiting “overshooting”. The highest species richness was found for the largest island at the largest initial population size. Accordingly, speciation is accelerated by the large population size of depauperate biota, whereas it is decelerated with increasing species richness from the decreasing population size. Explosive radiation without ecological divergence can occur in depauperate environments via neutral stochastic processes.

Darwinian Dynamics of Intratumoral Heterogeneity: Not Solely Random Mutations but Also Variable Environmental Selection Forces.

Spatial heterogeneity in tumors is generally thought to result from branching clonal evolution driven by random mutations that accumulate during tumor development. However, this concept rests on the implicit assumption that cancer cells never evolve to a fitness maximum because they can always acquire mutations that increase proliferative capacity. In this study, we investigated the validity of this assumption. Using evolutionary game theory, we demonstrate that local cancer cell populations will rapidly converge to the fittest phenotype given a stable environment. In such settings, cellular spatial heterogeneity in a tumor will be largely governed by regional variations in environmental conditions, for example, alterations in blood flow. Model simulations specifically predict a common spatial pattern in which cancer cells at the tumor-host interface exhibit invasion-promoting, rapidly proliferating phenotypic properties, whereas cells in the tumor core maximize their population density by promoting supportive tissue infrastructures, for example, to promote angiogenesis. We tested model predictions through detailed quantitative image analysis of phenotypic spatial distribution in histologic sections of 10 patients with stage 2 invasive breast cancers. CAIX, GLUT1, and Ki67 were upregulated in the tumor edge, consistent with an acid-producing invasive, proliferative phenotype. Cells in the tumor core were 20% denser than the edge, exhibiting upregulation of CAXII, HIF-1α, and cleaved caspase-3, consistent with a more static and less proliferative phenotype. Similarly, vascularity was consistently lower in the tumor center compared with the tumor edges. Lymphocytic immune responses to tumor antigens also trended to higher level in the tumor edge, although this effect did not reach statistical significance. Like invasive species in nature, cancer cells at the leading edge of the tumor possess a different phenotype from cells in the tumor core. Our results suggest that at least some of the molecular heterogeneity in cancer cells in tumors is governed by predictable regional variations in environmental selection forces, arguing against the assumption that cancer cells can evolve toward a local fitness maximum by random accumulation of mutations. Cancer Res; 76(11); 3136-44. ©2016 AACR.

Evolution of Pseudomonas aeruginosa Antimicrobial Resistance and Fitness under Low and High Mutation Rates.

Pseudomonas aeruginosa, a major cause of nosocomial and chronic infections, is considered a paradigm of antimicrobial resistance development. However, the evolutionary trajectories of antimicrobial resistance and the impact of mutator phenotypes remain mostly unexplored. Therefore, whole-genome sequencing (WGS) was performed in lineages of wild-type and mutator (ΔmutS) strains exposed to increasing concentrations of relevant antipseudomonal agents. WGS provided a privileged perspective of the dramatic effect of mutator phenotypes on the accumulation of random mutations, most of which were transitions, as expected. Moreover, a frameshift mutagenic signature, consistent with error-prone DNA polymerase activity as a consequence of SOS system induction, was also seen. This effect was evidenced for all antibiotics tested, but it was higher for fluoroquinolones than for cephalosporins or carbapenems. Analysis of genotype versus phenotype confirmed expected resistance evolution trajectories but also revealed new pathways. Classical mechanisms included multiple mutations leading to AmpC overexpression (ceftazidime), quinolone resistance-determining region (QRDR) mutations (ciprofloxacin), oprD inactivation (meropenem), and efflux pump overexpression (ciprofloxacin and meropenem). Groundbreaking findings included gain-of-function mutations leading to the structural modification of AmpC (ceftazidime), novel DNA gyrase (GyrA) modification (ciprofloxacin), and the alteration of the β-lactam binding site of penicillin-binding protein 3 (PBP3) (meropenem). A further striking finding was seen in the evolution of meropenem resistance, selecting for specific extremely large (>250 kb) genomic deletions providing a growth advantage in the presence of the antibiotic. Finally, fitness and virulence varied within and across evolved antibiotic-resistant populations, but mutator lineages showed a lower biological cost for some antibiotics.

Development of a Genome-Wide Random Mutagenesis System Using Proofreading-Deficient DNA Polymerase δ in the Methylotrophic Yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha is attracting interest as a potential strain for the production of recombinant proteins and biofuels. However, only limited numbers of genome engineering tools are currently available for H. polymorpha. In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase δ of H. polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3'-->5' exonuclease activity. The resulting HpPOL3* gene encoding the error-prone proofreading-deficient DNA polymerase δ was cloned under a methanol oxidase promoter to construct the mutator plasmid pHIF8, which also contains additional elements for site-specific chromosomal integration, selection, and excision. In a H. polymorpha mutator strain chromosomally integrated with pHIF8, a URA3(-) mutant resistant to 5-fluoroorotic acid was generated at a 50-fold higher frequency than in the wild-type strain, due to the dominant negative expression of HpPOL3*. Moreover, after obtaining the desired mutant, the mutator allele was readily removed from the chromosome by homologous recombination to avoid the uncontrolled accumulation of additional mutations. Our mutator system, which depends on the accumulation of random mutations that are incorporated during DNA replication, will be useful to generate strains with mutant phenotypes, especially those related to unknown or multiple genes on the chromosome.

AZT Treatment Increases mtDNA Mutations in HepG2 and CCD-1112Sk Cells

Mitochondrial dysfunction is linked to disease, but it remains unclear whether accumulation of random mutations in the mitochondrial genome is the cause of dysfunction. Using digital or near-digital LATE-PCR with Lights-On/Lights- Off probes we have measured the mutational load in mitochondrial genomes. Exposure of HepG2 and CCD-1112Sk cells to AZT for thirty days caused a significant increase in mutations in the three mitochondrial loci examined. These results demonstrate the utility of our method for analysis of mutational load without sequencing and reinforce the fact that mitochondrial DNA damage due to drugs, aging, and disease should be studied in detail.

Life history trade-offs and relaxed selection can decrease bacterial virulence in environmental reservoirs.

Pathogen virulence is usually thought to evolve in reciprocal selection with the host. While this might be true for obligate pathogens, the life histories of opportunistic pathogens typically alternate between within-host and outside-host environments during the infection-transmission cycle. As a result, opportunistic pathogens are likely to experience conflicting selection pressures across different environments, and this could affect their virulence through life-history trait correlations. We studied these correlations experimentally by exposing an opportunistic bacterial pathogen Serratia marcescens to its natural protist predator Tetrahymena thermophila for 13 weeks, after which we measured changes in bacterial traits related to both anti-predator defence and virulence. We found that anti-predator adaptation (producing predator-resistant biofilm) caused a correlative attenuation in virulence. Even though the direct mechanism was not found, reduction in virulence was most clearly connected to a predator-driven loss of a red bacterial pigment, prodigiosin. Moreover, life-history trait evolution was more divergent among replicate populations in the absence of predation, leading also to lowered virulence in some of the ‘predator absent’ selection lines. Together these findings suggest that the virulence of non-obligatory, opportunistic bacterial pathogens can decrease in environmental reservoirs through life history trade-offs, or random accumulation of mutations that impair virulence traits under relaxed selection.

Somatic mutations in aging, cancer and neurodegeneration

The somatic mutation theory of aging posits that the accumulation of mutations in the genetic material of somatic cells as a function of time results in a decrease in cellular function. In particular, the accumulation of random mutations may inactivate genes that are important for the functioning of the somatic cells of various organ systems of the adult, result in a decrease in organ function. When the organ function decreases below a critical level, death occurs. A significant amount of research has shown that somatic mutations play an important role in aging and a number of age related pathologies. In this review, we explore evidence for increases in somatic nuclear mutation burden with age and the consequences for aging, cancer, and neurodegeneration. We then review evidence for increases in mitochondrial mutation burden and the consequences for dysfunction in the disease processes.

Endless Forms Most Viral

Endless Forms Most Viral

Caterpillars evolved from onychophorans by hybridogenesis

I reject the Darwinian assumption that larvae and their adults evolved from a single common ancestor. Rather I posit that, in animals that metamorphose, the basic types of larvae originated as adults of different lineages, i.e., larvae were transferred when, through hybridization, their genomes were acquired by distantly related animals. "Caterpillars," the name for eruciforms with thoracic and abdominal legs, are larvae of lepidopterans, hymenopterans, and mecopterans (scorpionflies). Grubs and maggots, including the larvae of beetles, bees, and flies, evolved from caterpillars by loss of legs. Caterpillar larval organs are dismantled and reconstructed in the pupal phase. Such indirect developmental patterns (metamorphoses) did not originate solely by accumulation of random mutations followed by natural selection; rather they are fully consistent with my concept of evolution by hybridogenesis. Members of the phylum Onychophora (velvet worms) are proposed as the evolutionary source of caterpillars and their grub or maggot descendants. I present a molecular biological research proposal to test my thesis. By my hypothesis 2 recognizable sets of genes are detectable in the genomes of all insects with caterpillar grub- or maggot-like larvae: (i) onychophoran genes that code for proteins determining larval morphology/physiology and (ii) sequentially expressed insect genes that code for adult proteins. The genomes of insects and other animals that, by contrast, entirely lack larvae comprise recognizable sets of genes from single animal common ancestors.

High tolerance for ionizable residues in the hydrophobic interior of proteins

Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups. The 25 internal positions in staphylococcal nuclease were substituted one at a time with Lys, Glu, or Asp without abolishing enzymatic activity and without detectable changes in the conformation of the protein. Similar results with substitutions of 6 randomly chosen internal positions in ribonuclease H with Lys and Glu suggest that the ability of proteins to tolerate internal ionizable groups might be a property common to many proteins. Eighty-six of the 87 substitutions made were destabilizing, but in all but one case the proteins remained in the native state at neutral pH. By comparing the stability of each variant protein at two different pH values it was established that the pK(a) values of most of the internal ionizable groups are shifted; many of the internal ionizable groups are probably neutral at physiological pH values. These studies demonstrate that special structural adaptations are not needed for ionizable groups to exist stably in the hydrophobic interior of proteins. The studies suggest that enzymes and other proteins that use internal ionizable groups for functional purposes could have evolved through the random accumulation of mutations that introduced ionizable groups at internal positions, followed by evolutionary adaptation and optimization to modulate stability, dynamics, and other factors necessary for function.

Jack-of-All-Trades “Supergene” Controls Butterfly Wing Pattern Diversity

While studying the local flora and fauna of the Amazon jungle in the 1860s, Henry Walter Bates made a striking discovery. Butterflies inhabiting a particular geographic region sported the same wing patterns—even though they were unrelated species. Bates proposed that nonpoisonous species had mimicked the patterns of noxious species that predators avoided, thus gaining a selective advantage. (This adaptation is viewed as one-sided; if predators eat the foul-tasting “model” butterfly, they learn to shun all butterflies with that pattern. But if they sample a palatable mimic, they’re likely to stop avoiding butterflies with that pattern—until they relearn their lesson.) Today, scientists can use the tools of genomics and genetics to investigate the mechanism of convergent evolution—the emergence of similar physical traits (or phenotypes) in unrelated species. Such empirical studies have provided insight into a longstanding controversy spawned by evolutionary theorists over the origin of mimicry: does it arise gradually through the accumulation of random mutations by selection or in “phenotypic leaps” through the constraining influence of shared developmental pathways with a bias toward a particular phenotype? Recent molecular studies found evidence that regulation or recruitment of the same genes or gene variants may explain convergent evolution. In a new study, Mathieu Joron, Chris Jiggins, and colleagues took a different approach by investigating the molecular basis of both convergent and divergent phenotypes. The involvement of the same genomic loci in convergent phenotypes suggests that developmental constraints give rise to these shared phenotypes. The presence of a multitude of convergent and divergent phenotypes in the wing patterns of Heliconius butterflies allowed the researchers to test the possibility that mimetic convergence results from constraints in the regulation of butterfly color patterns. The researchers worked with three species of Heliconius butterflies, including Mullerian mimics (all mimetic species are poisonous, to mutual benefit). Two species, H. melpomene and H. erato, are distantly related yet have identical wing patterns. Both species radiated into over 30 races (or subspecies) in parallel, with the two species (“co-mimics) displaying a single pattern locally. The third species, H. numata, is closely related to H. melpomene but has radically different wing patterns, with up to seven different variations in a single region. Each of these variations mimics a different species of another butterfly genus, Melinaea. In H. melpomene, variation in white and yellow patterns is controlled by at least three genomic loci—N, Yb, and Sb—that are tightly linked, or inherited together. Red pattern elements are controlled by another linked loci pair—B and D. In H. erato, the Cr locus produces patterning effects similar to the interaction of N, Yb, and Sb in H. melpomene, while an unlinked locus, D, appears to control red pattern variation much like the B–D pair does in H. melpomene. In polymorphic H. numata, all the mimicked color patterns derive from a single locus, P, thought to be a “supergene” (a tightly linked cluster of individual genes). The researchers crossed different races of each of the three species to explore the genetic basis of the variations. For example, two different subspecies of H. melpomene from different regions in Ecuador were crossed with an H. melpomene subspecies stock from French Guiana to produce second-generation offspring. Offspring were then genotyped to identify genes responsible for the resulting color patterns and to map the relevant major color-patterning loci—N, Yb, and Sb loci for H. melpomene crosses, Cr for H. erato, and P for H. numata—in individual offspring. Using molecular markers developed in the region of the pattern genes, they found that the three loci controlling color pattern variation for each species inhabit the same genomic location. Indeed, the elements controlling white and yellow pattern variation in H. melpomene (N, Yb, and Sb) and H. erato (Cr) are tightly linked to genetic markers that occupy the same position in both species. Similarly, the locus P, which controls whole-wing variation in H. numata, is also linked to the same series of markers. These results, Joron et al. conclude, suggest that a single conserved locus is responsible for producing wing pattern diversity in Heliconius butterflies. Rather than a constraining role, this locus provides what the researchers call a “jack-of-all-trades flexibility.” It presumably functions as a “developmental switching mechanism” for natural selection, they explain, by responding to a wide range of mimetic pressures to produce radically divergent, locally adapted wing patterns. Now researchers can begin to identify and determine the modus operandi of the genes at the center of what has been called a “developmental hotspot” to better understand how they drive the adaptive evolution of mimetic color pattern shifts. For more on the evolution of mimicry in butterflies, see the Primer (DOI: 10.1371/journal.pbio.0040341 in the October 2006 issue).

Prehistory in blood and bone: An essay on the reconstruction of the past from genetics and morphology

Recent advances in genetic studies of modern humans have proved to be invaluable in tracking the relationships of living human populations through time. This has led to the impression in some quarters that osteological study of fossils has become an outmoded field and has been superceded by genetics. Certainly the use of non-adaptive DNA sequences to record the accumulation of random mutations over time has resulted in an objective method of measuring genetic distances between populations, but there is still much that can be learnt from the study of archaeological bones. Carbon isotope variation reveals prehistoric diets, while samples from cemetery sites provide crucial demographic information, and close study of the individual skeletons can disclose features of growth, life habit and palaeopathology. Ancient DNA is a relatively new field that has yet to make its mark in South African science. The apparent disjunction between the archaeological appearance of modern morphological patterns in KhoiSan populations and the great antiquity of these same populations according to serogenetic data implies that our understanding of the nuclear genome and how it codes for morphology remains far from complete. Until the morphological controls of nuclear genomics are better understood, morphometrics of archaeological specimens will remain the best method of reconstructing prehistoric population patterns from excavated samples.

Healthy ageing: a question of stress, damage and repair. Meeting on mechanisms of biological ageing.

A group of unstressed individuals from different age groups discussed the links between stress and ageing on the Greek island of Spetses, May 18–22, 2002. The conference was sponsored by EURESCO and entitled ‘Biological Agening: 2nd EuroConference on Normal Ageing, Longevity and Age‐Related Diseases’. ![][1] The recent EURESCO meeting on biological ageing provided an excellent overview of several trends emerging in this field of research. After many years during which the field has been dominated by controversy over the role of predetermined genetic programmes versus stochastic damage, it is now becoming clear that ageing is the result of a combination of mechanisms that are genetically modulated via pathways of maintenance and repair. This realization raises many important challenges. T. Kirkwood (Newcastle‐upon‐Tyne, UK) illustrated this with data from a range of model systems (e.g. Caenorhabditis elegans , Drosophila melanogaster , mice, humans) that showed how intrinsic stochastic factors, such as the accumulation of random mutations, combine with genetic and environmental factors, such as the age‐related expression of specific ‘ageing genes’, to shape the individual phenotype. In fact, the challenge of dealing with the heterogeneity of ageing at the molecular, cellular and organismal levels proved to be a recurring theme at this meeting. Integrating these multiple mechanisms also calls for new multidisciplinary approaches, as illustrated by the Biology of Ageing e‐Science Integration and Simulation (BASIS) system being developed in Newcastle. This is planned to be a user‐friendly interactive Internet interface that will involve collaborators around the world. Its main goal is to link theoretical ageing models with experimental data. For example, the ‘virtual ageing cell’ will be a module within which different elements can be activated or suppressed by the user in order to explore predicted outcomes. This module will focus on exploring the mechanisms of deterioration and damage resulting in age‐related impairment … [1]: /embed/graphic-1.gif

How environmental factors regulate mutagenesis and gene transfer in microorganisms

This review is focused on the physiological and evolutionary strategies of the processes occurring during the entry of microbial cells into stationary phase and the subsequent period of stasis. The molecular mechanisms adapting microorganisms from exponential growth to a static state involve activation and complex regulation of the stationary factor Sigma-S, which directs RNA polymerase to the specific promoters. As a result the static cells acquire general resistance (simultaneous tolerances) to different environmental stresses. In parallel with the physiological adaptation to stasis, diverse genetical processes are aimed towards resuming the growth of the static cells. Different types of mutagenesis occur: (i) in cells entering stasis and (ii) in static cells (adaptive mutagenesis). Cessation of growth induces the transient hypermutator state resulting in the accumulation of random mutations in the subpopulation of the static cells. If by chance, one or a few of such mutations lead to resumption of division, the growing cell will return to a normal mechanism of spontaneous mutagenesis. Another mechanism for generating genetical variability in stressed cells involves transposons and conjugative plasmids. Stresses can stimulate the excision of some transposons, which, in turn, can generate chromosomal mutations and activate intracellular mechanisms of mutagenesis. Under stress some conjugative plasmids activate genes encoding antirestriction proteins that repress restriction-modification systems of the recipient cells. Moreover, under stress special cellular mechanisms decrease (alleviate) the activity of restriction-modification systems which, in turn, enhance the probability of gene transfer into the stressed cells. Under stress, the efficiency of inter-species genetical barriers also decreases. This, stimulates inter-species gene transfer and may lead to a burst of incipient speciation in the population of non-growing cells. After resumption of growth the genetical barriers leading to isolation will be restored. In general, the cessation of growth “switches on”, and resumption of growth “switches off”, a set of special processes that are responsible for generating bursts of genetical variability in populations of microorganisms.

A new AluI satellite DNA in the root-knot nematode Meloidogyne fallax: relationships with satellites from the sympatric species M. hapla and M. chitwoodi.

A highly abundant satellite DNA comprising 20% of the Meloidogyne fallax (Nematoda, Tylenchida) genome was cloned and sequenced. The satellite monomer is 173 bp long and has a high A + T content of 72.3%, with frequent runs of A's and T's. The sequence variability of the monomers is 2.7%, mainly due to random distribution of single-point mutations. A search for evidence of internal repeated subunits in the monomer sequence revealed a 6-bp motif (AAATTT) for which five degenerated repeats, differing by just a single base pair, could be identified. Pairwise comparison of the M. fallax satellite with those from the sympatric species Meloidogyne chitwoodi and Meloidogyne hapla revealed a high sequence similarity (68.39%) with one satellite DNA subfamily in M. chitwoodi, which indicated an unexpected close relationship between them. Given the high copy number and the extreme sequence homogeneity among monomeric units, it may be assumed that the satellite DNA of M. fallax could have evolved through some recent and extensive amplification burst in the nematode genome. In this case, its relatively short life would not yet have allowed the accumulation of random mutations in independent amplified repeats. Considering the morphological resemblance between the two species and their ability to produce interspecific fertile hybrids under controlled conditions, these results indicate that M. fallax may share a common ancestor with M. chitwoodi, from which it could have diverged recently. All these data suggest that M. fallax could be the result of a recent speciation process and show that Meloidogyne satellite DNAs may be of interest to resolve phylogenetic relationships among closely related species from this genus.