Sensitive detection of RNA using strand-specific M13 probes

We have extended the method of Hu and Messing (Gene 17 (1982) 271-277) to prepare highly radioactive M13 probes suitable for use in RNA-DNA hybridization experiments. Single strands of M13 DNA carrying cloned sequences are rendered partially double-stranded by primed synthesis using a synthetic oligonucleotide primer complementary to a region 5' to the cloning site. The newly synthesized radioactive complementary strand is then covalently cross-linked to the M13 phage DNA by UV irradiation in the presence of 4,5,8-trimethylpsoralen (trioxsalen). Since the cross-linked probe is stable to heat denaturation, and the region of cloned sequence is kept single-stranded, these complexes may be used as strand-specific hybridization probes to detect RNA sequences under conditions which would denature DNA-DNA duplexes.     

Formation of Template-Switching Artifacts by Linear Amplification

Linear amplification is a method of synthesizing single-stranded DNA from either a single-stranded DNA or one strand of a double-stranded DNA. In this protocol, molecules of a single primer DNA are extended by multiple rounds of DNA synthesis at high temperature using thermostable DNA polymerases. Although linear amplification generates the intended full-length single-stranded product, it is more efficient over single-stranded templates than double-stranded templates. We analyzed linear amplification over single- or double-stranded mouse H-ras DNA (exon 1–2 region). The single-stranded H-ras template yielded only the intended product. However, when the double-stranded template was used, additional artifact products were observed. Increasing the concentration of the double-stranded template produced relatively higher amounts of these artifact products. One of the artifact DNA bands could be mapped and analyzed by sequencing. It contained three template-switching products. These DNAs were formed by incomplete DNA strand extension over the template strand, followed by switching to the complementary strand at a specific Ade nucleotide within a putative hairpin sequence, from which DNA synthesis continued over the complementary strand.     

Influence of template strandedness on in vitro replication of mutagen-damaged DNA

We analyzed the ability of DNA polymerases to bypass damage on single- and double-stranded templates. In vitro DNA synthesis was studied on UV-irradiated and polyaromatic hydrocarbon reacted (benzo[a]pyrenediol epoxide and oxiranylpyrene) double-stranded templates by a protocol involving initiation on a uniquely nicked circular double-stranded template. The template was prepared by treating single-stranded (+)M13mp2 circular strands with mutagen and then hybridizing with restricted M13 RFmp2, followed by isolation of the nicked RFII forms. The protocol permits either (+), (-), or both strands to carry lesions. We found that the rules for termination and bypass of lesions previously observed with single-stranded DNA templates also hold for double-stranded templates. Termination of synthesis occurs primarily one nucleotide 3' to the lesion in the template strand. Bypass of UV-induced lesions can be followed in a series of three partial reactions in the presence of Mn2+ and dGMP, which relax the specificity of nucleotide insertion and 3'----5' exonuclease activity, respectively. There is no evidence for greater permissivity of bypass in double-as opposed to single-stranded templates. As with single-stranded templates, purines and preferentially deoxyadenosine (dA) are inserted opposite lesions. Lesions in the nontemplate strand elicit neither termination nor pausing. The addition of Rec A protein resulted in a measurable increase of bypass in this system.     

Adenovirus DNA replication in vitro: duplication of single-stranded DNA containing a panhandle structure

Adenovirus DNA replicates by displacement of one of the parental strands followed by duplication of the displaced parental single strand (complementary strand synthesis). Displacement synthesis has been performed in a reconstituted system composed of viral and cellular proteins, employing either the viral DNA-terminal protein complex as template or linearized plasmids containing the origin. Previously, evidence was obtained that in vivo complementary strand synthesis requires formation of a panhandle structure originating from hybridization of the inverted terminal repeats. To study the conditions for complementary strand synthesis in vitro, we have constructed an artificial panhandle molecule that contains a double-stranded inverted terminal repetition (ITR) region and a single-stranded loop derived from the left and right terminal XmaI fragments of Ad2. Such a molecule appeared to be an efficient template and could initiate by the same protein-priming mechanism as double-stranded DNA, employing the precursor terminal protein. The efficiency of both types of template was comparable. Like for replication of the duplex molecule initiation of panhandle replication was stimulated by nuclear factors I and III, proteins that bind to specific double-stranded regions of the ITR. The Ad DNA-binding protein is essential and the 39 kDa C-terminal domain of this protein that harbors the DNA-binding properties is sufficient for its function. These results support the hypothesis that panhandle formation is required for duplication of the displaced strand.     

A short primer for sequencing DNA cloned in the single-stranded phage vector M13mp2.

In this paper we describe the synthesis and cloning of a short segment of DNA complementary to the region immediately adjacent to the EcoRI insertion site in the single-stranded bacteriophage vector M13mp2. This segment is useful as a "universal" primer for DNA sequencing by the dideoxynucleotide chain termination method; the template can be any DNA species cloned in M13mp2 or its derivatives. The primer has been cloned into the tetracycline resistance gene of plasmid pBR322 as one strand of a 26 bp EcoRI/BamHI fragment. This fragment may be readily prepared from an EcoRI + BamHI restriction digest of the parent plasmid (designated pSP14) by a simple size fractionation.     

Presynapsis and synapsis of DNA promoted by the STP alpha and single-stranded DNA-binding proteins from Saccharomyces cerevisiae

We previously purified an activity from meiotic cell extracts of Saccharomyces cerevisiae that promotes the transfer of a strand from a duplex linear DNA molecule to complementary circular single-stranded DNA, naming it Strand Transfer Protein alpha (STP alpha) (Sugino, A., Nitiss, J., and Resnick, M. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3683-3687). This activity requires no nucleotide cofactor but is stimulated more than 10-fold by the addition of yeast single-stranded DNA-binding proteins (ySSBs). In this paper, we describe the aggregation and strand transfer of double-stranded and single-stranded DNA promoted by STP alpha and ySSB. There is a good correlation between the aggregation induced by various DNA-binding proteins (ySSBs, DBPs and histone proteins) and the stimulation of STP alpha-mediated DNA strand transfer. This implies that the stimulation by ySSBs and other binding proteins is probably due to the condensation of single-stranded and double-stranded DNA substrates into coaggregates. Within these coaggregates there is a higher probability of pairing between homologous double-stranded and single-stranded DNA, favoring the initiation of strand transfer. The aggregation reaction is rapid and precedes any reactions related to DNA strand transfer. We propose that condensation into coaggregates is a presynaptic step in DNA strand transfer promoted by STP alpha and that pairing between homologous double- and single-stranded DNA (synapsis) occurs in these coaggregates. Synapsis promoted by STP alpha and ySSBs also occurs between covalently closed double-stranded DNA and single-stranded linear DNA as well as linear double-stranded and linear single-stranded DNAs in the absence of any nucleotide cofactors.     

Initiation signals for complementary strand DNA synthesis on single-stranded plasmid DNA.

The bacteriophage 0X174 origin for (+) strand DNA synthesis, when inserted in a plasmid, is in vivo a substrate for the initiator A protein, that is produced by infecting phages. The result of this interaction is the packaging of single-stranded plasmid DNA into preformed phage coats. These plasmid particles can transduce 0X-sensitive cells; however, the transduction efficiency depends strongly on the presence in the packaged DNA strand of an initiation signal for complementary strand DNA synthesis. A plasmid with the complementary (-) strand origin of 0X inserted in the same strand as the viral (+) origin transduces 50-100 times more efficient than the same plasmid without the (-) origin of 0X. The transduction efficiency of such a particle is comparable to the infection efficiency of the phage particle. It is shown that in this system the 0X (-) origin can be replaced by the complementary strand origins of the bacteriophages G4 and M13. We have used this system to isolate sequences, from E. coli plasmids (pACYC177, CloDF13, miniF and OriC) and from the E. coli chromosome that can function as initiation signals for the conversion of single-stranded plasmid DNA to double-stranded DNA. All isolated origins were found to be dependent for their activity on the dnaB, dnaC and dnaG proteins. We conclude that these signals were all primosome-dependent origins and that primosome priming is the major mechanism for initiation of the lagging strand DNA synthesis in E. coli. The assembly of the primosome depends on the sequence-specific interaction of the n' protein with single-stranded DNA. We have used the isolated sequences to deduce a consensus recognition sequence for the n' protein. The role of a possible secondary structure in this sequence is discussed.     

A fast and simple method for sequencing DNA cloned in the single-stranded bacteriophage M13.

The chain termination DNA sequencing procedure of Sanger et al. (1977) requires single-stranded DNA as template. M13 phage DNA exists as a single strand and therefore every DNA sequence cloned in M13 can be easily obtained in this form. Here we show that M13 single-stranded DNA pure enough to be used as a template for sequence determination can be prepared by simple centrifugation of the phage particle and extraction with phenol.     

The making of strand-specific M13 probes

A novel approach has been developed for the preparation of highly radioactive, strand-specific M13 probes. A universal primer, complementary to the region 5' to the multiple cloning sites of M13mp7, was used to initiate the DNA synthesis of the complementary strand of the M13 sequence downstream from the inserted sequence. The synthesis of the (?) strand, which was labeled with a radioactively labeled precursor, did not proceed to completion so that the inserted sequence was kept single-stranded. Thus, a partially double-stranded probe that had the specificity of this inserted sequence was obtained. As an example for the application of single-stranded specific hybridization probes, an M13mp7 subclone of a zein cDNA clone of maize (A30) was labeled and used in a dot hybridization test to select from the hundreds of M13mp7 subclones of the zein genomic clone, 24, the sequences complementary to the probe. The specificity of the probe was confirmed by dideoxy chain terminator sequencing experiments.     

Cloning of a functional replication origin of phage G4 into the genome of phage M13.

A chimeric single-stranded DNA phage, M13Gori1, has been formed as a result of the in vitro insertion of a 2216 base-pair HaeII fragment of bacteriophage G4 replicative form DNA into the replicative form DNA of bacteriophage M13. The inserted G4 DNA carries the dnaG-dependent origin for G4 complementary strand synthesis. The cloned G4 origin functions both in vivo and in vitro in the conversion of M13Gori1 single-stranded viral DNA to the duplex replicative form by a rifampicin-resistant mechanism. Labelling of the 3′ terminus of the single discontinuity in M13Gori1 replicative form II molecules synthesized in crude extracts and subsequent restriction analysis indicate that M13Gori1 complementary strand synthesis can be initiated at either the RNA polymeraseprimed M13 origin or at the dnaG-primed G4 origin. The M13Gori1 complementary strand initiated at the G4 origin terminates in the vicinity of the G4 origin after progressing around the circular template and traversing the M13 origin region, indicating the absence of a specific nucleotide sequence in the M13 origin for termination of the newly formed complementary strand. The ability of this chimeric phage to utilize the cloned G4 origin in vivo even in the presence of the presumed M13 pilot protein (gene 3 protein) indicate that the nucleotide sequence of the replication origin is sufficient for recognizing the appropriate initiation enzymes. Since decapsidation of M13 is tightly coupled to replicative form formation, initiation at the G4 origin, located over 1000 nucleotides from the M13 complementary strand origin, indicates that widely separated nucleotide sequences contained in the filamentous virion can be exposed to the cell cytoplasm during eclipse.     

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2).     

In vitro cell cycle arrest induced by using artificial DNA templates.

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.     

Signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A

In this work, a new signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A (OTA) is reported. OTA aptamer (DNA1) and OTA aptamer complementary (DNA2) were immobilized onto a magnetic bead (MB). In the presence of OTA, DNA2 was dissociated and released from the MB. The released DNA2 then hybridized with DNA3, which was linked at the 5' terminus of the amplification template and can extend along the template in the presence of Phi 29 DNA polymerase. The formed double-stranded DNA was cleaved by nicking endonuclease Nb.BbvCI and produced a short single-stranded DNA. The cleaved DNA strand generated a new site by Phi 29 DNA polymerase and the process of extension and cleavage was cyclical. Thus, a amount of the short single-stranded DNA were produced. Using DNA and ABEI labeled carboxylic silica nanoparticles chemiluminescence (CL) probe, the short single-stranded DNA could be sensitively detected. The CL intensity (ΔI) versus the concentration of OTA was linear in the range from 1.0×10(-12) to 5.0×10(-8)gmL(-1). The detection limit was 3.0×10(-13)gmL(-1), and the RSD was 3.4% at 1.0×10(-10)gmL(-1) (n=7). The developed method has been applied to detect OTA in naturally contaminated wheat samples. Due to its simplicity, sensitivity and no need of specific recognition of aptamer for cleavage, this CL bioassay offers a promising approach for the detection of OTA and other biomolecules.     

The process of infection with bacteriophage φX174: XXXIX. The structure of a DNA form with restricted binding of intercalating dyes observed during synthesis of φX single-stranded DNA

A DNA form with restricted binding of intercalating dyes (propidium iodide or ethidium bromide) has been found in bacteriophage φX-infected cells during the period of single-stranded DNA synthesis. In the electron microscope, this DNA form is seen to be a double-stranded DNA ring with two single-stranded DNA tails protruding from the same portion of the ring; it is composed of a linear φX DNA strand, longer than one φX genome, and a single-stranded ring complementary to φX DNA. Base-pairing of these two tails in partially complementary regions restricts unwinding of the double-stranded DNA ring and consequently intercalation and binding of the dyes. It is postulated that these molecules originate from a previously reported precursor of φX DNA, namely a double-stranded ring with a single-stranded tail, by branch migration.     

An efficient and site-specific gene trimming method

Trimming a DNA strand into a precisely determined fragment can be carried out efficiently by an improved method involving a site-specific trim-primer and a single-stranded DNA template which is generated from a multifunctional vector, pTZ18R, and linearized by using an Eco RI-pTZ18R splinter. A complementary DNA strand is synthesized by DNA polymerase I (Klenow), and the 3'-end of the template upstream from the annealed primer is trimmed by subsequent T4 DNA polymerase reaction. An ATG translation initiator codon or a termination codon can be incorporated into the trim-primer, providing versatility to this single-stranded DNA-initiated gene trimming method that can be applied to subcloning and expression of any DNA fragment with known terminal sequences.     

Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA.

The opposite strands of the ColE1 and ColE3 plasmids were isolated as circular single-stranded DNA molecules. These molecules were compared with M13 and phi X174 viral DNA with respect to their capacity to function as templates for in vitro DNA synthesis by a replication enzyme fraction from Escherichia coli. It was found for both ColE plasmids that the conversion of H as well as L strands to duplex DNA molecules closely resembles phi X174 complementary strand synthesis and occurs by a rifampicin-resistant priming mechanism involving the dnaB, dnaC, and dnaG gene products. Restriction analysis of partially double-stranded intermediates indicates that preferred start sites for DNA synthesis are present on both strands of the ColE1 HaeII-C fragment. Inspection of the nucleotide sequence of this region reveals structural similarities with the origin of phi X174 complementary strand synthesis. We propose that the rifampicin-resistant initiation site (rri) in the ColE1 L strand is required for the priming of discontinuous lagging strand synthesis during vegetative replication and that the rri site in the H strand is involved in the initiation of L strand synthesis during conjugative transfer.     

Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.