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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.

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.

Background— Long-QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene ( hERG ). More than 30% of the LQT2 mutations result in premature termination codons. Degradation of premature termination codon–containing mRNA transcripts by nonsense-mediated mRNA decay is increasingly recognized as a mechanism for reducing mRNA levels in a variety of human diseases. However, the role of nonsense-mediated mRNA decay in LQT2 mutations has not been explored. Methods and Results— We examined the expression of hERG mRNA in lymphocytes from patients carrying the R1014X mutation using a technique of allele-specific transcript quantification. The R1014X mutation led to a reduced level of mutant mRNA compared with that of the wild-type allele. The decrease in mutant mRNA also was observed in the LQT2 nonsense mutations W1001X and R1014X using hERG minigenes expressed in HEK293 cells or neonatal rat ventricular myocytes. Treatment with the protein synthesis inhibitor cycloheximide or RNA interference–mediated knockdown of the Upf1 protein resulted in the restoration of mutant mRNA to levels comparable to that of the wild-type minigene, suggesting that hERG nonsense mutations are subject to nonsense-mediated mRNA decay. Conclusions— These results indicate that LQT2 nonsense mutations cause a decrease in mutant mRNA levels by nonsense-mediated mRNA decay rather than production of truncated proteins. Our findings suggest that the degradation of hERG mutant mRNA by nonsense-mediated mRNA decay is an important mechanism in LQT2 patients with nonsense or frameshift mutations.

Cystic fibrosis (CF) is the most common lethal genetic disease among the Western population caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The development of effective therapeutic agents against CF requires a molecular understanding of how mutation-related structural alterations lead to impaired functioning of CFTR. The main cause for CF are processing mutations frequently found within CFTR-transmembrane (TM) segments which affect co-translational folding and insertion. However, the underlying molecular mechanisms for misfolding remain obscure, mainly because of the unavailability of high-resolution structures and a lack of methods suitable for studying membrane protein structure-function relationships.Devising a divide-and-conquer approach in combination with single-molecule FRET (smFRET), we present here a strategy to gain novel insights into the structural basis for conformational misfolding in CFTR. Using this approach, we study the CF-phenotypic polar mutation V232D located within the fourth TM helix (TM4) of CFTR. In vivo folding and insertion of TM4 is functionally coupled with TM3 where both TM segments are co-translationally inserted into the membrane as a TM helix-loop-helix hairpin motif (TM3/4). Utilizing this minimal folding unit, we employ smFRET as a molecular ruler to readout structural alterations imposed on the helical packing of the TM3/4 hairpin upon mutation. In contrast to earlier findings that suggested a TM lock between TM3 and TM4 by a non-native H-bond, our results reveal that V232D TM3/4 associates with membranes in an open conformation. This implies that the charged residue imposes an energy penalty on membrane insertion of the hairpin. We propose a model for CF pathogenesis where partitioning of V232D TM3/4 into the interfacial region leads to misfolding of CFTR during co-translational membrane insertion and inhibits maturation by trapping the protein as a partially folded intermediate.

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.

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 …

Two suppressor mutations of the temperature-sensitive DNA primase mutant dnaG2903 have been characterized. The gene responsible for suppression, era, encodes an essential GTPase of Escherichia coli. One mutation, rnc-15, is an insertion of an IS1 element within the leader region of the rnc operon and causes a polar defect on the downstream genes of the operon. A previously described polar mutation, rnc-40, was also able to suppress dnaG2903. The other mutation, era-1, causes a single amino acid substitution (P17R) in the G1 region of the GTP-binding domain of Era. Analysis of the GTPase activity of the Era-1 mutant protein showed a four- to five-fold decrease in the ability to convert GTP to GDP. Thus, lowered expression of wild-type Era caused by the polar mutations and reduced GTPase activity caused by the era-1 mutation suppresses dnaG2903 as well as a second dnaG allele, parB. Phenotypic analysis of the era-1 mutant at 25 degrees C showed that 10% of the cells contain four segregated nucleoids, indicative of a delay in cell division. Possible mechanisms of suppression of dnaG and roles for Era are discussed.

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

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.

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.

Vibrio anguillarum possesses at least two N -acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi . In this study, we have cloned an additional gene of this circuit, vanT , encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum ΔvanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ . Three lacZ fusions ( serA :: lacZ , hpdA-hgdA :: lacZ, and sat-vps73 :: lacZ) were identified which exhibited decreased expression in the ΔvanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an l -tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum ΔvanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.

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.

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.

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.

Nonsense mutations create a premature signal for the termination of translation of messenger RNA. Such mutations have been observed to cause a severe reduction in the amount of mutant allele transcript or to generate a peptide truncated at the carboxyl end. Analysis of fibrillin transcript from a patient with Marfan syndrome revealed the skipping of a constitutive exon containing a nonsense mutation. Similar results were observed for two nonsense mutations in the gene encoding ornithine delta-aminotransferase from patients with gyrate atrophy. All genomic DNA sequences flanking these exons that are known to influence RNA splicing were unaltered, which suggests that nonsense mutations can alter splice site selection in vivo.

The gal3 mutation in Escherichia coli, located in the operator-promoter region of the gal operon, is identified as an IS2 insertion in the polar orientation I relative to the direction of transcription. This mutation, which may be considered the earliest example of a polar mutation caused by an IS insertion, is shown by heteroduplex analysis of phage lambdagal3 to be located about 170 base pairs from the promoter-proximal end of the chlD-pgl deletion in lambdagal8. It appears indistinguishable in position, sequence and orientation from the IS2 insertion carried by lambdagal8-490. The endpoints of the bacterial DNA segments in lambdagal3 and lambdagal8 are physically mapped in relation to attL.

Summary: Four patients with hereditary glucose phosphate isomerase (GPI) deficiency and their parents have been studied by means of various enzymatic, immunologic, electrophoretic, and stability methods. The four defective mutants enzymes (identified here as “GPI Barcelona,” “GPI Utrecht,” “GPI Nijmegen,” and “GPI Kortrijk”) were antigenically identical with normal enzyme as judged by double immunodiffusion and microcomplement fixation tests. Immunologic specific activity (i.e., the ratio of enzyme activity to antigen concentration) was slightly lowered for three variants (between 60 and 75% of normal). The various methods used allowed us to establish that all of the patients were heterozygous for two different mutated alleles inherited from each parent. One of them was a silent allele which did not seem to code for any enzymatic cross-reacting material; the other mutated gene coded for a structurally modified glucose phosphate isomerase subunit. In two parents heterozygous for a structurally modified gene, only two enzymatic forms were detected, even in the young cells synthesizing actively proteins such as granulocytes: one was the normal homodimer, and the other corresponded to the heterodimer “normal subunit-mutant subunit”; the mutant homodimer was not detected. The assumption that, in these heterozygotes, the mutant subunit dimerized more easily with a normal subunit than with another mutant subunit could be proposed. In contrast, the three expected enzyme forms were detected in the leukocytes from a woman heterozygous for GPI Nijmegen. From these results the defect in activity observed in the patients could be explained in the following way: 50% of the defect was due to the inheritance of a silent gene. The decrease below 50% of the residual activity in red cells was due to the molecular instability of the mutant enzyme associated in three cases (GPI Barcelona, GPI Utrecht, and GPI Kortrijk) with the decreased immunologic specific activity. Speculation: In spite of the relatively high frequency of the mutations which are responsible for a silent GPI structural gene, GPI deficiency due to the inheritance of two silent genes has never been described. It can be speculated that the homozygous state of such a mutation, which would lead to null enzyme activity in all the tissues, could be lethal. As in most cases of genetic disorders due to silent genes, the nature of the mutation involved is unknown. A deletion, a polar mutation, a structural mutation leading to a highly labile product, or a cis-dominant regulator mutation are the different genetic hypotheses which could be put forward.

The phenotype of Escherichia coli K-12 carrying rho-15 in the genetic background DW319 ilv lacZ: :IS1 is described. Seventy-eight percent (70/90) of Ilv+ transductants acquired the following phenotype: temperature-sensitive growth on minimal salts medium, Ts+ growth on complex medium and suppression of the lac polar mutation. At 42° on minimal medium, the rho-15 transductants were cross-fed by a substance diffusing from Rho+ transductants or controls. The requirement for this substance was satisfied by methionine or cystathionine, but not by any other single amino acid or combination of amino acids, by spermidine, or by mono- or divalent cationic salts.—Transduction of rho-15 into four other Ilv- recipients revealed two phenotypic patterns. Recipients with rpsL or rpsE ribosomes yielded rho-15 transductants that were Ts on all media, or Ts on minimal medium whether or not methionine was present. The effect of the ribosome on expression of rho15 was confirmed by transduction of appropriate rps alleles into DW319, followed by co-transduction of rho-15 with Ilv+. The growth rate of double rho-15 rpsL or rho-15 rpsE strains was severely reduced at 42° in comparison with strains carrying any of these single mutations. Models for rho and ribosome interaction are presented.

Four closely-linked genes of Escherichia coli, comprising the deoxyribonucleoside (deo) operon, viz. dra, tpp, drm, and pup†, which code for enzymes involved in the catabolism of nucleosides and deoxynucleosides, are known to form two transcriptional units, one initiated from a promoter to the left of dra and one initiated between tpp and drm. Thus a potentially polar mutation induced by phage Mu-1 in tpp has no effect on the level of drm gene product. We show here, however, that if the level of transcription of all four genes is increased by the insertion of a regulatory mutation (deoR− or cytR−) then a Mu-induced polar mutation in tpp does in fact drastically reduce the level of drm gene product. The interpretation of this result is that two species of mRNA are produced; one is tetracistronic, being transcribed from a promoter located to the left of dra, and one is dicistronic, being transcribed from a promoter lying between tpp and drm. The two distal genes are therefore transcribed into two separate mRNA species. In the wild-type (deoR+ cytR+) most of the drm and pup gene products are synthesized from the short message and thus there is no polar effect of a Mu-1-induced tpp mutation on drm. The long message is controlled by the deoR and cytR gene products, whereas the remaining inducibility of drm in deoR− cytR− strains with a polar tpp mutation suggests that the short message is controlled by another, unknown, regulatory system.

A number of premature translation termination mutations (nonsense mutations) have been described in the human alpha- and beta-globin genes. Studies on mRNA isolated from patients with beta zero-thalassemia have shown that for both the beta-17 and the beta-39 mutations less than normal levels of beta-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human beta-globin mRNA.) In vitro studies using the cloned beta-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. We have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, we have studied the effect of five nonsense mutations and two missense mutations on the expression of human beta-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation.

Complementation tests performed between the rifampicin resistance transducing phage λcI857s7drif d18 and a polar amber mutation in RNA polymerase affecting the synthesis of both the β and β′ subunits, show that this phage carries both of these genes. In contrast, a second rif transducing phage, λcI857s7drif d12, fails to complement the polar mutation, but does complement a non-polar amber mutation affecting the synthesis of only the β subunit. Synthesis of β and β′ in the strain containing λcI857s7drif d18 and the polar amber mutation proceeds at the normal rate, suggesting that this phage also carries the promoter region for this set of bacterial genes. Replication of the same lysogen after heat induction produces a moderate gene dosage effect on subunit synthesis.

Suppressors specific for frameshift mutations have been studied. Of 21 frameshift mutations of the histidine operon selected for study, 13 gave rise to ICR † -induced external suppressors. Five of these mutants also gave rise to nitrosoguanidine-induced suppressors. Two frameshift mutations in the tryptophan operon were found which could be suppressed externally along with certain his frameshift mutations. Most of the externally suppressible mutations ( 12 15 ) are those tentatively classified as (+1) frameshifts. Forty-eight independent suppressor mutations were placed in 9 groups on the basis of their ability to suppress 13 his frameshift mutations. The 13 suppressible mutations are apparently of two general types. No cross-suppression of mutations of these two types has been observed. The efficiency of suppression was estimated for at least one suppressor from each of 8 groups. The efficiency of suppression ranges from 1% to about 15%.

Regulatory mutants of the histidine operon contain very high levels of the histidine biosynthetic enzymes. One consequence of this de-repression of the operon is that these regulatory mutants do not grow at 43 °C. A reversion of this temperature sensitivity can be achieved by a true back-mutation of the regulatory lesion or by a secondary polar mutation in the operon causing decreased levels of histidine biosynthetic enzymes. In the case of the operator-constitutive strain, hisO1242 , none of the temperature-resistant revertants is a true back mutation to hisO + . The majority of the revertants contain a mutation in the histidine operon which causes a strong polarity. These polar mutations are nonsense and frameshift mutations which map in the structural genes of the histidine operon. No completely polar mutations were isolated by this selection.

A set of lac − mutations has been isolated in cells of Escherichia coli after mutagenesis with ICR-191D, an acridine half-mustard known to cause frameshifts in bacteria. The mutations in the β-galactosidase gene ( z ) were studied and shown to have properties consistent with their assignment to the frameshift class: they are revertible by the acridine half-mustard, they are polar for expression of distal genes in the operon, they do not respond to any of the three types of nonsense (chain termination) suppressors tested and one of the mutations, ICR36, reverts to lac + by intragenic suppression. Three different internal suppressors of ICR36 were recovered from these revertants and also shown to be frameshifts. The 31 independently isolated frameshift mutations do not map randomly in z ; several “hot spots” for ICR-191D mutagenesis are located in the operator-proximal end of the gene.

What are polar mutations and polarity and what do they reveal about the functioning of operons ?

A comparison of the rates of synthesis of the tryptophan biosynthetic enzymes of Salmonella typhimurium under derepression showed that the genes of the trp operon can be expressed in a coordinate fashion in auxotrophs carrying nonpolar mutations. This coordination disappeared in trpA polar mutants. The loss of coordination affected only trpB , the second gene in the operon, which was always more drastically affected than the three distal genes. Polar mutations in trpA , the first gene of the trp operon, reduced the rates of synthesis of the tryptophan biosynthetic enzymes under conditions of derepression. When these rates were measured and correlated with the map position of each polar mutation, a polarity gradient of decreasing intensity (moving distally from the operator end of the gene) was obtained. Certain mutations (“unusual mutations”) mapping at the operator distal end of trpA , and considered by other workers to correspond to the operator proximal end of trpB , were found to be polar. The bearing of our observations on the question of coordinate versus semicoordinate expression of the trp genes and the status of the “unusual mutations” is discussed.

Two Escherichia coli suppressor genes, Su-4 and Su-5 , are characterized in the present report. These genes, which suppress the N1 class of phosphatase nonsense mutations, are genetically and physiologically distinct from the previously described suppressor genes for N1 mutations, Su-1 , Su-2 and Su-3 . The Su-4 gene is located in the tryptophan region of the bacterial chromosome, and the Su-5 gene in the galactose region. With the two new suppressor genes it has been possible to detect another class of suppressible phosphatase mutations, the N2 class, which does not respond to the other three suppressor genes. The N2 mutations appear to act as nonsense mutations in an Su − strain, since all unsuppressed N2 mutants fail to produce phosphatase enzymic activity or cross-reacting material. N2 mutations occur preferentially in codons that specify, in the standard phosphatase molecule, either basic or acidic amino acids, in contrast to N1 mutations which occur in codons that specify neutral amino acids. This suggests that the nonsense codon resulting from N2 mutations is different from the UAG nonsense codon resulting from N1 mutations. Other evidence for two different nonsense codons is the demonstration that an N2 codon can be transformed by mutation into an N1 codon. (In another report, the N2 codon has been identified as UAA.) When N1 or N2 mutations are suppressed by the Su-4 + gene, the phosphatase proteins produced appear, from electrophoretic analyses, to contain a neutral amino acid at the position in the molecule specified by the nonsense codon. When the same mutations are suppressed by the Su-5 + gene, there appears to be a bagic amino acid present at the corresponding positions. Thus, each suppressor gene causes a different amino acid to be specified by a nonsense codon. The Su-4 + gene is a moderately effective suppressor of N1 and N2 mutations. The highest level of suppression obtained, as measured by the amount of phosphatase cross-reacting material produced, is 16% of the amount in the standard P + strain. The Su-5 + gene suppresses N1 and N2 mutations less effectively, yielding a maximum of 6% of the standard amount of cross-reacting material. The N2 class of phosphatase mutations is similar in some characteristics, but not in all, to the ochre class of mutations in phage T4.

The only organism that has been used for the detailed analysis of the genes involved with dinitrogen fixation (nif genes) is Klebsiella pneumoniae. This organism is closely related to the well-studied Escherichia coli and Salmonella typhimurium. Unlike these latter species, certain K. pneumoniae strains are able to grow in media free of fixed nitrogen Compounds (Mahl et al., 1965). Such strains fix dinitrogen from the air. Dinitrogen fixation by K. pneumoniae only occurs anaerobically because oxygen is a potent inactivator of nitrogenase. Genetic techniques that have been developed for E. coli, such as generalized transduction by phage P1 or gene inactivation by random insertion of phage Mu have been used to develop the genetics of dinitrogen fixation in K. pneumonia (Streicher et al., 1971; Bachhuber et al., 1976; Rao, 1976; Elmerich et al., 1978). This chapter will focus on recent work that has been published from this laboratory. Earlier work and work from other laboratories have been reviewed (Brill, 1975; Elmerich 1979; Beringer, 1980).

We had previously demonstrated that several F specific polypeptide bands could be detected in the membranes of Flac, but not F- strains of Escherichia coli K12, (Moore et al. 1981). One of these polypeptides co-migrated with F-pilin protein on polyacrylamide gels. We have now analyzed 35[S]methionine labelled membrane preparations from a series of strains containing Flac tra mutant plasmids. The F-pilin polypeptide was absent from preparations of strains containing all traA mutants tested, confirming the importance of the traA gene on F-pilin biosynthesis. A polypeptide which migrate in the F-pilin position was still present, however, in membranes prepared from Flac strains carrying mutations in traL, traE, traK, traB, traV, traW, traC, traU, traF, traH or traG despite the inability of these mutants to elaborate F-pili filaments. Thus, all of these gene products may be concerned with F-pilus assembly and outgrowth rather than biosynthesis of the F-pilin subunit. The polar mutation tra-4 did, however, prevent the appearance of pilin polypeptide, indicating that at least one unidentified gene in the region between traE and traG must also be required in F-pilin biosynthesis. Our analysis also permitted the identification of a 100,000 dalton membrane protein as the product of traG. The appearance of an F specific 12,000 dalton protein was prevented by traD amber mutants. As expected, traJ mutants prevented the expression of all the tra operon products detected except the product of traT. The traT product band was reduced only to 50 - 60% of its normal intensity.

Mutants of S. typhimurium with a defect in the first structural gene of the trp operon can utilize anthranilic acid (AA) as a growth factor. Among a group of 5-methyltryptophan (MT) resistant derivatives of trpA mutants we encountered several with a novel phenotype: they actually grew better in the presence of MT than in its absence. Normally MT inhibits growth of S. typhimurium at the concentration we employed due to its ability to act as co-repressor of the trp operon and as a feedback inhibitor of anthranilate synthetase (AS) the first enzyme for tryptophan biosynthesis. Mutations to MT-dependence were only found in strains carrying extremely polar trpA mutations. In all cases analyzed, mutations causing MT-dependence mapped at the extreme operator distal end of trpA. The mutation trpA515 responsible for MT-dependence in strain SO61 (genotype trpA49trpA515) was recombined away from the polar mutation. The strain thus obtained, SO495 was totally dependent on MT for growth on AA supplement. Strain SO495 lacks AS and under repressing growth conditions synthesizes the trp enzymes constitutively at 2--3 times the basal level. Under derepression, while the levels of the distal enzymes, as represented by tryptophan synthetase--beta subunit (TSbeta), did not increase there was a marked drop in the activity of anthranilate-PRPP phosphoribosyltransferase, (PRT) the enzyme catalyzing the second step of tryptophan biosynthesis. trpA515 was found to revert to prototrophy at a low frequency (about 10(-8)) which was not increased by chemical mutagens or ultraviolet radiation. In contrast, it was found to revert to MT-independence (growth on AA in the absence of MT) at a fairly high spontaneous frequency (about 10(-6)) and this frequency could be increased approximately tenfold by mutagens causing base substitutions or deletions but not by frameshift mutagens. About one hundred MT-independent revertants of trpA515 were mapped and found to fall into three general classes: (A) mutations at or near the trpA515 site (B) secondary mutations located upstream from trpA515, (C) deletions of various sizes. Based on a detailed genetic and physiological study of twelve representative MT-independent revertants, it appears that trpA515 may be caused by the insertion of a piece of DNA with some of the properties described for the IS elements found in Escherichia coli. The trpA515 insertion should contain (in this order), a transcription terminator, a low efficiency promoter and, probably, a translation start signal.

A tenfold increase in the spontaneous reversion frequency of two of six lacZ frameshift mutations tested was observed in strains containing the following DNA polymerase I mutations-polA1, polA5, polA6, polAex1, res-3 and resA1. Reconstruction experiments indicated that this increase was not the result of a selective effect. Only a fourfold increase in frameshift mutations was found in strains containing a polA107 mutation. Both the polAex1 and polA107 mutations result in defective 5′ to 3′ exonuclease activity and do not affect polymerizing activity, but have different effects on frameshift mutations. A polA mutation on the chromosome induced frameshift mutations in a gene on an F episome. None of three auxotrophic mutations studied showed high frequency reversion in the presence of the polA1 or polA6 mutations.

The temperature bacteriophage Mu can integrate at random into the genome of its host Escherichia coli K12 (Taylor, 1963). Mu is a very powerful tool for genetic manipulations: it can produce strongly polar mutations, deletions, integration of episomic elements into the chromosome and transposition of genes into episomes (Howe and Bade, 1975; Bukhari et al., 1977).

The bacterial chromosome, while evolving by point mutations in its DNA, is thought to be rather stable with regard to its gross organization. For example, even though the DNAs of Salmonella and Escherichia cross-hybridize only weakly due to divergent sequence evolution (Demerec and New, 1965; Brenner et al., 1969), the genetic maps of the two bacteria are rather similar with respect to the order of functions (Sanderson and Demerec, 1965).

We have examined the nucleotide sequences of a series of murine antibody genes derived from one kappa light chain gene in order to gain insight into the mechanism that specifically mutates variable genes. Six rearranged VK167 genes from hybridoma and myeloma cells were cloned from bacteriophage lambda libraries. The sequences were compared to the germ-line sequence of the VK167 gene, the JK genes, and the CK gene to identify sites of mutation. Four of six rearranged genes had extensive mutation which occurred exclusively in a 1-kilobase region of DNA centered around the V-J gene. No mutations were found at more distant sites in the intervening sequence or in the constant gene. The frequency of mutation was approximately 0.5% (32 mutations per 6,749 base pairs). Mutations were mostly due to nucleotide substitutions with no preference for transitions or transversions. The location of mutations around each gene indicates that they occur in clusters at random sites. The observation of mutations in the intervening sequence downstream from the JK5 gene rules out models for the mechanism of mutagenesis that rely solely on gene conversion or recombination. The distribution and high frequency of mutations are most easily explained by a mechanism of error-prone repair that occurs during several cycles of cell division.

A single site in the middle of the coding sequence of the hisG gene of Salmonella is required for most of the polar effect of mutations in this gene. Nonsense and insertion mutations mapping upstream of this point in the hisG gene all have strong polar effects on expression of downstream genes in the operon; mutations mapping promotor distal to this site have little or no polar effect. Two previously known hisG mutations, mapping in the region of the polarity site, abolish the polarity effect of insertion mutations mapping upstream of this region. New polarity site mutations have been selected which have lost the polar effect of upstream nonsense mutations. All mutations abolishing the function of the site are small deletions; three are identical, 28-bp deletions which have arisen independently. A fourth mutation is a deletion of 16 base pairs internal to the larger deletion. Several point mutations within this 16-bp region have no effect on the function of the polarity site. We believe that a small number of polarity sites of this type are responsible for polarity in all genes. The site in the hisG gene is more easily detected than most because it appears to be the only such site in the hisG gene and because it maps in the center of the coding sequence.

The chromosome of Escherichia coli is a circular double-stranded DNA molecule with a length of about 1 mm, which is folded in a unique way. This DNA molecule has a coding capacity of some 3000–4000 average-sized polypeptide chains. The genes coding for these proteins are thought to be arranged in the chromosome in a sequence characteristic for a given bacterial species.

The presence of IS elements in the chromosome of E.coli was originally revealed by the transposition of these DNA elements from their natural positions into indicator systems, resulting in a recognizable mutant phenotype. If, for example, the gal operon is used as an indicator system, mutations can be isolated in which not only one of the three structural genes of the galactose operon is inactivated, but the expression of the Promoter distal genes is also abolished. These mutations therefore are strongly polar. The analysis of the nature of such mutations is facilitated by the existence of the gal transducing phage λ and the development of techniques for examining heteroduplex DNA in the electron microscope. With the help of these tools it is possible to inspect hybrid DNA molecules consisting of one DNA strand carrying the strongly polar mutation and a complementary strand of λ dgal in the electron microscope. The strongly polar mutation is seen as a single stranded DNA loop emerging from a position of the double stranded heteroduplex molecule, which corresponds to the map position of the mutation. Analysing various independently isolated strongly polar mutations with the above technique, revealed the existence of different categories of IS elements.

Strains with an amber, polar mutation in the dra 1 gene have been isolated. The mutation was introduced into a set of isogenic strains, wild type or with concurrent regulatory mutations, and further characterized by suppression and heat inactivation experiments. The effect of the polar dra mutation on the three remaining genes of the deo operon, the tpp, drm and pup genes, was determined by estimating the enzyme levels in the various dra-mutants. The effect was found to be non-coordinate, indicating the formation in the cells of two types of transcripts: A tetracistronic unit, containing the message from all four genes, and a dicistronic unit, covering the two distal genes only.

Internal re-initiation polypeptides which are products of the lacZ gene of Escherichia coli have been synthesized in a DNA-directed cell-free protein synthesis system. Some of the properties of these protein fragments have been characterized. The de novo synthesized re-initiation proteins, unlike both in vitro synthesized wild-type β-galactosidase and nonsense termination fragments, are insoluble when synthesized at 37 °C, but soluble if synthesis takes place at 25 °C. The same re-initiation proteins which are made in vivo have been detected in vitro. Unlike their in vivo counterparts, which are degraded rapidly, the in vitro synthesized restart proteins are completely stable for at least one hour following their synthesis. Both in vivo and in vitro, the re-initiation proteins are not synthesized from DNA containing a wild-type Z gene, but require a specific nonsense mutation in order to be expressed. Furthermore, the location of the mutation within the Z gene is very important in determining whether or not re-initiation will occur at a given site. Several nonsense mutations which do not result in the synthesis of detectable amounts of restart protein in vivo produce specific re-initiation polypeptides in vitro. These restart proteins display many of the same properties as do those which are made both in vivo and in vitro: they are not made from wild-type DNA, and they are only made from DNA containing a specific nonsense mutation. One of these mutations is 118, which is an extreme polar mutation in vivo. Another is 545, which synthesizes a restart protein in vivo if, and only if, it is coupled with a secondary mutation, π(1). π(1) thus appears to be necessary for the synthesis of a particular re-initiation protein in vivo but unnecessary for the synthesis of the same protein in vitro. The efficiencies of re-initiation vary at the different sites, but in all cases are less than the initiation frequency at the start of the gene. The experiments thus show that when complicating factors, such as polarity and protein degradation, are eliminated, translational re-initiation can be detected at many sites in the lacZ gene.