Novel site-specific DNA modification in Streptomyces: analysis of preferred intragenic modification sites present in a 5.7 kb amplified DNA sequence.

Both Streptomyces lividans and Streptomyces avermitilis encode similar systems of post-replicative DNA modification which act site-specifically on closely opposed guanines on either strand. The modifications can be detected since they react in vitro with an oxidative derivative of Tris, resulting in strand cleavage. Previous analysis of the preferred modification site of plasmid pIJ101 indicated that extensive amounts of flanking sequence, including direct and inverted repeat structures, are required to direct modification in vivo within a central 6 bp palindrome. We have now examined the preferred modification sites of a chromosomal element, the 5.7 kb amplified DNA sequence (ADS5.7) found in certain S. lividans mutants. In contrast to the pIJ101 site, each of the ADS5. 7sites is intragenic and modified with a 10-fold reduced frequency. However, similar extents of flanking sequence are required for authentic double-strand modification; deletion mutants exhibited different modification profiles, including displaced double-stranded or single-stranded modi-fication. Comparison of different modification sites reveals conservation of the central core sequence, but no significant similarities between flanking sequences. Enhanced modification was detected in a cloned region of the ADS5.7, suggesting that local DNA topology, probably influenced by both DNA supercoiling and the nature of flanking sequences, can influence the modifying activity.     

Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumour-inducing plasmid

During crown gall tumorigenesis a specific segment of the Agrobacterium tumefaciens tumour-inducing (Ti) plasmid, the T-DNA, integrates into plant nuclear DNA. Similar 23-bp direct repeats at each end of the T region signal T-DNA borders, and T-DNA transmission (transfer and integration) requires the right-hand direct repeat. A chemically synthesized right border repeat in its wild-type orientation promotes T-DNA transmission at a low frequency; Ti plasmid sequences which normally flank the right repeat greatly stimulate the process. To identify flanking sequences required for full right border activity, we tested the activity of a border repeat surrounded by different amounts of normal flanking sequences. Efficient T-DNA transmission required a conserved sequence (5' TAAPuTPy-CTGTPuT-TGTTTGTTTG 3') which lies to the right of the two known right border repeats. In either orientation, a synthetic oligonucleotide containing this conserved sequence greatly stimulated the activity of a right border repeat, and a deletion removing 15 bp from the right end of this sequence destroyed it stimulatory effect. Thus, wild-type T-DNA transmission required both the 23-bp right border repeat and a conserved flanking sequence which we call overdrive.     

Streptomyces coelicolor A3(2) lacks a genomic island present in the chromosome of Streptomyces lividans 66

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage phiHAU3 resistance (phiHAU3r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.     

Site-specific deletion at the replication origin of the antibiotic resistance factor R1

Summary The recombinant plasmid pRK101 carrying the complete replication origin of the antibiotic resistance factor R1 suffers frequently a deletion of 218 base pairs, removing parts or all of the origin sequence. This deletion seems to occur always when the Pst-E fragment carrying the replication origin is inserted into the cloning vector pBR322 in an orientation where the direction of R1 replication is the same as that of the vector plasmid and frequently when it is inserted in the opposite direction. DNA sequence analysis around the junction site generated by the deletion in three independently isolated deletion mutants reveals that the deletion occurs at a specific site, namely the end of a 22 bp sequence which is repeated almost identically at the other end of a segment of 197 bp. During the deletion one repeat unit is removed whereas the other is retained. The DNA sequence included by the two repeats contains high symmetric structures, i.e. inverted repeats, direct repeats and palindromes which may represent regulatory sites of the origin.     

Stability of an inverted repeat in a human fibrosarcoma cell.

Deletions and rearrangements of DNA sequences within the genome of human cells result in mutations associated with human disease. We have developed a selection system involving a neo gene containing a DNA sequence inserted into the NcoI site that can be used to quantitatively assay deletion of this sequence from the chromosome. The spontaneous deletion from the neo gene of a 122 bp inverted repeat occurred at a rate of 2.1 x 10(-8) to <3.1 x 10(-9) revertants/cell/generation in three different cell lines. Deletion of the 122 bp inverted repeat occurred between 6 bp flanking direct repeats. Spontaneous deletion of a 122 bp non-palindromic DNA sequence flanked by direct repeats was not observed, indicating a rate of deletion of <3.1 x 10(-9) revertants/cell/generation. This result demonstrates that a 122 bp inverted repeat can exhibit a low level of instability in some locations in the chromosome of a human cell line.     

Two distinct models account for short and long deletions within sequence repeats in Escherichia coli.

In Escherichia coli, (GpC)n sequences cloned into plasmid DNA molecules are deletion-prone with the occurrence of both short (<2 bp) and long (>2 bp) deletion events. These repetitive tracts can be stabilized by interrupting the strict monotony of the repetition with a variant dinucleotide sequence. The stabilization of short deletion events that is mediated by the variant sequence is completely lost in E. coli mismatch repair-deficient strains. In contrast, this repair pathway has no influence on the frequency of occurrence of long deletion events, even in sequences containing the variant repeat. These results lead us to propose two distinct models to account for short and long deletions within repetitive sequences in E. coli. Furthermore, this study reveals that the deletions occur preferentially at the end of the repeat sequence that is distal with respect to the origin of replication.     

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IneW plasmid R388 and comparison with the related IncN plasmid R46

Summary We cloned and sequenced a 402 by DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 by of oriT was sufficient for efficient mobilization. The first 86 by of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 by perfect inverted repeat. Deletion of the first 95 by reduced the frequency of transfer by a hundred-fold. The sequence between by 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 by segment (from by 176–236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between by 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, whch coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.     

The DNA between Rz and cosR in bacteriophage lambda is nonessential

Near the right end of phage lambda DNA, between gene Rz and the cos site, are 2050 bp of apparently non-coding DNA. We have cloned a lambda DNA fragment containing this DNA into a plasmid and constructed a deletion, omega l, extending from a site within the Rz gene to a site about 560 bp from cos. This deletion could be recombined into viable lambda phage at a frequency equal to that observed for the undeleted sequence. Recombinant phage lambda carrying the omega l deletion were demonstrated to have the same burst size and kinetics of phage production as undeleted lambda. The omega l deletion can be used to extend the capacity of lambda cloning vectors and to provide a region for the insertion of heterologous DNA which should exhibit controllable high level expression from the lambda late promoter, p'R.     

DNA modification by sulfur: analysis of the sequence recognition specificity surrounding the modification sites

The Dnd (DNA degradation) phenotype, reflecting a novel DNA modification by sulfur in Streptomyces lividans 1326, was strongly aggravated when one (dndB) of the five genes (dndABCDE) controlling it was mutated. Electrophoretic banding patterns of a plasmid (pHZ209), reflecting DNA degradation, displayed a clear change from a preferential modification site in strain 1326 to more random modifications in the mutant. Fourteen randomly modifiable sites on pHZ209 were localized, and each seemed to be able to be modified only once. Residues in a region (5′-c–cGGCCgccg-3′) including a highly conserved 4-bp central core (5′-GGCC-3′) in a well-documented preferential modification site were assessed for their necessity by site-directed mutagenesis. While the central core (GGCC) was found to be stringently required in 1326 and in the mutant, ‘gccg’ flanking its right could either abolish or reduce the modification frequency only in the mutant, and two separate nucleotides to the left had no dramatic effect. The lack of essentiality of DndB for S-modification suggests that it might only be required for enhancing or stabilizing the activity of a protein complex at the required preferential modification site, or resolving secondary structures flanking the modifiable site(s), known to constitute an obstacle for efficient modification.     

The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro

Summary The broad host range plasmid R1162 contains a directly repeated, 20 bp DNA sequence in the region of the plasmid required in cis for replication and maintenance. This sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility. The sequence also inhibits replication of R1162 DNA in a cell-free system. The strengths of both these effects are determined by the number of direct repeats (DRs) present, and are also affected to similar degrees by different mutations within the repeated sequence. Several of the mutations were tested for their effect in cis on plasmid maintenance in the cell, and one was found to cause an increase in plasmid copy number. The results suggest that the direct repeats exert incompatibility by inhibiting DNA replication, presumably because they are the binding sites for a limiting essential protein.Abbreviations bp base pairs - Cb^r, Km^r, Sm^r resistance to carbenicillin, kanamycin, streptomycin, respectively - DR direct repeat     

Efficient resolution of replicated poxvirus telomeres to native hairpin structures requires two inverted symmetrical copies of a core target DNA sequence.

The terminal hairpin sequences of the linear double-stranded DNA genome of the leporipoxvirus Shope fibroma virus (SFV) has been cloned in Saccharomyces cerevisiae and in recombination-deficient Escherichia coli as a palindromic insert within circular plasmid vectors. This sequence configuration is equivalent to the inverted repeat structure detected as a telomeric replicative intermediate during poxvirus replication in vivo. Previously, it has been shown that when circular plasmids containing this palindromic insert were transfected into SFV-infected cells, efficient replication and resolution generated linear minichromosomes with bona fide viral hairpin termini (A. M. DeLange, M. Reddy, D. Scraba, C. Upton, and G. McFadden, J. Virol. 59:249-259, 1986). To localize the minimal target DNA sequence required for efficient resolution, a series of staggered unidirectional deletions were constructed at both ends of the inverted repeat. Analyses of the resolution efficiencies of the various clones indicate that up to 240 base pairs (bp) centered at the symmetry axis were required for maximal resolution to minichromosomes. To investigate the role of the AT-rich central axis sequences, which in SFV include 8 nonpalindromic bp, a unique AflII site at the symmetry axis was exploited. Bidirectional deletions extending from this AflII site and insertions of synthetic oligonucleotides into one of the deletion derivatives were constructed and tested in vivo. The efficiency with which these plasmids resolved to linear minichromosomes with hairpin termini has enabled us to define the minimal target DNA sequence as two inverted copies of an identical DNA sequence between 58 and 76 bp in length. The nonpalindromic nucleotides, which, after resolution, constitute the extrahelical residues characteristic of native poxviral telomeres, were not required for resolution. The close resemblance of the SFV core target sequence to the analogous region from the orthopoxvirus vaccinia virus is consistent with a conserved mechanism for poxviral telomere resolution.     

DNA tertiary structures formed in vitro by misaligned hybridization of multiple tandem repeat sequences.

DNA tertiary structures are shown to be formed by denaturation and reannealing in vitro of molecularly-cloned DNA containing multiple tandem repeat sequences. Electron microscopy of homoduplex DNA molecules containing the human c-Harvey-ras gene revealed knot-like structures which mapped to the position of the 812 bp variable tandem repeat (VTR) sequence. We propose that the structures result from slipped-strand mispairing within the VTR and hybridisation of homologous repetitive sequences in the single-stranded loops so produced. Similar structures were also found in freshly-linearized supercoiled plasmids. More complex knot-like structures were found in homoduplexes of a 4 kb tandem array from the hypervariable region 3' to the human alpha-globin locus. Formation of such DNA tertiary structures in vitro also provides a practical method for identifying and mapping direct tandem repeat arrays that are at least 800 bp long.     

Palindromy and the Location of Deletion Endpoints in Escherichia Coli

The contributions of direct and inverted repeats to deletion formation were studied by characterizing Ampr revertants of plasmids with a series of insertion mutations at a specific site in the pBR322 ampicillin resistance (amp) gene. The inserts at this site are palindromic, variable in length, and bracketed by 9- or 10-bp direct repeats of amp sequence. There is an additional direct repeat composed of 4 bp within the insert and 4 bp of adjoining amp sequence. DNA sequencing and colony hybridization of Ampr revertants showed that they contained either the parental amp sequence, implying deletion endpoints in the flanking 9- or 10-bp repeats, or a specific 1-bp substitution, implying endpoints in the 4-bp repeats. Although generally direct repeats seem to be used as deletion endpoints with a frequency proportional to their lengths, we found that with uninterrupted palindromes longer than 32 bp, the majority of deletions ended in the 4 bp, not the 9- or 10-bp repeats. This preferential use of the shorter direct repeats associated with palindromes is interpreted according to a DNA synthesis-error model in which hairpin structures formed by intrastrand pairing foster the slippage of nascent strands during DNA synthesis.     

The infectivity of adenovirus genomes lacking DNA sequences from their left-hand termini.

Deletions extending various distances into the left-hand terminal DNA sequences of the adenovirus type 2 (Ad2) genome were generated in a plasmid containing a cloned fragment spanning from 0 to 4.9 map units. The altered Ad2 DNA sequences were introduced into viral genomes by ligating a plasmid-derived fragment, which included the sequences extending to 3.8 map units, to the 3.8-100 map unit fragment generated by XbaI cleavage of the DNA of the Ad5 variant, d1309 (N.Jones and T.Shenk, Cell 17 683-689, 1979). The infectivity of the ligation products was studied by transfection of line 293 cells. Genomes lacking 11, 40, or 51 nucleotides from their left-hand termini, or containing an additional 18dG residues linked to this position were infectious, and analysis of the progeny virus genomes demonstrated that the structure of these modified termini had been restored to normal. In contrast, genomes from which the first 160 base pairs (bp), including the entire 102 bp left hand inverted terminal repeat (ITR), had been removed were non-infectious. The results indicate that the ITRs present at the opposite ends of transfecting DNA molecules are able to interact in vivo, and enable the production of viable viruses containing corrected left-hand terminal sequences. Possible mechanisms for this interaction are discussed.     

Analysis of replication region of the cryptic plasmid pAG20 from Acetobacter aceti 3620

The DNA sequence of small cryptic plasmid pAG20 in Acetobacter aceti was determined at 3064 bp with 51.6% GC pairs. The plasmid encoded a 186 amino acid protein which is important for plasmid replication in Gram-negative bacteria except Escherichia coli. Two 21 bp large direct repeat sequence 1 and two 13 bp direct repeat sequence 2 were determined in the regulation region upstream from gene encoded Rep protein. Vector pAG24 with kanamycin gene and two deletion derivatives pAG25 and pAG26 without rep gene from plasmid pAG20 were constructed. Plasmid pAG24 was replicated in a broad host range like E. coli, Acetobacter pasteurianus, A. aceti, Comanomonas spp., Serratia marcescens, and Shigella spp.     

On the Deletion of Inverted Repeated DNA in Escherichia Coli: Effects of Length, Thermal Stability, and Cruciform Formation in Vivo

We have studied the deletion of inverted repeats cloned into the EcoRI site within the CAT gene of plasmid pBR325. A cloned inverted repeat constitutes a palindrome that includes both EcoRI sites flanking the insert. In addition, the two EcoRI sites represent direct repeats flanking a region of palindromic symmetry. A current model for deletion between direct repeats involves the formation of DNA secondary structure which may stabilize the misalignment between the direct repeats during DNA replication. Our results are consistent with this model. We have analyzed deletion frequencies for several series of inverted repeats, ranging from 42 to 106 bp, that were designed to form cruciforms at low temperatures and at low superhelical densities. We demonstrate that length, thermal stability of base pairing in the hairpin stem, and ease of cruciform formation affect the frequency of deletion. In general, longer palindromes are less stable than shorter ones. The deletion frequency may be dependent on the thermal stability of base pairing involving approximately 16-20 bp from the base of the hairpin stem. The formation of cruciforms in vivo leads to a significant increase in the deletion frequency. A kinetic model is presented to describe the relationship between the physical-chemical properties of DNA structure and the deletion of inverted repeats in living cells.     

Nucleotide sequence, genomic organization and evolution of a major repetitive DNA family in tilapia (Oreochromis mossambicus/hornorum).

A highly repetitive DNA sequence from tilapia (Oreochromis mossambicus/hornorum) has been cloned and sequenced. It is a tandemly arrayed sequence of 237 bp and constitutes 7% of the fish genome. The copy number of the repeat is approximately 3 x 10(5) per haploid genome. DNA sequence analysis of 7 cloned repeats revealed a high degree of conservation of the monomeric unit. Within the monomeric unit, a 9 bp AT rich motif is regularly spaced approximately 30 bp apart and may represent the progenitor of the amplified sequence. One cloned repeat, Ti-14, contained a 30 bp deletion at a position flanked by a 7 bp direct repeat. The Ti-14 sequence appears to have been amplified independently of the major 237 bp tandem array. A higher-order repeat unit, defined by longer-range periodicities revealed by restriction endonuclease digestion, is further imposed on the tandem array.