A model of DNA sequence evolution

Statistical studies of gene populations on the purine/pyrimidine alphabet have shown that the mean occurrence probability of thei-motif YRY(N) _i YRY (R=purine, Y=pyrimidine, N=R or Y) is not uniform by varyingi in the range [1,99], but presents a maximum ati=6 in the following populations: protein coding genes of eukaryotes, prokaryotes, chloroplasts and mitrochondria, and also viral introns, ribosomal RNA genes and transfer RNA genes (Arquès and Michel, 1987b,J. theor. Biol. 128, 457–461). From the “universality” of this observation, we suggested that the oligonucleotide YRY(N)₆ is a primitive one and that it has a central function in DNA sequence evolution (Arquès and Michel, 1987b,J. theor. Biol. 128, 457–461). Following this idea, we introduce a concept of a model of DNA sequence evolution which will be validated according to a shema presented in three parts. In the first part, using the last version of the gene database, the YRY(N)₆YRY preferential occurrence (maximum ati=6) is confirmed for the populations mentioned above and is extended to some newly analysed populations: chloroplast introns, chloroplast 5′ regions, mitochondrial 5′ regions and small nuclear RNA genes. On the other hand, the YRY(N)₆YRY preferential occurrence and periodicities are used in order to classify 18 gene populations. In the second part, we will demonstrate that several statistical features characterizing different gene populations (in particular the YRY(N)₆YRY preferential occurrence and the periodicities) can be retrieved from a simple Markov model based on the mixing of the two oligonucleotides YRY(N)₆ and YRY(N)₃ and based on the percentages of RYR and YRY in the unspecified trinucleotides (N)₃ of YRY(N)₆ and YRY(N)₃. Several properties are identified and prove in particular that the oligonucleotide mixing is an independent process and that several different features are functions of a unique parameter. In the third part, the return of the model to the reality shows a strong correlation between reality and simulation concerning the presence of large alternating purine/pyrimidine stretches and of periodicities. It also contributes to a greater understanding of biological reality, e.g. the presence or the absence of large alternating purine/pyrimidine stretches can be explained as being a simple consequence of the mixing of two particular oligonucleotides. Finally, we believe that such an approach is the first step toward a unified model of DNA sequence evolution allowing the molecular understanding of both the origin of life and the actual biological reality.     

A study of the purine/pyrimidine codon occurrence with a reduced centered variable and an evaluation compared to the frequency statistic

With the three-letter alphabet [R,Y,N] (R = purine, Y = pyrimidine, N = R or Y), there are 26 codons (NNN being excluded): RNN,...,NNY (six codons at two unspecified bases N), RRN,...,NYY (12 codons at one unspecified base N), RRR,...,YYY (eight specified codons). A statistical methodology that uses the codon frequency and a reduced centered variable leads to similar results for a codon occurrence study, regardless of gene function and regardless of a particular protein coding gene taxonomic population. Therefore, this variable can be considered a new codon usage index, whose use removes certain nonsignificant results found with the frequency statistic. This methodology identifies the common and rare codons (i.e., the codons having the highest and lowest occurrence) and leads to a model of codon evolution at three successive states: RNN, then RNY, and finally RYY. Some biological relations between this model and the YRY(N)6YRY preferential occurrence are also presented.     

A purine-pyrimidine motif verifying an identical presence in almost all gene taxonomic groups

A statistical parameter identifies, with a high degree of significance, a motif which is present in protein-coding sequences of eukaryotes, prokaryotes, chloroplasts, mitochondria, viral introns, ribosomal RNA genes, and transfer RNA genes. The random probability of occurrence of such a situation is 10(-12). This motif has the following properties: (i) its significant presence in almost all present-day genes explains why it can be considered as primitive oligonucleotide, (ii) its nucleotide order is: YRY (N)6YRY, R being a purine base, Y a pyrimidine one and N any base, (iii) its length and its terminal trinucleotides YRY suggest a primordial function related to the spatial structure of the DNA sequences. This motif is found in some viral protein-coding genes, but not in eukaryotic introns.     

Analytical expression of the purine/pyrimidine codon probability after and before random mutations

Recently, we proposed a new model of DNA sequence evolution (Arquès and Michel. 1990b.Bull. math. Biol. 52, 741–772) according to which actual genes on the purine/pyrimidine (R/Y) alphabet (R=purine=adenine or guanine, Y=pyrimidine=cytosine or thymine) are the result of two successive evolutionary genetic processes: (i) a mixing (independent) process of non-random oligonucleotides (words of base length less than 10: YRY(N)₆, YRYRYR and YRYYRY are so far identified; N=R or Y) leading to primitive genes (words of several hundreds of base length) and followed by (ii) a random mutation process, i.e. transformations of a base R (respectively Y) into the base Y (respectively R) at random sites in these primitive genes. Following this model the problem investigated here is the study of the variation of the 8 R/Y codon probabilities RRR,..., YYY under random mutations. Two analytical expressions solved here allow analysis of this variation in the classical evolutionary sense (from the past to the present, i.e. after random mutations), but also in the inverted evolutionary sense (from the present to the past, i.e. before random mutations). Different properties are also derived from these formulae. Finally, a few applications of these formulae are presented. They prove the proposition in Arquès and Michel (1990b.Bull. math. Biol. 52, 741–772), Section 3.3.2, with the existence of a miximal mean number of random mutations per base of the order 0.3 in the protein coding genes. They also confirm the mixing process of oligonucleotides by excluding the purine/pyrimidine contiguous and alternating tracts from the formation process of primitive genes.     

Periodicities in coding and noncoding regions of the genes

Gene population statistical studies of protein coding genes and introns have identified two types of periodicities on the purine/pyrimidine alphabet: (i) the modulo 3 periodicity or coding periodicity (periodicity P3) in protein coding genes of eukaryotes, prokaryotes, viruses, chloroplasts, mitochondria, plasmids and in introns of viruses and mitochondria, and (ii) the modulo 2 periodicity (periodicity P2) in the eukaryotic introns. The periodicity study is herein extended to the 5' and 3' regions of eukaryotes, prokaryotes and viruses and shows: (i) the periodicity P3 in the 5' and 3' regions of eukaryotes. Therefore, these observations suggest a unitary and dynamic concept for the genes as for a given genome, the 5' and 3' regions have the genetic information for protein coding genes and for introns: (1) In the eukaryotic genome, the 5' (P2 and P3) and 3' (P2 and P3) regions have the information for protein coding genes (P3) and for introns (P2). The intensity of P3 is high in 5' regions and weak in 3' regions, while the intensity of P2 is weak in 5' regions and high in 3' regions. (2) In the prokaryotic genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3). (3) In the viral genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3) and for introns (P3). The absence of P2 in viral introns (in opposition to eukaryotic introns) may be related to the absence of P2 in 5' and 3' regions of viruses.     

Trypanosoma brucei RNA editing protein TbMP42 (band VI) is crucial for the endonucleolytic cleavages but not the subsequent steps of U-deletion and U-insertion

Trypanosome mitochondrial mRNAs achieve their coding sequences through RNA editing. This process, catalyzed by approximately 20S protein complexes, involves large numbers of uridylate (U) insertions and deletions within mRNA precursors. Here we analyze the role of the essential TbMP42 protein (band VI/KREPA2) by individually examining each step of the U-deletional and U-insertional editing cycles, using reactions in the approximately linear range. We examined control extracts and RNA interference (RNAi) extracts prepared soon after TbMP42 was depleted (when primary effects should be most evident) and three days later (when precedent shows secondary effects can become prominent). This analysis shows TbMP42 is critical for cleavage of editing substrates by both the U-deletional and U-insertional endonucleases. However, on simple substrates that assess cleavage independent of editing features, TbMP42 is similarly required only for the U-deletional endonuclease, indicating TbMP42 affects the two editing endonucleases differently. Supplementing RNAi extract with recombinant TbMP42 partly restores these cleavage activities. Notably, we find that all the other editing steps (the 3'-U-exonuclease [3'-U-exo] and ligation steps of U-deletion and the terminal-U-transferase [TUTase] and ligation steps of U-insertion) remain at control levels upon RNAi induction, and hence are not dependent on TbMP42. This contrasts with an earlier report that TbMP42 is a 3'-U-exo that may act in U-deletion and additionally is critical for the TUTase and/or ligation steps of U-insertion, observations our data suggest reflect indirect effects of TbMP42 depletion. Thus, trypanosomes require TbMP42 for both endonucleolytic cleavage steps of RNA editing, but not for any of the subsequent steps of the editing cycles.     

Developing a mathematical method to search for latent periodicity in protein amino-acid sequences with deletions and insertions

A mathematical method has been developed in order to search for latent periodicity in protein amino-acid and other symbolical sequences using dynamic programming and random matrices. The method allows the detection of the latent periodicity with insertions and deletions at positions that are unknown beforehand. The developed method has been applied to search for the periodicity in the amino-acid sequences of several proteins and in the euro/dollar exchange rate since 2001. The presence of a long period with insertions and deletions in amino-acid sequences is shown. The period length of seven amino acids is observed in the proteins that contain supercoiled regions (a coiled-coil structure) as well as of six, five, or more amino acids. The existence of the period length of 6 and 7 days, as well as 24 and 25 h in the analyzed financial time series is observed; note that this periodicity is detectable only for insertions and deletions. The causes that underlie the occurrence of the latent periodicity with insertions and deletions in amino-acid sequences and financial time series are discussed.     

Amplification of the sequences displaying the pattern RNY in the RNA world: the translation --> translation/replication hypothesis

Based on previous considerations published in J. theor. Biol., new analyses of the organization of the genetic system are reported in this paper. We show that theoretical considerations about the order observed in the genetic code table support the idea of a primitive self-aminoacylation process achieved by primordial tRNAs. The physico-chemical constraints connected with this process may explain why a primitive genetic system predominantly uses sequences with the codonic pattern RNN (R=purine; Y=pyrimidine; N=any of the four bases) to polymerize the amino acids into peptides through translation. These considerations lead us to propose the Translation --> Translation/Replication hypothesis, which may explain why only RNA sequences with the pattern RNY, instead of less restrictive RNN, are susceptible to amplification. Using these ideas, supported by properties of symmetry, features of the genetic code may be connected with the replication of specific RNA sequences in the RNA world.     

Transglycosylation: a mechanism for RNA modification (and editing?)

The vast majority of the ca. 100 chemically distinct modified nucleosides in RNA appear to arise via the chemical transformation of a genetically encoded nucleoside. Two notable exceptions are queuosine and pseudouridine, which are incorporated into tRNA via transglycosylation. Transglycosylation is an extremely efficient process for incorporating highly modified bases such as queuine into RNA. Transglycosylation is also a requisite process for "isomerizing" an N-nucleoside into a C-nucleoside as is the case for pseudouridine formation. Finally, transglycosylation is an attractive possibility for certain RNA editing events (e.g., pyrimidine to purine conversions) that cannot occur via the known, more straightforward enzymatic reactions (e.g., deaminations). This review discusses what is known about the mechanisms of transglycosylation for the queuine and pseudouridine RNA modifications and will speculate about a potential role for transglycosylation in certain RNA editing events.     

Structural rearrangements and insertions of dispersed elements in pericentromeric alpha satellites occur preferably at kinkable DNA sites

Centromeric region of human chromosome 21 comprises two long alphoid DNA arrays: the well homogenized and CENP-B box-rich alpha21-I and the alpha21-II, containing a set of less homogenized and CENP-B box-poor subfamilies located closer to the short arm of the chromosome. Continuous alphoid fragment of 100 monomers bordering the non-satellite sequences in human chromosome 21 was mapped to the pericentromeric short arm region by fluorescence in situ hybridization (alpha21-II locus). The alphoid sequence contained several rearrangements including five large deletions within monomers and insertions of three truncated L1 elements. No binding sites for centromeric protein CENP-B were found. We analyzed sequences with alphoid/non-alphoid junctions selectively screened from current databases and revealed various rearrangements disrupting the regular tandem alphoid structure, namely, deletions, duplications, inversions, expansions of short oligonucleotide motifs and insertions of different dispersed elements. The detailed analysis of more than 1100 alphoid monomers from junction regions showed that the vast majority of structural alterations and joinings with non-alphoid DNAs occur in alpha satellite families lacking CENP-B boxes. Most analyzed events were found in sequences located toward the edges of the centromeric alphoid arrays. Different dispersed elements were inserted into alphoid DNA at kinkable dinucleotides (TG, CA or TA) situated between pyrimidine/purine tracks. DNA rearrangements resulting from different processes such as recombination and replication occur at kinkable DNA sites alike insertions but irrespectively of the occurrence of pyrimidine/purine tracks. It seems that kinkable dinucleotides TG, CA and TA are part of recognition signals for many proteins involved in recombination, replication, and insertional events. Alphoid DNA is a good model for studying these processes.     

Single-strand DNA triple-helix formation

Chemical modification studies provide evidence that single-stranded oligodeoxyribonucleotides can form stable intrastrand triple helices. Two oligonucleotides of opposite polarity were synthesized, each composed of a homopurine-homopyrimidine hairpin stem linked to a pyrimidine sequence which is capable of folding back on the hairpin stem and forming specific Hoogsteen hydrogen bonds. Using potassium permanganate as a chemical modification reagent, we have found that two oligodeoxyribonucleotides of sequence composition type 5'-(purine)8(N)4(pyrimidine)8(N)6(pyrimidine)8-3' and 5'-(pyrimidine)8N6(pyrimidine)8N4(purine)8-3' undergo dramatic structural changes consistent with intrastrand DNA triple-helix formation induced by lowering the pH or raising the Mg2+ concentration. The intrastrand DNA triple helix is sensitive to base mismatches.     

Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is incapable of de novo purine synthesis, and thus, purine acquisition from the host is an indispensable nutritional requirement. This purine salvage process is initiated by the transport of preformed purines into the parasite. We have identified a gene encoding a nucleoside transporter from P. falciparum, PfNT1, and analyzed its function and expression during intraerythrocytic parasite development. PfNT1 predicts a polypeptide of 422 amino acids with 11 transmembrane domains that is homologous to other members of the equilibrative nucleoside transporter family. Southern analysis and BLAST searching of The Institute for Genomic Research (TIGR) malaria data base indicate that PfNT1 is a single copy gene located on chromosome 14. Northern analysis of RNA from intraerythrocytic stages of the parasite demonstrates that PfNT1 is expressed throughout the asexual life cycle but is significantly elevated during the early trophozoite stage. Functional expression of PfNT1 in Xenopus laevis oocytes significantly increases their ability to take up naturally occurring D-adenosine (K(m) = 13.2 microM) and D-inosine (K(m) = 253 microM). Significantly, PfNT1, unlike the mammalian nucleoside transporters, also has the capacity to transport the stereoisomer L-adenosine (K(m) > 500 microM). Inhibition studies with a battery of purine and pyrimidine nucleosides and bases as well as their analogs indicate that PfNT1 exhibits a broad substrate specificity for purine and pyrimidine nucleosides. These data provide compelling evidence that PfNT1 encodes a functional purine/pyrimidine nucleoside transporter whose expression is strongly developmentally regulated in the asexual stages of the P. falciparum life cycle. Moreover, the unusual ability to transport L-adenosine and the vital contribution of purine transport to parasite survival makes PfNT1 an attractive target for therapeutic evaluation.     

Dynamics and competition of CRISPR–Cas9 ribonucleoproteins and AAV donor-mediated NHEJ,MMEJ and HDR editing

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T₅₀ (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.