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PubMed2001

Aggresome formation in liver cells in response to different toxic mechanisms: role of the ubiquitin-proteasome pathway and the frameshift mutant of ubiquitin.

Aggresomes form in cells when intracellular proteins undergo conformational changes, as in so-called conformational diseases. This phenomenon has been observed in the liver and brain and in cell culture in response to abnormal protein formation, such as mutant proteins. In the case of the brain the frameshift mutant ubiquitin (UBB+1) is involved. Mallory body formation in the liver is one example of this phenomenon in vivo. Mallory body formation is common in a variety of liver diseases of diverse pathogenesis. The study of the Mallory body forming model indicated that drug-conditioned hepatocytes form Mallory bodies when mice are given colchicine, ethanol, okadaic acid, or exposure to heat shock. These findings suggest that aggresome formation is a common pathway of liver injury due to diverse mechanisms. To further characterize the role of this common pathway, drug-primed mice were exposed to different types of liver injury, i.e., using such drugs as thioacetamide, galactosamine, tautomycin, and the proteasome inhibitor PS341. Mallory body formation was induced by treatment with all the toxins tested, giving credence to the proposal that aggresome formation in the liver is a common pathway in response to different primary mechanisms of liver injury. The frameshift mutant UBB+1 was invariably found to colocalize with ubiquitin in the Mallory body, indicating its essential involvement in the mechanism of MB formation.

Animals↗
PubMed1998

A second mammalian antizyme: conservation of programmed ribosomal frameshifting.

A second mammalian ornithine decarboxylase antizyme was discovered. The deduced protein sequence of the human antizyme2 is 54% identical and 67% similar to human antizyme1 but 99.5% identical to mouse antizyme2. Polyamine-regulated programmed ribosomal frameshifting is used in decoding antizyme2 mRNA as it is for antizyme1 mRNA. The mRNA signals for the programmed frameshifting are similar in the mRNAs for the two antizymes. However, in the stimulatory pseudoknot 3' of the shift site, while the sequences of the stems are highly conserved, the sequences of the loops are divergent. Functional distinctions between antizymes seem likely, but no distinction in the tissue distribution of human antizyme1 and 2 mRNAs was distinguished, though antizyme2 mRNA is 16-fold less abundant than its antizyme1 counterpart. In addition to the previously characterized human antizyme1 mRNA, a second antizyme1 mRNA with an additional 160 nucleotides at its 3' end was identified, and it has a tissue distribution different from that of the shorter antizyme1 mRNA.

Amino Acid Sequence↗
PubMed1994

Genetic evidence for IS1 transposition regulated by InsA and the delta InsA-B'-InsB species, which is generated by translation from two alternative internal initiation sites and frameshifting.

Insertion sequence IS1 contains two reading frames, insA and B'-insB, which are responsible for its transposition, and was previously shown to express two proteins. The first, InsA, is the product of insA. The second, InsA-B'-InsB is a fusion of InsA with the product of B'-insB. Synthesis of this protein occurs by a -1 frameshift from the 3' region of the insA frame to the open reading frame B', extending from the 5' end of the insB frame. Here, I have shown genetically that IS1 encodes the third species delta InsA-B'-InsB: delta InsA-B'-InsB uses two alternative initiation codons in the middle of the insA frame, and is produced by a frameshift mechanism similar to that used in InsA-B'-InsB expression. Deletion of the small region preceding these initiation codons resulted in decreased expression of delta InsA-B'-InsB, suggesting that the small region play some role in the translation initiation. Surprisingly, it was found that delta InsA-B'-InsB has a transposase-like function and InsA can stimulate the transposition promoted by delta InsA-B'-InsB, while delta InsA-B'-InsB seemed to bind to the left terminal inverted repeat (IRL) of IS1 and inhibit transposition when it was present in excess, as well as InsA represses transposition. It is likely that IS1 transposition activity depends on the ratio of InsA to delta InsA-B'-InsB. A double missense mutation of the internal initiation codons resulted in decreased cointegration activity, showing that delta InsA-B'-InsB is responsible for transposition but InsA-B'-InsB is probably not. Some IS elements, which also contain two tandem, out-of-phase, overlapping genes, appear to express deleted fusion proteins like delta InsA-B'-InsB, but the functions are unknown. The complex phenomena of transposition and its control found in IS1 may be more general in the other mobile DNAs.

Amino Acid Sequence↗
PubMed1998

tRNA imbalance promotes -1 frameshifting via near-cognate decoding.

tRNAGly1 is the Escherichia coli glycine tRNA specific for GGG codons. A genetic selection for multicopy suppressors of a frameshift mutation has shown that increased levels of wild-type tRNAGly1 causes -1 frameshifting. Analysis of the suppression spectrum of this multicopy suppressor and peptide sequencing of the suppressed protein product showed that it promoted GG doublet decoding at the near-cognate GGA codons. It is proposed that increasing the concentration of the GGG-specific tRNAGly1 relative to the cognate GGA-decoding tRNAGly2 allows the near-cognate tRNA to read GGA codons. Near-cognate decoding of GGA codons by tRNAGly1 can occur by a two-out-of-three reading mechanism, in which only the first two bases of the GGA codon are paired with the anticodon, thus permitting doublet translocations. In mycoplasmas, a single tRNA typically decodes all four triplets of a codon family and introduction of a feature of the Mypoplasma mycoides tRNAGly responsible for non-discriminate decoding, a C at position 32, into the anticodon E. coli tRNAGly1, enhanced the efficiency of doublet decoding.

Escherichia coli↗
PubMed1999

A three-way junction and constituent stem-loops as the stimulator for programmed -1 frameshifting in bacterial insertion sequence IS911.

Several signals are required for the programmed frameshifting in translation of IS911 mRNA. These include a Shine Dalgarno (SD)-like sequence, a slippery sequence of six adenine residues and a guanine residue (A6G) and a 3' secondary structure. The structure of the mRNA containing these elements was investigated using chemical and enzymatic probing. The probing data show that the 3' structure is a three-way junction of stems. The function of the three-way junction was investigated by mutagenesis. Disrupting the stability of the structure greatly affects frameshifting and transposition levels as tested by separate in vivo assays. Structural probing and thermal melting profiles indicate that the disrupted three-way junctions have altered structures.

Aldehydes↗
PubMed2000

Energetics of a strongly pH dependent RNA tertiary structure in a frameshifting pseudoknot.

Retroviruses employ -1 translational frameshifting to regulate the relative concentrations of structural and non-structural proteins critical to the viral life cycle. The 1.6 A crystal structure of the -1 frameshifting pseudoknot from beet western yellows virus reveals, in addition to Watson-Crick base-pairing, many loop-stem RNA tertiary structural interactions and a bound Na(+). Investigation of the thermodynamics of unfolding of the beet western yellows virus pseudoknot reveals strongly pH-dependent loop-stem tertiary structural interactions which stabilize the molecule, contributing a net of DeltaH approximately -30 kcal mol(-1) and DeltaG degrees (37) of -3.3 kcal mol(-1) to a total DeltaH and DeltaG degrees (37) of -121 and -16 kcal mol(-1), respectively, at pH 6.0, 0.5 M K(+) by DSC. Characterization of mutant RNAs supports the presence of a C8(+).G12-C26 loop 1-stem 2 base-triple (pK(a)=6.8), protonation of which contributes nearly -3.5 kcal mol(-1) in net stability in the presence of a wild-type loop 2. Substitution of the nucleotides in loop 2 with uridine bases, which would eliminate the minor groove triplex, destroys pseudoknot formation. An examination of the dependence of the monovalent ion and type on melting profiles suggests that tertiary structure unfolding occurs in a manner quantitatively consistent with previous studies on the stabilizing effects of K(+), NH(4)(+) and Na(+) on other simple duplex and pseudoknotted RNAs.

Base Pairing↗
PubMed2001

Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting.

RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49 degrees, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.

Adenine↗
PubMed1997

Chimeric HIV-1 virus-like particles containing gp120 epitopes as a result of a ribosomal frameshift elicit Gag- and SU-specific murine cytotoxic T-lymphocyte activities.

Insect cell expression of the HIV-1 Gag precursor protein by recombinant baculoviruses results in the assembly and budding of noninfectious virus-like particles (VLPs). The VLPs resemble immature virus in ultrastructural morphology and can be purified by conventional retroviral techniques. The virus-like appearance of the particles suggested that they could be used to package additional peptides. The retroviral frameshift mechanism was used to translate the pol gene products by expressing additional genetic information as chimeric Gag-Pol fusion proteins. Sequences encoding the carboxyl 65% of the HIV-1 surface glycoprotein (gp120, SU) were inserted into the Gag-Pol reading frame immediately downstream of the Gag stop codon. The assembly and budding of large quantities of Gag and chimeric Gag-SU VLPs were observed by standard transmission electron microscopy. The presence of gp120 epitopes in the Gag-SU VLPs was confirmed by immunoelectron microscopy and Western blot analysis using monoclonal anti-gp120 antibodies. Mice inoculated with the Gag-SU pseudovirions developed cytotoxic lymphocyte responses to both HIV-1 Gag and Env epitopes yet humoral immune responses only to Gag epitopes. The chimeric Gag-SU particles may have applications as vaccines or immunotherapeutic treatments for HIV-1 infection. In addition, the frameshift mechanism can be applied to the packaging of other viral or cellular proteins.

Animals↗
PubMed2001

Structure of the malondialdehyde deoxyguanosine adduct M1G when placed opposite a two-base deletion in the (CpG)3 frameshift hotspot of the Salmonella typhimurium hisD3052 gene.

Malondialdehyde (MDA) is a toxic and mutagenic metabolite produced by lipid peroxidation, and prostaglandin biosynthesis. MDA induces frameshift mutations in tester strains of Salmonella typhimurium. It reacts with DNA, and at physiological pH the major adduct is a pyrimidopurinone formed by reaction with guanine: M1G [3-(2'-deoxy-beta-D-erythropentofuranosyl)pyrimido[1,2-alpha]-purin-10(3H)-one]. When site-specifically incorporated into a duplex oligodeoxynucleotide containing a frameshift-prone (CG)3 repeat derived from the Salmonella typhimurium hisd3052 gene, spontaneous opening of M1G to the N2-(3-oxo-1-propenyl)-dG species occurred. In this work d(ATCGCMCGGCATG), (M=M1G) was annealed to d(CATGCCGCGAT) to model the putative strand slippage intermediate which would precede a two base deletion in the (CG)3 iterated repeat. 1H NMR studies indicate that in contrast to the duplex DNA structure, M1G remains intact. A single bulge conformation exists. M1G and its 3'-neighbor cytosine are unpaired. The M1G is intrahelical and stacked, whereas the unpaired cytosine is poorly stacked and appears to be extrahelical.

Base Pairing↗
PubMed1991

Characterization of a novel open reading frame, urf a, in the mitochondrial genome of fission yeast: correlation of urf a mutations with a mitochondrial mutator phenotype and a possible role of frameshifting in urf a expression.

Between the genes for tRNA(gin) and tRNA(ile) an open reading frame of 227 amino acids has been identified which is unique among known mitochondrial genomes and which has been termed urf a (Lang et al. 1983; Kornrumpf et al. 1984). It uses the "mitochondrial" genetic code, i.e., it contains a TGA codon, whereas all other protein-encoding genes, and all but one intronic open reading frame, use the "standard" genetic code (UGG for tryptophan). A previous paper has demonstrated that "mutator" strains show an increased formation of mitochondrial drug-resistant and respiration-deficient mutants (including deletions). In this paper we show that the mutator activity is correlated with mutations in urf a. A detailed analysis of one urf a mutant is presented (anar-6), where the deletion of an A residue leads to a frameshift mutation and consequently to premature termination of the putative protein. The phenotype of colonies originating from a single mutant clone varies from no growth up to full growth on non-fermentable substrate. This phenomenon of phenotypic segregation can be explained by the ability of the cell to perform translational frameshifting. A detailed analysis of the DNA sequence and the putative urf a protein will be presented and a possible function of the protein will be discussed.

Amino Acid Sequence↗
PubMed1993

Two novel frameshift mutations associated with the presence of direct repeats of the LDL receptor gene in familial hypercholesterolemia.

Two novel frameshift mutations were detected in the mutant LDL receptor genes responsible for familial hypercholesterolemia. One was a 5-bp insertion at codon 395 in exon 9, and the other was a one nucleotide deletion at codon 531 in exon 11. Both mutations alter the reading frame and consequently produce a premature stop codon in the region of the mature LDL receptor homologous to the epidermal growth factor (EGF) precursor. With regard to the mechanism responsible for the generation of these frameshift mutations, strand slipped mispairing mediated by short direct repeats is considered to be the most likely. The findings seem to support the hypothesis that a short direct repeat in DNA sequence can have a profound influence on the stability of a given gene and promote human gene mutations.

Adolescent↗
PubMed2006

MEN1 family with a novel frameshift mutation.

Multiple endocrine neoplasm type 1 (MEN1) syndrome predisposes to the development of endocrine and non-endocrine tumors with an autosomal dominant pattern of inheritance. Different mutations have been found throughout the gene with a variable phenotype expression. The proband, a Caucasian man, was admitted to our department in 2001, at the age of 51 because of a 1-yr history of diarrhoea and hypertension. He reported a previous intestinal resection for bowel occlusion with a histological diagnosis of unspecified mesenchymal neoplasia. He had also undergone a left adrenalectomy for a large nonfunctioning adrenal adenoma. Subsequently, he had suffered from gastralgia and melena; a gastroduodenoscopy showed an erosive gastritis. His family history was negative for endocrine disorders. On physical examination, multiple abdominal cutaneous lipomas and facial angiofibromas were observed. Biochemical screening revealed a primary hyperparathyroidism and an increase in circulating levels of PRL, chromogranin-A, gastrin and glucagon. The whole body computed tomography (CT) scan, the 111In-octreotide scan and the pituitary magnetic resonance imaging (MRI) did not reveal any abnormality. The presence of small neuroendocrine tumors was suspected by a positron emission tomography uptake in the epigastric region. The endoscopic ultrasound revealed a pancreatic lesion sized 1.1 cm that is under evaluation. Direct DNA sequencing analysis of the proband MEN1 gene revealed the 579delG frameshift mutation in the exon 3. The genetic screening of the family revealed the same mutation in 3 out of 5 offspring. The biochemical screening revealed some features of the MEN1 syndrome in all three of them. In conclusion, a novel frameshift MEN1 mutation was found in kindred with an apparently negative family history. Our experience confirms that MEN1 syndrome is a complex and underestimated condition, unless specifically investigated by trained specialists.

Adult↗
PubMed2001

A TGF betaRII frameshift-mutation-derived CTL epitope recognised by HLA-A2-restricted CD8+ T cells.

Microsatellite instability (MSI) is recognised as genome-wide alterations in repetitive DNA sequences caused by defects in the DNA mismatch repair machinery. Such mutation patterns have been found in almost all analysed malignancies from patients with hereditary non-polyposis colorectal cancer, and in approximately 15% of sporadic colorectal cancers. In cancers with the MSI phenotype, microsatellite-like sequences in coding regions of various cancer-related genes, including transforming growth factor beta receptor type II (TGF betaRII), are targets for mutations. The TGF betaRII gene harbours a 10-bp polyadenine tract, and mutations within this region are found in 90% of colorectal cancers with MSI. The frameshift mutations result in new amino acid sequences in the C-terminal part of the proteins, prematurely terminating where a novel stop codon appears. In this study we have defined new cytotoxic T lymphocyte (CTL) epitope (RLSSCVPVA), carrying a good HLA-A*0201 binding motif, and resulting from the most common frameshift mutation in TGF betaRII. A CTL line and several CTL clones were generated from an HLA-A2+ normal donor by repeated stimulation of T cells with dendritic cells pulsed with the peptide. One of the CTL clones was able to kill an HLA-A2+ colon cancer cell line harbouring mutant TGF betaRII. This epitope may be a valuable component in cancer vaccines directed at MSI-positive carcinomas.

Amino Acid Sequence↗
PubMed1998

Suppression of a +1 T mutation by a nearby substitution in the mitochondrial cox1 gene of Chlamydomonas reinhardtii: a new type of frameshift suppression in an organelle genome.

In Chlamydomonas reinhardtii, mutants defective in the cytochrome pathway of respiration lack the capacity to grow under heterotrophic conditions (in darkness on acetate). In the dark- strain duM18, a + 1 T addition in a run of four Ts, located at codon 145 of the mitochondrial cox1 gene encoding subunit I of cytochrome c oxidase, is responsible for the mutant phenotype. A leaky revertant (su11) that grows heterotrophically at a lower rate than wild-type cells was isolated from dum18. Its respiration sensitivity to cyanide was low and its cytochrome c oxidase activity was only 4% of that of the wild-type enzyme. Meiotic progeny obtained from crosses between revertant and wild-type cells inherited the phenotype of the mt- parent, showing that the suppressor mutation, like dum18 itself, is located in the mitochondrial genome. In order to map the su11 mutation relative to dum18, a recombinational analysis was performed on the diploid progeny. It demonstrated that su11 was very closely linked to the dum18 mutation less than 20-30 bp away. The cox1 gene of the su11 revertant was then sequenced. In addition to the + 1 T frameshift mutation still present at codon 145, an A-->C substitution was found at codon 146, leading to the replacement of a glutamic acid by an alanine in the polypeptide chain. No other mutations were detected in the cox1 coding sequence. As the new GCG codon (Ala) created at position 146 is very seldom used in the mitochondrial genome of C. reinhardtii, we suggest that the partial frameshift suppression by the nearby substitution is due to an occasional abnormal translocation of the ribosome (+ 1 base shift) facilitated both by the run of Ts and the low level of weak interaction of alanyl-tRNA.

Amino Acid Sequence↗
PubMed1996

Transcript analysis of CFTR frameshift mutations in lymphocytes using the reverse transcription-polymerase chain reaction technique and the protein truncation test.

mRNA transcripts of the cystic fibrosis transmembrane conductance regulator (CFTR) gene were analyzed from lymphocytes of two cystic fibrosis compound heterozygotes (394delTT/3195del6 and 1215delG/ 2423delG), of five related carriers heterozygous for one of these mutations, and of five normal individuals. After reverse transcription of total RNA and amplification by the polymerase chain reaction, fragments were investigated by sequencing and by the protein truncation test (PTT). The three frameshift mutants were correctly detected by PTT, as they introduced a premature termination codon resulting in shortened protein products. The PTT approach thus provides a simple and reliable alternative method for detecting frameshift, nonsense, or splice site mutations, and for ascertaining their putative effect on the reading frame of the mRNA. In addition, we have identified 6 alternatively spliced forms of CFTR mRNA, two of which (transcripts lacking 4 + 5 or 17B) have not been described previously.

Adolescent↗
PubMed1991

Programmed translational frameshifting and initiation at an AUU codon in gene expression of bacterial insertion sequence IS911.

The proteins expressed by insertion sequence IS911, a member of the widespread IS3 family of elements, have been analyzed. The results indicate that three major species are produced from two consecutive reading frames. A protein of Mr 11,500, ORFA, is synthesized from an upstream reading frame. A larger protein, ORFAB, uses the same initiation codon and is produced by a -1 programmed translational frameshift between orfA and a downstream frame, orfB, whose amino acid sequence shows significant homology with retroviral integrase proteins. The orfB frame is also expressed independently in two alternative forms: the first uses a rare AUU initiation codon in the orfB phase whereas the second appears to initiate in the orfA phase and is produced by a -1 frameshift mechanism similar to that used in ORFAB expression. A specific IS911 integration reaction using a minimal active junction composed of 51 base-pairs of the right inverted repeat and a flanking phase lambda sequence resembling a second end in inverted orientation has been developed to analyze the functions of these proteins by transcomplementation in vivo. The orfA and orfB frames are shown to be essential and production of ORFAB is shown to stimulate integration in this system, suggesting that this fusion protein is the IS911 transposase.

Amino Acid Sequence↗
PubMed1993

A proflavin-induced frameshift hotspot in the thymidylate synthase gene of bacteriophage T4.

Twenty-one independent thymidylate synthase deficient (td) mutants were isolated after proflavin mutagenesis of T4D0 phage. A strikingly high proportion of these mutations (17 of 21; 80%) mapped in a small 122 nucleotide (nt) region which spans the 5' splice site of this intron-containing gene. This region comprises only 14% of the total td exon sequence. RNA sequence analysis of these mutants identified a series of frameshift insertion/deletion mutations and indicated a hotspot for proflavin-induced mutations in the 3' end of exon I of the td gene. The mutant sequences at the hotspot site fully support a previously proposed mutagenic mechanism for proflavin-induced mutations in which frameshifts are produced as a consequence of exonuclease or DNA polymerase activity at the 3' ends of nicks in the DNA produced by perturbation of the T4-encoded type II topoisomerase activity by the acridine. Sixteen of the seventeen DNA mutations in the hotspot region can be explained by the model as a consequence of enzymatic processing of nicks at two phosphodiester bonds staggered by 4 base pairs (bp) and located on opposite strands of the DNA. Thus, these mutants exhibit precisely the symmetry expected of topoisomerase-mediated mutagenesis. The DNA sequences of the td hotspot mutants, when considered with the sequences of proflavin-induced mutants in the T4 rIIB and lysozyme genes, confirm the view that proflavin-induced mutations in diverse bacteriophage T4 DNA sequences are all produced by the topoisomerase-dependent mechanisms and do not support the view that classical misalignments in DNA repeats are hotspots for proflavin-induced mutagenesis in T4.

Bacteriophage T4↗
PubMed1996

Frameshift mutagenesis by 9-aminoacridine: antimutagenic effects of adenosine compounds.

It has been shown that frameshift mutagenesis by 9-aminoacridine (9AA) in Salmonella typhimurium is significantly inhibited if glucose is present while cells are being treated in liquid defined medium. We suggested that this effect might be a result of glucose-provoked alterations of cAMP levels within the cell. We therefore sought to investigate the effects of exogenous cAMP on mutagenesis by 9-aminoacridine in both Salmonella typhimurium and Escherichia coli. Contrary to expectation, we found that frameshift mutagenesis was significantly depressed when high concentrations of cAMP were added to the defined medium during liquid treatment with 9AA. Other adenosine 5'-phosphates such as adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) added to the liquid medium during 9AA treatment also substantially decreased the reversion rate to prototrophy in both S. typhimurium and E. coli, as did adenosine itself. Further experiments showed that neither influx nor efflux of 9-aminoacridine molecules were greatly affected by adenosine compounds, and that although cAMP and adenosine exerted similar antimutagenic effects on 9AA-treated stationary phase cells, their effects on log phase cells were quite different. The antimutagenic effect of a representative adenosine compound (ATP) was found to persist for some time after stationary phase cells had been washed, with maximal mutability being regained only after about 3 h.

Adenine Nucleotides↗