Amplification-mutagenesis: evidence that "directed" adaptive mutation and general hypermutability result from growth with a selected gene amplification

When a particular lac mutant of Escherichia coli starves in the presence of lactose, nongrowing cells appear to direct mutations preferentially to sites that allow growth (adaptive mutation). This observation suggested that growth limitation stimulates mutability. Evidence is provided here that this behavior is actually caused by a standard Darwinian process in which natural selection acts in three sequential steps. First, growth limitation favors growth of a subpopulation with an amplification of the mutant lac gene; next, it favors cells with a lac(+) revertant allele within the amplified array. Finally, it favors loss of mutant copies until a stable haploid lac(+) revertant arises and overgrows the colony. By increasing the lac copy number, selection enhances the likelihood of reversion within each developing clone. This sequence of events appears to direct mutations to useful sites. General mutagenesis is a side-effect of growth with an amplification (SOS induction). The F' plasmid, which carries lac, contributes by stimulating gene duplication and amplification. Selective stress has no direct effect on mutation rate or target specificity, but acts to favor a succession of cell types with progressively improved growth on lactose. The sequence of events--amplification, mutation, segregation--may help to explain both the origins of some cancers and the evolution of new genes under selection.     

Convergent neofunctionalization by positive Darwinian selection after ancient recurrent duplications of the xanthine dehydrogenase gene

Gene duplication is a primary source of molecular substrate for the emergence of evolutionary novelties. The chances for redundant gene sequences to evolve new functions are small compared with the probability that the copies become disabled by deleterious mutations. Functional divergence after gene duplication can result in two alternative evolutionary fates: one copy acquires a novel function (neofunctionalization), or each copy adopts part of the tasks of their parental gene (subfunctionalization). The relative prevalence of each outcome is unknown. Similarly unknown is the relative importance of positive selection versus random fixation of neutral mutations. Aldehyde oxidase (Ao) and xanthine dehydrogenase (Xdh) genes encode two complex members of the xanthine oxidase family of molybdo-flavoenzymes that carry different functions. Ao is known to have originated from a duplicate of an Xdh gene in eukaryotes, before the origin of multicellularity. We show that (i) Ao evolved independently twice from two different Xdh paralogs, the second time in the chordates, before the diversification of vertebrates; (ii) after each duplication, the Ao duplicate underwent a period of rapid evolution during which identical sites across the two molecules, involving the flavin adenine dinucleotide and substrate-binding pockets, were subjected to intense positive Darwinian selection; and (iii) the second Ao gene likely endured two periods of redundancy, initially as a duplicate of Xdh and later as a functional equivalent of the old Ao, which is currently absent from the vertebrate genome. Caution is appropriate in structural genomics when using sequence similarity for assigning protein function.     

Multiple pathways of selected gene amplification during adaptive mutation

In a phenomenon referred to as "adaptive mutation," a population of bacterial cells with a mutation in the lac operon (lac-) accumulates Lac+ revertants during prolonged exposure to selective growth conditions (lactose). Evidence was provided that selective conditions do not increase the mutation rate but instead favor the growth of rare cells with a duplication of the leaky lac allele. A further increase in copy number (amplification) improves growth and increases the likelihood of a sequence change by adding more mutational targets to the clone (cells and lac copies per cell). These duplications and amplifications are described here. Before selection, cells with large (134-kb) lac duplications and long junction sequences (>1 kb) were common (0.2%). The same large repeats were found after selection in cells with a low-copy-number lac amplification. Surprisingly, smaller repeats (average, 34 kb) were found in high-copy-number amplifications. The small-repeat duplications form when deletions modify a preexisting large-repeat duplication. The shorter repeat size allowed higher lac amplification and better growth on lactose. Thus, selection favors a succession of gene-amplification types that make sequence changes more probable by adding targets. These findings are relevant to genetic adaptation in any biological systems in which fitness can be increased by adding gene copies (e.g., cancer and bacterial drug resistance).     

Buffering of crucial functions by paleologous duplicated genes may contribute cyclicality to angiosperm genome duplication

Genome duplication followed by massive gene loss has permanently shaped the genomes of many higher eukaryotes, particularly angiosperms. It has long been believed that a primary advantage of genome duplication is the opportunity for the evolution of genes with new functions by modification of duplicated genes. If so, then patterns of genetic diversity among strains within taxa might reveal footprints of selection that are consistent with this advantage. Contrary to classical predictions that duplicated genes may be relatively free to acquire unique functionality, we find among both Arabidopsis ecotypes and Oryza subspecies that SNPs encode less radical amino acid changes in genes for which there exists a duplicated copy at a "paleologous" locus than in "singleton" genes. Preferential retention of duplicated genes encoding long complex proteins and their unexpectedly slow divergence (perhaps because of homogenization) suggest that a primary advantage of retaining duplicated paleologs may be the buffering of crucial functions. Functional buffering and functional divergence may represent extremes in the spectrum of duplicated gene fates. Functional buffering may be especially important during "genomic turmoil" immediately after genome duplication but continues to act approximately 60 million years later, and its gradual deterioration may contribute cyclicality to genome duplication in some lineages.     

Emergence of primate genes by retrotransposon-mediated sequence transduction

Gene duplication is one of the most important mechanisms for creating new genes and generating genomic novelty. Retrotransposon-mediated sequence transduction (i.e., the process by which a retrotransposon carries flanking sequence during its mobilization) has been proposed as a gene duplication mechanism. L1 exon shuffling potential has been reported in cell culture assays, and two potential L1-mediated exon shuffling events have been identified in the genome. SVA is the youngest retrotransposon family in primates and is capable of 3' flanking sequence transduction during retrotransposition. In this study, we examined all of the full-length SVA elements in the human genome to assess the frequency and impact of SVA-mediated 3' sequence transduction. Our results showed that approximately 53 kb of genomic sequences have been duplicated by 143 different SVA-mediated transduction events. In particular, we identified one group of SVA elements that duplicated the entire AMAC gene three times in the human genome through SVA-mediated transduction events, which happened before the divergence of humans and African great apes. In addition to the original AMAC gene, the three transduced AMAC copies contain intact ORFs in the human genome, and at least two are actively transcribed in different human tissues. The duplication of entire genes and the creation of previously undescribed gene families through retrotransposon-mediated sequence transduction represent an important mechanism by which mobile elements impact their host genomes.     

Amplification of N-myc oncogene in human melanoma cells

Tissue specimens from two melanoma patients and two patients with laryngeal carcinoma were studied for the expression of c-myc, N-myc and v-src oncogenes. Out of three different probes, only the N-myc probe one signalled amplification. While the two patients with laryngeal carcinoma showed only single copy of the N-myc gene in tumor cells, amplification of this gene was found in both two melanoma patients. The patients with 1 extra copy of the N-myc gene and its protein product had an early recurrence but is still alive, while the other melanoma patient with 4 extra copies of the gene, relapsed very early and died of melanoma within 7 months after diagnosis. Thus a higher amplification (4 extra copies) seems to correlate with very poor outcome of the disease.     

Heterogeneous lengths of copy number mutations in human coagulopathy revealed by genome-wide high-density SNP array

Background

The recent advent of genome-wide molecular platforms has facilitated our understanding of the human genome and disease, particularly copy number aberrations. We performed genome-wide single nucleotide polymorphism-array in hereditary coagulopathy to delineate the extent of copy number mutations and to assess its diagnostic utility.

Design and Methods

The study subjects were 17 patients with hereditary coagulopathy from copy number mutations in coagulation genes detected by multiple ligation-dependent probe amplification. Eleven had hemophilia (7 hemophilia A and 4 hemophilia B) and 6 had thrombophilia (4 protein S deficiency and 2 antithrombin deficiency). Single nucleotide polymorphism-array experiments were performed using Affymetrix Genome-Wide Human SNP arrays 6.0.

Results

Copy number mutations were identified by single nucleotide polymorphism-array in 9 patients, which ranged in length from 51 Kb to 6,288 Kb harboring 2 to ~160 genes. Single nucleotide polymorphism-array showed a neutral copy number status in 8 patients including 7 with either a single-exon copy number mutation or duplication mutations of PROS1.

Conclusions

This study revealed unexpectedly heterogeneous lengths of copy number mutations underlying human coagulopathy. Single nucleotide polymorphism-array had limitations in detecting copy number mutations involving a single exon or those of a gene with homologous sequences such as a pseudogene.     

Newly arisen DNA repeats in primate phylogeny.

We discovered the presence of an Alu and an Xba repetitive DNA element within introns 4 and 7, respectively, of the human alpha-fetoprotein (AFP) gene; these elements are absent from the same gene in the gorilla. The Alu element is flanked by 12-base-pair direct repeats, AGGATGTTGTGG ... (Alu) ... AGGATGTTGTGG, which presumably arose by way of duplication of the intronic target site AGGATGTTGTGG at the time of the Alu insertion. In the gorilla, only a single copy of the unoccupied target site is present, which is identical to the terminal repeat flanking the human Alu element. There are two copies of an Xba repeat in the human AFP gene, apparently the only two in the genome. Xba1 and Xba2, located within introns 8 and 7, respectively, differ from each other at 3 of 303 positions. Xba1 is referred to as the old (ancestral) repeat because it lacks direct repeats. The new (derived) Xba2 is flanked by direct repeats, TTTCTTTTT ... (Xba) ... TTTCTTCTT, and is thought to have arisen as a result of transposition of Xba1. The ancestral Xba1 and a single copy of the Xba2 target site are present at orthologous positions in the gorilla, but the new Xba2 is absent. We conclude that the Alu and Xba DNA repeats emerged in the human genome at a time postdating the human-gorilla divergence and became established as genetic novelties in the human lineage. We submit that the chronology of divergence of primate lines of evolution can be correlated with the timing of insertion of new DNA repeats into the genomes of those primates.     

AML1 gene amplification: a novel finding in childhood acute lymphoblastic leukemia

BACKGROUND AND OBJECTIVE: We previously found a high-level amplification in chromosomal region 21q22 in two children with acute lymphoblastic leukemia (ALL) using comparative genomic hybridization. The same region harbors the AML1 gene. The aim of the present study was to investigate whether AML1 is a target gene in these amplifications. DESIGN AND METHODS: Bone marrow samples were obtained from 112 childhood ALL patients. The copy number of AML1 was studied using fluorescent in situ hybridization with a dual color DNA probe specific for the AML1 and TEL genes. RESULTS: Three of the patients had 3-to-8 fold amplification of AML1 and showed a high-level amplification of 21q22 by comparative genomic hybridization. In two of them the extra copies were shown to be located tandemly in a derivative of chromosome 21. Thirty-seven of the patients (33%) had 1-to-2 extra copies of AML1, most probably reflecting the incidence of trisomy 21 and tetrasomy 21. The TEL-AML1 fusion was less frequent in the patients with extra copies of AML1 (7/40; 18%) than in the patients with no extra copy (24/72; 33%). None of the three patients with 3-to-8 fold amplification of AML1 showed the fusion or loss of TEL. INTERPRETATION AND CONCLUSIONS: Our findings suggest that the AML1 gene is a target gene in the 21q22 amplicon in childhood ALL. To understand the role, if any, of the AML1 amplification in leukemogenesis, further studies are needed.     

Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli

Duplicated genes provide an important raw material for adaptive evolution. However, the relationship between gene duplication and the emergence of new biochemical functions is complicated, and it has been difficult to quantify the likelihood of evolving novelty in any systematic manner. Here, we describe a comprehensive search for artificially amplified genes that are able to impart new phenotypes on Escherichia coli, provided their expression is up-regulated. We used a high-throughput, library-on-library strategy to screen for resistance to antibiotics and toxins. Cells containing a complete E. coli ORF library were exposed to 237 toxin-containing environments. From 86 of these environments, we identified a total of 115 cases where overexpressed ORFs imparted improved growth. Of the overexpressed ORFs that we tested, most conferred small but reproducible increases in minimum inhibitory concentration (≤16-fold) for their corresponding antibiotics. In many cases, proteins were acting promiscuously to impart resistance. In the absence of toxins, most strains bore no fitness cost associated with ORF overexpression. Our results show that even the genome of a nonpathogenic bacterium harbors a substantial reservoir of resistance genes, which can be readily accessed through overexpression mutations. During the growth of a population under selection, these mutations are most likely to be gene amplifications. Therefore, our work provides validation and biochemical insight into the innovation, amplification, and divergence model of gene evolution under continuous selection [Bergthorsson U, Andersson DI, Roth JR (2007) Proc Natl Acad Sci USA 104:17004-17009], and also illustrates the high frequency at which novel traits can evolve in bacterial populations.     

DNA methylation prevents the amplification of TROP1, a tumor-associated cell surface antigen gene.

We tested the hypothesis that different genes can have different abilities to be amplified after transfection under comparable selection conditions. DNA from human lymphoid or choriocarcinoma cell lines was transfected into L cells. Transfectants for CD5, CD8A, TROP1, and TROP2, genes expressed on lymphocytes or trophoblast and carcinomas, were selected by fluorescence-activated cell sorting. To select for amplification of the transfected gene we cloned twice by fluorescence-activated cell sorting the transfectants with the highest expression. We analyzed a total of 38 families (1768 clones) derived from the original transfectants. We then analyzed by Southern blotting the clones with the highest increase in surface expression and determined the copy number of each transfected gene. CD5, CD8A, and TROP2 were amplified with high frequency and progressively, whereas TROP1 essentially was not amplified at all. We examined the hypothesis that DNA methylation prevents the amplification of the TROP1 gene by treating JAR choriocarcinoma cells with 5-azacytidine to decrease DNA methylation. DNA extracted at different times after the treatment was used for transfection. When DNA that showed demethylation of the TROP1 gene was used, 16 Trop-1 transfectants were obtained and 6 of them were found to contain up to 40 copies of the TROP1 gene per haploid genome. Thus, we showed that transfectants obtained from a demethylated TROP1 gene were amplified efficiently and progressively. We propose that DNA methylation affects DNA amplification either by altering the recognition of methylated DNA sequences or by changing the conformation of the chromatin of methylated segments. We speculate that DNA methylation is a determinant of gene amplification in vivo, for example in tumor cells.     

Genomic environment of variant surface antigen genes of Trypanosoma equiperdum.

Expression of variant antigen genes in Trypanosoma equiperdum is accompanied by the duplication of a silent basic copy gene and the transposition of the copy to an expression site elsewhere in the genome. We have analyzed the genomic locations of both the basic and expression-linked copies of the T. equiperdum gene for variable surface glycoprotein VSG-1. Both copies are situated proximal to termini in both extracted DNA and in chromatin. The regions between the VSG-1 genes and the termini have a very high buoyant density in CsCl and contain an unidentified nucleoside that replaces deoxycytidine.     

Decoupled evolution of coding region and mRNA expression patterns after gene duplication: implications for the neutralist-selectionist debate

The neutralist perspective on molecular evolution maintains that the vast majority of mutations affecting gene function are neutral or deleterious. After a gene duplication where both genes are retained, it predicts that original and duplicate genes diverge at clock-like rates. This prediction is usually tested for coding sequences, but can also be applied to another important aspect of gene function, the genes' expression pattern. Moreover, if both sequence and expression pattern diverge at clock-like rates, a correlation between divergence in sequence and divergence in expression patterns is expected. Duplicate gene pairs with more highly diverged sequences should also show more highly diverged expression patterns. This prediction is tested for a large sample of duplicated genes in the yeast Saccharomyces cerevisiae, using both genome sequence and microarray expression data. Only a weak correlation is observed, suggesting that coding sequence and mRNA expression patterns of duplicate gene pairs evolve independently and at vastly different rates. Implications of this finding for the neutralist-selectionist debate are discussed.     

Massive amplification of rolling-circle transposons in the lineage of the bat Myotis lucifugus

Rolling-circle (RC) transposons, or Helitrons, are a newly recognized group of eukaryotic transposable elements abundant in the genomes of plants, invertebrates, and zebrafish. We provide evidence for the colonization of a mammalian genome by Helitrons, which has not been reported previously. We identified and characterized two families of Helitrons in the little brown bat Myotis lucifugus. The consensus sequence for the first family, HeliBat1, displays the hallmarks of an autonomous Helitron, including coding capacity for an approximately 1,500-aa protein with an RC replication motif and a region related to the SF1 superfamily of DNA helicases. The HeliBatN1 family is a nonautonomous Helitron family that is only distantly related to HeliBat1. The two HeliBat families have attained high copy numbers (approximately 15,000 and > 100,000 copies, respectively) and make up at least approximately 3% of the M. lucifugus genome. Sequence divergence and cross-species analyses indicate that both HeliBat families have amplified within the last approximately 30-36 million years and are restricted to the lineage of vesper bats. We could not detect the presence of Helitrons in any other order of placental mammals, despite the broad representation of these taxa in the databases. We describe an instance of HeliBat-mediated transduction of a host gene fragment that was subsequently dispersed in approximately 1,000 copies throughout the M. lucifugus genome. Given the demonstrated propensity of RC transposons to mediate the duplication and shuffling of host genes in bacteria and maize, it is tempting to speculate that the massive amplification of Helitrons in vesper bats has influenced the evolutionary trajectory of these mammals.     

Glucocerebrosidase recombinant allele: molecular evolution of the glucocerebrosidase gene and pseudogene in primates

Glucocerebrosidase is a lysosomal enzyme that hydrolyses the beta-glycosidic linkage of glucocerebroside, a ubiquitous sphingolipid present in the plasma membrane of mammalian cells. Deleterious mutations in the glucocerebrosidase gene result in Gaucher disease, the most prevalent lysosomal storage disease. Humans have one glucocerebrosidase functional gene and pseudogene that were located 16 kb apart on chromosome 1q21 and share 96% overall sequence similarity. Recombination between the two genes creates a 'complex allele' that renders glucocerebrosidase non-functional and accounts for >20% of the total Gaucher disease mutations in some population. The glucocerebrosidase pseudogene is absent in all other mammalian species surveyed so far. In order to learn more about the molecular evolution of the glucocerebrosidase functional gene and pseudogene, we have sequenced approximately 1.1 kb of the C-terminal region of these genes that encodes the enzyme catalytic site, from PCR-amplified genomic DNA of gorilla, chimpanzee, orangutan (the great apes), and squirrel monkey (a new-world monkey). In gorilla, chimpanzee, and orangutan, there are two copies of the glucocerebrosidase gene while the squirrel monkey possesses only a single copy. Similar to human, the second copy of glucocerebrosidase gene in gorilla and chimpanzee is non-functional because of a 55-bp deletion in exon 9, while that in orangutan appears to be unaffected and may still be functional. These data suggest that the glucocerebrosidase gene duplication event occurred after squirrel monkey divergence from the great apes, and that the exon 9 deletion that rendered the second copy of the glucocerebrosidase gene non-functional occurred prior to the divergence of gorilla and chimpanzee but after the divergence of orangutan from their common ancestor to human. The two genes in each species are least similar in gorilla and chimpanzee (97.8%) and most similar in orangutan (99.5%). None of the nucleotide variations in the GBA gene among the primates correspond to known mutations in Gaucher disease. Phylogenetic tree analysis using DNAstar and PAUP4.0 software indicates that gene conversion caused the evolution of glucocerebrosidase functional gene and pseudogene to be concerted.     

Comparing sequenced segments of the tomato and Arabidopsis genomes: large-scale duplication followed by selective gene loss creates a network of synteny

A 105-kilobase bacterial artificial chromosome (BAC) clone from the ovate-containing region of tomato chromosome 2 was sequenced and annotated. The tomato BAC sequence was then compared, gene by gene, with the sequenced portions of the Arabidopsis thaliana genome. Rather than matching a single portion of the Arabidopsis genome, the tomato clone shows conservation of gene content and order with four different segments of Arabidopsis chromosomes 2-5. The gene order and content of these individual Arabidopsis segments indicate that they derived from a common ancestral segment through two or more rounds of large-scale genome duplication events-possibly polyploidy. One of these duplication events is ancient and may predate the divergence of the Arabidopsis and tomato lineages. The other is more recent and is estimated to have occurred after the divergence of tomato and Arabidopsis approximately 112 million years ago. Together, these data suggest that, on the scale of BAC-sized segments of DNA, chromosomal rearrangements (e.g., inversions and translocations) have been only a minor factor in the divergence of genome organization among plants. Rather, the dominating factors have been repeated rounds of large-scale genome duplication followed by selective gene loss. We hypothesize that these processes have led to the network of synteny revealed between tomato and Arabidopsis and predict that such networks of synteny will be common when making comparisons among higher plant taxa (e.g., families).     

Invasion and maintenance of a gene duplication.

The ubiquity of multigene families is evidence for the frequent occurrence of gene duplication, but the origin of multigene families from a single gene remains a little-studied aspect of genome evolution. Although it is clear that a duplication can arise and become fixed in a population purely by random genetic drift and that the rate of fixation is accelerated if the duplication is directly advantageous, the nature of gene duplication suggests that other factors may influence the fate of a novel duplication. In the face of disadvantageous loss-of-function mutations, duplication of a functional gene may provide a buffer against such mutations. Here the conditions for invasion of a rare duplication starting from a mutation-selection balance are derived with formal population genetic models in both haploids and diploids. Recurrent duplication protects the duplicated chromosome from loss and can be very effective in increasing its frequency in a population. In the absence of recurrent duplication, one might suppose that a duplication would be favored by natural selection because it can mask the effects of deleterious mutations. However, the models show that a duplication can invade only if it provides a direct advantage to the organism. This result is closely related to recent theoretical work on the evolution of ploidy.