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Background The thalassemia syndromes are classified according to the globin chain or chains whose production is affected. β-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the β-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level. Methods Two cases of Polish patients with hereditary hemolytic anemia suspected of thalassemia were studied. DNA sequencing and mRNA quantification were performed. Stable human cell lines which express wild-type HBB and mutated versions were used to verify that detected mutation are responsible for mRNA degradation. Results We identified two different frameshift mutations positioned in the third exon of HBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although detected mutations are not expected to induce NMD, the mutant version of mRNA is not detectable. Restoring of the open reading frame brought back the RNA to that of the wild-type level. Conclusion Our results show that a lack of natural stop codon due to the frameshift in exon 3 of β-globin gene causes rapid degradation of its mRNA and indicate existence of novel surveillance pathway. Electronic supplementary material The online version of this article (doi:10.1186/s12881-017-0428-1) contains supplementary material, which is available to authorized users.

Clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible., Summary: A new CRISPR/Cas9-based method of fluorescent reporter technology in zebrafish to confirm loss of gene function in this model.

Frameshift mutations in the last coding exon of the 5-aminolevulinate synthase (ALAS) 2 gene were described to activate the enzyme causing increased levels of zinc- and metal-free protoporphyrin in patients with X-linked dominant protoporphyria (XLDPP). Only two such so-called gain-of-function mutations have been reported since the description of XLDPP in 2008. In this study of four newly identified XLDPP families, we identified two novel ALAS2 gene mutations, a nonsense p.Q548X and a frameshift c.1651-1677del26bp, along with a known mutation (delAGTG) found in two unrelated families. Of relevance, a de novo somatic and germinal mosaicism was present in a delAGTG family. Such a phenomenon may explain the high proportion of this mutation in XLDPP worldwide. Enhancements of over 3- and 14-fold in the catalytic rate and specificity constant of purified recombinant XLDPP variants in relation to those of wild-type ALAS2 confirmed the gain of function ascribed to these enzymes. The fact that both p.Q548X and c.1651-1677del26bp are located in close proximity and upstream from the two previously described mutations led us to propose the presence of a large gain-of-function domain within the C-terminus of ALAS2. To test this hypothesis, we generated four additional nonsense mutants (p.A539X, p.G544X, p.G576X and p.V583X) surrounding the human XLDPP mutations and defined an ALAS2 gain-of-function domain with a minimal size of 33 amino acids. The identification of this gain-of-function domain provides important information on the enzymatic activity of ALAS2, which was proposed to be constitutively inhibited, either directly or indirectly, through its own C-terminus.

Mycobacterial pathogens use the ESAT-6 system 1 (Esx-1) exporter to promote virulence. Previously, we used gene disruption and complementation to conclude that the MMAR_0039 gene in Mycobacterium marinum is required to promote Esx-1 export. Here we applied molecular genetics, proteomics, and whole-genome sequencing to demonstrate that the MMAR_0039 gene is not required for Esx-1 secretion or virulence. These findings suggest that we initially observed an indirect mechanism of genetic complementation. We identified a spontaneous nonsense mutation in a known Esx-1-associated gene which causes a loss of Esx-1 activity. We show that the Esx-1 function was restored by nonsense suppression. Moreover, we identified a polar mutation in the ppsC gene which reduced cellular impermeability but did not impact cytotoxicity in macrophages. Our studies reveal insight into Esx-1 export, nonsense suppression, and cell envelope lipid biogenesis.

Frameshift mutations at coding mononucleotide repeats (cMNR) are frequent in high-microsatellite instability (MSI-H) cancers. Frameshift mutations in cMNR result in the formation of a premature termination codon (PTC) in the transcribed mRNA, and these abnormal mRNAs are generally degraded by nonsense mediated mRNA decay (NMD). We have identified novel genes that are frequently mutated at their cMNR by blocking NMD in two MSI-H cancer cell lines. After blocking NMD, we screened for differentially expressed genes using DNA microarrays, and then used database analysis to select 28 candidate genes containing cMNR with more than 9 nucleotide repeats. cMNR mutations have not been previously reported in MSI-H cancers for 15 of the 28 genes. We analyzed the cMNR mutation of each of the 15 genes in 10 MSI-H cell lines and 21 MSI-H cancers, and found frequent mutations of 12 genes in MSI-H cell lines and cancers, but not in microsatellite stable (MSS) cancers. Among these genes, the most frequently mutated in MSI-H cell lines were MLL3 (70%), PHACTR4 (70%), RUFY2 (50%) and TBC1D23 (50%). MLL3, which has already been implicated in cancer, had the highest mutation frequency in MSI-H cancers (48%). Our combined approach of NMD block, database search, and mutation analysis has identified a large number of genes mutated in their cMNR in MSI-H cancers. The identified mutations are expected to contribute to MSI-H tumorigenesis by causing an absence of gene expression or low gene dosage effects.

Ralstonia solanacearum strain OE1-1 causes bacterial wilt on tobacco plants. The popA-mutant 31b, derived from OE1-1 by insertion of transposon Tn4431, did not cause wilt on tobacco plants inoculated through the roots. However, when 31b was directly inoculated into xylem vessels, the tobacco plants wilted, similarly to those inoculated with OE1-1. 31b retained its exopolysaccharide productivity and its type-III secretion function. Furthermore, 31b grew in intercellular spaces and systemically infected tobacco plants, similarly to OE1-1. popA consists of an operon with popB and popC, and suppression of popB and popC expression resulting from polar mutation by transposon insertion did not affect the virulence of 31b. The mutated popA (popA31b) was composed of 960 nucleotides, including 39 derived from Tn4431. A recombinant mutant from OE1-1, where popA31b was introduced by marker exchange, showed the same phenotype as 31b. PopA31b protein was extracellularly secreted by 31b co-cultured with Arabidopsis thaliana. These results suggest that PopA31b extracellularly secreted by 31b in intercellular spaces may be implicated in suppression of disease development, leading to inability of the bacteria to induce wilt on plants. Taken together, interactions between host plants and R. solanacearum existing in intercellular spaces immediately after invasion may be involved in disease development.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disorder caused by mutations in the NF1 gene. In this paper we report our experience using the cDNA-SSCP/HD analysis as a mutational screening approach and the double characterization of all mutations at the DNA and RNA levels. Two hundred and eighty-two different mutations (in 374 independent patients) were identified, 140 of which were novel in our population. Most of these mutations are unique and distributed along the gene. However, we also detected 37 recurrent mutations. Our approach is limited with respect to the detection of single base substitutions, but it is highly effective in the detection of frameshift mutations and mutations that affect the correct splicing. Due to this bias we focus here in the characterization of these two types of mutations. Forty-seven percent of mutations found were frameshift mutations, with small deletions being 2.3 times more common than small insertions. At the mRNA level, 44% of mutations affected the correct splicing, 80% of them located in the consensus sequences, with the donor site being much more frequently involved. The remaining 20% consisted of mutations located in deep intronic sites and mutations located in the coding region. In general the latter group produces different types of mutated transcripts with specific proportions for each mutation. The double characterization of mutations at the DNA and RNA levels enables to detect a broader spectrum of mutations than any single level approach, and provides a greater understanding of their molecular pathogenesis. © 2008 Wiley-Liss, Inc.

Nonsense-mediated decay (NMD) is a eukaryotic cellular RNA surveillance and quality-control mechanism that degrades mRNA containing premature stop codons (nonsense mutations) that otherwise may exert a deleterious effect by the production of dysfunctional truncated proteins. Collagen X (COL10A1) nonsense mutations in Schmid-type metaphyseal chondrodysplasia are localized in a region toward the 3′ end of the last exon (exon 3) and result in mRNA decay, in contrast to most other genes in which terminal-exon nonsense mutations are resistant to NMD. We introduce nonsense mutations into the mouse Col10a1 gene and express these in a hypertrophic-chondrocyte cell line to explore the mechanism of last-exon mRNA decay of Col10a1 and demonstrate that mRNA decay is spatially restricted to mutations occurring in a 3′ region of the exon 3 coding sequence; this region corresponds to where human mutations have been described. This localization of mRNA-decay competency suggested that a downstream region, such as the 3′ UTR, may play a role in specifying decay of mutant Col10a1 mRNA containing nonsense mutations. We found that deleting any of the three conserved sequence regions within the 3′ UTR (region I, 23 bp; region II, 170 bp; and region III, 76 bp) prevented mutant mRNA decay, but a smaller 13 bp deletion within region III was permissive for decay. These data suggest that the 3′ UTR participates in collagen X last-exon mRNA decay and that overall 3′ UTR configuration, rather than specific linear-sequence motifs, may be important in specifying decay of Col10a1 mRNA containing nonsense mutations.

Erwinia carotovorasubsp.carotovorastrain ATTn10 produces theβ-lactam antibiotic 1-carbapen-2-em-3-carboxylic acid (carbapenem) by expressing thecarABCDEFGHoperon. Mutants exhibiting increased carbapenem gene transcription were positively selected using an engineered strain with a functionalβ-lactamase translational fusion incarH, the last gene of the operon. However, spontaneous ampicillin-resistant mutants were isolated even when transcription ofcarH : : blaMwas blocked by a strongly polar mutation incarE. The mechanism of resistance was shown to be due to cryptic IS10elements transposing upstream ofcarH : : blaM, thereby providing new promoters enablingcarH : : blaMtranscription. Southern blots showed that IS10was present in multicopy in ATTn10. In addition, a Tn10genetic remnant was discovered. The results offer insights into the genetic archaeology of strain ATTn10 and highlight the powerful impacts of cryptic IS elements in bacterial adaptive evolution.

In the present work we have characterized for the first time non-lethal nonsense mutations in the essential gene SUP35, which codes for the translation termination factor eRF3 in Saccharomyces cerevisiae. The screen used was based on selection for simultaneous suppression of two auxotrophic nonsense mutations. Among 48 mutants obtained, sixteen were distinguished by the production of a reduced amount of eRF3, suggesting the appearance of nonsense mutations. Fifteen of the total mutants were sequenced, and the presence of nonsense mutations was confirmed for nine of them. Thus a substantial fraction of the sup35 mutations recovered are nonsense mutations located in different regions of SUP35, and such mutants are easily identified by the fact that they express reduced amounts of eRF3. Nonsense mutations in the SUP35 gene do not lead to a decrease in levels of SUP35 mRNA and do not influence the steady-state level of eRF1. The ability of these mutations to complement SUP35 gene disruption mutations in different genetic backgrounds and in the absence of any tRNA suppressor mutation was demonstrated. The missense mutations studied, unlike nonsense mutations, do not decrease steady-state amounts of eRF3.

It was thought that polycistronic transcription is a characteristic of bacteria and archaea, where many of the genes are clustered in operons composed of two to more than ten genes. By contrast, the genes of eukaryotes are generally considered to be monocistronic, each with its own promoter at the 5' end and a transcription terminator at the 3' end; however, it has recently become clear that not all eukaryotic genes are transcribed monocistronically. Numerous instances of polycistronic transcription in eukaryotes, from protists to chordates, have been reported. These can be divided into two broad types. Dicistronic transcription units specify a messenger RNA (mRNA) encoding two separate genes that is transported to the cytoplasm and translated in that form. Presumably, internal ribosome entry sites (IRES), or some form of translational re-initiation following the stop codon, are responsible for allowing translation of the downstream gene. In the other type, the initial transcript is processed by 3' end cleavage and trans-splicing to create monocistronic mRNAs that are transported to the cytoplasm and translated. Like bacterial operons, eukaryotic operons often result in co-expression of functionally related proteins.

Numerous studies have established important roles for microRNAs (miRNAs) in regulating gene expression. Here, we report that miRNAs also serve as a surveillance system to repress the expression of nonsense mRNAs that may produce harmful truncated proteins. Upon recognition of the premature termination codon by the translating ribosome, the downstream portion of the coding region of an mRNA is redefined as part of the 3′ untranslated region; as a result, the miRNA-responsive elements embedded in this region can be detected by miRNAs, triggering accelerated mRNA deadenylation and translational inhibition. We demonstrate that naturally occurring cancer-causing APC (adenomatous polyposis coli) nonsense mutants which escape nonsense-mediated mRNA decay (NMD) are repressed by miRNA-mediated surveillance. In addition, we show that miRNA-mediated surveillance and exon–exon junction complex-mediated NMD are not mutually exclusive and act additively to enhance the repressive activity. Therefore, we have uncovered a new role for miRNAs in repressing nonsense mutant mRNAs. DOI: http://dx.doi.org/10.7554/eLife.03032.001, eLife digest To produce a protein from a gene, the sequence of the gene must be transcribed to produce a molecule of messenger RNA (mRNA). The sequence of the mRNA is then read in groups of three letters at a time (called codons), and each codon instructs for a particular amino acid to be added into the protein. Some codons, however, do not code for an amino acid and instead these ‘stop codons’ mark the end of a protein. If a DNA letter is added, lost, or changed, this mutation can sometimes produce a stop codon too early in the mRNA sequence. This is called a nonsense mutation, and produces truncated proteins that either work incorrectly or do not work at all, which can harm the organism. For example, people with a nonsense mutation in the human tumor suppressor gene called APC—which normally stops uncontrolled cell growth and division—are more likely to develop colon cancer than people without this mutation. Cells in the body employ several different surveillance mechanisms to detect nonsense mutations. The best-known mechanism involves a large protein group called the exon–exon junction complex (EJC), which binds to sites within the mRNA. The cellular translation machinery removes all the EJCs bound to a normal mRNA during the production of proteins. If the translation machinery reaches a stop codon too early, so that EJCs located downstream of it are not removed, the mRNA molecule is destroyed. However, this mechanism does not work for all genes—including APC. Very short sections of RNA called microRNAs regulate protein production by causing mRNAs to degrade and by inhibiting their translation, and Zhao et al. have now found that microRNAs also act as a defense against nonsense mutations in the APC gene. A premature stop codon exposes sites further along the mRNA molecule that microRNA molecules bind to, which triggers the breaking down of the mRNA and inhibits its translation. The microRNA surveillance system works independently of the system involving the EJC. However, both mechanisms can work in parallel alongside each other, which provides extra protection against nonsense mutations. Zhao et al. also found that microRNAs can protect against nonsense mutations in several other types of gene found in human cells. Therefore, microRNA surveillance is likely to be a common method employed by cells to restrict the production of potentially harmful truncated proteins. DOI: http://dx.doi.org/10.7554/eLife.03032.002

Using a pair of plasmids carrying the rpsL target sequence in different orientations to the replication origin, we analyzed a large number of forward mutations generated in wild-type and mismatch-repair deficient (MMR(-)) Escherichia coli cells to assess the effects of directionality of replication-fork movement on spontaneous mutagenesis and the generation of replication error. All classes of the mutations found in wild-type cells but not MMR(-) cells were strongly affected by the directionality of replication fork movement. It also appeared that the directionality of replication-fork movement governs the directionality of sequence substitution mutagenesis, which occurred in wild-type cells at a frequency comparable to base substitutions and single-base frameshift mutations. A very strong orientation-dependent hot-spot site for single-base frameshift mutations was discovered and demonstrated to be caused by the same process involved in sequence substitution mutagenesis. It is surprising that dnaE173, a potent mutator mutation specific for sequence substitution as well as single-base frameshift, did not enhance the frequency of the hot-spot frameshift mutation. Furthermore, the frequency of the hot-spot frameshift mutation was unchanged in the MMR(-) strain, whereas the mutHLS-dependent mismatch repair system efficiently suppressed the generation of single-base frameshift mutations. These results suggested that the hot-spot frameshift mutagenesis might be initiated at a particular location containing a DNA lesion, and thereby produce a premutagenic replication intermediate resistant to MMR. Significant numbers of spontaneous single-base frameshift mutations are probably caused by similar mechanisms.

The SUF13 and SUF14 genes were identified among extragenic suppressors of +1 frameshift mutations. SUF13 is synonymous with MBF1, a single-copy nonessential gene coding for a POLII transcription factor. The suf13-1 mutation is a two-nucleotide deletion in the SUF13/MBF1 coding region. A suf13::TRP1 null mutant suppresses +1 frameshift mutations, indicating that suppression is caused by loss of SUF13 function. The suf13-1 suppressor alters sensitivity to aminoglycoside antibiotics and reduces the accumulation of his4-713 mRNA, suggesting that suppression is mediated at the translational level. The SUF14 gene is synonymous with RPS3, a single-copy essential gene that codes for the ribosomal protein S3. The suf14-1 mutation is a missense substitution in the coding region. Increased expression of S3 limits the accumulation of SUF14 mRNA, suggesting that expression is autoregulated. A frameshift mutation in SUF14 that prevents full-length translation eliminated regulation, indicating that S3 is required for regulation. Using CUP1-SUF14 and SUF14-lacZ fusions, run-on transcription assays, and estimates of mRNA half-life, our results show that transcription plays a minor role if any in regulation and that the 5′-UTR is necessary but not sufficient for regulation. A change in mRNA decay rate may be the primary mechanism for regulation.

Special search for frameshift mutations, which are suppressed by the cytoplasmic [PSI] factor and by omnipotent nonsense suppressors (recessive mutations in theSUP35and SUP45genes), partially inactivating a translation termination complex, was initiated in theLYS2gene in the yeast Saccharomyces cerevisiae.Mutations were obtained after exposure to UV light and treatment with a mixture of 1,6- and 1,8-dinitropyrene (DNP). This mixture was shown to induce mutations of the frameshift type with a high frequency. The majority of these mutations were insertions of one A or T, which is in good agreement with the data obtained in studies of DNP-induced mutagenesis in other eukaryotes. Frameshift suppression was shown on the example of the mutation obtained in this work (lys2-90), which carried the insertion of an extra T in the sequence of five T. This frameshift suppression was first shown to occur in the presence of the [PSI] factor (i.e., due to the prionization of the translation release factor eRF3) and as a result of mutations in genes SUP35orSUP45, which partially inactivate translation termination factors eRF3 and eRF1, respectively. Alternative mechanisms of programmed translational frameshifting in the course of translation and the possibility of enhancing the effectiveness of such frameshifting in the presence of the [PSI] factor are considered.

Editor—Mitochondrial DNA (MtDNA) is highly polymorphic. Each person is estimated to differ from another on average at about 25 base pairs among the 16 569 that comprise the mitochondrial genome.1 Thus, only a small fraction of mtDNA variants are likely to be of pathogenic significance. Criteria currently used for determining the likelihood that a missense mutation is pathogenic include heteroplasmy (the percentage of mtDNA molecules within a cell or tissue harbouring a mutation), evolutionary conservation of the altered amino acid, a maternal inheritance pattern, absence of the mutation in controls, clinical features commonly linked to known pathogenic mtDNA mutations, and defects in mitochondrial morphologies and enzyme activities.1 However, these criteria are inadequate for several reasons. Many mitochondrial missense mutations are homoplasmic. Pathogenic mtDNA mutations are typically characterised by incomplete penetrance, even when homoplasmic, presumably reflecting interactions with environmental and genetic factors.2 As a result, inherited mtDNA mutations may manifest as “sporadic” disorders rather than with the classical maternal inheritance pattern. Biochemical assays may also be inconclusive, as the expression of a defect in mitochondrial function depends on the nuclear background and tissue type in which the mutation is studied.3 ,4 As a result, mtDNA mutations identified in rare families or subjects with a putative mitochondrial genetic disorder are often of uncertain pathogenic significance. Over 100 point mutations have been identified in mitochondrial genes in association with human disease, at least 45 of which are missense mutations in protein encoding genes.5 However, frameshift mtDNA mutations are exceedingly rare. An acquired frameshift 4 bp deletion mutation was identified in the cytochrome b gene at nucleotide position (np) 14 787-14 790 in a patient with parkinsonism-MELAS overlap syndrome6 and somatic mutations including frameshift mutations have been found in human cancers.7 ,8In contrast, inherited frameshift …

Rhodobacter capsulatus ORF1696 mutant strains were created by insertion of antibiotic resistance cartridges at different sites within the ORF1696 gene in a strain that lacks the light-harvesting II (LHII) complex. Steady-state absorption spectroscopy profiles and the kinetics of the light-harvesting I (LHI) complex assembly and decay were used to evaluate the function of the ORF1696 protein in various strains. All of the mutant strains were found to be deficient in the LHI complex, including one (ΔNae) with a disruption located 13 codons before the 3′ end of the gene. A 5′-proximal disruption after the 31st codon of ORF1696 resulted in a mutant strain (ΔMun) with a novel absorption spectrum. The two strains with more 3′ disruptions (ΔStu and ΔNae) were restored nearly to the parental strain phenotype when trans complemented with a plasmid expressing the ORF1696 gene, but ΔMun was not. The absorption spectrum of ΔMun resembled that of a strain which had a polar mutation in ORF1696 . We suggest that a rho -dependent transcription termination site exists between the Mun I and proximal Stu I sites of ORF1696 . A comparison of LHI complex assembly kinetics showed that assembly occurred 2.6-fold faster in the parental strain than in strain ΔStu. In contrast, LHI complex decay occurred 1.7-fold faster in the ORF1696 parental strain than in ΔStu. These results indicate that the ORF1696 protein has a major effect on LHI complex assembly, and models of ORF1696 function are proposed.

Mutations in the PKD2 gene on the long arm of chromosome 4 are responsible for ~ 15% of cases of polycystic kidney disease. Perhaps the only difference from the more common ADPKD1 cases is the rate of progression of cystic changes, and the age of onset, which is 10-15 years later for the ADPKD2 form. In Cyprus there are at least three large families, documented by molecular linkage analysis, that map to the PKD2 locus. For two of them the defects were recently shown to be nonsense mutations at positions arginine 742 and glutamine 405. In this report, we describe the mutation in the third family, CY1602. For this, the entire coding sequence was systematically screened by single strand conformation analysis and heteroduplex formation. A novel mutation was identified in exon 2 where a new cytosine residue was inserted immediately after codon 231 (231insC). It causes a translation frameshift and is expected to lead to the introduction of 37 novel amino acids before the translation reaches a new STOP codon. It is the most amino terminal mutation reported to date, and based on the protein's modeled structure, is predicted to be within the first transmembrane domain. It is the fourth PKD2 mutation reported thus far, and the first which is not a nonsense mutation. 6 949 952 Cited By :22

Small regulatory RNAs (sRNAs) are influential post-transcriptional modulators of gene expression in bacteria. They regulate gene expression by base pairing to target mRNAs, leading to inhibition of translation and/or alteration of mRNA stability. Recently, several sRNAs have been discovered to regulate genes encoded in operons. In some cases, these sRNAs regulate all the genes encoded by the polycistronic mRNA (coordinate regulation) while in other cases, only a select subset of cistrons is controlled by the sRNA (discoordinate regulation). In this point of view, mechanisms of regulation and characteristics of sRNA-mRNA interactions involving polycistronic mRNAs are described. The diversity in mechanisms represented by these few characterized examples suggests that we still have much to learn about sRNA regulation of long polycistronic messages.

X-linked chronic granulomatous disease (CGD) is due to mutations in the gp91phox gene on Xp21.1. Studies in white and Japanese X-linked CGD patients have shown mutations in nearly every exon. We studied the molecular defect of seven Chinese patients with X-linked CGD from six unrelated families. Mutations were located by single-strand conformation polymorphism and then defined by sequence analysis. The mutations were two different amino acid substitutions, a nonsense mutation, an in-frame trinucleotide deletion, a single A insertion causing a frameshift, and a premature stop. Lastly, a rare splice site mutation caused by G to A transition at the terminal nucleotide of exon 3, resulting in the skipping of exon 3, was found. The possible effects of these mutations on protein structure-function or splicing were discussed. Together with previous reports, the A insertion in the run of six As from nucleotide 749 to 754 and the G to A transition at the terminal position of exon 3 may be mutation hotspots of the gp91phox gene. The extreme heterogeneous mutations found in our patients suggest the absence of ethnic group-specific mutation.

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

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.

We screened a total of 92 unrelated patients with neurofibromatosis type 1 (NF1) for mutations in exon 37 of the NF1 gene, by using temperature gradient gel electrophoresis. Two novel mutations were found: a 4 bp deletion in a so-called quasi-symmetric element (6789del-TTAC) and a recurrent nonsense mutation, which was identified in two unrelated patients, at codon 2264 (C6792A). The independent origin of the latter mutation in two families was confirmed by haplotype analysis. The nonsense mutation and the 4 bp deletion are both predicted to lead to a truncated protein product lacking the C-terminal 20% (approximately) of its sequence. The occurrence of three independent mutations among 92 NF1 patients at codons 2263-2264 (exon 37) suggests that a specific search for mutations in this area should be undertaken in screening programs for NF1 mutations.

Cancer genomes contain vast amounts of somatic mutations, many of which are passenger mutations not involved in oncogenesis. Whereas driver mutations in protein-coding genes can be distinguished from passenger mutations based on their recurrence, non-coding mutations are usually not recurrent at the same position. Therefore, it is still unclear how to identify cis-regulatory driver mutations, particularly when chromatin data from the same patient is not available, thus relying only on sequence and expression information. Here we use machine-learning methods to predict functional regulatory regions using sequence information alone, and compare the predicted activity of the mutated region with the reference sequence. This way we define the Predicted Regulatory Impact of a Mutation in an Enhancer (PRIME). We find that the recently identified driver mutation in the TAL1 enhancer has a high PRIME score, representing a “gain-of-target” for MYB, whereas the highly recurrent TERT promoter mutation has a surprisingly low PRIME score. We trained Random Forest models for 45 cancer-related transcription factors, and used these to score variations in the HeLa genome and somatic mutations across more than five hundred cancer genomes. Each model predicts only a small fraction of non-coding mutations with a potential impact on the function of the encompassing regulatory region. Nevertheless, as these few candidate driver mutations are often linked to gains in chromatin activity and gene expression, they may contribute to the oncogenic program by altering the expression levels of specific oncogenes and tumor suppressor genes., Author Summary Precise regulation of gene expression is controlled by cis-regulatory modules (CRM) containing binding sites for transcription factors (TF). The genome-wide location of all TF binding sites can often be obtained by ChIP-seq (chromatin immunoprecipitation followed by deep sequencing), yet in most cases only a minority of the binding peaks actually represent functional CRMs that control the transcription initiation of a bona fide TF target gene. Here, we investigated for 45 cancer-related TFs how machine-learning approaches can be used to predict functional TF target CRMs. After careful evaluation of their performance, we used these TF-target classifiers to predict which cis-regulatory mutations may have a significant impact on gene regulation by evaluating whether the mutation causes a significant gain or loss in the probability that the CRM is a functional TF target. We found that Random Forest classifiers can achieve more than 100-fold higher specificity for mutation prediction compared to the simple approaches based on scanning with position weight matrices. By scanning somatic mutations in breast cancer genomes and in the HeLa genome, we finally show that our TF-target classifiers can identify high impact non-coding mutations that are associated with concordant TF binding, gene expression changes and chromatin activity. In conclusion, TF-specific Random Forest classifiers can be used to prioritize cis-regulatory mutations in cancer genomes with high accuracy.

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.

Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis (CF) patients. Previously, we reported that ShvR, a LysR regulator, influences colony morphology, virulence, and biofilm formation and regulates the expression of an adjacent 24-kb genomic region encoding 24 genes. In this study, we report the functional characterization of selected genes in this region. A Tn 5 mutant with shiny colony morphology was identified with a polar mutation in BCAS0208, predicted to encode an acyl-coenzyme A dehydrogenase. Mutagenesis of BCAS0208 and complementation analyses revealed that BCAS0208 is required for rough colony morphology, biofilm formation, and virulence on alfalfa seedlings. It was not possible to complement with BCAS0208 containing a mutation in the catalytic site. BCAS0201, encoding a putative flavin adenine dinucleotide (FAD)-dependent oxidoreductase, and BCAS0207, encoding a putative citrate synthase, do not influence colony morphology but are required for optimum levels of biofilm formation and virulence. Both BCAS0208 and BCAS0201 contribute to pellicle formation, although individual mutations in each of these genes had no appreciable effect on pellicle formation. A mutant with a polar insertion in BCAS0208 was significantly less virulent in a rat model of chronic lung infection as well as in the alfalfa model. Genes in this region were shown to influence utilization of branched-chain fatty acids, tricarboxylic acid cycle substrates, l -arabinose, and branched-chain amino acids. Together, our data show that the ShvR-regulated genes BCAS0208 to BCAS0201 are required for the rough colony morphotype, biofilm and pellicle formation, and virulence in B. cenocepacia .

A host-inducible lacZ fusion was mapped down-stream of the nodYABCSUIJ operon in Bradyrhizobium japonicum strain USDA110. Sequencing of this region identified three novel genes, nolMNO. RNA dot blot analysis showed that nolO transcription is nodD1-dependent and that a polar mutation in nodS, located 5 kilobases upstream of nolO, blocks the transcription of nolO. Coupled with the host-inducible nature of nolO expression, these results indicate that nolMNO are part of a 9-kilobase operon, nodYABCSUIJnolMNO. The lipo-oligosaccharide nodulation signals produced by strains SL67 (nolO-) and SL65(nolNO-) were purified, and their chemical structures were determined. In addition to the wild-type signal molecules, both mutants produced modified compounds that are not produced by the parent strain USDA110. The most prevalent difference observed was the absence of the 2-O-methylfucosyl residue from the mutant structures. In addition, metabolites were found in which the N-acetylglucosamine residue at the reducing end was glycosidically linked to glycerol. These alterations in the profiles of nodulation signals produced by strains SL67 and SL65 were accompanied by reduced nodulation efficiency on all hosts tested.

The mRNAs of six C. elegans genes are known to be trans-spliced to SL2. We report here that a similarly oriented gene is located 100-300 bp upstream of each. We present evidence that the genes in these clusters are cotranscribed and downstream mRNAs are formed by cleavage at the polyadenylation site and trans-splicing. From one three-gene cluster we isolated cDNA clones representing both polycistronic RNAs and mRNAs polyadenylated at the free 3' end created by trans-splicing, suggesting that polycistronic RNAs can be processed by trans-splicing. Several experiments indicate that SL2 trans-splicing is a consequence of a gene's downstream location in an operon. In particular, when an SL1-accepting gene was moved to a downstream location, its mRNA was trans-spliced largely to SL2. The possible regulatory significance of cotranscription of C. elegans genes is discussed.

We report genomic linkage of a pair of tandem, identical ubiquitin-extension protein 52 (EP52) genes, a novel EF-hand superfamily member gene (EFH5), and the calmodulin gene cluster in Trypanosoma brucei. The intergenic regions of these four genes are short: about 108 bp between the calmodulin gene C and the EFH5 gene, about 111 bp between the EFH5 gene and the ubiquitin-EP52/1 gene, and about 116 bp between the ubiquitin-EP52/1 and -EP52/2 genes. RNA molecules that span these three intergenic regions have been detected by polymerase chain reaction, which suggests that the genes are transcribed in a polycistronic manner. Transcription of the calmodulin, EFH5, and ubiquitin-EP52 genes in isolated nuclei is rapidly inactivated by UV irradiation, which further strengthens the hypothesis that this cluster of three different genes is transcribed in a polycistronic manner and suggests that they are under the control of a single distant upstream promoter. These results suggest that polycistronic transcription is common in trypanosomes and will probably be found for most, if not all, protein-encoding genes. The presence of at least three housekeeping genes with different known or potential regulatory functions within a polycistronic unit suggests that regulation of transcription initiation plays an important role in the coordinated expression of housekeeping genes in trypanosomes.

Trans-splicing of one of two short leader RNAs, SL1 or SL2, occurs at the 5′ ends of pre-mRNAs of many C. elegans genes. We have exploited RNA-sequencing data from the modENCODE project to analyze the transcriptome of C. elegans for patterns of trans-splicing. Transcripts of ∼70% of genes are trans-spliced, similar to earlier estimates based on analysis of far fewer genes. The mRNAs of most trans-spliced genes are spliced to either SL1 or SL2, but most genes are not trans-spliced to both, indicating that SL1 and SL2 trans-splicing use different underlying mechanisms. SL2 trans-splicing occurs in order to separate the products of genes in operons genome wide. Shorter intercistronic distance is associated with greater use of SL2. Finally, increased use of SL1 trans-splicing to downstream operon genes can indicate the presence of an extra promoter in the intercistronic region, creating what has been termed a “hybrid” operon. Within hybrid operons the presence of the two promoters results in the use of the two SL classes: Transcription that originates at the promoter upstream of another gene creates a polycistronic pre-mRNA that receives SL2, whereas transcription that originates at the internal promoter creates transcripts that receive SL1. Overall, our data demonstrate that >17% of all C. elegans genes are in operons.

Nonsense mutations account for approximately 11% of all described gene lesions causing human inherited disease and approximately 20% of disease-associated single-basepair substitutions affecting gene coding regions. Pathological nonsense mutations resulting in TGA (38.5%), TAG (40.4%), and TAA (21.1%) occur in different proportions to naturally occurring stop codons. Of the 23 different nucleotide substitutions giving rise to nonsense mutations, the most frequent are CGA --TGA (21%; resulting from methylation-mediated deamination) and CAG --TAG (19%). The differing nonsense mutation frequencies are largely explicable in terms of variable nucleotide substitution rates such that it is unnecessary to invoke differential translational termination efficiency or differential codon usage. Some genes are characterized by numerous nonsense mutations but relatively few if any missense mutations (e.g., CHM) whereas other genes exhibit many missense mutations but few if any nonsense mutations (e.g., PSEN1). Genes in the latter category have a tendency to encode proteins characterized by multimer formation. Consistent with the operation of a clinical selection bias, genes exhibiting an excess of nonsense mutations are also likely to display an excess of frameshift mutations. Tumor suppressor (TS) genes exhibit a disproportionate number of nonsense mutations while most mutations in oncogenes are missense. A total of 12% of somatic nonsense mutations in TS genes were found to occur recurrently in the hypermutable CpG dinucleotide. In a comparison of somatic and germline mutational spectra for 17 TS genes, approximately 43% of somatic nonsense mutations had counterparts in the germline (rising to 98% for CpG mutations). Finally, the proportion of disease-causing nonsense mutations predicted to elicit nonsense-mediated mRNA decay (NMD) is significantly higher (P=1.56 x 10(-9)) than among nonobserved (potential) nonsense mutations, implying that nonsense mutations that elicit NMD are more likely to come to clinical attention.

Neurofibromatosis type 1 (NF1) is caused by mutations in the neurofibromin (NF1) gene. Mutation analysis of NF1 is complicated by its large size, the lack of mutation hotspots, pseudogenes and frequent de novo mutations. Additionally, the search for NF1 mutations on the mRNA level is often hampered by nonsense-mediated mRNA decay (NMD) of the mutant allele. In this study we searched for mutations in a cohort of 38 patients and investigated the relationship between mutation type and allele-specific transcription from the wild-type versus mutant alleles. Quantification of relative mRNA transcript numbers was done by Pyrosequencing, a novel real-time sequencing method whose signals can be quantified very accurately. We identified 21 novel mutations comprising various mutation types. Pyrosequencing detected a definite relationship between allelic NF1 transcript imbalance due to NMD and mutation type in 24 of 29 patients who all carried frame-shift or nonsense mutations. NMD was absent in 5 patients with missense and silent mutations, as well as in 4 patients with splice-site mutations that did not disrupt the reading frame. Pyrosequencing was capable of detecting NMD even when the effects were only moderate. Diagnostic laboratories could thus exploit this effect for rapid prescreening for NF1 mutations as more than 60% of the mutations in this gene disrupt the reading frame and are prone to NMD.

Operons are a major feature of all prokaryotic genomes, but how and why operon structures vary is not well understood. To elucidate the life-cycle of operons, we compared gene order between Escherichia coli K12 and its relatives and identified the recently formed and destroyed operons in E. coli. This allowed us to determine how operons form, how they become closely spaced, and how they die. Our findings suggest that operon evolution may be driven by selection on gene expression patterns. First, both operon creation and operon destruction lead to large changes in gene expression patterns. For example, the removal of lysA and ruvA from ancestral operons that contained essential genes allowed their expression to respond to lysine levels and DNA damage, respectively. Second, some operons have undergone accelerated evolution, with multiple new genes being added during a brief period. Third, although genes within operons are usually closely spaced because of a neutral bias toward deletion and because of selection against large overlaps, genes in highly expressed operons tend to be widely spaced because of regulatory fine-tuning by intervening sequences. Although operon evolution may be adaptive, it need not be optimal: new operons often comprise functionally unrelated genes that were already in proximity before the operon formed., Synopsis In bacteria, adjacent genes are often transcribed together in operons. Which genes are placed together in operons varies greatly across bacteria. This diversity of operon structure can be used to predict the function of genes: genes that are sometimes in an operon are likely to have related functions, even if they are transcribed separately in the organism of interest. However, it has not been clear why this diversity exists or what its consequences are. This work reconstructs evolutionarily recent changes to operon structures in the well-studied bacterium Escherichia coli. Changes in operon structure are shown to be associated with changes in gene expression patterns, so the diversity in operon structure may reflect adaptation to differing lifestyles. Indeed, some of these changes appear to be beneficial to the organism. This work also reconstructs the molecular mechanisms of operon evolution. Understanding these mechanisms should aid other analyses of bacterial genomes. For example, new operons often arise by deleting the DNA between functionally unrelated genes that happen to be near each other. Thus, recently evolved operons should not be used to infer their genes' function. Overall, this work provides a framework for understanding the evolutionary life-cycle of operons.

The organization of bacterial genes into operons was originally ascribed to the benefits of co-regulation. More recently, the “selfish operon” model, in which operons are formed by repeated gain and loss of genes, was proposed. Indeed, operons are often subject to horizontal gene transfer (HGT). On the other hand, non-HGT genes are particularly likely to be in operons. To clarify whether HGT is involved in operon formation, we identified recently formed operons in Escherichia coli K12. We show that genes that have homologs in distantly related bacteria but not in close relatives of E. coli—indicating HGT—form new operons at about the same rates as native genes. Furthermore, genes in new operons are no more likely than other genes to have phylogenetic trees that are inconsistent with the species tree. In contrast, essential genes and ubiquitous genes without paralogs—genes believed to undergo HGT rarely—often form new operons. We conclude that HGT is not a cause of operon formation but instead promotes the prevalence of pre-existing operons. To explain operon formation, we propose that new operons reduce the amount of regulatory information required to specify optimal expression patterns and infer that operons should be more likely to evolve than independent promoters when regulation is complex. Consistent with this hypothesis, operons have greater amounts of conserved regulatory sequences than do individually transcribed genes.

To investigate the interaction between DNA abnormalities, p53 and p16 gene mutations, and methylation and protein expression, 20 cancer cell lines were examined by Western blotting. A clear relation was found to exist between p53 accumulation and mutation status. Of 20 cell lines examined, 14 demonstrated p53 homozygous mutations in exons 3-10, including 12 missense mutations, one nonsense mutation, and one frameshift mutation. Overexpression of p53 was always linked to missense mutations in exons 6-8. Intermediate expression of p53 was noted in cells with missense mutations or polymorphism to proline at codon 72 in exons 4-5, whereas there was slight or no visible expression in wild type cells and in cells with nonsense and frameshift mutations. DNA aberration in the p16 promoter gene correlated significantly with protein expression of the p16 suppressor gene. Overexpression was noted in six cell lines, intermediate expression in two, and slight or no visible expression in 12. Methylation-caused disappearance of p16 protein was noted in 40% (8/20) of the cell lines. Of six cell lines overexpressing p16 protein, two could be amplified with primers for both unmethylated and methylated forms in a methylation-specific RCP analysis. One cell line with no visible expression could also be amplified with both primers. Overexpression or disappearance of p16 protein may readily occur when one of two alleles has been methylated.

To date, approximately 200 different mutations in the MECP2 gene have been identified. We analyzed the entire coding sequence of the MECP2 gene and the X-chromosome inactivation pattern in 42 sporadic cases of Rett syndrome. Of the 42 patients, 30 had pathogenic mutations, including 14 different mutations: 9 missense mutations, 4 nonsense mutations, and 1 frameshift mutation. One was a novel mutation (S134P). There was a tendency for patients who had a nonsense mutation in the transcriptional repression domain region to show earlier onset of regression and more severe language retardation than patients with a mutation in the methyl-CpG binding domain region. However, the parameters of clinical severity were variable among patients with the same type of mutation, depending on the pattern of X-chromosome inactivation. This study suggests that the X-chromosome inactivation pattern can modify the phenotype of Rett syndrome, which is primarily determined by the type and site of MECP2 gene mutation. ( J Child Neurol 2004;19:503—508).

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.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs containing premature translation termination codons. In mammalian cells, a termination codon is ordinarily recognized as "premature" if it is located greater than 50-54 nucleotides 5' to the final exon-exon junction. We have described a set of naturally occurring human beta-globin gene mutations that apparently contradict this rule. The corresponding beta-thalassemia genes contain nonsense mutations within exon 1, and yet their encoded mRNAs accumulate to levels approaching wild-type beta-globin (beta(WT)) mRNA. In the present report we demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are intermediate between beta(WT) mRNA and beta-globin mRNA carrying a prototype NMD-sensitive mutation in exon 2 (codon 39 nonsense; beta 39). Functional analyses of these mRNAs with 5'-proximal nonsense mutations demonstrate that their relative resistance to NMD does not reflect abnormal RNA splicing or translation re-initiation and is independent of promoter identity and erythroid specificity. Instead, the proximity of the nonsense codon to the translation initiation AUG constitutes a major determinant of NMD. Positioning a termination mutation at the 5' terminus of the coding region blunts mRNA destabilization, and this effect is dominant to the "50-54 nt boundary rule." These observations impact on current models of NMD.

The Holt-Oram syndrome (OMIM 142900) is an autosomal dominant disorder with clinical features characterised by a variety of skeletal malformations and congenital heart defects.1–5 The gene for Holt-Oram syndrome has been identified as TBX5 on chromosome 12q24.6–11 TBX5 encodes a protein of 518 amino acids that belongs to the family of the T box transcriptional factors,10,11 and is expressed in embryonic heart and limb tissues, consistent with its involvement in development of the heart and skeletal structures.12–17 TBX5 contains a highly conserved DNA binding domain, the T box domain. Recently, three groups have provided direct evidence that TBX5 can bind to DNA and activate transcription of its target genes including the gene for atrial natriuretic factor ( ANF ).17–19 Furthermore, TBX5 can interact directly with the cardiac homeobox protein NKX2.5 synergistically to activate transcription of ANF .17,18 To date, 22 different non-translocation TBX5 mutations have been reported. These include five truncations (nonsense mutations), three splicing changes, six frameshift mutations, seven missense mutations, and one large deletion involving exons 3-9.20–22 Frameshift, splicing, and nonsense mutations are expected to produce truncated TBX5 or no TBX5 at all (for example, via nonsense mediated mRNA decay), which causes Holt-Oram syndrome by haploinsufficiency. However, the mechanisms by which the missense mutations cause Holt-Oram syndrome are not well understood. Ghosh et al 19 found that missense mutations G80R and R237Q eliminated DNA binding, whereas mutations G169R and S252I did not affect DNA binding. Hiroi et al 18 analysed the effect of G80R and R237Q on transcription activity of the ANF promoter. Mutation G80R caused significant reduction of transcriptional activation activity of TBX5 and loss of synergistic activation with NKX2.5. Surprisingly, mutation R237Q, which eliminated DNA binding, had minor effects on transcriptional activation.18 …

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.

Hereditary and sporadic gastrointestinal cancer of the microsatellite mutator phenotype (MMP) is characterized by a remarkable genomic instability at simple repeated sequences. The genomic instability is often caused by germline and somatic mutations in DNA mismatch repair (MMR) genes hMSH2 and hMLH1. The MMP can be also caused by epigenetic inactivation of hMLH1. The MMP generates many somatic frameshift mutations in genes containing mononucleotide repeats. We previously reported that in MMP tumors the hMSH6 and hMSH3 MMR genes often carry frameshift mutations in their (C)(8) and (A)(8) tracks, respectively. We proposed that these 'secondary mutator mutations' contribute to a gradual manifestation of the MMP. Here we report the detection of other frameshift, nonsense, and missense mutations in these genes in colon and gastric cancers of the MMP. A germline frameshift mutation was found in hMSH6 in a colon tumor harboring another somatic frameshift mutation. Several germline sequence variants and somatic missense mutations at conserved residues were detected in hMSH6 and only one was detected in hMSH3. Of the three hMSH6 germline variants in conserved residues, one coexisted with a somatic mutation at the (C)(8) track and another had a somatic missense mutation. We suggest that some of these germline and somatic missense variants are pathogenic. While biallelic hMSH6 and hMSH3 frameshift mutations were found in some tumors, many tumors seemed to contain only monoallelic mutations. In some tumors, these somatic monoallelic frameshift mutations at the (C)(8) and (A)(8) tracks were found to coexist with other somatic mutations in the other allele, supporting their functionality during tumorigenesis. However, the low incidence of these additional somatic mutations in hMSH6 and hMSH3 leaves many tumors with only monoallelic mutations. The impact of the frameshift mutations in gene expression was studied by comparative analysis of RNA and protein expression in different tumor cell clones with different genotypes. The results show that the hMSH6 (C)(8) frameshift mutation abolishes protein expression, ruling out a dominant negative effect by a truncated protein. We suggest the functionality of these secondary monoallelic mutator mutations in the context of an accumulative haploinsufficiency model.

We demonstrated the clinical characteristics of each member of a family from Oki Island in western Japan, whose members have familial amyotrophic lateral sclerosis (FALS) with a 2-base pair (bp) deletion at codon 126 of Cu/Zn superoxide dismutase (SOD1) gene. Mean disease duration among the Oki family members was about 2 years. Long-term survivors with respiratory support presented disturbances in eye movement and urination toward the end stages of the disease. In addition, we focused on in-vitro instabilities in the frameshift and nonsense mutations, including the 2-bp deletion, as well as some deletional, insertional and intronic mutations. These mutations were all found within exon 4, exon 5 and intron 4. As for the durations of illness, there were no significant differences between FALS patients with these SOD1 mutations and those with point mutations, although the former cases were likely to have shorter disease durations. In cell culture experiments, SOD1 proteins with frameshift and nonsense mutations were extremely unstable and showed very short half-lives. We postulated that the in-vitro instability of the mutant SOD1 might be related to the pathogenesis of FALS, e.g. through the mechanism of copper release.

The ability of Gram-negative phytopathogenic bacteria to cause disease is determined by hrp/hrc genes, which encode a type-III secretion system, and hop genes, which produce the secreted effector proteins (5). A >24-kb cluster of hrp/hop genes is necessary for Pantoea stewartii subsp. stewartii to incite Stewart’s Wilt on corn (1,3). This study describes some aspects of the genetic organization and function of the regulatory region in the hrp/hop cluster of P. stewartii involved in sensing environmental signals.

Publisher Summary This chapter provides an introduction to gene fusion. The development of gene fusion approaches came from studies on the lac operon of E. coli . The first fusions of lac were obtained unwittingly as revertants of strong polar mutations in the lacZ gene. Selection for restoration of the activity of the downstream lacY gene yielded many deletions that removed the polar mutation site, the promoter of lac, and fused the lacY gene to an upstream promoter of an unknown neighboring gene. Another important step in the evolution of uses of gene fusions came with the concept of signal sequence traps. The first development of this concept came out of the recognition that the bacterial enzyme alkaline phosphatase is active when it is exported to the periplasm but inactive when it is retained in the cytoplasm. Thus, alkaline phosphatase without its signal sequence provides an assay for export signals via gene fusion approaches—that is, alkaline phosphatase will only be active if one attaches a region of DNA that encodes a signal sequence, thus reallowing its export. Extending beyond the differentiation of exported vs cytosolic proteins, gene fusion techniques can be evolved to determine the subcellular localization of proteins more generally. The use of green fluorescent protein (GFP) fusions enhances this ability. In addition, reporter proteins that sense the specific features of organelle environment may provide a tool for detecting location and genetically manipulating signals for the localization process.

Neurofibromatosis type 1 (NF1) is caused by mutations in a tumour suppressor gene located on chromosome 17 (17q11.2). Disease causing mutations are dispersed throughout the gene, which spans 350 kilobases and includes 59 exons. A common consequence of NF1 mutations is introduction of a premature stop codon, and the majority of mutant genes encode truncated forms of neurofibromin. We used a protein truncation assay to screen for mutations in 15 NF1 patients and obtained positive results in 11 of them (73%). Sequencing of cDNA and genomic DNA yielded identification of 10 different mutations, including four splicing errors, three small deletions, two nonsense mutations, and one small insertion. Nine mutations were predicted to cause premature termination of translation, while one mutation caused in frame deletion as a result ofexon skipping. In one other case involving abnormal splicing, five different aberrantly spliced transcripts were detected. One germline nonsense mutation (R1306X, 3916C>T) corresponded to the same base change that occurs by mRNA editing in normal subjects. The second nonsense mutation (R2496X) was the sole germline mutation that has been previously described. The subjects studied represented typically affected NF1 patients and no correlations between genotype and phenotype were apparent. A high incidence of ocular hypertelorism was observed.

With the sequence of two bacterial genomes complete (Fleischmann et al., 1995; Fraser et al., 1995) and several more near completion, it is now possible to view the entire genetic structure of an organism with a new perspective. Yet this perspective is not a static one. As we learn more about various bacterial genomes, the number of characterized bacterial insertion sequences (ISs) continues to increase. Examples are known from a wide range of Gram-negative and Gram-positive bacterial species as well as from the archaebacteria (reviewed by Galas and Chandler, 1989; Murphy, 1989; Charlebois and Doolittle, 1989). ISs are normal constituents not only of many bacterial chromosomes but also of some plasmids and bacteriophages. The prevalence of transposable elements (ISs and transposons) with their capacity for moving from one site in the genome to another, for modifying gene expression, and for promoting genome rearrangements, contributes significantly to a genome in a state of continuous change.

The genomes of most eukaryotes are composed of genes arranged on the chromosomes without regard to function, with each gene transcribed from a promoter at its 5′ end. However, the genome of the free-living nematode Caenorhabditis elegans contains numerous polycistronic clusters similar to bacterial operons in which the genes are transcribed sequentially from a single promoter at the 5′ end of the cluster. The resulting polycistronic pre-mRNAs are processed into monocistronic mRNAs by conventional 3′ end formation, cleavage, and polyadenylation, accompanied by trans-splicing with a specialized spliced leader (SL), SL2. To determine whether this mode of gene organization and expression, apparently unique among the animals, occurs in other species, we have investigated genes in a distantly related free-living rhabditid nematode in the genus Dolichorhabditis (strain CEW1). We have identified both SL1 and SL2 RNAs in this species. In addition, we have sequenced a Dolichorhabditis genomic region containing a gene cluster with all of the characteristics of the C. elegans operons. We show that the downstream gene is trans-spliced to SL2. We also present evidence that suggests that these two genes are also clustered in the C. elegans and Caenorhabditis briggsae genomes. Thus, it appears that the arrangement of genes in operons pre-dates the divergence of the genus Caenorhabditis from the other genera in the family Rhabditidae, and may be more widespread than is currently appreciated.

Using a genetic selection for suppressors of a UGA nonsense mutation in trpA, we have isolated a G to A transition mutation at position 1491 in the decoding region of 16S rRNA. This suppressor displayed no codon specificity, suppressing UGA, UAG and UAA nonsense mutations and +1 and -1 frameshift mutations in lacZ. Subsequent examination of a series of mutations at G1491 and its base-pairing partner C1409 revealed various effects on nonsense suppression and frameshifting. Mutations that prevented Watson-Crick base pairing between these residues were observed to increase misreading and frameshifting. However, double mutations that retained pairing potential produced an antisuppressor or hyperaccurate phenotype. Previous studies of antibiotic resistance mutations and antibiotic and tRNA footprints have placed G1491 and C1409 near the site of codon-anticodon pairing. The results of this study demonstrate that the nature of the interaction of these two residues influences the fidelity of tRNA selection.