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Genes involved in the determination of the rate of inversions at short inverted repeats
Genes to Cells
Abstract
Not all of the enzymatic pathways involved in genetic rearrangements have been elucidated. While some rearrangements occur by recombination at areas of high homology, others are mediated by short, often interrupted homologies. We have previously constructed an Escherichia coli strain that allows us to examine inversions at microhomologies, and have shown that inversions can occur at short inverted repeats in a recB,C-dependent fashion.
Here, we report on the use of this strain to define genetic loci involved in limiting rearrangements on an F' plasmid carrying the lac genes. Employing mini-Tn10 derivatives to generate insertions near or into genes of interest, we detected three loci (rmuA,B,C) that, when mutated, increase inversions. We have mapped, cloned and sequenced these mutator loci. In one case, inactivation of the sbcC gene leads to an increase in rearrangements, and in another, insertions near the recE gene lead to an even larger increase. The third gene involved in limiting inversions, rmuC, has been mapped at 86 min on the E. coli chromosome and encodes a protein of unknown function with a limited homology to myosins, and some of the SMC (structural maintenance of chromosomes) proteins.
This work presents the first example of an anti-mutator role of the sbcC,D genes, and defines a new gene (rmuC) involved in DNA recombination.
Supplementary resource (1)
Nucleotide Sequence
August 2000
... COG1322/pfam02646 is represented by the RmuC protein from Escherichia coli, which has been implicated in limiting inversions at short-inverted repeats [33]. It has been speculated that RmuC exhibits "limited homology" to human Rad50 protein, centrosome protein pericentrin, nuclear mitotic apparatus proteins and the SbcC proteins, and therefore it may be a structural protein that protects DNA against nuclease action or be itself involved in DNA cleavage at the regions of DNA secondary structure [33]. ...
... COG1322/pfam02646 is represented by the RmuC protein from Escherichia coli, which has been implicated in limiting inversions at short-inverted repeats [33]. It has been speculated that RmuC exhibits "limited homology" to human Rad50 protein, centrosome protein pericentrin, nuclear mitotic apparatus proteins and the SbcC proteins, and therefore it may be a structural protein that protects DNA against nuclease action or be itself involved in DNA cleavage at the regions of DNA secondary structure [33]. Our secondary structure predictions revealed that RmuC contains an N-terminal transmembrane helix (aa 1–25), ), a globular α/β domain (aa 200–360), and a disordered C-terminus (aa 420–475). ...
... Analysis of the multiple sequence alignment (Figure 2) reveals that proteins from the RmuC/COG1322/ pfam02646 family exhibit a hallmark PD-(D/E)XK motif associated with the characteristic pattern of predicted secondary structures, which strongly suggests that RmuC is a nuclease that may cleave DNA structures arising during the recombination of short-inverted repeats and thereby thwart the inversion of the internal sequence. We suspect that the initial predictions of homology between RmuC and proteins involved in the structural maintenance of chromosome (Rad50 and SbcC) [33] was due to a spurious similarity in the regions of low sequence complexity, e.g. coiled coils. ...
The PD-(D/E)XK nuclease superfamily, initially identified in type II restriction endonucleases and later in many enzymes involved in DNA recombination and repair, is one of the most challenging targets for protein sequence analysis and structure prediction. Typically, the sequence similarity between these proteins is so low, that most of the relationships between known members of the PD-(D/E)XK superfamily were identified only after the corresponding structures were determined experimentally. Thus, it is tempting to speculate that among the uncharacterized protein families, there are potential nucleases that remain to be discovered, but their identification requires more sensitive tools than traditional PSI-BLAST searches.
The low degree of amino acid conservation hampers the possibility of identification of new members of the PD-(D/E)XK superfamily based solely on sequence comparisons to known members. Therefore, we used a recently developed method HHsearch for sensitive detection of remote similarities between protein families represented as profile Hidden Markov Models enhanced by secondary structure. We carried out a comparison of known families of PD-(D/E)XK nucleases to the database comprising the COG and PFAM profiles corresponding to both functionally characterized as well as uncharacterized protein families to detect significant similarities. The initial candidates for new nucleases were subsequently verified by sequence-structure threading, comparative modeling, and identification of potential active site residues.
In this article, we report identification of the PD-(D/E)XK nuclease domain in numerous proteins implicated in interactions with DNA but with unknown structure and mechanism of action (such as putative recombinase RmuC, DNA competence factor CoiA, a DNA-binding protein SfsA, a large human protein predicted to be a DNA repair enzyme, predicted archaeal transcription regulators, and the head completion protein of phage T4) and in proteins for which no function was assigned to date (such as YhcG, various phage proteins, novel candidates for restriction enzymes). Our results contributes to the reduction of "white spaces" on the sequence-structure-function map of the protein universe and will help to jump-start the experimental characterization of new nucleases, of which many may be of importance for the complete understanding of mechanisms that govern the evolution and stability of the genome.
... The protein products of both rpoB and rmuC interact closely with genomic DNA. RmuC is a regulator which can prevent sequence inversion during replication [34]. RmuC has also been identified as a possible multi-drug resistance (MDR) gene in other Gram-negative bacterial species [35,36]. ...
Bacterial exposure to antibiotic concentrations below the minimum inhibitory concentration (MIC) may result in a selection window allowing for the rapid evolution of resistance. These sub-MIC concentrations are commonly found in soils and water supplies in the greater environment. This study aimed to evaluate the adaptive genetic changes in Klebsiella pneumoniae 43816 after prolonged but increasing sub-MIC levels of the common antibiotic cephalothin over a fourteen-day period. Over the course of the experiment, antibiotic concentrations increased from 0.5 μg/mL to 7.5 μg/mL. At the end of this extended exposure, the final adapted bacterial culture exhibited clinical resistance to both cephalothin and tetracycline, altered cellular and colony morphology, and a highly mucoid phenotype. Cephalothin resistance exceeded 125 μg/mL without the acquisition of beta-lactamase genes. Whole genome sequencing identified a series of genetic changes that could be mapped over the fourteen-day exposure period to the onset of antibiotic resistance. Specifically, mutations in the rpoB subunit of RNA Polymerase, the tetR/acrR regulator, and the wcaJ sugar transferase each fix at specific timepoints in the exposure regimen where the MIC susceptibility dramatically increased. These mutations indicate that alterations in the secretion of colanic acid and attachment of colonic acid to LPS may contribute to the resistant phenotype. These data demonstrate that very low sub-MIC concentrations of antibiotics can have dramatic impacts on the bacterial evolution of resistance. Additionally, this study demonstrates that beta-lactam resistance can be achieved through sequential accumulation of specific mutations without the acquisition of a beta-lactamase gene.
... In this respect, the complex SbcDC, present in SA211, acts as part of the SOS pathway in bacteria, being very active in high UV exposure conditions in DNA repair processes (Chen et al., 2007). Furthermore, proteins such as RecA, LexA, RecFOR, RecN, UvrABC system, and RmuC (coded in SA211's genome) have been widely characterized in bacteria as active in DNA repair processes (Estévez Castro et al., 2018;McCready and Marcello, 2003;Repar et al., 2010;Slupska et al., 2000). Being highly conserved, these proteins would also participate in repairing DNA when damages occur as a consequence of hostile environmental conditions. ...
Micrococcus luteus SA211, isolated from the Salar del Hombre Muerto in Argentina, developed responses that allowed its survival and growth in presence of high concentrations of lithium chloride (LiCl). In this research, analysis of total genome sequencing and a comparative proteomic approach were performed to investigate the responses of this bacterium to the presence of Li. Through proteomic analysis, we found differentially synthesized proteins in growth media without LiCl (DM) and with 10 (D10) and 30 g/L LiCl (D30). Bi-dimensional separation of total protein extracts allowed the identification of 17 over-synthesized spots when growth occurred in D30, five in D10, and six in both media with added LiCl. The results obtained showed different metabolic pathways involved in the ability of M. luteus SA211 to interact with Li. These pathways include defense against oxidative stress, pigment and protein synthesis, energy production, and osmolytes biosynthesis and uptake. Furthermore, mono-dimensional gel electrophoresis revealed differential protein synthesis at equivalent NaCl and LiCl concentrations, suggesting that this strain would be able to develop different responses depending on the nature of the ion. Moreover, the percentage of proteins with acidic pI predicted and observed was highlighted, indicating an adaptation to saline environments. To the best of our knowledge, this is the first report showing the relationship between protein synthesis and genome sequence analysis in response to Li, showing the great biotechnological potential that native microorganisms present, especially those isolated from extreme environments.
... The first selected gene family encode RmuC, of which the precise function is unknown, but that has been associated with DNA recombination processes (Slupska et al., 2000). Intriguingly, the RmuC gene was detected only in 16SrI and 16SrXII phytoplasma groups, as briefly mentioned by Kube Saccardo et al. (2012), whereas it is readily detectable in other genera of the Mollicutes class, such as Acholeplasma and Spiroplasma, as well as many bacterial genera outside Mollicutes. ...
Although phytoplasma studies are still hampered by the lack of axenic cultivation methods, the availability of genome sequences allowed dramatic advances in the characterization of the virulence mechanisms deployed by phytoplasmas, and highlighted the detection of signal peptides as a crucial step to identify effectors secreted by phytoplasmas. However, various signal peptide prediction methods have been used to mine phytoplasma genomes, and no general evaluation of these methods is available so far for phytoplasma sequences. In this work, we compared the prediction performance of SignalP versions 3.0, 4.0, 4.1, 5.0 and Phobius on several sequence datasets originating from all deposited phytoplasma sequences. SignalP 4.1 with specific parameters showed the most exhaustive and consistent prediction ability. However, the configuration of SignalP 4.1 for increased sensitivity induced a much higher rate of false positives on transmembrane domains located at N-terminus. Moreover, sensitive signal peptide predictions could similarly be achieved by the transmembrane domain prediction ability of TMHMM and Phobius, due to the relatedness between signal peptides and transmembrane regions. Beyond the results presented herein, the datasets assembled in this study form a valuable benchmark to compare and evaluate signal peptide predictors in a field where experimental evidence of secretion is scarce. Additionally, this study illustrates the utility of comparative genomics to strengthen confidence in bioinformatic predictions.
... cleavage in regions of secondary structure (Slupska et al. 2000). UvrA, or exinuclease A subunit, like the RecA protein, was very abundant in C/wStr1 cells, and the B subunit (UvrB) was also highly represented (Table S1). ...
Wolbachia is an obligate intracellular bacterium that has undergone extensive genomic streamlining in its arthropod and nematode hosts. Because the gene encoding the bacterial DNA recombination/repair protein RecA is not essential in Escherichia coli, abundant expression of this protein in a mosquito cell line persistently infected with Wolbachia strain wStri was unexpected. However, RecA’s role in the lytic cycle of bacteriophage lambda provides an explanation for retention of recA in strains known to encode lambda-like WO prophages. To examine DNA recombination/repair capacities in Wolbachia, a systematic examination of RecA and related proteins in complete or nearly complete Wolbachia genomes from supergroups A, B, C, D, E, F, J and S was undertaken. Genes encoding proteins including RecA, RecF, RecO, RecR, RecG and Holliday junction resolvases RuvA, RuvB and RuvC are uniformly absent from Wolbachia in supergroup C and have reduced representation in supergroups D and J, suggesting that recombination and repair activities are compromised in nematode-associated Wolbachia, relative to strains that infect arthropods. An exception is filarial Wolbachia strain wMhie, assigned to supergroup F, which occurs in a nematode host from a poikilothermic lizard. Genes encoding LexA and error-prone polymerases are absent from all Wolbachia genomes, suggesting that the SOS functions induced by RecA-mediated activation of LexA do not occur, despite retention of genes encoding a few proteins that respond to LexA induction in E. coli. Three independent E. coli accessions converge on a single Wolbachia UvrD helicase, which interacts with mismatch repair proteins MutS and MutL, encoded in nearly all Wolbachia genomes. With the exception of MutL, which has been mapped to a eukaryotic association module in Phage WO, proteins involved in recombination/repair are uniformly represented by single protein annotations. Putative phage-encoded MutL proteins are restricted to Wolbachia supergroups A and B and show higher amino acid identity than chromosomally encoded MutL orthologs. This analysis underscores differences between nematode and arthropod-associated Wolbachia and describes aspects of DNA metabolism that potentially impact development of procedures for transformation and genetic manipulation of Wolbachia.
... This further suggests that nonsymmetric inversions may be more common than currently realized, but only symmetric inversions are pervasively retained through evolution due to their low fitness cost. In studies selecting for inversion within a gene bearing 12-or 23-bp regions of inverted homology, Miller and co-workers estimated inversion rates to be 4 × 10 −9 to 1 × 10 −7 per generation [34,35]. Inversion rates will depend on many factors, including the length of homology at the inversion break-points [36]. ...
Replication-transcription conflicts promote mutagenesis and give rise to evolutionary signatures, with fundamental importance to genome stability ranging from bacteria to metastatic cancer cells. This review focuses on the interplay between replication-transcription conflicts and the evolution of gene directionality. In most bacteria, the majority of genes are encoded on the leading strand of replication such that their transcription is co-directional with the direction of DNA replication fork movement. This gene strand bias arises primarily due to negative selection against deleterious consequences of head-on replication-transcription conflict. However, many genes remain head-on. Can head-on orientation provide some benefit? We combine insights from both mechanistic and evolutionary studies, review published work, and analyze gene expression data to evaluate an emerging model that head-on genes are temporal targets for adaptive mutagenesis during stress. We highlight the alternative explanation that genes in the head-on orientation may simply be the result of genomic inversions and relaxed selection acting on nonessential genes. We seek to clarify how the mechanisms of replication-transcription conflict, in concert with other mutagenic mechanisms, balanced by natural selection, have shaped bacterial genome evolution.
... Himar1 intragenic insertion mutants were also identified in genes for vitamin synthesis [thiamine biosynthesis protein ThiC (Lawhorn et al., 2004) and dethiobiotin synthase (Otsuka et al., 1988)], mutation repair [RmuC (Slupska et al., 2000), DNA mismatch repair proteins MutS and MutL (Mansour et al., 2001)], transcriptional regulation [DNA-3-methyladenine glycosylase (Wyatt et al., 1999), transcriptional regulator NrdR (Torrents et al., 2007)], nutrient transport [sodium:alanine symporter family protein (Khani et al., 2018), and two MFS transporters (Quistgaard et al., 2016)], bacterial division and chromosomal segregation [chromosome partitioning ATPase ParA (Lutkenhaus, 2012), cell division ZapA family protein (Small et al., 2007)], and others [amidophosphoribosyltransferase ComF (Bhagavan and Ha, 2015), tellurium resistance protein TerC (Turkovicova et al., 2016), RDD family protein (Shao et al., 2018), glutathione S-transferase (Nebert and Vasiliou, 2004), aspartate kinase (Min et al., 2015), gamma carbonic anhydrase family protein (Hewett-Emmett and Tashian, 1996), octaprenyldiphosphate synthase (Ashby and Edwards, 1990)] ( Table 1). These genes are apparently not required for HF strain infection of macrophages. ...
Ehrlichia spp. are emerging tick-borne obligatory intracellular bacteria that cause febrile and sometimes fatal diseases with abnormal blood cell counts and signs of hepatitis. Ehrlichia HF strain provides an excellent mouse disease model of fatal human ehrlichiosis. We recently obtained and established stable culture of Ehrlichia HF strain in DH82 canine macrophage cell line, and obtained its whole genome sequence and annotation. To identify genes required for in vivo virulence of Ehrlichia, we constructed random insertional HF strain mutants by using Himar1 transposon-based mutagenesis procedure. Of total 158 insertional mutants isolated via antibiotic selection in DH82 cells, 74 insertions were in the coding regions of 55 distinct protein-coding genes, including TRP120 and multi-copy genes, such as p28/omp-1, virB2, and virB6. Among 84 insertions mapped within the non-coding regions, seven are located in the putative promoter region since they were within 50 bp upstream of the seven distinct genes. Using limited dilution methods, nine stable clonal mutants that had no apparent defect for multiplication in DH82 cells, were obtained. Mouse virulence of seven mutant clones was similar to that of wild-type HF strain, whereas two mutant clones showed significantly retarded growth in blood, livers, and spleens, and the mice inoculated with them lived longer than mice inoculated with wild-type. The two clones contained mutations in genes encoding a conserved hypothetical protein and a staphylococcal superantigen-like domain protein, respectively, and both genes are conserved among Ehrlichia spp., but lack homology to other bacterial genes. Inflammatory cytokine mRNA levels in the liver of mice infected with the two mutants were significantly diminished than those infected with HF strain wild-type, except IL-1β and IL-12 p40 in one clone. Thus, we identified two Ehrlichia virulence genes responsible for in vivo infection, but not for infection and growth in macrophages.
... The PBC-5 DNA motif. This candidate motif (Additional file 10: Figure S8c) has 98 unique examples, and is often found upstream of rmuC, a DNA recombination protein predicted to cleave ssDNA [23,24]. The structure of this motif contains three hairpins with the sequence GTTTAGA appearing in the stems of the first two (orange shading). ...
... DNA lesion is commonly found in L. lactis under stress conditions, which cause a high genetic instability. Proteins related to DNA repair such as Phosphohydrolase, MutT/nudix and RmuC were more induced in NAE at 37 • C. MutT acts in the prevention of errors in DNA replication by hydrolyzing mutagenic nucleotide substrates as 8-oxo-dGTP (Hamm et al., 2016), while, RmuC is part of the rmuABC loci, which prevents DNA inversions at short inverted repeats (Slupska et al., 2000). Moreover, uracil-DNA glycosylase was less induced in NAE (37 • C: 30 • C); this enzyme hydrolyzes the N-glycosidic bond to remove uracil in DNA and initiates the base excision repair process (Schormann et al., 2014). ...
Lactococcus lactis is a gram positive facultative anaerobe widely used in the dairy industry and human health. L. lactis subsp. lactis NCDO 2118 is a strain that exhibits anti-inflammatory and immunomodulatory properties. In this study, we applied a label-free shotgun proteomic approach to characterize and quantify the NCDO 2118 proteome in response to variations of temperature and oxygen bioavailability, which constitute the environmental conditions found by this bacterium during its passage through the host gastro-intestinal tract and in other industrial processes. From this proteomic analysis, a total of 1,284 non-redundant proteins of NCDO 2118 were characterized, which correspond to approximately 54% of its predicted proteome. Comparative proteomic analysis identified 149 and 136 proteins in anaerobic (30°C and 37°C) and non-aerated (30°C and 37°C) conditions, respectively. Our label-free proteomic analysis quantified a total of 1,239 proteins amongst which 161 proteins were statistically differentially expressed. Main differences were observed in cellular metabolism, stress response, transcription and proteins associated to cell wall. In addition, we identified six strain-specific proteins of NCDO 2118. Altogether, the results obtained in our study will help to improve the understanding about the factors related to both physiology and adaptive processes of L. lactis NCDO 2118 under changing environmental conditions.
... The PBC-5 DNA motif. This candidate motif (Additional file 10: Figure S8c) has 98 unique examples, and is often found upstream of rmuC, a DNA recombination protein predicted to cleave ssDNA [23,24]. The structure of this motif contains three hairpins with the sequence GTTTAGA appearing in the stems of the first two (orange shading). ...
Background
Structured noncoding RNAs (ncRNAs) play essential roles in many biological processes such as gene regulation, signaling, RNA processing, and protein synthesis. Among the most common groups of ncRNAs in bacteria are riboswitches. These cis-regulatory, metabolite-binding RNAs are present in many species where they regulate various metabolic and signaling pathways. Collectively, there are likely to be hundreds of novel riboswitch classes that remain hidden in the bacterial genomes that have already been sequenced, and potentially thousands of classes distributed among various other species in the biosphere. The vast majority of these undiscovered classes are proposed to be exceedingly rare, and so current bioinformatics search techniques are reaching their limits for differentiating between true riboswitch candidates and false positives.
Results
Herein, we exploit a computational search pipeline that can efficiently identify intergenic regions most likely to encode structured ncRNAs. Application of this method to five bacterial genomes yielded nearly 70 novel genetic elements including 30 novel candidate ncRNA motifs. Among the riboswitch candidates identified is an RNA motif involved in the regulation of thiamin biosynthesis.
Conclusions
Analysis of other genomes will undoubtedly lead to the discovery of many additional novel structured ncRNAs, and provide insight into the range of riboswitches and other kinds of ncRNAs remaining to be discovered in bacteria and archaea.
Electronic supplementary material
The online version of this article (10.1186/s12866-019-1433-7) contains supplementary material, which is available to authorized users.
... Finally, the clone 42−1 (REL2047) from the 42°C treatment has a nonsynonymous mutation in rmuC (yigN). Mutations in this gene have been reported to increase the rates of inversions and other chromosomal rearrangements (27). Although there was a 37,530-bp amplification in 42−1, two of the other five 42°C genomes each had more large chromosomal rearrangements than did 42−1, suggesting that the rmuC mutation did not increase the rate at which these mutations accumulated. ...
Significance
Organisms evolve and adapt via changes in their genomes that improve survival and reproduction in the context of their environment. Few experiments have examined how these genomic signatures of adaptation, which may favor mutations in certain genes or molecular pathways, vary across a set of similar environments that have both shared and distinctive characteristics. We sequenced complete genomes from 30 Escherichia coli lineages that evolved for 2,000 generations in one of five environments that differed only in the temperatures they experienced. Particular “signature” genes acquired mutations in these bacteria in response to selection imposed by specific temperature treatments. Thus, it is sometimes possible to predict aspects of the environment recently experienced by microbial populations from changes in their genome sequences.
... Par 20 ailleurs, la protéine SbcC clive les structures secondaires au niveau des fourches de réplication. Lorsque le gène sbcC est muté, un état mutateur se met en place, caractérisé par la production à haute fréquence d'événements d'inversion de courtes séquences homologues (Slupska et al., 2000). (Wright, 1997). ...
In Streptomyces ambofaciens, mutants generated by genetic instability and named Pig-pap were characterised. Analyses of the chromosome of the 29C1 strain revealed a new type of rearrangement associated to the production of a green pigment. The Pig-pap mutants, pigment and spore deficient, derived from papillae on the colonies. The introduction of the whiG gene coding a sigma factor restored sporulation and pigmentation. In these mutants, this gene is not mutated and is transcribed. whiH gene whose transcription depends on WhiG is not transcribed. WhiG would be not fonctional and a post-transcriptional modification could be responsible of the Pig-pap phenotype. Study of mutants derived from a transconjugant showed that the whiG transgene constitute a target of mutations. More, the production of mutants is increased during a amino acids limitation and a relA mutant does not produce papilla, implicating the stringent response in this phenomenon.
... Spd_1778 is a member of the bacterial RmuC family of DNA recombination proteins. RmuC of E. coli limits the rate of DNA inversions at short inverted repeats (Slupska et al., 2000), suggesting a potential role in DNA recombination. Furthermore, RmuC is a component of the E. coli LexA regulon, which controls bacterial response to DNA damage (van Dyk et al., 2001). ...
The competence regulon of Streptococcus pneumoniae (pneumococcus) is crucial for genetic transformation. During competence development, the alternative sigma factor ComX is activated, which in turn, initiates transcription of 80 "late" competence genes. Interestingly, only 16 late genes are essential for genetic transformation. We hypothesized that these late genes that are dispensable for competence are beneficial to pneumococcal fitness during infection. These late genes were systematically deleted, and the resulting mutants were examined for their fitness during mouse models of bacteremia and acute pneumonia. Among these, 14 late genes were important for fitness in mice. Significantly, deletion of some late genes attenuated pneumococcal fitness to the same level in both wild-type and ComX-null genetic backgrounds, suggesting that the constitutive baseline expression of these genes was important for bacterial fitness. In contrast, some mutants were attenuated only in the wild-type genetic background but not in the ComX-null background, suggesting that specific expression of these genes during competence state contributed to pneumococcal fitness. Increased virulence during competence state was partially caused by the induction of allolytic enzymes that enhanced pneumolysin release. These results distinguish the role of basal expression versus competence induction in virulence functions encoded by ComX-regulated late competence genes.
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... However, SbcCD and homologous recombination may limit the frequency of these deletions (388). Supporting this hypothesis are the facts that RecBCD and SbcCD decrease the level of homology-facilitated illegitimate recombination in Acinetobacter baylyi (389) and that rates of illegitimate inversions are elevated in E. coli sbcC mutants (390). ...
SUMMARY Bacterial genomes are remarkably stable from one generation to the next but are plastic on an evolutionary time scale, substantially shaped by horizontal gene transfer, genome rearrangement, and the activities of mobile DNA elements. This implies the existence of a delicate balance between the maintenance of genome stability and the tolerance of genome instability. In this review, we describe the specialized genetic elements and the endogenous processes that contribute to genome instability. We then discuss the consequences of genome instability at the physiological level, where cells have harnessed instability to mediate phase and antigenic variation, and at the evolutionary level, where horizontal gene transfer has played an important role. Indeed, this ability to share DNA sequences has played a major part in the evolution of life on Earth. The evolutionary plasticity of bacterial genomes, coupled with the vast numbers of bacteria on the planet, substantially limits our ability to control disease.
... Even if the correlation between hairpin stability and SIs can be perfectly explained in the context of the hairpin hypothesis, alternative explanations are also possible. For some of the molecular mechanisms proposed to explain SI occurrence (double-stranded slippage, Leach in Slupska et al., 2000 2000). The action of intramolecular or intermolecular recombination in the DNA inversion process is a common characteristic of the no-hairpin proposals. ...
Small inversions (SIs) in the chloroplast genome of angiosperms are ubiquitous. These inversions are always flanked by inverted repeats (palindromes or quasipalindromes) between approximately 8 and 50 bp long that form a hairpin structure when the DNA is single-stranded. We evaluated different methodological and empirical issues about SI evolution. As a case study, we analysed an SI recently discovered in the psbC–trnS intergenic region of Prosopis (Fabaceae). First, we analysed how inversions can be optimized in cases where the inverted segment also shows indels and substitutions, proposing a method based on Fixed States Optimization. Second, we evaluated the occurrence of this inversion on a phylogeny that includes the major lineages of angiosperms. Finally, we assessed whether the occurrence of this inversion was related to the thermodynamic stability of the hairpin structure (measured by its corresponding free energy) and/or the length of the palindromes by using a modified version of Maddison’s Concentrated Changes Test. Hairpin structure was conserved in most of the 154 sequences analysed, with the inversion taking place at least 10 times in different lineages (monocots, magnoliids, rosids). As was previously proposed for other SIs, our analysis strongly suggests that the occurrence of this inversion is correlated with higher hairpin stability. In contrast, we found no evidence of a correlation with longer palindromes. Our results are in agreement with the hypothesis that hairpin formation is a requisite for SI occurrence. However, alternative explanations cannot be discarded.
© The Willi Hennig Society 2008.
... If mutations occur to revert lacZ back to the wild type, blue papillae will form on the surface of white parental colonies on glucose minimal plates containing X-Gal and P-Gal. Correlation between the rate of papillation and the rate of mutation allows one to readily detect mutators that have elevated mutation rates by monitoring the levels of papillae formation Nghiem et al., 1988;Michaels et al., 1990;Miller, 1996;Miller and Michaels, 1996;Slupska et al., 2000;Miller et al., 2002). Papillation assays have also been successfully used in other areas, such as recombination (Konrad, 1977), adaptive mutagenesis (for reviews, see Foster, 1993;, screening for transformation-deficient mutants (Lacks and Greenberg, 1977) and hyperactive transposase mutants (Lampe et al., 1999), as well as elucidating mechanisms of human activation-induced deaminase expressed in E. coli (Wang et al., 2009). ...
Colonies of Bacillus anthracis Sterne allow the growth of papillation after 6 days of incubation at 30°C on Luria-Bertani medium. The papillae are due to mutations that allow the cells to overcome the barriers to continued growth. Cells isolated from papillae display two distinct gross phenotypes (group A and group B). We determined that group A mutants have mutations in the nprR gene including frameshifts, deletions, duplications and base substitutions. We used papillation as a tool for finding new mutators as the mutators generate elevated levels of papillation. We discovered that disruption of yycJ or recJ leads to a spontaneous mutator phenotype. We defined the nprR/papillation system as a new mutational analysis system for B. anthracis. The mutational specificity of the new mutator yycJ is similar to that of mismatch repair-deficient strains (MMR⁻) such as those with mutations in mutL or mutS. Deficiency in recJ results in a unique specificity, generating only tandem duplications.
... Notably, this may be the case for the R strain MGA_1107 gene, which again is adjacent to transposase genes and is absent in the F strain. MGA_1107 contains a domain shared among proteins involved in DNA metabolism, including proteins similar to the putative nuclease RmuC, thought to affect DNA recombination of short inverted repeat sequences in E. coli (31,65). MGA_1107 also shares a relatively high level of amino acid identity (92%) with M. synoviae MS53_0172, indicating that the MGA_1107 gene, and perhaps flanking transposon sequences, have involved or mediated a horizontal gene transfer (HGT) event, consistent with previous observation of a likely vlhA HGT between M. synoviae and M. gallisepticum (48,70). ...
Mycoplasma gallisepticum is a significant respiratory and reproductive pathogen of domestic poultry. While the complete genomic sequence of the virulent,
low-passage M. gallisepticum strain R (Rlow) has been reported, genomic determinants responsible for differences in virulence and host range remain to be completely
identified. Here, we utilize genome sequencing and microarray-based comparative genomic data to identify these genomic determinants
of virulence and to elucidate genomic variability among strains of M. gallisepticum. Analysis of the high-passage, attenuated derivative of Rlow, Rhigh, indicated that relatively few total genomic changes (64 loci) occurred, yet they are potentially responsible for the observed
attenuation of this strain. In addition to previously characterized mutations in cytadherence-related proteins, changes included
those in coding sequences of genes involved in sugar metabolism. Analyses of the genome of the M. gallisepticum vaccine strain F revealed numerous differences relative to strain R, including a highly divergent complement of vlhA surface lipoprotein genes, and at least 16 genes absent or significantly fragmented relative to strain R. Notably, an Rlow isogenic mutant in one of these genes (MGA_1107) caused significantly fewer severe tracheal lesions in the natural host compared
to virulent M. gallisepticum Rlow. Comparative genomic hybridizations indicated few genetic loci commonly affected in F and vaccine strains ts-11 and 6/85,
which would correlate with proteins affecting strain R virulence. Together, these data provide novel insights into inter-
and intrastrain M. gallisepticum genomic variability and the genetic basis of M. gallisepticum virulence.
... Two perfect inverted repeats of 5 bp were detected in B. cetaceae B202R flanking the DNA inversion. Inverted repeats have been shown to be involved in the mechanism of DNA inversion [29,30] and might also be responsible for the DNA inversion that occurred in B. cetaceae strains B1/94 and B202R. The left inverted repeat is also conserved in B. melitensis 16M (Fig. 3C) and B. suis 1330 (data not shown), but it is not the case for the 5-nucleotide region corresponding to the right inverted repeat that differs in both B. melitensis 16M (Fig. 3C) and B. suis 1330 (data not shown). ...
Five genes homologous to the well-known omp25 and omp31 genes, that code for two major Brucella spp. outer membrane proteins (OMPs), have been detected in the genome of Brucella melitensis 16M and Brucella suis 1330. In this work we have determined the nucleotide sequence of these five genes, named omp31b, omp25b, omp25c, omp25d and omp22, in the six classical Brucella species reference strains and in representative strains of the recently proposed species Brucella cetaceae and Brucella pinnipediae that classify the Brucella strains isolated in the last years from marine mammals. Although these genes are quite conserved in the genus Brucella, several important differences have been found between species (i) omp31b contains a premature stop codon in B. canis and B. ovis truncating the encoded protein; (ii) the 5' end of omp31b is deleted in the three biovars of B. melitensis which probably prevents synthesis of Omp31b in this species; (iii) only B. melitensis, B. suis and B. neotomae would be able to synthesize the Omp25b protein with the characteristics shared by the Omp25/Omp31 group of proteins (characteristic signal sequence and C-terminal phenylalanine); (iv) a DNA inversion of 1747 bp including omp25b was detected in B. cetaceae strains; (v) a DNA deletion of about 15 kb was detected in all the six B. ovis strains tested. This deletion in B. ovis includes, among other genes, omp25b and wboA, a gene that has been shown to be required for the synthesis of the O-polysaccharide chain of the Brucella spp. smooth lipopolysaccharide. Several features of the DNA region absent from B. ovis suggest that this DNA fragment is a genomic island acquired by the Brucella ancestor by horizontal transfer and later deleted from B. ovis. The DNA polymorphism we have found in this work within the genus Brucella might be involved in the differences in pathogenicity and host preference displayed by the Brucella species.
... Experimentally well characterized proteins encoded by this family are involved in limiting inversions (in genetic rearrangements). Furthermore, the sequences of these proteins show limited homology to myosins and to some of the SMC (structural maintenance of chromosomes) proteins [16]. ...
There is an increasing interest to better understand endosymbiont capabilities in insects both from an ecological point of view and for pest control. Blochmannia floridanus provides important nutrients for its host, the ant Camponotus, while the bacterium in return is provided with a niche to proliferate. Blochmannia floridanus proteins and metabolites are difficult to study due to its endosymbiontic life style; however, its complete genome sequence became recently available.
Improved sequence analysis algorithms, databanks and gene and pathway context methods allowed us to reveal new information on various enzyme and pathways from the Blochmannia floridanus genome sequence [EMBL-ID BX248583]. Furthermore, these predictions are supported and linked to experimental data for instance from structural genomics projects (e.g. Bfl341, Bfl 499) or available biochemical data on proteins from other species which we show here to be related. We were able to assign a confirmed or at least a putative molecular function for 21 from 27 previously conserved hypothetical proteins. For 48 proteins of 66 with a previous putative assignment the function was further clarified. Several of these proteins occur in many proteobacteria and are found to be conserved even in the compact genome of this endosymbiont. To extend and re-test predictions and links to experimentally verified protein functions, functional clusters and interactions were assembled. These included septum initiation and cell division (Bfl165, Bfl303, Bfl248 et al.); translation; transport; the ubiquinone (Bfl547 et al.), the inositol and nitrogen pathways.
Taken together, our data allow a better and more complete description of the pathway capabilities and life style of this typical endosymbiont.
The JCVI-Syn3 organism is a minimal organism derived from Mycoplasma mycoides capri, which is capable of self-replication. While the ancestor has 863 genes, the synthetic progeny has only 473, with 434 of these coding for proteins. Despite initial efforts to understand all functions of the organism, a significant number of these protein-coding genes still have unknown functions, and subsequent studies have been only partially successful in elucidating their roles. In this study, we employ our innovative method PROST to identify homologs and better understand these previously unidentified genes. PROST employs protein language embeddings and enables the identification of remote homologs with as low as 16% sequence identity. PROST successfully finds functionally annotated homologs for 93% of the minimal genome with a high level of accuracy, both confirming previously identified functions, as well as proposing new functions for others. The results of our study can be accessed at https://bit.ly/prost-syn3a.
Competitive bacteria-bacteriophage interactions have resulted in the evolution of a plethora of bacterial defense systems preventing phage propagation. In recent years, computational and bioinformatic approaches have underpinned the discovery of numerous novel bacterial defense systems. Anti-phage systems are frequently encoded together in genomic loci termed defense islands. Here we report the identification and characterisation of a novel anti-phage system, that we have termed Shield, which forms part of the Pseudomonas defensive arsenal. The Shield system comprises the core component ShdA, a membrane-bound protein harboring an RmuC domain. Heterologous production of ShdA alone is sufficient to mediate bacterial immunity against several phages. We demonstrate that Shield and ShdA confer population-level immunity and that they can also decrease transformation efficiency. We further show that ShdA homologues can degrade DNA in vitro and, when expressed in a heterologous host, can alter the organisation of the host chromosomal DNA. Use of comparative genomic approaches identified how Shield can be divided into four subtypes, three of which contain additional components that in some cases can negatively affect the activity of ShdA and/or provide additional lines of phage defense. Collectively, our results identify a new player within the Pseudomonas bacterial immunity arsenal that displays a novel mechanism of protection, and reveals a role for RmuC domains in phage defense.
Mutations are changes in the genome sequence of an organism. Mutations are the driving force of evolution. Under selection, mutations resulting in adaptive traits are favored, while those resulting in maladaptive traits are selected against. Mutations can be spontaneous, occurring through errors in DNA replication, recombination and repair. Mutations can also be induced by a variety of agents including chemical mutagens, UV irradiation, and exposure to radioactive material. Other induced mutations occur by the activity, insertion or removal of active DNA elements such as transposons and viruses.
DNA inversion duplications are genome rearrangements observed in cancer. In this issue, Deng et al. (2015) demonstrate that in S. cerevisiae RPA and Mre11-Sae2 cooperate to prevent the formation of inversion duplications initiated at short DNA secondary structures.
Proteins belonging to PD-(D/E)XK phosphodiesterases constitute a functionally diverse superfamily with representatives involved in replication, restriction, DNA repair and tRNA-intron splicing. Their malfunction in humans triggers severe diseases, such as Fanconi anemia and Xeroderma pigmentosum. To date there have been several attempts to identify and classify new PD-(D/E)KK phosphodiesterases using remote homology detection methods. Such efforts are complicated, because the superfamily exhibits extreme sequence and structural divergence. Using advanced homology detection methods supported with superfamily-wide domain architecture and horizontal gene transfer analyses, we provide a comprehensive reclassification of proteins containing a PD-(D/E)XK domain. The PD-(D/E)XK phosphodiesterases span over 21,900 proteins, which can be classified into 121 groups of various families. Eleven of them, including DUF4420, DUF3883, DUF4263, COG5482, COG1395, Tsp45I, HaeII, Eco47II, ScaI, HpaII and Replic_Relax, are newly assigned to the PD-(D/E)XK superfamily. Some groups of PD-(D/E)XK proteins are present in all domains of life, whereas others occur within small numbers of organisms. We observed multiple horizontal gene transfers even between human pathogenic bacteria or from Prokaryota to Eukaryota. Uncommon domain arrangements greatly elaborate the PD-(D/E)XK world. These include domain architectures suggesting regulatory roles in Eukaryotes, like stress sensing and cell-cycle regulation. Our results may inspire further experimental studies aimed at identification of exact biological functions, specific substrates and molecular mechanisms of reactions performed by these highly diverse proteins.
Coxiella burnetii is the bacterial agent of Q fever in humans. Here, we describe a unique, ~7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry. blast analyses with CBU1224a showed no significant similarity to sequences outside strains of C. burnetii. Additional in silico analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by >95 % on FW blots done in the presence of EDTA, imidazole, Ni(2+) or Zn(2+), and roughly halved in the presence of Co(2+) or Fe(3+). The limB gene was maximally expressed at 3-7 days post-infection in Coxiella-infected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (~7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (~8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique Coxiella lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.
Almost all cellular organisms employ RecA orthologues to guide the strand invasion reactions necessary for DNA recombination and repair. One of the few exceptions to this orthodoxy is a group of gamma-proteobacteria flourishing in obligate intracellular symbiosis with insects and deep-sea clams. The apparent inability of these bacteria to commence the recombinational exchange process seems to confer genetic stability by preventing any further rearrangements or lateral transfer events. Although debate has centred on the absence of selected recombination functions and their impact on a fixed genomic architecture, no explanation has been offered for how bacteria survive the loss of such an integral DNA repair system. This question is addressed here by speculating on how the current repertoire of recombinases in symbiotic bacteria could enable recovery from potentially lethal injuries to the DNA template. Depending on which functions remain, several different options are plausible. The possibility that specific defects in recombination encourage radical genome erosion in mutualistic endosymbionts and other intracellular bacteria is discussed.
Structural maintenance of chromosomes (SMC) proteins are known to be essential for chromosome segregation in some prokaryotes and in eukaryotes. A systematic search for the distribution of SMC proteins in prokaryotes with fully or partially sequenced genomes showed that they form a larger family than previously anticipated and raised the number of known prokaryotic homologs to 54. Secondary structure predictions revealed that the length of the globular N-terminal and C-terminal domains is extremely well conserved in contrast to the hinge domain and coiled-coil domains which are considerably shorter in several bacterial species. SMC proteins are present in all gram-positive bacteria and in nearly all archaea while they were found in less than half of the gram-negative bacteria. Phylogenetic analyses indicate that the SMC tree roughly resembles the 16S rRNA tree, but that cyanobacteria and Aquifex aeolicus obtained smc genes by lateral transfer from archaea. Fourteen out of 22 smc genes located in fully sequenced genomes seem to be co-transcribed with a second gene out of six different gene families, indicating that the deduced gene products might be involved in similar functions. The SMC proteins were compared with other prokaryotic proteins with long coiled-coil domains. The lengths of different protein domains and signature sequences allowed to differentiate SMCs, MukBs, which were found to be confined to gamma proteobacteria, and two subfamilies of COG 0419 including the SbcC nuclease from E. coli. A phylogenetic analysis was performed including the prokaryotic coiled-coil proteins as well as SMCs and Rad18 proteins from selected eukaryotes.
Mre11-Rad50 (MR) proteins are encoded by bacteriophage, eubacterial, archeabacterial and eukaryotic genomes, and form a complex with a remarkable protein architecture. This complex is capable of tethering the ends of DNA molecules, possesses a variety of DNA nuclease, helicase, ATPase and annealing activities, and performs a wide range of functions within cells. It is required for meiotic recombination, double-strand break repair, processing of mis-folded DNA structures and maintaining telomere length. This article reviews current knowledge of the structure and enzymatic activities of the MR complex and attempts to integrate biochemical information with the roles of the protein in a cell.
Nucleoside diphosphate (NDP) kinase is one of the enzymes that maintains triphosphate pools. Escherichia coli strains (ndk) lacking this enzyme have been shown to be modest base substitution mutators, and two members of the human family of NDP kinases act as tumor suppressors. We show here that in E. coli strains lacking NDP kinase high levels of mispairs are generated, but most of these are corrected by the mismatch-repair system. Double mutants that are ndk mutS, lacking both the NDP kinase and mismatch repair, have levels of base substitutions 15-fold higher and levels of certain frameshifts up to 10-fold higher than those of the respective mutations in mutS strains that are NDP kinase proficient. A sequence analysis of the specificity of base substitution mutations generated in ndk and ndk mutS backgrounds as well as other experiments suggests that NDP kinase deficiency stimulates polymerase errors that lead to A:T --> G:C transitions and that the editing capacity of cells may be affected, leading to additional uncorrected mispairs and to A:T --> T:A transversions.
Plasmid F′128 was formed by an exchange between chromosomal Rep sequences that placed lac near dinB between many pairs of Rep sequences. Plasmid F′128 is critical for selection-enhanced lac reversion (adaptive mutation), which requires prior lac amplification. The structure of F′128 supports the idea that amplification is initiated by Rep-Rep recombination and that general mutagenesis requires coamplification
of dinB (error-prone polymerase) with lac.
Restriction endonucleases and other nucleic acid cleaving enzymes form a large and extremely diverse superfamily that display
little sequence similarity despite retaining a common core fold responsible for cleavage. The lack of significant sequence
similarity between protein families makes homology inference a challenging task and hinders new family identification with
traditional sequence-based approaches. Using the consensus fold recognition method Meta-BASIC that combines sequence profiles
with predicted protein secondary structure, we identify nine new restriction endonuclease-like fold families among previously
uncharacterized proteins and predict these proteins to cleave nucleic acid substrates. Application of transitive searches
combined with gene neighborhood analysis allow us to confidently link these unknown families to a number of known restriction
endonuclease-like structures and thus assign folds to the uncharacterized proteins. Finally, our method identifies a novel
restriction endonuclease-like domain in the C-terminus of RecC that is not detected with structure-based searches of the existing
PDB database.
The SbcCD complex and its homologues play important roles in DNA repair and in the maintenance of genome stability. In Escherichia coli, the in vitro functions of SbcCD have been well characterized, but its exact cellular role remains elusive. This work investigates
the regulation of the sbcDC operon and the cellular localization of the SbcC and SbcD proteins. Transcription of the sbcDC operon is shown to be dependent on starvation and RpoS protein. Overexpressed SbcC protein forms foci that colocalize with
the replication factory, while overexpressed SbcD protein is distributed through the cytoplasm.
The RecT protein of Escherichia coli is a DNA-pairing protein required for the RecA-independent recombination events promoted by the RecE pathway. The RecT protein was found to bind to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in the absence of Mg2+. In the presence of Mg2+, RecT binding to dsDNA was inhibited drastically, whereas binding to ssDNA was inhibited only to a small extent. RecT promoted the transfer of a single-stranded oligonucleotide into a supercoiled homologous duplex to form a D (displacement)-loop. D-loop formation occurred in the absence of Mg2+ and at 1 mM Mg2+ but was inhibited by increasing concentrations of Mg2+ and did not require a high energy cofactor. Strand transfer was mediated by a RecT-ssDNA nucleoprotein complex reacting with a naked duplex DNA and was prevented by the formation of RecT-dsDNA nucleoprotein complexes. Finally, RecT mediated the formation of joint molecules between a supercoiled DNA and a linear dsDNA substrate with homologous 3'-single-stranded tails. Together these results indicate that RecT is not a helix-destabilizing protein promoting a reannealing reaction but rather is a novel type of pairing protein capable of promoting recombination by a DNA strand invasion mechanism. These results are consistent with the observation that RecE (exonuclease VIII) and RecT can promote RecA-independent double-strand break repair in E. coli.
Using site-specific mutagenesis in vitro, we have constructed Escherichia coli strains that allow the detection of the inversion of an 800-bp segment in the lac region. The invertible segment is bounded by inverted repeats of either 12 or 23 bp. Inversions occurring at these inverted repeats will restore the Lac+ phenotype. Inversions can be detected at both short homologies at frequencies ranging from 0.5 x 10(-8) to 1 x 10(-7). These events, which have been verified by DNA sequence analysis, are reduced up to 1000-fold in strains deficient for either RecA, RecB or RecC. They are not reduced in strains deficient in the RecF, J pathway. These results show that the RecB,C,D system can mediate rearrangements at short sequence repeats, and probably plays a major role in cellular rearrangements.
The function of an open reading frame (orf-45) located upstream of the sbcC gene of Escherichia coli was investigated. Mutations that inactivate sbcC improve the ability to propagate lambda red gam phage that carry a palindromic sequence in their DNA. They also act with sbcB mutations as cosuppressors of the defects in recombination, DNA repair, and cell viability associated with recBC mutations. A 1,282-bp cassette encoding resistance to kanamycin was used to disrupt orf-45. The mutation, which has a polar effect on the expression of sbcC, allowed stable propagation of palindromic lambda phage even when the sbcC gene product was provided in trans. Additional nonpolar mutations in orf-45 were isolated on the basis of their ability to improve the growth of recBC sbcB strains. These mutations also confer resistance to mitomycin C, allow efficient recombination in Hfr crosses, and facilitate stable propagation of palindromic phage. It is concluded that the products of orf-45 and sbcC are functionally related. The orf-45 gene is therefore renamed sbcD.
Frequent alterations in the structure of the complement component C1 inhibitor gene have been found in patients affected by the common variant of hereditary angioedema, characterized by low plasma levels of C1 inhibitor. This control protein limits the enzymic activity of the first component of complement and of other plasma serine proteases. Sequence comparisons of a 4.6-kilobase-long segment of the normal gene and the corresponding gene segments isolated from two patients carrying family-specific DNA deletions point to unusually long clusters of tandem repeats of the Alu sequence family as a source of genetic instability in this locus. Unequal crossovers, in a variety of registers, among Alu sequences of the clusters result in deletions of variable length that encompass exon 4. In a third family, exon 4 was instead found to be duplicated along with the same tracts of flanking introns lost in one of the deletions. In addition to undergoing Alu-mediated partial deletions and duplications, the gene is also a target for more recent retroposition events. Gross alterations in the C1 inhibitor gene account for about 20% of the hereditary angioedema chromosomes and consequently make this gene a prime example of the mutagenic liability of Alu repeats.
Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-linked gene encoding the lysosomal enzyme, alpha-galactosidase A (EC 3.2.1.22). Six alpha-galactosidase A gene rearrangements that cause Fabry disease were investigated to assess the role of Alu repetitive elements and short direct and/or inverted repeats in the generation of these germinal mutations. The breakpoints of five partial gene deletions and one partial gene duplication were determined by either cloning and sequencing the mutant gene from an affected hemizygote, or by polymerase chain reaction amplifying and sequencing the genomic region containing the novel junction. Although the alpha-galactosidase A gene contains 12 Alu repetitive elements (representing approximately 30% of the 12-kilobase (kb) gene or approximately 1 Alu/1.0 kb), only one deletion resulted from an Alu-Alu recombination. The remaining five rearrangements involved illegitimate recombinational events between short direct repeats of 2 to 6 base pairs (bp) at the deletion or duplication breakpoints. Of these rearrangements, one had a 3' short direct repeat within an Alu element, while another was unusual having two deletions of 1.7 kb and 14 bp separated by a 151-bp inverted sequence. These findings suggested that slipped mispairing or intrachromosomal exchanges involving short direct repeats were responsible for the generation of most of these gene rearrangements. There were no inverted repeat sequences or alternating purine-pyrimidine regions which may have predisposed the gene to these rearrangements. Intriguingly, the tetranucleotide CCAG and the trinucleotide CAG (or their respective complements, CTGG and CTG) occurred within or adjacent to the direct repeats at the 5' breakpoints in three and four of the five alpha-galactosidase A gene rearrangements, respectively, suggesting a possible functional role in these illegitimate recombinational events. These studies indicate that short direct repeats are important in the formation of gene rearrangements, even in human genes like alpha-galactosidase A that are rich in Alu repetitive elements.
The sbcC gene product of Escherichia coli interferes with the growth of a λred gam phage carrying a long palindrome in its DNA. This phenotype was used to identify recombinant plasmids harbouring the wild-type
gene and to isolate sbcC mutant derivatives carrying Tn1000 insertions. Analysis of these plasmids located sbcC between proC and phoR at a slightly different position from that reported before (Lloyd, R. G. and Buckman, C. 1985, J. Bacteriol. 164, 836–844).
Nucleotide sequencing revealed that the gene spans a DNA segment of 3.3 kb that encodes a poorly expressed protein of 118
kDa and which lies downstream of a gene of unknown function that encodes a polypeptide of 45 kDa. The amino acid sequence
of SbcC contains a nucleotide binding fold similar to that in RecB and other recombination proteins.
Differential gene expression in the mother cell chamber of sporulating cells of Bacillus subtilis is determined in part by an RNA polymerase sigma factor called sigma K (or sigma 27). The sigma K factor was assigned as the product of the sporulation gene spoIVCB on the basis of the partial aminoterminal amino acid sequence of the purified protein. The spoIVCB gene is now shown to be a truncated gene capable of specifying only the amino terminal half of sigma K. The carboxyl terminal half is specified by another sporulation gene, spoIIIC, to which spoIVCB becomes joined inframe at an intermediate stage of sporulation by site-specific recombination within a 5-base pair repeated sequence. Juxtaposition of spoIVCB and spoIIIC need not be reversible in that the mother cell and its chromosome are discarded at the end of the developmental cycle. The rearrangement of chromosomal DNA could account for the presence of sigma K selectively in the mother cell and may be a precedent for the generation of cell type-specific regulatory proteins in other developmental systems where cells undergo terminal differentiation.
The nucleotide sequences of a small gene, racC, and the adjacent N-terminal half of the wild-type recE gene are presented. A frameshift mutation, recE939, inactivating recE and preventing synthesis of the active recE enzyme, exonuclease VIII, was identified. The endpoints of five deletion mutations suppressing recE939 were sequenced. All five delete the frameshift site. Two are intra-recE deletions and fuse the N- and C-terminal portions of recE in frame. Three of the deletions remove the entire N-terminal portion of recE, fusing the C-terminal portion to N-terminal portions of racC in frame. These data indicate that about 70% of the N-terminal half of recE is not required to encode a hypothesized protein domain with exonuclease VIII activity.
The role of recBC functions has been tested for three types of chromosomal recombination events: (1) recombination between direct repeats to generate a deletion, (2) recombination between a small circular fragment and the chromosome, and (3) recombination between inversely oriented repeats to form an inversion. Deletion formation by recombination between direct repeats, which does not require a fully reciprocal exchange, is independent of recBC function. Circle integration and inversion formation are both stimulated by the recBC function; these events require full reciprocality. The results suggest that half-reciprocal exchanges can occur without recBC, but recBC functions greatly stimulate completion of a fully reciprocal exchange. We propose that chromosomal recombination is a two-step process, and recBC functions are primarily required for the second step.
Recombinant DNA libraries generated in vitro should in theory contain all of the sequences of the genomes from which they are derived. However, the literature is dotted with reports of sequences that cannot be recovered, are under-represented, or are highly unstable. In particular, long palindromic nucleotide sequences of perfect or near-perfect symmetry are either lethal to the vector or suffer deletions or other rearrangements that remove symmetry [Collins, Cold Spring Harbor Symp. Quant. Biol. 45 (1981) 409-416; Collins et al., Gene 19 (1982) 139-146; Hagan and Warren, Gene 24 (1983) 317-326]. We report here that mutation of a single gene, namely sbcC, can overcome this inviability and allow for the stable propagation of a 571-bp nearly perfect palindrome in Escherichia coli. This has implications for the choice of strains used for the recovery and analysis of cloned nucleotide sequences.
We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli. Detection relied on a papillation technique that uses a combination of beta-galactosides to reveal blue Lac+ papillae. One class of mutators is specific for the G.C----T.A transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the lacI gene. The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E. coli. The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in A.T----C.G transversions.
We describe gene fusions that result from genetic duplications of 5-20 kb, which are amplified 50- to 100-fold. Because one end point of the fusion lies within the sequenced lacI gene, the new junctures created by the duplications are readily identified. Using a procedure for dideoxy sequencing of double-stranded DNA, we show that the duplications occur almost exclusively at short sequence repeats (less than 15 bp), sometimes involving broken homologies, in the 30 cases examined. Most of the duplications place the lacI-Z encoded hybrid repressor-beta-galactosidase protein under the control of a downstream promoter, resulting in the production of a more complex hybrid protein with beta-galactosidase activity. In some cases the fusion occurs with the lacY or the lacA gene, which suggests that silent promoters can be uncovered by gene fusion and subsequent amplification. In some ways this system represents a bacterial analog to chromosomal rearrangements of oncogenes in higher cells, since here the expression of a silent gene is the result of a genetic rearrangement that is followed by amplification during selected growth.
Isogenic Escherichia coli strains carrying single DNA-repair mutations were compared for their capacity for (i) the repair of X-ray-induced DNA double-strand breaks (DSB) as measured using neutral sucrose gradients; (ii) medium-dependent resistance, i.e., a recA-dependent X-ray survival phenomenon that correlates closely with the capacity for repairing DSB; and (iii) the growth medium-dependent, recA-dependent repair of X-ray-induced DNA single-strand breaks (SSB) as measured using alkaline sucrose gradients (about 80% of these SSB are actually parts of DSB). These three capacities were measured to quantitate more accurately the involvement of the various genes in the repair of DSB over a wide dose range. The mutations tested were grouped into five classes according to their effect on the repair of X-ray-induced DSB: (I) the recA, recB, recC, and lexA mutants were completely deficient; (II) the radB and recN mutants were about 90% deficient; (III) the recF and recJ mutants were about 70% deficient; (IV) the radA and uvrD mutants were about 30% deficient; and (V) the umuC mutant resembled the wild-type strains in its capacity for the repair of DSB.
Evidence is presented to show that Escherichia coli JC7618, JC7621, and JC7623, previously regarded as having a recB recC sbcB genotype, carry an additional mutation in a new gene designated sbcC at minute 9 on the standard genetic map. In the absence of the sbcC mutation these strains are sensitive to mitomycin C and have a reduced efficiency of recombination. Cultures of recBC sbcB (sbcC+) strains grow slowly, contain many inviable cells, and rapidly accumulate fast-growing variants due to mutation of sbcC. sbcC has been identified on recombinant plasmids and tentatively located by Tn1000 mutagenesis to a 0.9-kilobase DNA section between proC and phoR.
Mutations in sbcB inactivate exonuclease I and suppress the UV-sensitive, mitomycin-sensitive, recombination-deficient phenotypes associated with recB and recC mutations. Mapping experiments have located sbcB about 0.4 minutes from the his operon at 38.0 on the standard map of E. coli. This places sbcB between supD and his. A four-point cross shows that sbcB lies between P2 attH and his. P2 eduction deleting the his operon beginning with P2 attH also deletes sbcB and produces the expected exonuclease I deficiency and suppression of recB(-). The occurrence of chemical-mutagen-induced and spontaneous mutations indirectly suppressing recB(-) and recC(-) is examined. Three lines of strains produce only sbcA mutations while only sbcB mutations occur in a fourth line. Explanations for this behavior are proposed in light of the ability of the first three lines to express sbcB mutations which they inherit by transduction.
Under physiological conditions the initial action of the RecBC enzyme (exonuclease V) on duplex DNA is unwinding of the DNA strands. We have examined by electron microscopy the initial products of this unwinding reaction. When such reactions are carried out in the presence of DNA binding protein, unwinding structures are seen both at the terminus of the duplex DNA and at locations remote from the ends of the DNA molecule. Both terminal and internal unwinding structures proceed along DNA at about 300 nucleotides per second, and the single-stranded loops in both types of structure enlarge at about 100 nucleotides per second. In the internal unwindings DNA must be rewound behind the enzyme at about 200 nucleotides per second. The structures do not occur on supercoiled or nicked circular DNA, indicating that free ends are needed for their formation. In the absence of DNA binding protein only internal unwinding structures are seen, suggesting that the internal structures are formed from the terminal unwindings by base-pairing of their unwound single-strand tails. We present a model which incorporates these structures and is consistent with previous observations on the unwinding and degradative actions of the enzyme. In this model the enzyme travels through duplex DNA by unwinding the DNA ahead of itself and rewinding it behind itself. The internal unwindings produced by the RecBC enzyme could be active in initial synapsis step in genetic recombination.
Exonuclease VIII from Escherichia coli was shown to preferentially degrade linear duplex DNA, although a limited amount of activity with single-stranded linear DNA substrates was detected. No nucleolytic activity was observed with double-stranded circular substrates containing single strand breaks or gaps. Exonuclease VIII was shown to degrade linear duplex DNA from the 5' termini of the molecules and proceed in the 5' to 3' direction via a processive reaction mechanism. Initiation could occur from either a 5'-hydroxyl or 5'-phosphate residue at equal rates. The products of degradation of linear duplex DNA were an equivalent amount of 5'-mononucleotides and single-stranded DNA. Possible roles for exonuclease VIII in genetic recombination are discussed.
Using site-specific mutagenesis in vitro, we have constructed Escherichia coli strains that allow the detection of the inversion of an 800-bp segment in the lac region. The invertible segment is bounded by inverted repeats of either 12 or 23 bp. Inversions occurring at these inverted repeats will restore the Lac+ phenotype. Inversions can be detected at both short homologies at frequencies ranging from 0.5 x 10(-8) to 1 x 10(-7). These events, which have been verified by DNA sequence analysis, are reduced up to 1000-fold in strains deficient for either RecA, RecB or RecC. They are not reduced in strains deficient in the RecF, J pathway. These results show that the RecB,C,D system can mediate rearrangements at short sequence repeats, and probably plays a major role in cellular rearrangements.
Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination.
Genetic studies in yeast have indicated a role of the RAD50 and MRE11 genes in homologous recombination, telomere length maintenance, and DNA repair processes. Here, we purify from nuclear extract of Raji cells a complex consisting of human Rad50, Mre11, and another protein factor with a size of about 95 kDa (p95), which is likely to be Nibrin, the protein encoded by the gene mutated in Nijmegen breakage syndrome. We show that the Rad50-Mre11-p95 complex possesses manganese-dependent single-stranded DNA endonuclease and 3' to 5' exonuclease activities. These nuclease activities are likely to be important for recombination, repair, and genomic stability.
During the course of studies on the growth of gamma λ mutants in rec
+
and rec
-
cells, L. W. Enquist and I (Enquist and Skalka, 1973) observed a growth defect in λ red mutants that was independent of the rec condition of the host. Red mutants synthesize their DNA at only 1/3 to 1/2 the wild-type rate, and concatemers formed at late times are on the average shorter than normal. Red
-
DNA also seems to be packaged with somewhat less than the wild-type efficiency, and phage bursts are only about 30 to 40% that of wild type (Table 1). The importance of red genes to λ growth is further emphasized by the occurrence of a number of bacterial mutants on which λ red mutants do not plate at all. This class, called feb
-
, includes polA- and lig
-
strains (Zissler et al., 1971). The following section summarizes results from studies of DNA synthesis and phage production in red
-
and gam
-
infection of these feb
-
hosts.
This chapter reviews the somatic generation of antibody diversity. It was shown that a typical antibody molecule is composed of two identical light chains and two identical heavy chains. It had also been found that each of these two types of chain exhibits great sequence variability in the amino terminal region between one antibody molecule and the next and no sequence variability in the carboxyl terminal regions. Use of restriction enzymes and recombinant DNA methods allowed resolution of a long-standing and central issue in immunology, the genetic origins of antibody diversity. It turned out that an organism does not inherit even a single complete gene for antibody polypeptide chains. Rather, the genetic information is transmitted in germline as no more than several hundred gene segments. Through a series of specialized somatic recombination occurring specifically during the differentiation of B lymphocytes, these gene segments are assembled into tens of thousands of complete genes. In the immune system, organisms have exploited two major processes for modification of DNA, recombination and mutation, as a means to diversify somatically the limited amount of inherited genetic information to cope with the vastly diverse antigen universe. Somatic diversification allows the individual organism to generate a virtually limitless number of lymphocyte variants.
Hybridization of lambda-mRNAs to excess liver DNA yielded results compatible with gene reiteration frequencies of three or less. Purified mRNA from tumors producing structurally different lambda chains were used in competition hybridization experiments. An unlabeled lambda-mRNA competed with another, labelled mRNA to the same extent as homologous unlabelled lambda-mRNA. Mouse DNA was digested with Eco R-I restriction endonuclease and fractionated by gel electrophoresis. A DNA fragment carrying the V lambda-gene(s) was indentified in this digest. This fragment hybridized with lambda-mRNAs coding for two different lambdaV regions equally well. These results indicate that base sequence homology among lambda-mRNAs is so high that any lambda-mRNA should cross-hybridize with all or most of germ line V lambda genes. From amino acid sequence data, it is argued that there are probably more than 25 different lambdaV regions. Hence it is concluded that the number of germ line genes is too small to account for the diversity of lambda chains.
We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli. Detection relied on a papillation technique that uses a combination of beta -galactosides to reveal blue Lac+ papillae. One class of mutators is specific for the G\cdot C -> T\cdot A transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the lacI gene. The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E. coli. The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in A\cdot T -> C\cdot G transversions.
We report here that homologous recombination functions are required for the viability of Escherichia coli cells maintaining a 240 bp chromosomal inverted repeat (palindromic) sequence. Wild-type cells can successfully replicate this palindrome but recA, recB or recC mutants carrying the palindrome are unviable. The dependence on homologous recombination for cell viability is overcome in sbcC mutants. Directly repeated copies of the DNA containing the palindrome are rapidly resolved to single copies in wild-type cells but not in sbcC mutants. Our results suggest that double-strand breaks introduced at the palindromic DNA sequence by the SbcCD nuclease are repaired by homologous recombination. The repair is conservative and the palindrome is retained in the repaired chromosome. We conclude that SbcCD can attack secondary structures but that repair conserves the DNA sequence with the potential to fold.
Endothelium-derived relaxing factor (EDRF) is a labile humoral agent which mediates the action of some vasodilators. Nitrovasodilators, which may act by releasing nitric oxide (NO), mimic the effect of EDRF and it has recently been suggested by Furchgott that EDRF may be NO. We have examined this suggestion by studying the release of EDRF and NO from endothelial cells in culture. No was determined as the chemiluminescent product of its reaction with ozone. The biological activity of EDRF and of NO was measured by bioassay. The relaxation of the bioassay tissues induced by EDRF was indistinguishable from that induced by NO. Both substances were equally unstable. Bradykinin caused concentration-dependent release of NO from the cells in amounts sufficient to account for the biological activity of EDRF. The relaxations induced by EDRF and NO were inhibited by haemoglobin and enhanced by superoxide dismutase to a similar degree. Thus NO released from endothelial cells is indistinguishable from EDRF in terms of biological activity, stability, and susceptibility to an inhibitor and to a potentiator. We suggest that EDRF and NO are identical.
140 independently occurring spontaneous mutations in the lacI gene of Escherichia coli have been examined genetically and physically. DNA sequence analysis of a genetic “hotspot” shows that the tandemly repeating sequence 5′-C-T-G-G-C-T-G-G-C-T-G-G-3′ generates mutations at a high rate, either deleting or adding one unit of four nucleotides (C-T-G-G). Twelve larger deletion mutations have also been sequenced; seven of these were formed by eliminating segments between repeated sequences of five or eight nucleotides, one copy of the repeated sequence remaining after the deletion. Possible mechanisms accounting for the involvement of repeated sequences in the creation of spontaneous mutations are considered.
A model is described for the repair of induced double-strand breaks in DNA which involves a proposed heteroduplex intermediate and normally occurring repair enzymes. The model predicts that this repair can lead to non-reciprocal and/or reciprocal recombination. The evidence which has been evaluated from other studies indicates a clear correspondence between the production of double-strand breaks and the occurrence of intragenic recombinants. In addition, the model describes how enzymatically-induced double-strand breaks could initiate recombination without concomitant loss of chromosomal segments.
A key step in homologous recombination is the alignment and pairing of homologous DNAs. The Escherichia coli RecA protein initiates pairing by binding to single-strand DNA, forming a helical nucleoprotein filament. We demonstrate that in the presence of the nonhydrolyzable ATP analogue adenosine 5'-[gamma-thio]triphosphate and ADP, RecA can pair a homologous oligonucleotide 15 bases long with a duplex DNA to yield synaptic complexes consisting of the oligonucleotide and duplex DNA stabilized by RecA. RecA can pair as few as eight bases of homology to form such synaptic complexes. The homologous DNAs remain paired to each other upon removal of RecA provided that the length of shared homology is at least 26 base pairs. Based on our findings and the work of others, we propose that in vitro, one helical turn of a RecA nucleoprotein filament containing approximately six RecA monomers and 15 bases of single-strand DNA is the functional unit sufficient to carry out the homology search.
This chapter discusses the uses of transposons with emphasis on Tn 10. As transposable elements have become indispensable tools for bacterial genetics, many different types of specialized transposon derivatives have been constructed. The most widely used constructs are derived from insertion sequence (IS)-based elements (Tn10 and Tn5) or from bacteriophage Mu; constructs based on cointegrate-forming elements are also available. This chapter discusses the major types of transposon constructs available and describes a method to best choose the most appropriate construct for the desired application. The chapter also discusses methods used for Tnl0-derived transposon vehicles and the most recent set of useful Tnl0 vehicles..
In terms of evolution and fitness, the most significant spontaneous mutation rate is likely to be that for the entire genome (or its nonfrivolous fraction). Information is now available to calculate this rate for several DNA-based haploid microbes, including bacteriophages with single- or double-stranded DNA, a bacterium, a yeast, and a filamentous fungus. Their genome sizes vary by approximately 6500-fold. Their average mutation rates per base pair vary by approximately 16,000-fold, whereas their mutation rates per genome vary by only approximately 2.5-fold, apparently randomly, around a mean value of 0.0033 per DNA replication. The average mutation rate per base pair is inversely proportional to genome size. Therefore, a nearly invariant microbial mutation rate appears to have evolved. Because this rate is uniform in such diverse organisms, it is likely to be determined by deep general forces, perhaps by a balance between the usually deleterious effects of mutation and the physiological costs of further reducing mutation rates.
The muscle mitochondria of a patient with Kearns-Sayre/chronic external ophthalmoplegia plus syndrome were found to be completely deficient in respiratory complex I activity and partially deficient in complex IV and V activities. Treatment of the patient with coenzyme Q10 and succinate resulted in clinical improvement of respiratory function, consistent with the respiratory deficiencies. Restriction enzyme analysis of the muscle mtDNA revealed a 4.9-kilobase deletion in 50% of the mtDNA molecules. Polymerase chain reaction analysis demonstrated that the deletion was present in the patient's muscle but not in her lymphocytes or platelets. Furthermore, the deletion was not present in the muscle or platelets of two sisters. Hence, the mutation probably occurred in the patient's somatic cells. Direct sequencing of polymerase chain reaction-amplified DNA revealed a 4977-base-pair deletion removing four genes for subunits of complex I, one gene for complex IV, two genes for complex V, and five genes for tRNAs, which paralleled the respiratory enzymes affected in the disease. A 13-base-pair direct repeat was observed upstream from both breakpoints. Relative to the direction of heavy-strand replication, the first repeat was retained and the second repeat was deleted, suggesting a slip-replication mechanism. Sequence analysis of the human mtDNA revealed many direct repeats of 10 base pairs or greater, indicating that this mechanism could account for other reported deletions. We postulate that the prevalence of direct repeats in the mtDNA is a consequence of the guanine-cytosine bias of the heavy and light strands.
Lambda red gam phage carrying a 571 base-pair palindrome are unviable in wild-type Escherichia coli hosts. By using de-methylation to study the fate of DNA strands introduced into E. coli, we have demonstrated that a decrease in the yield of palindrome-containing molecules with two newly synthesized strands can occur without any concomitant loss of replicated molecules containing input strands. This implies that the palindrome-containing DNA is not being destroyed even as a consequence of replication, but rather that its replication rate is reduced. These results demonstrate that a palindrome can mediate unviability without directing cleavage of its carrier replicon.
Following the strategy outlined in an accompanying paper, we studied 32 X-linked muscular dystrophy families (29 Duchenne [DMD] and three Becker [BMD] type) for abnormalities of HindIII and BglII fragments detected by the entire dystrophin cDNA. Twenty-one different single-intragenic deletions, and no duplications, were identified. The deletion endpoints were precisely mapped on the published HindIII fragment map. Detailed analysis of overlapping deletions led to clarification of the fragment order for some previously unsettled regions of the HindIII map and to the construction of a partial map of exon-containing BglII fragments. For the regions involved in deletions, the corresponding HindIII and BglIII fragments could be identified. Noncontiguous comigrating fragments were detected in two regions by careful analysis of the patterns in deletion patients. Four of the 21 deletions generated novel restriction fragments that facilitated detection of female carriers in these families. Twelve of the deletions had a breakpoint in one of the two large introns known to be the sites of breakpoint clusters. By combining deletions and RFLP analyses, we unequivocally identified the gamete that first carried the mutation in 13 families: eight oocytes and five sperm. Germ-line mosaicism previously detected in one male was confirmed by cDNA studies. In two additional families gonadal mosaicism was found in females. As evidence is accumulating for frequent mitotic origin of these deletion mutations, this phenomenon has to be considered when postulating mutational mechanisms and in genetic counseling of DMD/BMD families.
To determine the minimum amount of homology required for efficient recombination in Escherichia coli, we measured recombination frequencies between bacteriophage λ and pBR322 derivatives containing λ DNA fragments of various sizes by assaying for phages that could transduce the bla and ori genes of pBR322. Efficient recombination required about 40 bp of homology; increases in homology above 40 bp resulted in proportionate increases in recombination, while decreases below 40 bp resulted in precipitous decreases in recombination. The recA
+ gene stimulated recombination over the entire range of homologies tested. Restriction enzyme digests of several recombinant DNA molecules indicated that they contained the complete plasmid DNA inserted in the λ genome as expected for a reciprocal crossover. Analysis of recombination frequencies in different recombination-deficient mutant strains indicated that the formation of λ-plasmid cointegrates by homologous recombination proceeded predominantly by the RecBC pathway and very inefficiently, if at all, by the RecE and RecF pathways.
We studied the in vivo recombination between homologous DNA sequences cloned in phage lambda and a pBR322-derived plasmid by assaying for the formation of phage-plasmid cointegrates by a single (or an odd number of) reciprocal exchange. (1) Recombination proceeds by the RecBC pathway in wild-type cells and by low levels of a RecF-dependent pathway in recBC- cells. The RecE pathway appears not to generate phage-plasmid cointegrates. (2) Recombination is linearly dependent on the length of the homologous sequences. In both RecBC and RecF-dependent pathways there is a minimal length, called the minimal efficient processing segment (MEPS), below which recombination becomes inefficient. The length of MEPS is between 23-27 base pairs (bp) and between 44-90 bp for the RecBC- and RecF-dependent pathways, respectively. A model, based on overlapping MEPS, of the correlation of genetic length with physical length is presented. The bases for the different MEPS length of the two pathways are discussed in relationship to the enzymes specific to each pathway. (3) The RecBC and the RecF-dependent pathways are each very sensitive to substrate homology. In wild-type E. coli, reduction of homology from 100% to 90% decreases recombinant frequency over 40-fold. The homology dependence of the RecBC and RecF-dependent pathways are similar. This suggests that a component common to both, probably recA, is responsible for the recognition of homology.
In the absence of a combined nitrogen source, such as ammonia, approximately every tenth vegetative cell along filaments of the cyanobacterium Anabaena develops into a heterocyst, a terminally differentiated cell that is morphologically and biochemically specialized for nitrogen fixation. At least two specific DNA rearrangements involving the nitrogen-fixation (nif) genes occur during heterocyst differentiation, one within the nifD gene and the other near the nifS gene. The two rearrangements have several properties in common. Both occur quantitatively in all heterocyst genomes, both occur at approximately the same developmental time, late in the process of heterocyst differentiation, and both result from site-specific recombination between short repeated DNA sequences. We report here the nucleotide sequences found at the site of recombination near the nifS gene. These sequences differ from those found previously for the nifD rearrangement, suggesting that the two rearrangements are catalysed by different enzymes and may be regulated independently. We also show that the nifS gene is transcribed only from rearranged genomes.
Thirty-four hundred lambda phage clones containing segments of the E. coli chromosome were isolated and used to construct a 4700 kb long integrated restriction map for eight six-base-recognizing enzymes by a rapid mass-analysis method. Our strategy was to measure the sizes of partial restriction enzyme digests by hybridization with a vector probe in a manner analogous to nucleotide sequencing. The data were sorted into groups by a computer program and the boundary clones were further correlated with each other using a mass hybridization method. These clones can be exploited for the isolation of any desired E. coli genes if their map positions are known. Also, the strategy is applicable to analyses of the genomes of other organisms.
Exonuclease VIII (exoVIII) of Escherichia coli has been purified from a strain carrying a plasmid-encoded recE gene by using a new procedure. This procedure yielded 30 times more protein per gram of cells, and the protein had a twofold higher specific activity than the enzyme purified by the previously published procedure (J. W. Joseph and R. Kolodner, J. Biol. Chem. 258:10411-10417, 1983). The sequence of the 12 N-terminal amino acids was also obtained and found to correspond to one of the open reading frames predicted from the nucleic acid sequence of the recE region of Rac (C. Chu, A. Templin, and A. J. Clark, manuscript in preparation). Polyclonal antibodies directed against purified exoVIII were also prepared. Cell-free extracts prepared from strains containing a wide range of chromosomal- or plasmid-encoded point, insertion, and deletion mutations which result in expression of exoVIII were examined by Western blot (immunoblot) analysis. This analysis showed that two point sbcA mutations (sbcA5 and sbcA23) and the sbc insertion mutations led to the synthesis of the 140-kilodalton (kDa) polypeptide of wild-type exoVIII. Plasmid-encoded partial deletion mutations of recE reduced the size of the cross-reacting protein(s) in direct proportion to the size of the deletion, even though exonuclease activity was still present. The analysis suggests that 39 kDa of the 140-kDa exoVIII subunit is all that is essential for exonuclease activity. One of the truncated but functional exonucleases (the pRAC3 exonuclease) has been purified and confirmed to be a 41-kDa polypeptide. The first 18 amino acids from the N terminus of the 41-kDa pRAC3 exonuclease were sequenced and fond to correspond to one of the translational start signals predicted from the nucleotide sequence of radC (Chu et al., in preparation).
All recB(-) and recC(-) mutants of E. coli carry out significant residual genetic recombination, whereas all recA(-) mutants form no recombinants. This observation suggests that an alternative minor pathway of recombination, independent of recB(+) and recC(+) products, may be operative in Escherichia coli. Rec(+) revertants of recB(-)recC(+), recB(+)recC(-), and recB(-) strains of E. coli have been isolated and are shown to fall into at least two major genotypic classes. One class carries revertant mutations which map in or very near the recB and recC genes. In this class an ATP-dependent DNase characteristic of wild type E. coli is restored. The reversions in this class are probably back-mutations or intragenic suppressor mutations. A second class carries revertant mutations which are located far from the recB and recC genes. In this class there is a high level of DNase activity which does not require ATP and is inactive on T4 DNA. Indirect and not informational suppression appears to be responsible for the second class of revertants. The suggestion is made that restoration of recombination by indirect suppression involves an activation or derepression of one or a series of enzymes, which participate in a pathway of recombination, alternative to the recB and recC pathway, but normally of minor importance. The ATP-independent DNase may be one of these enzymes.
Using lacl-Z fusion strains of Escherichia coli we have devised systems that detect deletions of varying lengths. We examined deletions 700-1000 base pairs long, and genetically characterized over 250 spontaneous deletions. Of these, we analyzed 24 by direct DNA sequencing and 18 by inspection of restriction fragment patterns. Deletions of this size occur almost exclusively at short repeated sequences in both (recA+ and recA- strain backgrounds, but are detected 25-fold more frequently in a recA+ background. The frequency of deletion formation correlates with the extent of homology between the short repeated sequences, although other factors may be involved. The largest hotspot, which accounts for 60% of the deletions detected, involves the largest homology in the system (14 of 17 base pairs). Altering a single base pair within this homology reduces deletion incidence by an order of magnitude. We discuss possible mechanisms of deletion formation and consider its relationship to the excision of transposable elements.
In the genome of a germ-line cell, the genetic information for an immunoglobulin polypeptide chain is contained in multiple gene segments scattered along a chromosome. During the development of bone marrow-derived lymphocytes, these gene segments are assembled by recombination which leads to the formation of a complete gene. In addition, mutations are somatically introduced at a high rate into the amino-terminal region. Both somatic recombination and mutation contribute greatly to an increase in the diversity of antibody synthesized by a single organism.