Which demonstrates an insertion mutation of the sequence

We calculated information content R based on Shannon entropy for each base pair, which measures conservation of a sequence position and has a maximum value of 2 bits if the position is fully conserved.

In taxa without the insertion Lepidosauria, crocodiles, certain turtles, and certain birds , the region around position from to bp of ND3 had a similar conservation level as the remainder of the gene Fig. In contrast, all turtle and bird species with the insertion had noticeably more conserved base pairs higher R values around position than in other regions of ND3 Fig. Specifically, when the insertion was present, nucleotides upstream of the insertion position — were highly conserved with some variability on the third codon positions Fig.

Nucleotide and codon usage variability in ND3 of Diapsida. A Information content R cubed for visualization across the ND3 sequence in different diapsid groups. The vertical red line marks the insertion at position The red shading highlights an area of high conservation high information content , which is only seen in birds and turtles that have the insertion. B and C Sequence conservation as a sequence logo from position to showing variability among species B with the insertion and C without the insertion.

Note that the frameshift correction is thought to occur at the following nucleotide, by skipping the nucleotide A at position D Circle packing showing the frequency of codon usage in species of birds and turtles that contain the insertion. The 2 options of the shifted and corrected reading frame following the insertion at position are shown. Circle diameters indicate prevalence of a specific codon, which are grouped into larger circles if codons are synonymous. Circle color indicates amino acid class.

We also analyzed the codon conservation in sequences containing the insertion, i. Notably, the green wood hoopoe Phoeniculus purpureus , Bucerotiformes deviated from this pattern with an ATC encoding for isoleucine.

Both leucine and isoleucine are nonpolar amino acids. In the 0 reading frame, the first 2 codons downstream of the insertion showed almost complete conservation to AGT encoding serine, a polar amino acid in the first codon following the insertion position — and AGC encoding serine, a polar amino acid in the second codon following the insertion position — Fig.

The only exception to this high codon conservation following the insertion was Baillon's crake Porzana pusilla , Gruiformes with a CGT codon encoding arginine, a basic amino acid in the first following codon. The second codon following the insertion position — was also conserved in coding for alanine a nonpolar amino acid , albeit with all 4 synonymous codons present Fig.

We investigated whether lineages containing the ND3 insertion showed differences in tRNA secondary structure, which could enhance programmed frameshifting. Leucine is the tRNAs decoding the codon where the insertion occurs serine and valine are the 2 tRNAs that compete for being decoded downstream of the insertion Fig.

The translational machinery of certain birds and turtles seems to enable programmed frameshifting in order to correct single-nucleotide insertions in coding regions, and additional frameshift locations could exist. Using a subset of Diapsida sequences with full mitochondrial genomes, we checked for other frameshifts in the ND3 gene Supplementary File S2.

The 2 Cuora turtles had frameshifts in different positions. For P. We could not perform this check for the frameshift insertions in the other 4 turtle species because no additional genomic or transcriptomic data was available. This study significantly expands the sampling of previous studies 61 species in [ 9 ], 34 species in [ 11 ] to provide a broader picture across vertebrates on one hand and more fine-scale resolution of sequence conservation on the other hand.

We confirm that the insertion is present only in turtles and birds [ 9 , 11 ], but the improved sampling shows that the insertion was both more frequently gained and lost than previously thought. Different from previous interpretations, which predicted the presence of this insertion in the common ancestor of turtles and birds Archelosauria , ML and MP ancestral state reconstruction suggested an independent evolution of this insertion in birds and turtles.

The insertion was retained in many lineages but lost in the common ancestor of Passeriformes 39—49 million years ago [ 20—22 ] and not regained since. In turtles, the most recent common ancestor was inferred to not have had the insertion in ND3, as opposed to previous ideas [ 9 , 11 ]. Our data included turtles, 93 more than in the last study on turtle mitochondrial genomes [ 11 ], which produced an alternative interpretation of the gain and loss patterns.

Within turtles, the insertion has been independently gained 1—3 times on the basis of our reconstructions. Within clades that have insertion, some lineages show mutations to other nucleotides. The quality of the ND3 sequences and the observed absence or presence of the insertion at position on these sequences is of crucial importance for our inferences. Most of the ND3 sequences used here originate from Sanger-sequenced ND3 genes and chromatograms may have been hand-curated for sequencing errors.

While insertions at position are likely to be genuine because they would have been flagged as problematic during submission to NCBI's Genbank and would require a special annotation to address the frameshift insertion often to [ 9 ] , the absence of the insertion may be overrepresented.

A frameshift insertion in ND3 may have been curated out of the sequence because such insertions in protein-coding sequences are extremely rare and could have been considered a sequencing error. It is therefore possible that the estimated number of loss events in turtles and birds is overestimated. Where losses were observed in multiple members of a clade, the most extreme case being the absence in all 2, included Passeriformes, the absence of the insertion is likely real.

To estimate the prevalence of this potential problem, we compared ND3 annotations for bird species that had ND3 sequences on NCBI's GenBank mostly Sanger-sequenced and also high-throughput sequenced mitochondrial genomes from the Bird 10, Genomes Project B10K , for which we have ourselves created the annotations and can therefore exclude manual modification. Reassuringly, we found that there were no cases in which the B10K dataset contained the insertion while the GenBank sequence did not contain it.

This assessment admittedly spans only a small fraction of the taxa investigated here but lends support that at least in birds, annotation errors may be limited. It is possible that submitters of bird and turtle sequences are more aware of the possibility of a frameshift insertion because the insertions have been reported from these taxa [ 9 , 11 ] than submitters of taxa in which the insertion has not been observed, such as Lepidosauria or Mammalia. It is intriguing that an insertion in exactly the same position of the ND3 gene has independently evolved 33 MP model to 37 times ML model across turtles and birds.

This site specificity points to an underlying common feature that causes the frameshift to occur in this position. One possibility is that there is an increased probability to produce indels in this specific position. Alternatively, insertions may appear at a normal rate but can only be tolerated if they are embedded in a specific sequence motif that allows ribosomes to conduct the frameshift correction. Intriguingly, this conservation is not found in birds and turtles without the insertion Fig.

This indicates that the heavily conserved sequence is needed for correcting the insertion and tolerating it in the mitochondrial genome. Most of the features observed in this study conformed with the "out-of-frame" frameshift model [ 11 ], which is characterized by a weak codon tRNA interaction in the P-site, a rare codon downstream, and an alternative codon if the reading frame is restored, which achieves a canonical Watson-Crick match with its tRNA [ 13 ].

Our extended sampling provides higher resolution of the sequence conservation features that may be involved in the programmed translational frameshift, adding examples of species that deviate from the most commonly used codons. This codon upstream from the translational frameshift position was proposed to produce a wobble pairing initiating a translation stall [ 11 ].

We found it to be highly conserved as a leucine codon CTN in all examined birds and turtles with the exception of 1 bird green wood hoopoe P. We further confirm that a serine codon AGT , a polar amino acid, was always observed after the insertion [ 11 ]. However, we also show that this pattern can be more flexible at least in Baillon's crake P. It is thought that rarely used codons, such as AGT being a rarely used codon for serine, promote the stall in the translation [ 11 ].

It is not known whether the observed variable codon CGT is also a rarely used codon for arginine in most vertebrates. In human mitochondria this seems to be the case because the CGT codon is only the third of 4 possible codons for arginine in codon preference [ 24 ]. If CGT was also rarely used in the other vertebrates, it could be an additional example of a rare codon enhancing the translational stall.

Regarding the insertion itself, we have observed all 4 nucleotides to be present in different frequencies. According to the out-of-frame frameshift model, wobble pairing between a sequence and the tRNA anticodon promotes frameshifting [ 11 ].

Consequently, the insertion should rarely occur as an adenosine because a CTA codon produces a perfect match with the tRNA-leucine anticodon [ 11 ]. In addition to Reeve's turtle Chinemys reevesi , which has previously been shown to contain an A-insertion [ 11 ], we found an independent occurrence of an A-insertion in a bird, Baillon's crake P. These 2 species may therefore be interesting candidates for further investigations on the programmed translational frameshift in the absence of wobble pairing.

In addition to the detailed investigation of position , we found 4 additional frameshifts in ND3 in 5 turtles. Of these, the African helmeted turtle P. These 2 species are Pelomedusidae and both Pelusios and Pelomedusa contain a number of species [ 25 ] that could be sequenced for ND3 to investigate whether the insertion is shared across Pelomedusidae or independently obtained in the 2 species.

The other 3 frameshifts are, to the best of our knowledge, potential new frameshifts in the ND3 gene. These findings and the ubiquitous presence of frameshifts in other mitochondrial genes of turtles [ 11 ] suggest a broad tolerance of turtles to frameshift insertions. Our study demonstrates that incorporating a large number of sequences can improve resolution in inferred evolutionary patterns and give additional power to investigate sequence conservation.

Our analyses suggest an independent origin of the frameshift insertion in both turtles and birds, and complex patterns of gains and losses within each group. The high sequence conservation surrounding the insertion suggests purifying selection retaining the sequence motifs needed for translational frameshifting.

Nonetheless, a few species deviate from the conserved pattern. Additional losses and gains of the insertion and other deviations from the conserved motifs will likely be found once more sequences become available, within birds and turtles, and possibly also in other groups. The present work advances our understanding of the distribution of the frameshift insertion in the mitochondrial gene ND3 across the vertebrate tree of life and identifies highly conserved sequence features that seem to be associated with its occurrence.

Slowly evolving sites i. Alternatively, the DNA sites that evolve at a rate similar to the synonymous sites in the coding region are not conserved and therefore give a lower LLR scores. The results of our analysis provide evidence that LLR score can also be used to measure the effect of indels that disrupt the conserved DNA sites.

A similar approach was previously used in [ 7 ] to assess the effect of SNPs in non-coding as well as the protein-coding regions [ 43 ]. We sought to test whether our scores for large-effect mutations reflected their functional impact. More deleterious variants are expected to segregate at lower frequencies in the population and occur at lower densities that would be expected of neutral variants [ 23 ]. Therefore, in a natural population, we expect mutations with larger predicted effects to segregate at lower frequencies and be found at lower densities than mutations with smaller predicted effects.

Using sequence data from a population of 39 strains of S. We first computed the derived allele frequency spectrum DAF and tested for a shift towards lower DAFs left of the spectrum [ 23 ], which is expected under purifying selection [ 8 ]. Spectrums of mutation allele frequencies. We compared this fraction between genes with higher score, i.

The paucity of high frequency alleles is consistent with stronger purifying selection on the mutations we predicted to be more deleterious using the score D. To test for the effects of selection on the density of variations, we compared the distribution of scores to that expected if the mutations were randomly placed. For this purpose, we generated sets of variations randomly placed on genes in our dataset and computed the information loss scores for them.

We then compared the distribution of the scores from the random datasets to the distribution of scores obtained from our yeast dataset. This suggests that purifying selection has acted to remove mutations with greater score D from the population.

Randomization experiments. For each type of variation, this distribution is different when compared to the variations with records of disease association green and variations that do not have such records blue. A set of randomly generated FS indels red shows a similar distribution to those that are associated with diseases.

In panels a and b, the "randomized" histogram bars represent the mean of random samplings of the data, and the error bars represent the standard deviation observed over the samplings, while in panels c-e the "randomized" histogram bars represent the mean of 50 random samplings of the data, and the error bars represent the standard deviation observed over the 50 samplings.

We identified indels that fall within the promoter regions bp upstream of genes in a population of 39 strains of S. To test for the effects of selection on the density of indels, we compared the distribution of DNA sites with respect to their LLR scores with what is expected if the indels were placed randomly.

For this purpose, we generated 50 sets of indels distributed randomly in the bp upstream of all genes in the reference S. This suggests that indels at highly conserved DNA sites have been removed from the population by purifying selection. Our method identifies candidates for new deleterious variations in a pool of genes with mutations.

We ranked the mutations in our yeast dataset in terms of their deleterious effects on the corresponding proteins. In the following, we study the top ranked FS indels and the NMs with lowest scores. We observed that three of the genes on the list, that are also essential to yeast [ 44 ], carry highly deleterious FS indels. We further studied possible association of these indels with yeast phenotypes using data from phenotypic experiments [ 45 ] as well as phenotype data from [ 46 ].

A reduction in the function of SMD1 is associated with a decrease in the resistance of yeast to the drug Tunicamycin [ 46 ]. We therefore considered the reproduction efficiency RE of yeast strains in the presence of 1. We observed that the RE of the 2 individuals carrying FS indels was lower compared to the population. A sample of ranked genes in terms of their information loss score D. One of the other top ranked indel predictions was in TFB3.

A reduction in the function of the gene TFB3 is associated with an increase in resistance to the same drug [ 46 ]. Interestingly, we observed the expected effects in the 2 individuals that carry the FS indel and therefore are predicted to lack the function for this gene. Because of the small number of individuals that carry the putatively deleterious FS indel alleles here 2 we were not able to test the significance of these phenotypic observations.

However, these examples show the practical uses of the proposed methods. We further studied the bottom 5 genes with NMs with lowest scores in our dataset. These mutations are located in the C-termini of these genes.

To test whether our methods can be applied to the variations in the human population we examined genes with FS indels and NMs reported in dbSNP [ 10 ]. We categorized the variations into two classes: variations that have records of diseases association in OMIM [ 11 ] and LSDB [ 12 ] and variations with no such records. We expect that the latter class i. We then sought to study the allele frequency spectrum of these variations.

Heterozygousity information was only available for the NMs with no disease association. We studied the segregation of NMs in the human population by comparing the spectrums of MAF of the NMs that cause greater information loss i.

Thus, these mutations appear to have more deleterious effects in the population. To study the effects of selection on mutation' density, we compared the observed distribution of scores with that expected if variations were randomly placed. To do so, we computed the score D for a large number of FS indels as well as NMs placed randomly on the human genes in our dataset. The significant abundance of mutations with lower deleterious effects in the data with no disease association, or in other words, the paucity of variations with higher information loss scores, indicates that purifying selection had acted on highly deleterious variations.

The abundance of variations in genes associated with diseases as well as a wide range of information loss they cause is overwhelming. As an example, consider the tumor repressor gene P53 and its protein product TP53 [ 47 ].

There are 95 somatic, 49 cell-line and 15 germline NMs as well as somatic, cell-line and 36 germline FS indels reported to have association with different types of cancer. While it is difficult to determine which mutations cause these diseases [ 48 , 49 ], different effect of these mutations on protein conservation suggests different roles they potentially play in damaging the protein function. Consistent with the prediction that these have little impact on protein function, these regions are not part of the so-called "hot spots" in this protein i.

Distribution of mutations on the human tumour repressor TP53 with respect to their respective loss of information scores. The upper panel shows the distribution of D for 95 somatic, 49 cell-line and 15 germline NM in the TP53 tumour suppressor. The lower panel show the distribution of D for somatic, cell-line and 36 germline FS indels reported for TP53 protein. These mutations are associated with a wide range of cancers. Our proposed methods are useful for practical purposes to sort a huge number of FS indels, NMs, as well as indels in non-coding DNA in terms of their deleterious effect.

It is important to note that our methods do not seek to classify variations into deleterious and non-deleterious but rather to rank their effect for further analysis and laboratory experiments. For the variations in the protein-coding DNA, the proposed score is built upon the principle assumption that the effect of nonsense or FS indel mutations on protein can be computed as sum of effects due to individual residues.

This is obviously an over-simplification that is widely accepted in statistically modeling the individual columns of an alignment independently.

A more complex method that considered correlations between each residue was also implemented using a profile-HMM based on a generative hidden-Markov model [ 51 ] data not shown here. The score S were computed as the likelihood of the sequence given the profile-HMM [ 52 ]. Similar prediction results were observed, i. We observed a strong correlation between the position of the nonsense or FS mutations and the loss of information they cause Additional file 1 , Figure S1.

We were not able to demonstrate that the D score outperforms the percentage of the protein that is truncated the "length lost".

When we compare the distribution of the length lost to the random expectation Additional file 1 , Figure S2 we find that the length-lost score appears to show less deviation from the random expectation than the D score for the human data Additional file 1 , Figure S2c,d. This is consistent with the hypothesis that the D score captures more information than the length lost.

While simply considering the number of residues affected provides a reasonable guess at the impact of mutations "on average", there are cases for which the position of the mutation does not reflect its effect on evolutionary conservation off-diagonal points in Additional file 1 , Figure S1. Furthermore, we believe that the D score represents a more principled approach to quantifying the importance of these variants because it directly measures evolutionary information, and because it is consistent with previous approaches to quantify the effects of variants, such as SIFT.

However, if multiple sequence alignments are not available, the length lost might also provide a reasonable substitute to quantify the effect of a FS indel or NM. Identification of causative mutations for diseases remains a challenge even for the case of single genes, let alone in cases where mutations are studied in a network of genes and regulatory elements e.

Due to the overwhelming abundance of variations, the information loss score, that captures the evolutionary conservation context of the sequences harbouring mutations, seems to be a good candidate for weighting variations in large-scale association analyses [ 53 , 54 ]. To obtain the information loss, D, we compute the scores of the WT and the mutant sequences against a position weight matrix as it is defined above. The mutant proteins with highest scores D are more likely to carry a highly deleterious mutation.

This is to maintain a minimum degree of diversity between sequences and to avoid biasing the estimation of the PWM towards closely related species. We exclude mutations on genes from our dataset in these cases. Any column of the PWM, f, is the maximum likelihood estimate of the distribution of amino acid residues observed in the alignment.

In ideal cases where there is sufficiently large number of sequences in the alignment, the PWM is simply a matrix with columns equal to the relative frequency of each observed amino acid residue at that column. However, in practice, due to a relatively large number of residues i. Problem 3. Problem 4. Problem 5. Problem 6. Problem 7. Problem 8. Problem 9. Problem Video Transcript this question asked, which demonstrates an insertion mutation of the following sequence.

Numerade Educator. Elements and Their Atoms In chemistry and physics, an element is a substance that cannot be broken do…. The Elements of Life In biology, the elements of life are the essential building blocks that make…. Which of the following pairs of sequences might be found at the ends of an i…. What type of mutation is depicted by the following sequences shown as mRNA …. Which of the following pairs of base sequences could form a short stretch….

What is the template …. In a direct repair, thymine dimers are directly broken down by the enzyme photolyase. It is used to identify mutants with restored biosynthetic activity. Why is it more likely that insertions or deletions will be more detrimental to a cell than point mutations? Envision that each is a section of a DNA molecule that has separated in preparation for transcription, so you are only seeing the template strand.

What type of mutation is each? Figure from: P arker, et al Microbiology from Openstax In recent years, scientific interest has been piqued by the discovery of a few individuals from northern Europe who are resistant to HIV infection. Back to the Top It is used to identify newly formed auxotrophic mutants. It is used to identify spontaneous mutants.

It is used to identify mutants lacking photoreactivation activity. Think about It Why is it more likely that insertions or deletions will be more detrimental to a cell than point mutations? World Health Organization. Accessed August 5, DNA oxidation.