Mechanisms of deletion formation in Escherichia coli plasmids. II. Deletions mediated by short direct repeats.

Summary A set of plasmids containing 42, 21 and 13 bp direct repeats was used to analyze the effect of repeat length on the frequencies of deletion formation and the structure of the deleted derivatives of different recombination-deficient Escherichia coli strains. Agarose gel electrophoresis of plasmid DNA demonstrated that the formation of deletions in these plasmids was associated with dimerization of plasmid DNA. Restriction analysis of the dimers showed that deletions at short direct repeats arose non-conservatively, that is, the formation of a deletion in one monomeric plasmid unit was not associated with a duplication in the other. Mutations in the recA, recF, recJ and recO genes had no marked effect on either the frequencies of deletion formation or the structure of dimers. In contrast, recB recC mutations greatly increased the frequencies of deletion formation, 6-fold for 42 bp, and 115-fold for 21 by direct repeats. Conversion of DNA replication to the rolling circle mode in a recB recC strain, resulting in the formation of double-stranded ends, is suggested as the stimulatory effector.     

Recombination between repeats in Escherichia coli by a recA-independent, proximity-sensitive mechanism

We have examined the influence of proximity on the efficiency of recombination between repeated DNA sequences in Escherichia coli. Our experiments have employed a plasmid-based assay to detect deletions between direct repeats of 100 bp. The rate of deletion of the juxtaposed direct repeats was reasonably high at 6 × 10^–5 per cell. A comparison of recA+ and recA mutant strains showed that these deletion events are primarily the result of recA-independent recombination at these homologous sequences. Random restriction fragments of yeast or E. coli genomic DNA were used to separate the two repeats. Deletion rates decreased over two orders of magnitude with increasing separation of up to 7 kb. There was a surprisingly strong effect of even short sequence separations, with insertions of a few hundred base pairs exhibiting 10-fold reductions of deletion rates. No effect of recA on the efficiency of deletion was observed at any distance between repeats.     

Mutational analysis of the CCR5 and CXCR4 genes (HIV-1 co-receptors) in resistance to HIV-1 infection and AIDS development among intravenous drug users

We analysed a group of Spanish intravenous drug users and controls to determine the role of mutations at the chemokine receptor-5/HIV-1 cofactor (CCR5), previously implicated in resistance to HIV-1 infection, and CXCR4 genes in susceptibility to HIV-1 infection. The complete coding sequence of both genes was amplified by the polymerase chain reaction from genomic DNA of 50 seropositive slow progressors and 10 long-term non-progressors, and analysed by the single-strand conformation polymorphism technique in a search for mutations. No mutation in CXCR4 was found, and Δccr5 was the only mutation identified at the CCR5 gene. We genotyped (Δccr5 allele) 150 HIV-1+ intravenous drug users and 250 healthy controls from the same population (Asturias, Northern Spain). Patients were divided into rapid progressors, presenting an event indicating progression to the acquired immunodeficiency syndrome (AIDS) in the 2 years after infection (100 patients), and slow progressors, remaining asymptomatic for 2–10 years (50 patients). The frequencies of the Δccr5 allele were 0.105 and 0.040 in controls and HIV-1+ patients, respectively. Eighteen per cent of the controls (45/250) and 8% (12/150) of the patients carried the Δccr-5 allele (P=0.013). The frequency of Δccr5 carriers among rapid and slow disease progressors was 3 and 15%, respectively. A highly significant difference was found between rapid progressors and controls (P=0.0014). No patient (0/150) was Δccr5 homozygous compared with 1% among controls. Thus, the Δccr5 allele (the only CCR5 mutation found in our HIV-1 patients) was rare among seropositive intravenous drug users, suggesting that the absence of this mutation confers an advantage to the virus when infecting cells in vivo. In addition, patients carrying the Δccr5 allele tend to show a slow progression towards HIV-1-related disease, remaining asymptomatic for longer periods of time. Received: 15 October 1997 / Accepted: 12 January 1998     

Reciprocal relationship between stem-loop potential and substitution density in retroviral quasispecies under positive Darwinian selection

Nucleic acids have the potential to form in trastrand stem-loops if complementary bases are suitably located. Computer analyses of poliovirus and retroviral RNAs have revealed a reciprocal relationship between statistically significant stem-loop potential and sequence variability. The statistically significant stem-loop potential of a nucleic acid segment has been defined as a function of the difference between the folding energy of the natural segment (FONS) and the mean folding energy of a set of randomized (shuffled) versions of the natural segment (FORS-M). Since FONS is dependent on both base composition and base order, whereas FORS-M is solely dependent on base composition (a genomic characteristic), it follows that statistically significant stem-loop potential (FORS-D) is a function of base order (a local characteristic). In retroviral genomes, as in all DNA genomes studied, positive FORS-D values are widely distributed. Thus there have been pressures on base order both to encode specific functions and to encode stem-loops. As in the case of DNA genomes under positive Darwinian selection pressure, in HIV-1 specific function appears to dominate in rapidly evolving regions. Here high sequence variability, expressed as substitution density (not indel density), is associated with negative FORS-D values (impaired base-order-dependent stem-loop potential). This suggests that in these regions HIV-1 genomes are under positive selection pressure by host defenses. The general function of stem-loops is recombination. This is a vital process if, from among members of viral quasispecies, functional genomes are to be salvaged. Thus, for rapidly evolving RNA genomes, it is as important to conserve base-order-dependent stem-loop potential as to conserve other functions.     

A Petunia hybrida chloroplast DNA region, close to one of the inverted repeats, shows sequence homology with the Euglena gracilis chloroplast DNA region that carries the putative replication origin

Summary Three distinct chloroplast (cp) DNA fragments from Petunia hybrida, which promote autonomous replication in yeast, were mapped on the chloroplast genome. Sequence analysis revealed that these fragments (called ARS A, B and C) have a high AT content, numerous short direct and inverted repeats and at least one yeast ARS consensus sequence 5A/TTTTATPuTTTA/T, essential for yeast ARS activity. ARS A and B also showed the presence of (semi-)conserved sequences, present in all Chlamydomanas reinhardii cpDNA regions that promote autonomous replication in yeast (ARS sequences) or in C. reinhardii (ARC sequences). A 431 bp BamHI/EcoRI fragment, close to one of the inverted repeats and adjacent to the ARS B subfragment contains an AT-rich stretch of about 100 nucleotides that show extensive homology with an Euglena gracilis cpDNA fragment which is part of the replication origin region. This conserved region contains direct and inverted repeats, stem-and-loop structures can be folded and it contains an ARS consensus sequence. In the near vicinity a GC-rich block is present. All these features make this cpDNA region the best candidate for being the origin of replication of P. hybrida cpDNA.     

Evaluation of FORS-D Analysis: A Comparison with the Statistically Significant Stem-loop Potential

The stem-loop potential of a nucleic acid segment (expressed as a FONS value), decomposes into base composition-dependent and base order-dependent components. The latter, expressed as a FORS-D value, is derived by subtracting the value of the base composition-dependent component (FORS-M) from the FONS value. FORS-D analysis is the use of FORS-D values to estimate the potential of local base order to contribute to a stem-loop structure, and it has been used to investigate the relationship between stem-loop structure and other selective pressures on genomes. In the present study, we evaluated the reliability of FORS-D analysis by comparing it with statistically significant stem-loop potential, another robust method developed by Le and Maizel for examining stem-loop structure. We found that FORS-M values calculated using 10 randomized sequences are as reliable as those calculated using 100 randomized sequences. The resulting FORS-D values have a similar trend and distribution as statistically significant stem-loop potential, implying that FORS-D analysis is as reliable as the latter in measuring the distribution of base order-dependent stem-loop potential. Since the calculation of the FORS-M values is time consuming, the integrated program Bodslp developed by us will become a convenient tool for large-scale FORS-D analysis. The results also suggest that for some purposes the online program SigStb developed by Le and Maizel may be used as an alternative tool for FORS-D analysis. C. Y. Zhang and J. F. Wei contributed equally to this paper.     

Replication of the broad-host-range plasmid RK2: isolation and characterization of a spontaneous deletion mutant that can replicate in Agrobacterium tumefaciens but not in Escherichia coli

Two spontaneous deletions of a derivative of the broad-host-range plasmid RK2 were isolated from Agrobacterium tumefaciens. The two deletions have lost 56 and 505 bp, respectively, near the origin of replication (oriV). Of the eight 17-bp repeats present in the RK2 oriV, the smaller deletion has lost the first two while the larger one has lost the first three. The deletions led to a significant increase (3- to 7-fold) in plasmid copy number in A. tumefaciens, indicating their importance in copy number control. While the smaller deletion could replicate in Escherichia coli, the larger one could not. The role of the oriV sequences in the replication of pRK2 in A. tumefaciens and in E. coli is discussed.     

The key role for local base order in the generation of multiple forms of China HIV-1 B'/C intersubtype recombinants

Background  

HIV-1 is a retrovirus with high rate of recombination. Increasing experimental studies in vitro indicated that local hairpin structure of RNA was associated with recombination by favoring RT pausing and promoting strand transfer. A method to estimate the potential to form stem-loop structure by calculating the folding of randomized sequence difference (FORS-D) has been used to investigate the relationship between secondary structure and evolutionary pressure in some genome. It showed that gene regions under strong positive "Darwinian" selection were associated with positive FORS-D values. In the present study, the sequences of HIV-1 subtypes B' and C, both of which represent the parent strains of CRF07_BC, CRF08_BC and China URFs, were selected to investigate the relationship between natural recombination and secondary structure by calculating the FORS-D values.     

Genetic deletions between directly repeated sequences in bacteriophage T7

Summary DNA sequence analysis of genetic deletions in bacteriophage T7 has shown that these chromosomal rearrangements frequently occur between directly repeated DNA sequences. To study this type of spontaneous deletion in more quantitative detail synthetic fragments of DNA, made by hybridizing two complementary oligonucleotides, were introduced into the non-essential T7 gene 1.3 which codes for T7 DNA ligase. This insert blocked synthesis of functional ligase and made the phage that carried an insert unable to form plaques on a host strain deficient in bacterial ligase. The sequence of the insert was designed so that after it is put into the T7 genome the insert is bracketed by direct repeats. Perfect deletion of the insert between the directly repeated sequences results in a wild-type phage. It was found that these deletion events are highly sensitive to the length of the direct repeats at their ends. In the case of 5 bp direct repeats excision from the genome occurred at a frequency of less than 10⁻¹⁰, while this value for an almost identical insert bracketed by 10 bp direct repeats was approximately 10⁻⁶. The deletion events were independent of a hostrecA mutation.     

Characterization and chromosome location of satellite DNA in the leaf beetle Chrysolina americana (Coleoptera, Chrysomelidae)

This paper is the first record of the satellite DNA of the specialized phytophagous genus Chrysolina. The satellite DNA of Chrysolina americana is organized in a tandem repeat of monomers 189 bp long, has a A + T content of 59.6 % and presents direct and inverted internal repeats. Restriction analysis of the total DNA with methylation sensitive enzymes suggests that this repetitive DNA is undermethylated. In situ hybridization with a biotinylated probe of the satellite DNA showed the pericentromeric localization of these sequences in all meiotic bivalents. The presence of this repetitive DNA in other species of the genus was also tested by Southern analysis. The results showed that this satellite DNA sequence is specific to the C. americana genome and has not been found in three other species of Chrysolina with a different choice of host plants than in the former. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of 54 large deletions/duplications in TSC1 and TSC2 using MLPA, and genotype-phenotype correlations

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by mutations in either of two genes, TSC1 and TSC2. Point mutations and small indels account for most TSC1 and TSC2 mutations. We examined 261 TSC DNA samples (209 small-mutation-negative and 52 unscreened) for large deletion/duplication mutations using multiplex ligation-dependent probe amplification (MLPA) probe sets designed to permit interrogation of all TSC1/2 exons, as well as 15–50 kb of flanking sequence. Large deletion/duplication mutations in TSC1 and TSC2 were identified in 54 patients, of which 50 were in TSC2, and 4 were in TSC1. All but two mutations were deletions. Only 13 deletions were intragenic in TSC2, and one in TSC1, so that 39 (73%) deletions extended beyond the 5′, 3′ or both ends of TSC1 or TSC2. Mutations were identified in 24% of small-mutation-negative and 8% of unscreened samples. Eight of 54 (15%) mutations were mosaic, affecting 34–62% of cells. All intragenic mutations were confirmed by LR-PCR. Genotype/phenotype analysis showed that all (21 of 21) patients with TSC2 deletions extending 3′ into the PKD1 gene had kidney cysts. Breakpoints of intragenic deletions were randomly distributed along the TSC2 sequence, and did not preferentially involve repeat sequence elements. Our own 20-plex probe sets gave more robust performance than the 40-plex probe sets from MRC-Holland. We conclude that large deletions in TSC1 and TSC2 account for about 0.5 and 6% of mutations seen in TSC patients, respectively, and MLPA is a highly sensitive and accurate detection method, including for mosaicism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of a chloroplast DNA sequence from Chlorella ellipsoidea that promotes autonomous replication in yeast

Summary An EcoRI 2.7 kbp fragment from Chlorella ellipsoidea chloroplast DNA (cpDNA) cloned in YIp5 was shown to promote autonomous replication in Saccharomyces cerevisiae. The fragment was localized in the small single copy region close to the inverted repeat. The ARS activity (autonomously replicating sequences in yeast) was found to be confined within a subclone of a ca. 300 bp HindIII fragment. Sequence analysis of this fragment revealed its high AT content and the presence of several direct and inverted repeats and a few elements that were related to the yeast ARS consensus sequence. Electron microscopic studies revealed that this sequence did not coincide with the primary replication origin of chloroplast DNA. The functioning of this sequence as a possible origin of plasmid replication in vivo is discussed. This is the first report on Chlorella cpDNA sequence. re]19850821 rv]19851211 ac]19851216     

Reductive genome evolution in chemoautotrophic intracellular symbionts of deep-sea <Emphasis Type="Italic">Calyptogena</Emphasis> clams

To understand reductive genome evolution (RGE), we comparatively analyzed the recently reported small genomes of two chemoautotrophic, intracellular symbionts of deep-sea clams, Calyptogena okutanii and C. magnifica. Both genomes lack most genes for DNA recombination and repair such as recA and mutY. Their genome architectures were highly conserved except one inversion. Many deletions from small (<100 bp) to large (1–11 kbp) sizes were detected and the deletion numbers decreased exponentially with size. Densities of deletions and short-repeats, as well as A+T content were higher in non-coding regions than in coding regions. Because Calyptogena symbiont genomes lack recA, we propose that deletions and the single inversion occurred by RecA-independent recombination (RIR) at short-repeats with simultaneous consumption of repeats, and that short-repeats were regenerated by accelerated mutations with enhanced A+T bias due to the absence of mutY. We further propose that extant Calyptogena symbiont genomes are in an actively reducing stage of RGE consisting of small and large deletions, and the deletions are caused by short-repeat dependent RIR along with regeneration of short-repeats. In future, the RGE rate will slowdown when the gene repertoires approach the minimum gene set necessary for intracellular symbiotic life. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. H. Kuwahara and Y. Takaki equally contributed to this work.     

Deletions in Plasmid Pbr322: Replication Slippage Involving Leading and Lagging Strands

We test here whether a class of deletions likely to result from errors during DNA replication arise preferentially during synthesis of either the leading or the lagging DNA strand. Deletions were obtained by reversion of particular insertion mutant alleles of the pBR322 amp gene. The alleles contain insertions of palindromic DNAs bracketed by 9-bp direct repeats of amp sequence; in addition, bp 2 to 5 in one arm of the palindrome form a direct repeat with 4 bp of adjoining amp sequence. Prior work had shown that reversion to Ampr results from deletions with endpoints in the 8- or 4-bp repeat, and that the 4-bp repeats are used preferentially because one of them is in the palindrome. To test the role of leading and lagging strand synthesis in deletion formation, we reversed the direction of replication of the amp gene by inverting the pBR322 replication origin, and also constructed new mutant alleles with a 4-bp repeat starting counterclockwise rather than clockwise of the insertion. In both cases the 4-bp repeats were used preferentially as deletion endpoints. A model is presented in which deletions arise during elongation of the strand that copies the palindrome before the adjoining 4-bp repeat, and in which preferential use of the 4-bp repeats independent of the overall direction of replication implies that deletions arise during syntheses of both leading and lagging strands.     

Direct and Inverted Repeats Elicit Genetic Instability by Both Exploiting and Eluding DNA Double-Strand Break Repair Systems in Mycobacteria

Repetitive DNA sequences with the potential to form alternative DNA conformations, such as slipped structures and cruciforms, can induce genetic instability by promoting replication errors and by serving as a substrate for DNA repair proteins, which may lead to DNA double-strand breaks (DSBs). However, the contribution of each of the DSB repair pathways, homologous recombination (HR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA), to this sort of genetic instability is not fully understood. Herein, we assessed the genome-wide distribution of repetitive DNA sequences in the Mycobacterium smegmatis, Mycobacterium tuberculosis and Escherichia coli genomes, and determined the types and frequencies of genetic instability induced by direct and inverted repeats, both in the presence and in the absence of HR, NHEJ, and SSA. All three genomes are strongly enriched in direct repeats and modestly enriched in inverted repeats. When using chromosomally integrated constructs in M. smegmatis, direct repeats induced the perfect deletion of their intervening sequences ∼1,000-fold above background. Absence of HR further enhanced these perfect deletions, whereas absence of NHEJ or SSA had no influence, suggesting compromised replication fidelity. In contrast, inverted repeats induced perfect deletions only in the absence of SSA. Both direct and inverted repeats stimulated excision of the constructs from the attB integration sites independently of HR, NHEJ, or SSA. With episomal constructs, direct and inverted repeats triggered DNA instability by activating nucleolytic activity, and absence of the DSB repair pathways (in the order NHEJ>HR>SSA) exacerbated this instability. Thus, direct and inverted repeats may elicit genetic instability in mycobacteria by 1) directly interfering with replication fidelity, 2) stimulating the three main DSB repair pathways, and 3) enticing L5 site-specific recombination.     

Increase in incidence of chromosome instability and non-conservative recombination between repeats in Saccharomyces cerevisiae hpr1Δ strains

Null hprl strains show a large increase (up to 2000-fold) over wild type in the frequency of occurrence of deletions between direct repeats on three different chromosomes. However, we show that hprl mutations have little or no effect on reciprocal exchange, gene conversion or unequal sister chromatid exchange, as determined using intrachromosomal, interchromosomal and plasmid-chromosome assay systems. A novel intrachromosomal recombination system has allowed us to determine that over 95% of deletions in hpr1 strains do not occur by reciprocal exchange. On the other hand, hpr1 strains show chromosome loss frequencies of up to 100 times the wild-type level. Our results suggest that yeast cells have a very efficient non-conservative recombination mechanism, dependent on RADI and RAD52, that causes deletions between direct DNA repeats, and this mechanism is strongly stimulated in hpr1 strains. The results indicate that the Hpr1 protein is required for stability of DNA repeats and chromosomes. We propose that in the absence of the Hprl protein the cell destabilizes the genome by allowing the initiation of events that lead to deletions of sequences between repeats, and to chromosome instability. We discuss the roles that proteins such as Hprl have in maintaining direct repeats and in preventing non-conservative recombination and consider the importance of these functions for chromosome stability.     

In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501

Summary The tobacco (Nicotiana tabacum) nuclear genome contains long tracts of DNA (i.e. in excess of 18 kb) with high sequence homology to the tobacco plastid genome. Five lambda clones containing these nuclear DNA sequences encompass more than one-third of the tobacco plastid genome. The absolute size of these five integrants is unknown but potentially includes uninterrupted sequences that are as large as the plastid genome itself. An additional sequence was cloned consisting of both nuclear and plastid-derived DNA sequences. The nuclear component of the clone is part of a family of repeats, which are present in about 400 locations in the nuclear genome. The homologous sequences present in chromosomal DNA were very similar to those of the corresponding sequences in the plastid genome. However significant sequence divergence, including base substitutions, insertions and deletions of up to 41 bp, was observed between these nuclear sequences and the plastid genome. Associated with the larger deletions were sequence motifs suggesting that processes such as DNA replication slippage and excision of hairpin loops may have been involved in deletion formation.     

Identification of Novel Non-autonomous CemaT Transposable Elements and Evidence of their Mobility within the C. elegans Genome

We describe here two new transposable elements, CemaT4 and CemaT5, that were identified within the sequenced genome of Caenorhabditis elegans using homology based searches. Five variants of CemaT4 were found, all non-autonomous and sharing 26 bp inverted terminal repeats (ITRs) and segments (152–367 bp) of sequence with similarity to the CemaT1 transposon of C. elegans. Sixteen copies of a short, 30 bp repetitive sequence, comprised entirely of an inverted repeat of the first 15 bp of CemaT4’s ITR, were also found, each flanked by TA dinucleotide duplications, which are hallmarks of target site duplications of mariner-Tc transposon transpositions. The CemaT5 transposable element had no similarity to maT elements, except for sharing identical ITR sequences with CemaT3. We provide evidence that CemaT5 and CemaT3 are capable of excising from the C. elegans genome, despite neither transposon being capable of encoding a functional transposase enzyme. Presumably, these two transposons are cross-mobilised by an autonomous transposon that recognises their shared ITRs. The excisions of these and other non-autonomous elements may provide opportunities for abortive gap repair to create internal deletions and/or insert novel sequence within these transposons. The influence of non-autonomous element mobility and structural diversity on genome variation is discussed.     

Genetic analysis of the minimal replicon of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid

Using a combination of mutagenesis with the transposon and polymerase chain reaction subcloning, the essential elements of the replication region of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid have been identified. An open reading frame, coding for a protein with homology to Rep proteins from other Lactococcus plasmids, is essential. This protein is trans-acting and could not be replaced by the Rep protein from another Lactococcus plasmid. A second open reading frame immediately downstream from the first could be removed or inactivated with no apparent effect on plasmid replication. A region containing two 10 by direct repeats and three tandem repeats of a 22 by sequence, immediately upstream of the essential open reading frame, is also essential and probably includes the origin of replication. A 181-bp DNA fragment containing this region was sufficient to allow replication in Lactococcus if the trans-acting protein was provided on another replicon. Single-stranded replication intermediates could not be detected, suggesting that the citrate plasmid uses theta replication rather than rolling-circle replication.     

Genetic variation at chemokine receptor CCR5 in leporids: alteration at the 2nd extracellular domain by gene conversion with CCR2 in Oryctolagus, but not in Sylvilagus and Lepus species

Whereas in its natural host (Sylvilagus sps.) the effects of myxoma virus infections are benign, in European rabbit (Oryctolagus cuniculus), it causes a highly infectious disease with very high mortality rate, known as myxomatosis. There is evidence that, as with HIV-1 virus in human, myxoma virus may use chemokine receptors such as CCR5 of the host target cell for entry and activation of pathways of immune avoidance. We have characterized and compared CCR5 genes of leporid species with different susceptibility levels to myxomatosis. The CCR5 protein of O. cuniculus differs markedly from all those known from other species. The most striking was the replacement of a specific peptide motif of the second extracellular loop (ECL2) by a motif, which in other species characterizes the CCR2 molecules. While absent in Sylvilagus and Lepus species, this CCR2 imposed CCR5–ECL2 alteration was observed in all genomes of 25 European rabbits, representing the subspecies O. cuniculus algirus and O. cuniculus cuniculus. Allelic variation at the rabbit CCR5 locus confirmed that the gene conversion predates the subspecies split (1–2 Ma).