Adaptive Mutagenesis Research Articles

Incorporation of chemical modulators to enhance algal biomass and bioproduct synthesis

To minimize the cost of algae-based biofuels and bioproducts, boosting biomass productivity and enhancing the stability of cultures in outdoor cultivation systems are imperative. Strain improvement strategies, mainly adaptive evolution, random mutagenesis, and genetic engineering, focus on perturbing the metabolism of algae to develop desired phenotypes. However, the aforementioned techniques are time- and labor-intensive with a possibility of reversion of the mutated/adapted phenotypes back to wild type. In this regard, the chemical genetics approach has emerged as a rapid and powerful approach to preferentially direct the metabolism of algae towards desired target products with the incorporation of small chemical modulators in a dose-dependent manner. In this review, we critically asses the use of chemical modulators to tweak the metabolism of several algal species and enhance biomass production, bioproduct yield, and resilience to abiotic stressors. The review highlights important categories of chemical modulators and the techniques employed to identify and screen such small effective molecules. Chemical modulators enable the re-design and fine-tuning of targeted algal metabolic pathways rendering chemical genetics a promising approach for enhancing algal productivity particularly in large-scale cultivation.

Analysis of translesion polymerases in colorectal cancer cells following cetuximab treatment: A network perspective.

Adaptive mutagenesis observed in colorectal cancer (CRC) cells upon exposure to EGFR inhibitors contributes to the development of resistance and recurrence. Multiple investigations have indicated a parallel between cancer cells and bacteria in terms of exhibiting adaptive mutagenesis. This phenomenon entails a transient and coordinated escalation of error-prone translesion synthesis polymerases (TLS polymerases), resulting in mutagenesis of a magnitude sufficient to drive the selection of resistant phenotypes. In this study, we conducted a comprehensive pan-transcriptome analysis of the regulatory framework within CRC cells, with the objective of identifying potential transcriptome modules encompassing certain translesion polymerases and the associated transcription factors (TFs) that govern them. Our sampling strategy involved the collection of transcriptomic data from tumors treated with cetuximab, an EGFR inhibitor, untreated CRC tumors, and colorectal-derived cell lines, resulting in a diverse dataset. Subsequently, we identified co-regulated modules using weighted correlation network analysis with a minKMEtostay threshold set at 0.5 to minimize false-positive module identifications and mapped the modules to STRING annotations. Furthermore, we explored the putative TFs influencing these modules using KBoost, a kernel PCA regression model. Our analysis did not reveal a distinct transcriptional profile specific to cetuximab treatment. Moreover, we elucidated co-expression modules housing genes, for example, POLK, POLI, POLQ, REV1, POLN, and POLM. Specifically, POLK, POLI, and POLQ were assigned to the "blue" module, which also encompassed critical DNA damage response enzymes, for example. BRCA1, BRCA2, MSH6, and MSH2. To delineate the transcriptional control of this module, we investigated associated TFs, highlighting the roles of prominent cancer-associated TFs, such as CENPA, HNF1A, and E2F7. We found that translesion polymerases are co-regulated with DNA mismatch repair and cell cycle-associated factors. We did not, however, identified any networks specific to cetuximab treatment indicating that the response to EGFR inhibitors relates to a general stress response mechanism.

Evolutionary engineering and chemical mutagenesis of Propionibacterium acidipropionici for improved propionic acid production from sugarcane-derived saccharides

Propionic acid (PA) is a 3-carbon carboxylic acid widely used in the chemical, food and pharmaceutical industries. Current production of PA is primarily via petrochemical routes and alternative sources using renewable feedstocks are desirable. The goal of the present study was to develop bacterial strains for efficient PA production by fermentation of sugarcane-derived saccharides in inverted sugarcane syrup. Adaptive evolution and random chemical mutagenesis of Propionibacterium acidipropionici were individually evaluated. Although evolutionary engineering proved to be a suitable strategy for producing strains tolerant to acid stress, chemical mutagenesis coupled with a novel screening methodology proved to be a superior strategy for strain improvement. The selected strain presented high PA productivity (0.55 g L−1 h−1) and titer (40 g L−1) in relation to the parental strain (0.38 g L−1 h−1 and 27 g L−1, respectively), in fermentations using inverted sugarcane syrup.

Abstract 2477: Exploring the interaction of DNA damage and transcription in the initiation of adaptive mutagenesis in Saccharomyces cerevisiae

Abstract 2477: Exploring the interaction of DNA damage and transcription in the initiation of adaptive mutagenesis in Saccharomyces cerevisiae

Mutational signatures impact the evolution of anti-EGFR antibody resistance in colorectal cancer.

Anti-EGFR antibodies such as cetuximab are active against KRAS/NRAS wild-type colorectal cancers (CRC) but acquired resistance invariably evolves. Which mutational mechanisms enable resistance evolution and whether adaptive mutagenesis, a transient cetuximab-induced increase in mutation generation, contributes in patients is unknown. Here, we investigate this in exome sequencing data of 42 baseline and progression biopsies from cetuximab treated CRCs. Mutation loads did not increase from baseline to progression and evidence for a contribution of adaptive mutagenesis was limited. However, the chemotherapy-induced mutational signature SBS17b was the main contributor of specific KRAS/NRAS and EGFR driver mutations that are enriched at acquired resistance. Detectable SBS17b activity before treatment predicted for shorter progression free survival and for the evolution of these specific mutations during subsequent cetuximab treatment. This suggests that chemotherapy mutagenesis can accelerate resistance evolution. Mutational signatures may be a new class of cancer evolution predictor.

The roles of replication-transcription conflict in mutagenesis and evolution of genome organization.

Replication-transcription conflicts promote mutagenesis and give rise to evolutionary signatures, with fundamental importance to genome stability ranging from bacteria to metastatic cancer cells. This review focuses on the interplay between replication-transcription conflicts and the evolution of gene directionality. In most bacteria, the majority of genes are encoded on the leading strand of replication such that their transcription is co-directional with the direction of DNA replication fork movement. This gene strand bias arises primarily due to negative selection against deleterious consequences of head-on replication-transcription conflict. However, many genes remain head-on. Can head-on orientation provide some benefit? We combine insights from both mechanistic and evolutionary studies, review published work, and analyze gene expression data to evaluate an emerging model that head-on genes are temporal targets for adaptive mutagenesis during stress. We highlight the alternative explanation that genes in the head-on orientation may simply be the result of genomic inversions and relaxed selection acting on nonessential genes. We seek to clarify how the mechanisms of replication-transcription conflict, in concert with other mutagenic mechanisms, balanced by natural selection, have shaped bacterial genome evolution.

Higher Germline Mutagenesis of Genes with Stronger Testis Expressions Refutes the Transcriptional Scanning Hypothesis.

Why are more genes expressed in the testis than in any other organ in mammals? The recently proposed transcriptional scanning hypothesis posits that transcription alleviates mutagenesis through transcription-coupled repair so has been selected in the testis to modulate the germline mutation rate in a gene-specific manner. Here, we show that this hypothesis is theoretically untenable because the selection would be too weak to have an effect in mammals. Furthermore, the analysis purported to support the hypothesis did not control known confounding factors and inappropriately excluded genes with no observed de novo mutations. After remedying these problems, we find the human germline mutation rate of a gene to rise with its testis expression level. This trend also exists for inferred coding strand-originated mutations, suggesting that it arises from transcription-associated mutagenesis. Furthermore, the testis expression level of a gene robustly correlates with its overall expression in other organs, nullifying the need to explain the testis silencing of a minority of genes by adaptive germline mutagenesis. Taken together, our results demonstrate that human testis transcription increases the germline mutation rate, rejecting the transcriptional scanning hypothesis of extensive gene expressions in the mammalian testis.

Connecting Microbial Genotype with Phenotype in the Omics Era.

Although rational design-based metabolic engineering has been applied widely to obtain promising microbial biocatalysts, conventional strategies such as adaptive laboratory evolution (ALE) and mutagenesis are still efficient approaches to improve microorganisms for exceptional features such as a broad spectrum of substrate utilization, robustness of cell growth, as well as high titer, yield, and productivity of the target products. In this chapter, we describe the procedure to generate mutant strains with desired phenotypes using ALE and a new mutagenesis approach of Atmosphere and Room Temperature Plasma (ARTP). In addition, we discuss the methodology to combine next-generation sequencing (NGS)-based genome-resequencing and RNA-Seq transcriptomics approaches to characterize the mutant strains and connect the phenotypes with their corresponding genotypic changes.

The main problem of studying biological evolution

The evolutionary aspect of the emergence problem is the main problem of studying the laws of biological evolution. A possible mechanism of how an ontogenetically modified phenotype (phenotypic modification, “morphosis”) is fixed in the genotype – the problem of “genetic assimilation” is considered. In particular, it is assumed that adaptive mutagenesis is involved in this, generating random multiple mutations that are “not Lamarckian”, but Darwinian, because they occur in random places in the genome. Stress-induced mutations that arise as a result of error-prone repair processes, while not targeting specific genes, are not randomly scattered around the genome. On the contrary, these mutations are concentrated around double-stranded DNA breaks caused by various stressors. It is assumed that the breaks occur with greater probability in actively transcribed DNA regions, reflecting the current activity of the organism and being the most open DNA regions. All this creates the conditions for the more likely appearance of useful mutations in the “trained” locus of the genome. Keywords: selectogenesis, nomogenesis, genetic assimilation, stress-mutagenesis.

Structural Evolution of Human-Specific SRGAP2 Proteins

In the development of the human brain, human-specific genes are considered to play key roles, bringing about its unique advantages and vulnerabilities. At the time when the Homo lineage diverged from Australopithecus, SRGAP2C gradually emerged (3.4-2.4 million years ago, mya) through a process of serial duplications and mutagenesis from the ancestral SRGAP2A. Remarkably, ectopic expression of SRGAP2C endowed mouse brain cultured cells, with human-specific morphologies. To understand the molecular mechanisms underlying SRGAP2A and SRGAP2C function, we have determined the structure of SRGAP2A by an exhaustive molecular symmetry search, utilizing the conserved biological symmetry of the F-BAR fold, and studied the interplay between SRGAP2A and SRGAP2C. We found that: (i) SRGAP2A homo-dimerizes through a large interface that includes an F-BAR, a newly identified F-BAR extension (Fx), and RhoGAP-SH3 domains. (ii) SRGAP2A has an unusual inverse geometry, which enables it to associate with lamellipodia and dendritic spine heads in vivo, and to scaffold membrane protrusions in cell culture. (iii) As a result of the initial partial duplication event (∼3.4 mya), SRGAP2C carries a defective Fx-domain that severely compromises its solubility and membrane scaffolding. Consistently, SRGAP2A:SRAGP2C hetero-dimers are insoluble and therefore likely to be inactive. (iv) The level of SRGAP2A inactivation depends on its hetero-dimerization with SRGAP2C. We found that the primal form of SRGAP2C (P-SRGAP2C, existing between ∼3.4-2.4 mya) is less effective in hetero-dimerizing with SRGAP2A than the modern form of the protein, which carries several substitutions (∼2.4 mya). Thus, the adaptive mutagenesis phase contributed to SRGAP2C activity by improving its ability to associate with SRGAP2A, which indicates a stepwise involvement of SRGAP2C in human evolutionary history.To the best of our knowledge, this is a first detailed structural description of a human specific protein.

Error-prone processing of apurinic/apyrimidinic (AP) sites by PolX underlies a novel mechanism that promotes adaptive mutagenesis in Bacillus subtilis.

In growing cells, apurinic/apyrimidinic (AP) sites generated spontaneously or resulting from the enzymatic elimination of oxidized bases must be processed by AP endonucleases before they compromise cell integrity. Here, we investigated how AP sites and the processing of these noncoding lesions by the AP endonucleases Nfo, ExoA, and Nth contribute to the production of mutations (hisC952, metB5, and leuC427) in starved cells of the Bacillus subtilis YB955 strain. Interestingly, cells from this strain that were deficient for Nfo, ExoA, and Nth accumulated a greater amount of AP sites in the stationary phase than during exponential growth. Moreover, under growth-limiting conditions, the triple nfo exoA nth knockout strain significantly increased the amounts of adaptive his, met, and leu revertants produced by the B. subtilis YB955 parental strain. Of note, the number of stationary-phase-associated reversions in the his, met, and leu alleles produced by the nfo exoA nth strain was significantly decreased following disruption of polX. In contrast, during growth, the reversion rates in the three alleles tested were significantly increased in cells of the nfo exoA nth knockout strain deficient for polymerase X (PolX). Therefore, we postulate that adaptive mutations in B. subtilis can be generated through a novel mechanism mediated by error-prone processing of AP sites accumulated in the stationary phase by the PolX DNA polymerase.

Novel autoproteolytic and DNA-damage sensing components in the bacterial SOS response and oxidized methylcytosine-induced eukaryotic DNA demethylation systems

The bacterial SOS response is an elaborate program for DNA repair, cell cycle regulation and adaptive mutagenesis under stress conditions. Using sensitive sequence and structure analysis, combined with contextual information derived from comparative genomics and domain architectures, we identify two novel domain superfamilies in the SOS response system. We present evidence that one of these, the SOS response associated peptidase (SRAP; Pfam: DUF159) is a novel thiol autopeptidase. Given the involvement of other autopeptidases, such as LexA and UmuD, in the SOS response, this finding suggests that multiple structurally unrelated peptidases have been recruited to this process. The second of these, the ImuB-C superfamily, is linked to the Y-family DNA polymerase-related domain in ImuB, and also occurs as a standalone protein. We present evidence using gene neighborhood analysis that both these domains function with different mutagenic polymerases in bacteria, such as Pol IV (DinB), Pol V (UmuCD) and ImuA-ImuB-DnaE2 and also other repair systems, which either deploy Ku and an ATP-dependent ligase or a SplB-like radical SAM photolyase. We suggest that the SRAP superfamily domain functions as a DNA-associated autoproteolytic switch that recruits diverse repair enzymes upon DNA damage, whereas the ImuB-C domain performs a similar function albeit in a non-catalytic fashion. We propose that C3Orf37, the eukaryotic member of the SRAP superfamily, which has been recently shown to specifically bind DNA with 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, is a sensor for these oxidized bases generated by the TET enzymes from methylcytosine. Hence, its autoproteolytic activity might help it act as a switch that recruits DNA repair enzymes to remove these oxidized methylcytosine species as part of the DNA demethylation pathway downstream of the TET enzymes.ReviewersThis article was reviewed by RDS, RF and GJ.

Selection-Driven Accumulation of Suppressor Mutants in Bacillus subtilis: The Apparent High Mutation Frequency of the Cryptic gudB Gene and the Rapid Clonal Expansion of gudB+ Suppressors Are Due to Growth under Selection

Soil bacteria like Bacillus subtilis can cope with many growth conditions by adjusting gene expression and metabolic pathways. Alternatively, bacteria can spontaneously accumulate beneficial mutations or shape their genomes in response to stress. Recently, it has been observed that a B. subtilis mutant lacking the catabolically active glutamate dehydrogenase (GDH), RocG, mutates the cryptic gudBCR gene at a high frequency. The suppressor mutants express the active GDH GudB, which can fully replace the function of RocG. Interestingly, the cryptic gudBCR allele is stably inherited as long as the bacteria synthesize the functional GDH RocG. Competition experiments revealed that the presence of the cryptic gudBCR allele provides the bacteria with a selective growth advantage when glutamate is scarce. Moreover, the lack of exogenous glutamate is the driving force for the selection of mutants that have inactivated the active gudB gene. In contrast, two functional GDHs are beneficial for the cells when glutamate was available. Thus, the amount of GDH activity strongly affects fitness of the bacteria depending on the availability of exogenous glutamate. At a first glance the high mutation frequency of the cryptic gudBCR allele might be attributed to stress-induced adaptive mutagenesis. However, other loci on the chromosome that could be potentially mutated during growth under the selective pressure that is exerted on a GDH-deficient mutant remained unaffected. Moreover, we show that a GDH-proficient B. subtilis strain has a strong selective growth advantage in a glutamate-dependent manner. Thus, the emergence and rapid clonal expansion of the active gudB allele can be in fact explained by spontaneous mutation and growth under selection without an increase of the mutation rate. Moreover, this study shows that the selective pressure that is exerted on a maladapted bacterium strongly affects the apparent mutation frequency of mutational hot spots.

Yeasts Acquire Resistance Secondary to Antifungal Drug Treatment by Adaptive Mutagenesis

Acquisition of resistance secondary to treatment both by microorganisms and by tumor cells is a major public health concern. Several species of bacteria acquire resistance to various antibiotics through stress-induced responses that have an adaptive mutagenesis effect. So far, adaptive mutagenesis in yeast has only been described when the stress is nutrient deprivation. Here, we hypothesized that adaptive mutagenesis in yeast (Saccharomyces cerevisiae and Candida albicans as model organisms) would also take place in response to antifungal agents (5-fluorocytosine or flucytosine, 5-FC, and caspofungin, CSP), giving rise to resistance secondary to treatment with these agents. We have developed a clinically relevant model where both yeasts acquire resistance when exposed to these agents. Stressful lifestyle associated mutation (SLAM) experiments show that the adaptive mutation frequencies are 20 (S. cerevisiae –5-FC), 600 (C. albicans –5-FC) or 1000 (S. cerevisiae – CSP) fold higher than the spontaneous mutation frequency, the experimental data for C. albicans –5-FC being in agreement with the clinical data of acquisition of resistance secondary to treatment. The spectrum of mutations in the S. cerevisiae –5-FC model differs between spontaneous and acquired, indicating that the molecular mechanisms that generate them are different. Remarkably, in the acquired mutations, an ectopic intrachromosomal recombination with an 87% homologous gene takes place with a high frequency. In conclusion, we present here a clinically relevant adaptive mutation model that fulfils the conditions reported previously.

The PAD region in the mycobacterial DinB homologue MsPolIV exhibits positional heterogeneity

Y-family DNA polymerases (dPols) have evolved to carry out translesion bypass to rescue stalled replication; prokaryotic members of this family also participate in the phenomenon of adaptive mutagenesis to relieve selection pressure imposed by a maladapted environment. In this study, the first structure of a member of this family from a prokaryote has been determined. The structure of MsPolIV, a Y-family dPol from Mycobacterium smegmatis, shows the presence of the characteristic finger, palm and thumb domains. Surprisingly, the electron-density map of the intact protein does not show density for the PAD region that is unique to members of this family. Analysis of the packing of the molecules in the crystals showed the existence of large solvent-filled voids in which the PAD region could be located in multiple conformations. In line with this observation, analytical gel-filtration and dynamic light-scattering studies showed that MsPolIV undergoes significant compaction upon DNA binding. The PAD region is known to insert into the major groove of the substrate DNA and to play a major role in shaping the active site. Comparison with structures of other Y-family dPols shows that in the absence of tertiary contacts between the PAD domain and the other domains this region has the freedom to adopt multiple orientations. This structural attribute of the PAD will allow these enzymes to accommodate the alterations in the width of the DNA double helix that are necessary to achieve translesion bypass and adaptive mutagenesis and will also allow regulation of their activity to prevent adventitious error-prone DNA synthesis.

A Dominant Role for an Error‐Prone DNA Polymerase in Recombination Drives Evolution

Homologous recombination (HR) repairs double‐strand breaks in DNA by directing replication to copy sequence information from a homologous donor DNA. Importantly, this mode of repair is considered error‐free which implies that only high‐fidelity DNA polymerases (pols) perform recombination‐directed replication (RDR). Yet in E. coli, error‐prone Y‐family translesion pols function with RecA recombinase, are highly upregulated in response to stress, and promote mutations following DSBs, which suggests a possible role for these mutagenic pols in HR. Here, we investigate E. coli translesion pol activity in HR by reconstituting RDR in vitro. We find that translesion pol IV is proficient in RDR even without the β‐processivity clamp, whereas translesion pol V lacks this activity. Notably, low‐fidelity pol IV initiates RDR more efficiently than high‐fidelity replicative pol III and is preferentially recruited to D‐loop recombination intermediates under stress response conditions. Unexpectedly, we find that pol III is inhibited in RDR due to its proofreading activity which is stimulated at D‐loops. Finally, we show that pol IV promotes mutations during RDR. These data demonstrate a dominant role for error‐prone pol IV in HR and elucidate the molecular basis of adaptive mutagenesis which facilitates evolution under conditions of stress.

Interaction of gene HSM3 with genes of the epistatic RAD6 group in yeast Saccharomyces cerevisiae

In eukaryotes, damage tolerance of matrix DNA is mainly determined by the repair pathway under the control of the RAD6 epistatic group of genes. T this pathway is also a main source of mutations generated by mutagenic factors. The results of our recent studies show that gene HSM3 participating in the control of adaptive mutagenesis increases the frequency of mutations induced by different mutagens. Mutations rad18, rev3, and mms2 controlling various stages of the RAD6 pathway are epistatic with mutation hsm3 that decreases UV-induced mutagenesis to the level typical for single radiation-sensitive mutants. The level of mutagenesis in the double mutant srs2 hsm3 was lower than in both single mutants. Note that a decrease in the level of mutagenesis relative to the single mutant srs2 depends on the mismatch repair, since this level in the triple mutant srs2 hsm3 pms 1 corresponds to that in the single mutant srs2. These data show that the mutator phenotype hsm3 is probably determined by processes occurring in a D loop. In a number of current works, the protein Hsm3 was shown to participate in the assembly of the proteasome complex S26. The assembly of proteasomes is governed by the N-terminal domain. Our results demonstrated that the Hsm3 protein contains at least two domains; the N-terminal part of the domain is responsible for the proteasome assembly, whereas the C-terminal portion of the protein is responsible for mutagenesis.

MsDpo4—a DinB Homolog fromMycobacterium smegmatis—Is an Error-Prone DNA Polymerase That Can Promote G:T and T:G Mismatches

Error-prone DNA synthesis in prokaryotes imparts plasticity to the genome to allow for evolution in unfavorable environmental conditions, and this phenomenon is termed adaptive mutagenesis. At a molecular level, adaptive mutagenesis is mediated by upregulating the expression of specialized error-prone DNA polymerases that generally belong to the Y-family, such as the polypeptide product of the dinB gene in case of E. coli. However, unlike E. coli, it has been seen that expression of the homologs of dinB in Mycobacterium tuberculosis are not upregulated under conditions of stress. These studies suggest that DinB homologs in Mycobacteria might not be able to promote mismatches and participate in adaptive mutagenesis. We show that a representative homolog from Mycobacterium smegmatis (MsDpo4) can carry out template-dependent nucleotide incorporation and therefore is a DNA polymerase. In addition, it is seen that MsDpo4 is also capable of misincorporation with a significant ability to promote G:T and T:G mismatches. The frequency of misincorporation for these two mismatches is similar to that exhibited by archaeal and prokaryotic homologs. Overall, our data show that MsDpo4 has the capacity to facilitate transition mutations and can potentially impart plasticity to the genome.

Roles of Endonuclease V, Uracil-DNA Glycosylase, and Mismatch Repair in Bacillus subtilis DNA Base-Deamination-Induced Mutagenesis

The disruption of ung, the unique uracil-DNA-glycosylase-encoding gene in Bacillus subtilis, slightly increased the spontaneous mutation frequency to rifampin resistance (Rif(r)), suggesting that additional repair pathways counteract the mutagenic effects of uracil in this microorganism. An alternative excision repair pathway is involved in this process, as the loss of YwqL, a putative endonuclease V homolog, significantly increased the mutation frequency of the ung null mutant, suggesting that Ung and YwqL both reduce the mutagenic effects of base deamination. Consistent with this notion, sodium bisulfite (SB) increased the Rif(r) mutation frequency of the single ung and double ung ywqL strains, and the absence of Ung and/or YwqL decreased the ability of B. subtilis to eliminate uracil from DNA. Interestingly, the Rif(r) mutation frequency of single ung and mutSL (mismatch repair [MMR] system) mutants was dramatically increased in a ung knockout strain that was also deficient in MutSL, suggesting that the MMR pathway also counteracts the mutagenic effects of uracil. Since the mutation frequency of the ung mutSL strain was significantly increased by SB, in addition to Ung, the mutagenic effects promoted by base deamination in growing B. subtilis cells are prevented not only by YwqL but also by MMR. Importantly, in nondividing cells of B. subtilis, the accumulations of mutations in three chromosomal alleles were significantly diminished following the disruption of ung and ywqL. Thus, under conditions of nutritional stress, the processing of deaminated bases in B. subtilis may normally occur in an error-prone manner to promote adaptive mutagenesis.

Analysis of miRNA duplication in the human genome and the role of transposon evolution in this process

Duplication of miRNAs is one of the modes of their evolution. This study demonstrates that human miRNAs arranged mostly in 5000-nt clusters and their copies are scattered randomly throughout the genome at an average distance of 4.3 × 106 bp. We compared the neighborhood of miRNAs copies with the transposable elements (TEs) and found that most miRNAs homologues (96%) propagate by DNA transposons and retroelements. Copies of primate-specific made1 elements containing the has-mir-548 miRNAs family contributed the most to this process. An analysis of made1’s evolution supports the earlier observation that these TEs emerged and multiplied explosively over the genome of a common ancestor and diverged later at average genomic rates. At that time, the modern copies of the has-mir-548 family appeared due to adaptive mutagenesis of some of the transposons copies; they were recruited into the silencing system. Nucleotide changes leading to transposon transformation into miRNAs genes and numerous miRNA copies may be helpful in predicting new miRNAs.