Using Triplet Periodicity of Nucleotide Sequences for Finding Potential Reading Frame Shifts in Genes

We introduce a novel approach for the detection of possible mutations leading to a reading frame (RF) shift in a gene. Deletions and insertions of DNA coding regions are considerable events for genes because an RF shift results in modifications of the extensive region of amino acid sequence coded by a gene. The suggested method is based on the phenomenon of triplet periodicity (TP) in coding regions of genes and its relative resistance to substitutions in DNA sequence. We attempted to extend 326 933 regions of continuous TP found in genes from the KEGG databank by considering possible insertions and deletions. We revealed totally 824 genes where such extension was possible and statistically significant. Then we generated amino acid sequences according to active (KEGG''s) and hypothetically ancient RFs in order to find confirmation of a shift at a protein level. Consequently, 64 sequences have protein similarities only for ancient RF, 176 only for active RF, 3 for both and 581 have no protein similarity at all. We aimed to have revealed lower bound for the number of genes in which a shift between RF and TP is possible. Further ways to increase the number of revealed RF shifts are discussed.     

Classification analysis of triplet periodicity in protein-coding regions of genes

We introduce a new concept of triplet periodicity class (TPC) and a measure of similarity between such classes. We performed classification of 472288 triplet periodicity (TP) regions found in 578868 genes from 29th release of KEGG databank. Totally 2520 classes were obtained. They contain 94% of 472288 found cases of TP. For 92% of TP regions contained in classes the same linkage of TP to open reading frame (ORF) is observed. For 8% of TP cases we revealed a shift between ORF of a gene and ORF common for majority of genes contained in a TPC. For these 8% of periodic regions the hypothetical amino acid sequences corresponding to ORF built by TPC were made. BLAST program has shown that 2679 hypothetical amino acid sequences have statistically significant similarity with proteins from UniProt databank. We suppose that 8% of TP regions contained in classes possess a mutation originating from ORF shift. Obtained TPCs can be used for identification of genes' coding regions as well as for searching for mutations arisen arising from ORF shift.     

Classification of triplet periodicity in DNA sequences of genes taken from KEGG databank

We conducted classification for 472,288 regions of triplet periodicity found in 578,868 genes from release 29 of KEGG databank. A new concept of triplet periodicity class and a measure of similarity between them are introduced. Totally 2520 classes were created that contain 94% of found triplet periodicity. For 92% of triplet periodicity regions contained in classes an identical linkage of triplet periodicity to reading frame is observed. For the rest triplet periodicity cases a shift between reading frame of a gene and reading frame common for majority of genes contained in a class of triplet periodicity was observed. These periodicity regions were encoded into hypothetical amino acid sequences in accordance with reading frame built by triplet periodicity class. By BLAST program it was shown that 2660 hypothetical amino acid sequences have statistically significant similarity with proteins from UniProt databank. We suppose that 8% of triplet periodicity regions that joined classes mutated by means of reading frame shift. Created classes of triplet periodicity can be used for identification of coding regions of genes as well as for searching for mutations arisen from reading frame shift.     

Estimating the frequency of events that cause multiple-nucleotide changes

Existing mathematical models of DNA sequence evolution assume that all substitutions derive from point mutations. There is, however, increasing evidence that larger-scale events, involving two or more consecutive sites, may also be important. We describe a model, denoted SDT, that allows for single-nucleotide, doublet, and triplet mutations. Applied to protein-coding DNA, the SDT model allows doublet and triplet mutations to overlap codon boundaries but still permits data to be analyzed using the simplifying assumption of independence of sites. We have implemented the SDT model for maximum-likelihood phylogenetic inference and have applied it to an alignment of mammalian globin sequences and to 258 other protein-coding sequence alignments from the Pandit database. We find the SDT model's inclusion of doublet and triplet mutations to be overwhelmingly successful in giving statistically significant improvements in fit of model to data, indicating that larger-scale mutation events do occur. Distributions of inferred parameter values over all alignments analyzed suggest that these events are far more prevalent than previously thought. Detailed consideration of our results and the absence of any known mechanism causing three adjacent nucleotides to be substituted simultaneously, however, leads us to suggest that the actual evolutionary events occurring may include still-larger-scale events, such as gene conversion, inversion, or recombination, or a series of rapid compensatory changes.     

Localizing triplet periodicity in DNA and cDNA sequences

Background  

The protein-coding regions (coding exons) of a DNA sequence exhibit a triplet periodicity (TP) due to fact that coding exons contain a series of three nucleotide codons that encode specific amino acid residues. Such periodicity is usually not observed in introns and intergenic regions. If a DNA sequence is divided into small segments and a Fourier Transform is applied on each segment, a strong peak at frequency 1/3 is typically observed in the Fourier spectrum of coding segments, but not in non-coding regions. This property has been used in identifying the locations of protein-coding genes in unannotated sequence. The method is fast and requires no training. However, the need to compute the Fourier Transform across a segment (window) of arbitrary size affects the accuracy with which one can localize TP boundaries. Here, we report a technique that provides higher-resolution identification of these boundaries, and use the technique to explore the biological correlates of TP regions in the genome of the model organism C. elegans.     

Classification of triplet periodicity in the DNA sequences of genes from KEGG databank

Totally, 472 288 regions of triplet periodicity were found in 578 868 genes from KEGG databank version 29 and classified. A new concept of triplet periodicity class and a measure of similarity between periodicity classes were introduced. Overall, 2520 classes were created and contained 94% of the triplet periodicity cases found. A similar correlation between the triplet periodicity and reading frame was observed for 92% of triplet periodicity regions contained in different classes. The remaining triplet periodicity regions displayed a shift of the reading frame relative to that common for the majority of genes belonging to the same triplet periodicity class. The hypothetical amino acid sequences were deduced from the periodicity regions according to the reading frame characteristic of the given triplet periodicity class. BLAST analysis demonstrated that 2660 hypothetical amino acid sequences display a statistically significant similarity to proteins from the Uni-Prot databank. It was supposed that 8% of the triplet periodicity regions contained in the classes have frameshift mutations. The triplet periodicity classes can be used to identify the coding regions in genes and to searching for frameshift mutations.     

An approach for searching insertions in bacterial genes leading to the phase shift of triplet periodicity

The concept of the phase shift of triplet periodicity (TP) was used for searching potential DNA insertions in genes from 17 bacterial genomes. A mathematical algorithm for detection of these insertions has been developed. This approach can detect potential insertions and deletions with lengths that are not multiples of three bases, especially insertions of relatively large DNA fragments (>100 bases). New similarity measure between triplet matrixes was employed to improve the sensitivity for detecting the TP phase shift. Sequences of 17,220 bacterial genes with each consisting of more than 1,200 bases were analyzed, and the presence of a TP phase shift has been shown in ～16% of analysed genes (2,809 genes), which is about 4 times more than that detected in our previous work. We propose that shifts of the TP phase may indicate the shifts of reading frame in genes after insertions of the DNA fragments with lengths that are not multiples of three bases. A relationship between the phase shifts of TP and the frame shifts in genes is discussed.     

A systematic search for protein signature sequences.

Signature sequences are contiguous patterns of amino acids 10-50 residues long that are associated with a particular structure or function in proteins. These may be of three types (by our nomenclature): superfamily signatures, remnant homologies, and motifs. We have performed a systematic search through a database of protein sequences to automatically and preferentially find remnant homologies and motifs. This was accomplished in three steps: 1. We generated a nonredundant sequence database. 2. We used BLAST3 (Altschul and Lipman, Proc. Natl. Acad. Sci. U.S.A. 87:5509-5513, 1990) to generate local pairwise and triplet sequence alignments for every protein in the database vs. every other. 3. We selected "interesting" alignments and grouped them into clusters. We find that most of the clusters contain segments from proteins which share a common structure or function. Many of them correspond to signatures previously noted in the literature. We discuss three previously recognized motifs in detail (FAD/NAD-binding, ATP/GTP-binding, and cytochrome b5-like domains) to demonstrate how the alignments generated by our procedure are consistent with previous work and make structural and functional sense. We also discuss two signatures (for N-acetyltransferases and glycerol-phosphate binding) which to our knowledge have not been previously recognized.     

Evolution of a D. melanogaster glutamate tRNA gene cluster

We have determined the DNA sequence of a cloned cluster of essentially identical glutamate tRNA genes of D. melanogaster. The cluster consists of five genes: a gene triplet spanning approximately 0.55 kb followed by a 0.45 kb gene doublet 3.0 kb downstream. The genes are all arranged with the same polarity, do not encode the tRNA CCA end and contain no intervening sequences. Examination of the 5' and 3' sequences immediately flanking each gene reveals a striking pattern of sequence homologies between certain of the genes, which suggests a possible evolutionary history of this gene cluster. We propose that two ancestral genes each gave rise to gene doublets by duplication, while one of these gene pairs then gave rise, in turn, to a trio of genes as a result of unequal crossover.     

Evidence for gene conversion between the phosphoglycerate kinase genes of Trypanosoma brucei

Trypanosoma brucei contains a tandem array of three genes for phosphoglycerate kinase (PGKase), genes A, B and C, each coding for a different protein. We have compared allelic variants of this gene array and find evidence for gene conversion between the three genes. Near the 3' end, the different alleles and gene B contain a variable sequence that is similar to the corresponding sequence in either gene A or gene C. This sequence is flanked by glycine triplets that are conserved in all PGKases from bacteria to mammals. The triplets are encoded by (GGT)n, resulting in sequences that resemble the recombination-promoting chi-sites of Escherichia coli. Upstream of the variable sequence, there is an area of 800 base-pairs in which genes A, B and C are highly homologous; in all three genes this region ends with a sharp boundary at which gene B again shows segmental homology with both genes A and C. These results suggest that repeated gene conversion events partially erase the differences between genes A, B and C that arise in evolution and suggest that chi-like sequences may act as recombinational hotspots in protozoa such as T. brucei.     

An Approach for Searching Insertions in Bacterial Genes Leading to the Phase Shift of Triplet Periodicity

The concept of the phase shift of triplet periodicity (TP) was used for searching potential DNA insertions in genes from 17 bacterial genomes. A mathematical algorithm for detection of these insertions has been developed. This approach can detect potential insertions and deletions with lengths that are not multiples of three bases, especially insertions of relatively large DNA fragments (>100 bases). New similarity measure between triplet matrixes was employed to improve the sensitivity for detecting the TP phase shift. Sequences of 17,220 bacterial genes with each consisting of more than 1,200 bases were analyzed, and the presence of a TP phase shift has been shown in ~16% of analysed genes (2,809 genes), which is about 4 times more than that detected in our previous work. We propose that shifts of the TP phase may indicate the shifts of reading frame in genes after insertions of the DNA fragments with lengths that are not multiples of three bases. A relationship between the phase shifts of TP and the frame shifts in genes is discussed.     

A simple model to explain three-base periodicity in coding DNA

A simple model is put forward to explain the long-known three-base periodicity in coding DNA. We propose the concept of same-phase triplet clustering, i.e. a condition wherein a triplet appears several times in one phase without interruption by the two other possible phases. For instance, in the sequence (i): NTT_GNN_NTT_GNN_NTT_GNN_NNN_NTT_GNN (where N is any nucleotide but combinations producing TTG are excluded) there would be clustering of same-phase TTG because this triplet appears uninterruptedly in phase 2. In contrast, in the sequence (ii): TTG_NTT_GNN_NNT_TGN_NNN_NTT_GNN there is no same-phase clustering because neighboring TTGs are all in different phases. Observe also that in sequence (i) TTG triplets are separated by 3, 3 and 6 nucleotides (3n distances), while in sequence (ii) they are separated by 1, 4 and 5 nucleotides (non-3n distances). In this work, we demonstrate that in coding DNA the 3n distances generated by (i)-type sequences proportionally outnumber the non-3n distances generated by (ii)-type sequences, this condition would be the basis of three-base periodicity. Randomized sequences had (i)- and (ii)-type sequences too but clustering was statistically different. To prove our model we generated (i)-type sequences in a randomized sequence by inducing clustering of same-phase triplets. In agreement with the model this sequence displayed three-base periodicity. Furthermore, two- and four-base periodicities could also be induced by artificially inducing clustering of duplets and tetraplets.     

Symmetric connectivity of secondary structure elements enhances the diversity of folding pathways

The influence of native connectivity of secondary structure elements (SSE) on folding is studied using coarse-grained models of proteins with mixed alpha and beta structure and the analysis of the structural database of wild-type proteins. We found that the distribution of SSE along a sequence determines the diversity of folding pathways. If alpha and beta SSE are localized in different parts of a sequence, the diversity of folding pathways is restricted. An even (symmetric) distribution of alpha and beta SSE with respect to sequence midpoint favors multiple folding routes. Simulations are supplemented by the database analysis of the distribution of SSE in wild-type protein sequences. On an average, two-thirds of wild-type proteins with mixed alpha and beta structure have symmetric distribution of alpha and beta SSE. The propensity for symmetric distribution of SSE is especially evident for large proteins with the number of SSE > or = 10. We suggest that symmetric SSE distribution in protein sequences may arise due to nearly random allocation of alpha and beta structure along wild-type sequences. The tendency of long sequences to misfold is perhaps compensated by the enhanced pathway diversity. In addition, folding pathways are shown to progress via hierarchic assembly of SSE in accordance with their proximity along a sequence. We demonstrate that under mild denaturation conditions folding and unfolding pathways are similar. However, the reversibility of folding/unfolding pathways is shown to depend on the distribution of SSE. If alpha and beta SSE are localized in different parts of a sequence, folding and unfolding pathways are likely to coincide.     

Human imprinted chromosomal regions are historical hot-spots of recombination

Human recombination rates vary along the chromosomes as well as between the two sexes. There is growing evidence that epigenetic factors may have an important influence on recombination rates, as well as on crossover position. Using both public database analysis and wet-bench approaches, we revisited the relationship between increased rates of meiotic recombination and genome imprinting. We constructed metric linkage disequilibrium (LD) maps for all human chromosomal regions known to contain one or more imprinted genes. We show that imprinted regions contain significantly more LD units (LDU) and have significantly more haplotype blocks of smaller sizes than flanking nonimprinted regions. There is also an excess of hot-spots of recombination at imprinted regions, and this is likely to do with the presence of imprinted genes, per se. These findings indicate that imprinted chromosomal regions are historical "hot-spots" of recombination. We also demonstrate, by direct segregation analysis at the 11p15.5 imprinted region, that there is remarkable agreement between sites of meiotic recombination and steps in LD maps. Although the increase in LDU/Megabase at imprinted regions is not associated with any significant enrichment for any particular sequence class, major sequence determinants of recombination rates seem to differ between imprinted and control regions. Interestingly, fine-mapping of recombination events within the most male meiosis-specific recombination hot-spot of Chromosome 11p15.5 indicates that many events may occur within or directly adjacent to regions that are differentially methylated in somatic cells. Taken together, these findings support the involvement of a combination of specific DNA sequences and epigenetic factors as major determinants of hot-spots of recombination at imprinted chromosomal regions.     

Entamoeba histolytica: construction and applications of subgenomic databases

Knowledge about the influence of environmental stress such as the action of chemotherapeutic agents on gene expression in Entamoeba histolytica is limited. We plan to use oligonucleotide microarray hybridization to approach these questions. As the basis for our array, sequence data from the genome project carried out by the Institute for Genomic Research (TIGR) and the Sanger Institute were used to annotate parts of the parasite genome. Three subgenomic databases containing enzymes, cytoskeleton genes, and stress genes were compiled with the help of the ExPASy proteomics website and the BLAST servers at the two genome project sites. The known sequences from reference species, mostly human and Escherichia coli, were searched against TIGR and Sanger E. histolytica sequence contigs and the homologs were copied into a Microsoft Access database. In a similar way, two additional databases of cytoskeletal genes and stress genes were generated. Metabolic pathways could be assembled from our enzyme database, but sometimes they were incomplete as is the case for the sterol biosynthesis pathway. The raw databases contained a significant number of duplicate entries which were merged to obtain curated non-redundant databases. This procedure revealed that some E. histolytica genes may have several putative functions. Representative examples such as the case of the delta-aminolevulinate synthase/serine palmitoyltransferase are discussed.     

Antisense overlapping open reading frames in genes from bacteria to humans.

Long Open Reading Frames (ORFs) in antisense DNA strands have been reported in the literature as being rare events. However, an extensive analysis of the GenBank database revealed that a substantial number of genes from several species contain an in-phase ORF in the antisense strand, that overlaps entirely the coding sequence of the sense strand, or even extends beyond. The findings described in this paper show that this is a frequent, non-random phenomenon, which is primarily dependent on codon usage, and to a lesser extent on gene size and GC content. Examination of the sequence database for several prokaryotic and eukaryotic organisms, demonstrates that coding sequences with in-phase, 100% overlapping antisense ORFs are present in every genome studied so far.     

Unusual DNA structures associated with germline genetic activity in Caenorhabditis elegans

We describe a surprising long-range periodicity that underlies a substantial fraction of C. elegans genomic sequence. Extended segments (up to several hundred nucleotides) of the C. elegans genome show a strong bias toward occurrence of AA/TT dinucleotides along one face of the helix while little or no such constraint is evident on the opposite helical face. Segments with this characteristic periodicity are highly overrepresented in intron sequences and are associated with a large fraction of genes with known germline expression in C. elegans. In addition to altering the path and flexibility of DNA in vitro, sequences of this character have been shown by others to constrain DNA::nucleosome interactions, potentially producing a structure that could resist the assembly of highly ordered (phased) nucleosome arrays that have been proposed as a precursor to heterochromatin. We propose a number of ways that the periodic occurrence of An/Tn clusters could reflect evolution and function of genes that express in the germ cell lineage of C. elegans.     

Origin and evolution of genes and genomes. Crucial role of triplet expansions

A novel concept on mechanisms of evolution of genes and genomes is suggested: the sequences evolve largely by local events of triplet expansion and subsequent mutational changes in the repeats. The immediate memory about the earlier expansion events still resides in the sequences, in form of the frequently occurring segments of tandemly repeating codons. Other predicted fossils of the original repeats are: (I) the expanding triplets should be accompanied by their point mutation derivatives and (II) the remaining excess of codons formerly belonging to the tandem repeats should be reflected in overall codon usage biases. Both predictions are confirmed by analysis of largest available database of non-redundant protein coding sequences, of total size ～5?×?10(9) codons. One important conclusion also follows from the results. Life which, presumably, started with replication of expanding triplets and their subsequent mutational changes, is continuing to emerge within the genes and genomes, in form of new events of triplet expansion.