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Frameshift mutations are generally considered to be deleterious and of little importance for the evolution of novel gene functions. However, by screening an exhaustive set of vertebrate gene families, we found that, when a second transcript encoding the original gene product compensates for this mutation, frameshift mutations can be retained for millions of years and enable new gene functions to be acquired.
Frameshift mutations generally result in loss-of-function changes since they drastically alter the protein sequence downstream of the frameshift site, besides creating premature stop codons. Here we present data suggesting that frameshift mutations in the C-terminal domain of specific ancestral MADS-box genes may have contributed to the structural and functional divergence of the MADS-box gene family. We have identified putative frameshift mutations in the conserved C-terminal motifs of the B-function DEF/AP3 subfamily, the A-function SQUA/AP1 subfamily and the E-function AGL2 subfamily, which are all involved in the specification of organ identity during flower development. The newly evolved C-terminal motifs are highly conserved, suggesting a de novo generation of functionality. Interestingly, since the new C-terminal motifs in the A- and B-function subfamilies are only found in higher eudicotyledonous flowering plants, the emergence of these two C-terminal changes coincides with the origin of a highly standardized floral structure. We speculate that the frameshift mutations described here are examples of co-evolution of the different components of a single transcription factor complex. 3' terminal frameshift mutations might provide an important but so far unrecognized mechanism to generate novel functional C-terminal motifs instrumental to the functional diversification of transcription factor families.
Microsatellite instability of DNA samples of 79 sporadic colon cancer patients were analyzed. These samples were also screened to search mutations in the repeat sequences in the gene for the type II receptor of transforming growth factor-beta (TGF-beta RII) using polymerase chain reaction (PCR), electrophoresis with urea gel, and PCR-single strand conformation polymorphism (PCR-SSCP) method. The incidence of microsatellite instability, defined as severe replication error phenotype (RER) with microsatellite alterations in more than three loci, was 6%. Deletion and insertion of an A residue in the (A)10 region, which cause frameshift mutation, were found in four samples and their incidence in the samples with microsatellite instability was 80%. A novel nucleotide substitution of T for G at 1918, which causes missense mutation of arginine to leucine at codon 528, was found in a sample with microsatellite instability. The mutation at 1918 was in highly conservative amino acid residue.
The rare lysosomal storage disease, fucosidosis results from an almost complete deficiency of alpha-L-fucosidase (EC 3.2.1.51). We have identified six new potential disease causing mutations detected by PCR amplification and sequencing of all 8 exons of the alpha-L-fucosidase gene FUCA1. (1) A C to T mutation (Q77X) in exon 1 of two Jewish-Italian siblings. This mutation was present in one allele and was found also in the mother who was of Italian origin. (2) A C to A mutation (W382X) in exon 6 in an Italian patient. This mutation was found in one allele and obliterates a unique Hphl site. (3) A C to A mutation (Y211X) in exon 3 in a Belgian patient. This mutation obliterates a unique Rsal site and was present in both alleles. (4) A homozygous single base (C) deletion in exon 2 in an Italian patient. This deletion results in a frameshift mutation (P141fs) and obliterates a unique Eael site. (5) A homozygous single base (C) deletion in exon 5 in a Portuguese patient, which also results in a frameshift mutation (S265fs). (6) A single base (A) deletion in exon 3 in a Canadian-Indian patient, which also results in a frameshift mutation (S216fs). The S216fs mutation was found in only one allele; the mutation in the other allele is not yet known.
A single-base-pair deletion in exon 7 of the human carbonic anhydrase II gene was found to be the molecular defect in a group of independently ascertained, clinically heterogeneous, Hispanic carbonic anhydrase II-deficient patients, all of whom had ancestors from the Caribbean islands. This mutation predicts a +1 frameshift at codon 227 and incorporation of 12 missense amino acids before an early stop codon at position 239 produces a 27-kDa truncated carbonic anhydrase II. Expression of the Hispanic mutant cDNA in bacteria produced predominantly the 27-kDa protein, which was inactive. However, a minor 29-kDa polypeptide species was also produced that had 10% the specific activity of the wild-type enzyme after affinity purification. Amino acid sequencing showed that the 29-kDa mutant protein was produced by two frameshift events: a +1 frameshift at codon 227 due to the single-base deletion and a -1 ribosomal frameshift at codon 237 that restored the original reading frame after 11 missense amino acids were incorporated. Antibody against the 11-amino acid frameshift peptide detected the 29-kDa mutant protein in lysates of transfected COS cells. These results indicate that ribosomal frameshift can partially rescue the human carbonic anhydrase II frameshift mutation and suggest a mechanism whereby a compensatory ribosomal frameshift can ameliorate the consequences of certain frameshift mutations. Whether individual differences in efficiency of ribosomal frameshift contribute to clinical heterogeneity in patients with such mutations deserves further study.
6 out of 14 uncharacterized beta-thalassemia alleles from 187 Thai beta-thalassemia/HbE patients were identified by direct sequencing of DNA amplified by polymerase chain reaction. A novel mutation occurring from an insertion of adenosine in codon 95, which results in a shift of the reading frame with terminator at the new codon 101, was detected in one patient. In addition, two frameshift mutations not previously reported among the Thai population were also detected in 3 patients: one with a deletion of thymidine in codon 15 and two with an insertion of cytidine in codons 27/28. A frameshift mutation that occurred from a cytidine deletion in codon 41 was also found in one patient in this study. The remaining case was an amber mutation, GAG-TAG, in codon 43 in exon 2 of the beta-globin gene. These mutations bring the number of mutations known to be present in the Thai population to a total of 20, 15 of which were detected in beta-thalassemia/HbE patients.
We describe a patient with severe generalized dystrophic epidermolysis bullosa (EBD) and a novel combination of compound heterozygous mutations in the COL7A1 gene. The maternal mutation was an A-to-G transition (425-A --> G) at position -2 of the donor splice site within exon 3 that causes aberrant splicing of two abnormal transcripts. One includes intron 3, and one excludes both exon 3 and intron 3. Both splice variants contained a premature termination of the translation. The paternal mutation is a 25-bp deletion in exon 20 (2638de125) that leads to a frameshift and a premature termination codon 133 bp downstream from the site of deletion. This combination of mutations allowed expression of collagen VII mRNA. Immunofluorescence staining of the patient's skin and cultured keratinocytes with domain-specific collagen VII antibodies, however, demonstrated markedly reduced levels of alpha1(VII) polypeptides, and no stable collagen VII protein could be extracted from the patient's cells. Electron microscopy showed severely hypoplastic fibrils below the lamina densa, without evidence of normal anchoring fibrils. The clinically unaffected parents were heterozygous for the mutations, suggesting that both COL7A1 gene defects were recessively inherited disease-causing mutations that are "silent" in heterozygous carriers but in combination can severely interfere with the dermal-epidermal adhesion and lead to severe EBD.
BACKGROUND: Liddle syndrome is an autosomal dominant form of salt-sensitive hypertension caused by mutations in the epithelial sodium channel expressed in the distal nephron playing an essential role in Na absorption. All reported mutations in Liddle syndrome are either missense mutations or frameshift mutations destroying the PY motif closer to the C-terminus of the beta or gamma subunits causing the situation that the epithelial sodium channels are not degraded and sodium is pooled and thus hypertension and hypokalemia are caused. METHODS: We sequenced the C-terminus of the beta or gamma subunits of the epithelial sodium channel in a Japanese family of a patient clinically diagnosed as having Liddle syndrome. RESULTS: As a result, we found in the proband, a frameshift mutation of the beta subunit caused by a single cytosine insertion at the codon 595, introducing a new stop codon at 605 and deleting the last 34 amino acids from the normally encoded protein. CONCLUSION: This mutation is carried by neither parent (with paternity proven) and hence confirms this has occurred as a event within this family.
The DNA sequences of 185 independent spontaneous frameshift mutations in the rIIB gene of bacteriophage T4 are described. Approximately half of the frameshifts, including those at hot spot sites, are fully consistent with classical proposals that frameshift mutations are produced by a mechanism involving the misaligned pairing of repeated DNA sequences. However, the remaining frameshifts are inconsistent with this model. Correlations between the positions of two base-pair frameshifts and the bases of DNA hairpins suggest that local DNA topology might influence frameshift mutation. Warm spots for larger deletions share the property of having endpoints adjacent to DNA sequences whose complementarity to sequences a few base-pairs away suggest that non-classical DNA misalignments may participate in deletion mutation. A model for duplication mutation as a consequence of strand displacement synthesis is discussed. In all, 15 frameshifts were complex combinations of frameshifts and base substitutions. Three of these were identical, and have extended homology to a sequence 256 base-pairs away that is likely to participate in the mutational event; the remainder are unique combinations of frameshifts and transversions. The frequency and diversity of complex mutants suggest a challenge to the assumption that the molecular evolution of DNA must depend primarily upon the accumulation of single nucleotide changes.
Homo-oligomeric runs were inserted into a spleen necrosis virus-based retrovirus vector to determine the nature and rate of mutations within runs of 10 to 12 identical nucleotides during a single replication cycle. Clones of helper cells containing integrated copies of retroviral vectors were used to produce virus for infection of target (nonhelper) cells. Proviral sequences from target cell clones were compared with proviral sequences from helper cell clones to study mutations that occurred during a single cycle of replication. In addition to the internal region spanning the homo-oligomeric inserts, a naturally occurring run of 10 T's in the long terminal repeat (LTR) also was sequenced. Rates of mutation ranged from < 0.01 to 0.38 frameshift mutations per run per cycle for different nucleotide runs. Frameshift mutations ranged from deletions of 2 bases to additions of 5 bases; the most common mutations were +1 and -1. Frameshift mutation rates did not increase as the run length increased from 10 to 12 bases. Rates of frameshift mutation for runs of T's and A's were significantly higher than rates for runs of C's and G's, and rates for runs of pyrimidines were significantly higher than those for runs of purines. Interestingly, the vast majority of frameshift mutations in the internal region (95%) were positive, suggesting that the primer strand tends to slip backward on the template in this region. LTR runs had a significantly lower number of positive frameshift mutations than the internal runs. By analyzing the types of frameshift mutations within runs and by comparing the patterns of frameshift mutations in the 5' and 3' LTRs of individual proviruses, we conclude that the majority of mutations observed in our system occurred during minus-strand DNA synthesis of reverse transcription.
The mismatch repair system of Escherichia coli is known to contribute to the fidelity of the replicational process. This system involves the functions of mutH, mutL, mutS and mutU (uvrD) loci which recognize mispaired bases as a consequence of errors due to the polymerase itself. Chemical modifications of DNA have also been suspected to create mispaired bases which, if the mispaired bases are removed, will lead to mutations by frameshift. Using the pBR322 plasmid DNA modified by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) we have investigated this possibility in a forward mutational assay (tetracycline sensitivity). This fluorene derivative has been shown to induce predominantly frameshift mutations. Our results show that: The sensitivity of the deficient strains mutH, mutL and mutS to the AAF adducts is similar to that of the corresponding wild-type strain. However, the mutU strain appears much more sensitive to those adducts although less than a uvrA, B or C-deficient strain. This suggests that the mutU gene product is involved in the repair of AAF adducts. For the four mut deficient strains, and as it was shown with the wild-type strain, AAF adducts induced mutations to tetracycline sensitivity are only observed when the SOS system of the host bacteria is induced by irradiation of the cells prior to transformation with the modified plasmid. The mutation frequencies depend upon the ultraviolet light doses and similar maxima were found for the four mut strains and the corresponding wild-type strain. In agreement with the results obtained with wild-type or uvrA strains we observe that AAF adducts induce mostly frameshift mutations in the mut strains. Two types of hot spots of mutagenesis were described in wild-type and uvrA strains occurring either at repetitive sequences or at sequences of the type 5' G-G-C-G-C-C 3' (NarI restriction enzyme recognition sequence). While the second type of mutational hot spot does exist in the mismatch repair-deficient strains, we observe that the repetitive sequences are no longer hot spots of mutations in these strains, suggesting that the mismatch repair protein complex is involved in the establishment of AAF-induced frameshift mutations at repetitive sequences.
ICR-induced frameshift mutations at the his4 locus in Saccharomyces cerevisiae have been classified into several groups on the basis of their reversion and suppression properties. One group of externally suppressible his4 mutations, designated Group II, have been shown to contain +1 G:C insertions in glycine codons and are suppressed by any one of five suppressor mutations described previously (SUF1, SUF3, SUF4, SUF5, and SUF6). The suppressor genes are believed to encode glycine tRNAs containing four base anticodons.--An analysis of spontaneous co-revertants of the Group II frameshift mutation his4-206 and leu2-3 has revealed the existence of eleven new Group II-specific suppressor genes (SUF15 through SUF25). The locations of the new suppressor loci on the yeast genetic map have been determined.--By comparing the ability or inability of Group II-specific suppressors mapping at 16 different Group II his4 mutations, two subclasses of suppressors have been defined. One subclass suppresses his4-38 and his4-519, which contain the altered four base mRNA codons 5'-GGGU-3' and 5'-GGGG-3', respectively. The other subclass suppresses his4-38, but fails to suppress his4-519. The mechanism of tRNA-mediated frameshift suppression and the molecular basis for this division of the suppressors into two subclasses is discussed.
Straightforward detectable Duchenne muscular dystrophy (DMD) gene rearrangements, such as deletions or duplications involving an entire exon or more, are involved in about 70% of dystrophinopathies. In the remaining 30% a variety of point mutations or "small" mutations are suspected. Due to their diversity and to the large size and complexity of the DMD gene, these point mutations are difficult to detect. To overcome this diagnostic issue, we developed and optimized a routine muscle biopsy-based diagnostic strategy. The mutation detection rate is almost as high as 100% and mutations were identified in all patients for whom the diagnosis of DMD and Becker muscular dystrophy (BMD) was clinically suspected and further supported by the detection on Western blot of quantitative and/or qualitative dystrophin protein abnormalities. Here we report a total of 124 small mutations including 11 nonsense and frameshift mutations detected in BMD patients. In addition to a comprehensive assessment of muscular phenotypes that takes into account consequences of mutations on the expression of the dystrophin mRNA and protein, we provide and discuss genomic, mRNA, and protein data that pinpoint molecular mechanisms underlying BMD phenotypes associated with nonsense and frameshift mutations.
Induction of back mutations to prototrophy by methylene blue (MB)-sensitized photodynamic (PD) treatment has been studied in wild-type and repair-deficient strains of Salmonella typhimurium carrying either the base-pair substitution mutation hisG46 or the frameshift mutation hisD30529 We found that reversion of the hisG46 mutation was increased in a strain carrying a uvrB deletion and decreased in a strain carrying a recA-type mutation. Reversion of the hisD3052 (frameshift) mutation, on the other hand, was decreased in both uvrB deletion and recA-type strains. The former results are consistent with the hypothesis that the majority of MB-sensitized PD-INDUCED BASE-PAIR SUBSTITUTION MUTATIONS ARIse by a mechanism similar to that currently believed to be involved in UV mutagenesis. The latter results suggest that PD-induced frameshift mutations may arise in some other way, and two possible mechanisms involving sequential action of the excision repair and recombinational repair pathways are considered.
BACKGROUND: TGF-beta type II receptor (TGF-beta RII) mutations associated with microsatellite instability(MSI) are characteristically frameshift mutations within a 10 bp poly-A tract. These frameshift mutations have been reported to be common in colorectal and gastric cancers with MSI, though, rarely reported in non-small cell lung cancer (NSCLC). MATERIALS AND METHOD: In this study, we analysed MSI and TGF-beta RII frameshift mutations in 7 NSCLC cell lines and 21 surgically resected NSCLC tissues. Determination of MSI in NSCLC was performed using primer sets for BAT-25, BAT-26 and BAT-40. In order to examine the presence of the frameshift mutations of TGF-beta RII in samples with MSI, sequencing for TGF-beta RII poly-A tract was performed. RESULTS: MSI was observed in 5 out of 7 NSCLC cell lines and 3 out of 21 NSCLC tissues. Six out of 8 samples with MSI(75%) showed frameshift mutations in TGF-beta RII poly-A tract. CONCLUSION: These results suggest that MSI is highly associated with TGF-beta RII frameshift mutations in NSCLC and further support the hypothesis that TGF-beta RII plays an important role in NSCLC carcinogenesis.
Compensated frameshift mutation is a modification of the reading frame of a gene that takes place by way of various molecular events. It appears to be a widespread event that is only observed when homologous amino acid and nucleodotide sequences are compared. To identify these mutation events, the sequence analysis rationale was based on the search for short regions that would have much lower degrees of conservation in protein, but not in DNA, in well-conserved beta-glucosidase families. We have restricted our study to a seed set of sequences of O-glycoside hydrolase families 1 and 3. We found compensated frameshift mutation in the family of 1 beta-glucosidases for the Erwinia herbicola, Cellulomonas fimi, and (non-cyanogenic) Trifolium repens gene sequences, and in the family of 3 beta-glucosidases for the Clostridium thermocellum and Clostridium stercorarium gene sequences. By computational treatment, the observed mutation events in the gene frameshifting sub-sequence have been neutralised. Each nucleotide insertion must be eliminated and each nucleotide deletion must be substituted by the symbol N (any nucleotide). When the frameshifting fragments of the amino acid sequences were substituted by the computationally neutralised subsequences, the beta-glucosidase alignments were improved. We also discuss the structural implications of the compensated frameshift mutations events.
The effects of the umuC36 mutation on the induction of base-change and frameshift mutations were studied. An active umuC gene was necessary in either the uvr+ strains of Escherichia coli K12 for UV- and X-ray-induced mutations to His+, ColER and SpcR, which are presumably base-change mutations, but it was not essential for ethyl methanesulphonate or N-methyl-N'-nitro-N-nitrosoguanidine-induced His+ mutations. In contrast, only 1 out of 13 trp- frameshift mutations examined was UV reversible, and the process of mutagenesis was umuC+-dependent, whereas a potent frameshift mutagen, ICR191, effectively induced Trp+ mutations in most of the strains regardless of the umu+ or umuC genetic background. These results suggest that base substitutions are a major mutational type derived from the umuC+-dependent pathway of error-prone repair.
Frameshift mutations occur by a number of mechanisms. To better understand the nature of these mechanisms, we determined the DNA sequence changes of 232 independent, spontaneous frameshift mutations in the HIS4 gene of REV1 and rev1-1 strains of Saccharomyces cerevisiae. All frameshift mutants were selected based on their ability to revert the +1 frameshift mutation his4-38. DNA sequence information was recovered using two approaches-the double-strand gap repair of plasmid pMP4, and the polymerase chain reaction (PCR). Using these techniques, saturated mutation spectra for the spontaneous reversion of his4-38 were generated. The most frequently occurring mutational events in both strains were -1 frameshifts, but +2 frameshifts, larger deletions, larger insertions and more complex mutations were also observed. Between the REV1 and rev1-1 strains, we noticed a significant difference in the distribution of -1 frameshift mutations. In addition, while for -1 frameshift events there was no significant difference between the reversion spectra determined by double-strand gap repair or PCR, there was a surprisingly significant difference between the types of frameshift mutations recovered by double-strand gap repair (only -1 frameshifts and one +2 frameshift), and those recovered using PCR (-1 frameshifts, +2 frameshifts, larger deletions and insertions, and more complex mutations). This difference may reflect a selectional mechanism inherent in double-strand break repair that avoids chromosomal sequences which include complex alterations.