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W Szybalski

Publications and source records attributed to W Szybalski.

At least 19 recordsLinked to original sources
PubMed1998

Genomic DNA sequencing by SPEL-6 primer walking using hexamer ligation.

DNA sequencing by SPEL-6 (Sequential Primer Elongation by Ligation of 6-mers) primer walking is based on the rapid assembly of true primers by ligation of several (three to 10) contiguous hexamers complementary to a DNA template saturated with Escherichia coli single-stranded DNA-binding protein. To prove the usefulness and to check the reliability of this method, a 3-kb DNA fragment carrying the genes encoding the EcoVIII restriction-modification (RM) system was sequenced with low redundancy (2.8). The use of both single-stranded (ss) and double-stranded (ds) DNA templates was compared. For this project, 27 primers were assembled by hexamer ligation to form 18-30-nt strings of three to five hexamers. Each primer was designed based on nucleotide sequence determined in a previous run, and was produced in a matter of minutes. The overall length of the easily readable sequencing ladders was about 300-450nt. We found that strong secondary structures in the ss DNA tend to interfere with its template function for the primer assembly by hexamer ligation, especially when they overlap the 3'-end of such a primer. This was easily overcome either by avoiding such hairpin regions or by using longer strings of hexamers, since we show that their ligation is highly cooperative, and ligation efficiency increases with the length of the string (). Some general rules for successful primer assembly and prospects for using the SPEL-6 method for large-scale, fully automated fluorescent sequencing of large genomes are discussed.

Base Sequence↗
PubMed1998

Cre/loxP-mediated in vivo excision of large segments from yeast genome and their amplification based on the 2microm plasmid-derived system.

In vivo excision and amplification of pre-determined, large genomic segments, directly from the genome of a natural host, provides an alternative to conventional cloning in foreign vectors. Using this approach, we have devised an in vivo procedure for excising large segments of Saccharomyces cerevisiae genome using Cre/loxP system of bacteriophage P1, followed by amplification of excised circles, as based on the yeast 2microm plasmid-derived ori and Flp/FRT machinery. To provide the excision and replication enzymes, trans-acting genes cre and FLP, which were under a very tight control of GAL1 and GAL10 promoters, respectively, were inserted by homologous recombination into the URA3 gene on chromosome V. Two parallel loxP sequences, which serve as the recognition sites for the Cre recombinase, were also integrated into the genome at pre-determined sites that are 50-100kb apart. Moreover, 2microm ori, REP3 and two inverted FRTs, which serve as a conditional replication system, were also integrated between the loxP sites. The strain carrying all these inserted elements was perfectly stable. Only after the induction by galactose of the Cre excision function, the genomic segment flanked by two loxP sites was excised and circularized. Applying this procedure, the 50-kb LEU2-YCR011c and 100-kb LEU2-YCR035c regions of chromosome III were successfully excised from the S. cerevisiae genome, whereas the 2microm ori, as aided by FRT/Flp, provided the amplification function. Such excised and amplified genomic segments can be used for the sequencing and functional analysis of any yeast genes.

Chromosomes↗
PubMed1998

Targeting and retrofitting pre-existing libraries of transposon insertions with FRT and oriV elements for in-vivo generation of large quantities of any genomic fragment.

A procedure is described that converts the pre-existing transposon insertion libraries to a collection of 'pop-out' strains, each allowing generation of 20- to 100-kb genomic fragments directly from the genome. The procedure consists of two steps: (1) single transposon insertions are targeted and retrofitted with excision and amplification elements (FRT and oriV), by homologous recombination with an FRT-oriV-carrying plasmid; and (2) two retrofitted neighbouring transposons are brought together by P1 transduction. From each strain, a 20- to 100-kb genomic fragment, bound by a pair of retrofitted transposons, could be excised and amplified upon supplying in trans the excision (Flp) and replication (TrfA) functions. To enhance the efficiency of crossing-in the FRT-oriV cassette, we transiently increased the copy number of our retrofitting plasmids using a temperature-sensitive TrfA-supplying helper plasmid. Using FRT-oriV and helper plasmids, we retrofitted four Tn10KmR and three Tn10CmR insertions. Subsequently, the FRT-oriV retrofitted insertions were crossed with each other in pairs (KmRxCmR), using P1 phage transductions. The resulting CmRFRT-[28-65-kb]-KmRFRT strains were transformed with a plasmid expressing FLP and trfA genes from the tightly controlled Ptet promoter. Induction of this tightly repressed promoter by autoclaved chlortetracycline (cTc) resulted in the efficient excision and amplification of genomic fragments located between FRT sites, but only in productive strains, i.e. having two parallel FRTs. We have shown that genomic fragments of 28-, 40-, 50- and 65-kb were efficiently excised and amplified. Furthermore, we could convert non-productive strains (having FRTs in non-parallel orientation), to productive combination of parallel FRTs, because one of the FRT elements was flanked by two convergent loxP sites, and thus could be inverted by the Cre function delivered either by the P1 phage or by a specially constructed temperature-sensitive Plac-cre plasmid. Although several microbial genomes were recently sequenced, the described method will help in supplying large quantities of any genomic fragment (prepared without the conventional cloning and its artifacts) for refined sequence comparison among strains and species, and for further analysis of uncharacterized ORFs, various mutations, and regulatory elements or functions. The excised and circularized DNA fragments (plasmids) could be propagated like any other large plasmids but only in hosts that could supply the appropriate Rep function. Our original 'pop-out' method [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398] was already employed for sequencing of the E. coli genome [Blattner et al. (1997) Science 277, 1453-1462]. Moreover, the Flp-mediated recombination between two FRT elements resulted in bacterial strains with large deletions (for parallel FRT orientations) or with large inversions (for inverted FRT orientations).

Cloning, Molecular↗
PubMed1998

Tightly controlled two-stage expression vectors employing the Flp/FRT-mediated inversion of cloned genes.

We have developed a tightly controlled, two-stage expression system. It is based on a single plasmid that carries the TetR repressor/Ptet promoter/Otet operator for the first-stage control, and the Flp recombinase/ FRT sites for the second-stage control. The gene to be expressed (GENE) is cloned in an inverted orientation (with respect to the stationary promoter) into a multiple-cloning site (MCS) located between two convergent FRT1 and FRT2 sites. In the OFF stage, no inadvertent transcription can enter the 5' end of cloned GENE because of four rrnBT1 terminators, located just outside the FRT1-MCS-FRT2 cassette and because the FRT2 construct was deprived of any promoter function. When using the lacZ reporter, it was shown that in their OFF stage our two-stage expression plasmids exhibit a significantly lower basal expression than the repressed single-stage tetR/PtetOtet-lacZ vectors. To enter the ON stage, the tetR/PtetOtet module is induced by adding autoclaved chlortetracycline (cTc), leading to synthesis of the Flp recombinase, which in turn, inverts the FRT1-MCS-FRT2 module together with the cloned GENE. This results in the massive GENE expression from one (pInvMS) or two (pImpMS) stationary promoters.

Bacterial Proteins↗
PubMed1997

RecA-mediated Achilles' heel cleavage.

The specific protection of only one of many restriction sites in a genome from inactivation by a cognate methyltransferase (MTase) creates a unique cleavage site - an Achilles' heel cleavage (AC) site. In the RecA-AC, or RARE, technique, such specific protection is provided by a synaptic complex composed of RecA protein, a gamma-S analog of ATP and a 30-60 nucleotide long oligodeoxynucleotide complementary or identical to the sequence-targeted site in which the protected restriction site is embedded. Upon methylation and the subsequent removal of the protective complex and MTase, the protected site is the only site cut by the cognate restriction enzyme. Two such targeted cuts permit the excision of a unique DNA fragment from the genome. Recent advances include the calibration of DNA clones, the mapping of gaps, and the determination of the sizes of excised fragments by pulsed-field gel electrophoresis, which allows one to measure distances between any two neighboring sequence-targeted sites, in the range of a few kilobases to 10 megabases, with the purpose of physically mapping the genome.

Binding Sites↗
PubMed1996

Co-operativity of hexamer ligation.

The SPEL-6 (sequential rimer elongation by ligation of 6-mers) procedure is based on the assembly of DNA primers by ligation of three or more hexamers taken from a library of 4096 hexamers. In this way, the synthesized primers enable DNA sequencing by primer walking. Ligation by both T4 DNA ligase and Rhodothermus marinus thermophilic DNA ligase is highly cooperative. Sequencing ladders obtained with 18-60-nucleotide (nt) primers (produced by ligation of three to ten hexamers using T4 DNA ligase) were all of high quality, with no spurious bands. R. marinus DNA ligase requires at least seven hexamers for successful primer synthesis. Long primers (up to 60 nt), which are easy to obtain, especially by automated ligation, offer a definite advantage in DNA priming in regions with pronounced secondary structure. Moreover, the SPEL-6 procedure for DNA sequencing reduces the sequencing effort manifold. An additional application of hexamer ligation is the detection of point mutants, as described here.

DNA Ligases↗
PubMed1996

Automated four-color DNA sequencing using primers assembled by hexamer ligation.

A procedure based on the assembly of sequencing primers by hexamer ligation and then using them in automated DNA sequencing is described. This method is based on a four-color fluorescent terminator chemistry. Sequencing ladders were analyzed using an ABI 373 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The best results were obtained for primers assembled by ligation of four to ten hexamers. The accuracy of the method was estimated to be 99.5% up to 400 nt of the read sequence, and somewhat lower at 400-600 nt.

Base Sequence↗
PubMed1996

A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50- to 100-kb genomic fragments for direct DNA sequencing.

A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity. Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome. This system, which employed the yeast Flp/FRT elements for excision and the plasmid R6K-based replication machinery for DNA amplification, permits one to bypass conventional cloning [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398]. To extend the applicability of such a system to many species, we describe here a broad-host-range (bhr) system in which the amplification of the excised DNA fragment depends on the oriV element and the Rep (TrfA) protein from the promiscuous RK2/RP4 plasmid. We have constructed insertion plasmids which carry the FRT and oriV sites. To introduce such plasmids into the appropriate position in the host genome, a short genomic sequence homologous to this position was cloned into the multiple cloning site (MCS) of the FRT/oriV insertion plasmid and then recombined into this position in the genome by RecA-mediated recombination. In such a manner, many strains with single FRT/oriV insertions at various positions could be generated. Subsequent genetic crosses or phage transduction allow two neighboring FRT/oriV sites (less than 150 kb apart) to be brought into a single genome. In the present report, the lacZ and phoB sites, which are 51 kb apart in the E. coli genome, were used for the introduction of the FRT/oriV sites. To deliver the Flp (excision) and Rep (amplification) functions in trans, the yeast FLP and RK2 plasmid trfA genes were placed under the control of the Ptet promoter/operator which is tightly regulated by the TetR repressor. The addition of heated chlortetracycline (cTc) inactivates TetR, turning on the synthesis of Flp and TrfA, which respectively, execute (i) excision of the 51-kb genomic segment between the two FRT sites (in lacZ and in phoB), and (ii) its amplification.

Chromosome Walking↗
PubMed1996

Structural requirements for FokI-DNA interaction and oligodeoxyribonucleotide-instructed cleavage.

The FokI restriction endonuclease recognizes the double-stranded (ds) 5'-GGATG-3' site and cuts at the 9th and 13th nucleotides downstream from the 5'-3' and 3'-5' strands, respectively. To elucidate the interaction between FokI and DNA, and the effect of Mg2+ on this interaction, we used FokI with various combinations of dsDNA, single-stranded (ss) DNA and oligodeoxyribonucleotides (oligos) containing a double-stranded hairpin carrying the FokI recognition site. Oligo- and dsDNA-FokI interactions showed that for fully effective recognition, two or more base-pairs were required outside the 5'-GGATG-3' site. When using FokI with ssDNA and oligos, precise cutting with no observable byproducts was observed at the 9th or 13th nucleotide. This was independent of whether the region between the recognition and cut sites was perfectly complementary or whether there were up to four mismatches in this region, or a single mismatch within the cut site. Moreover, FokI cleavage, when followed by step-wise filling-in of FokI cohesive ends in the dsDNA, allowed FokI to recleave such sites when two or more nucleotides were added, releasing 2-mer, 3-mer, or 4-mer single-stranded chains. Electrophoretic mobility shift assays showed that the DNA helix was bent when complexed with FokI (without Mg2+. Such a complex, when formed in the absence of Mg2+, did not accept the subsequently added Mg2+ for several minutes. This suggests a tight, diffusion-resistant contact between the enzyme and the cognate DNA sequence. In the presence of Mg2+, the half-life of the complex FokI and dsDNA was 12 minutes at 22 degrees C. In the absence of Mg2+, such a complex, possessing a terminally located 5'-GGATG-3' site, had a half-life of 1.5 to 2 minutes. However, if magnesium ions were present, this complex had a stability similar to that of a complex formed with dsDNA containing a centrally located 5'-GGATG-3' site.

Base Sequence↗
PubMed1995

Construction of lacIts and lacIqts expression plasmids and evaluation of the thermosensitive lac repressor.

To characterize a thermosensitive lacIts mutant isolated by Bukrinsky et al. [Gene 70 (1989) 415-417] and to adapt it as a convenient control element, we have (i) mapped the mutation to the inducer-binding domain of the LacIts repressor, (ii) determined that the mutation resulted in the loss of a HaeIII site in lacIts due to a G-->A transition, leading to a Gly187-->Ser substitution, (iii) removed extraneous lacZop DNA downstream of lacI, and (iv) cloned lacIts (in plasmids based on ori of either ColE1 or P15A) under control of the wild-type or lacIq promoters. The LacIts repressor is insensitive to IPTG. The repression of lacZop by LacIts is very efficient at 30 degrees C and total induction was achieved at 42 degrees C, providing that the LacIts concentrations are not excessive and that repressor-to-operator ratios are not too high.

Amino Acid Sequence↗
PubMed1994

Escherichia coli genome targeting, I. Cre-lox-mediated in vitro generation of ori- plasmids and their in vivo chromosomal integration and retrieval.

We have constructed a plasmid system designed for the insertion of cloned DNA (e.g., genes, gene fusions, regulatory elements, etc.) into the Escherichia coli genome. Its principal feature is the presence of two tandem lox sites on the plasmids, which upon Cre-mediated in vitro recombination resolve the plasmids into ori- and ori+ DNA circles. The non-replicating ori- circles contain the lambda attP site, several unique restriction sites for cloning, a NotI site and KmR, a kanamycin-resistance-encoding gene. The ori+ circles carry the origin of DNA replication (ori) together with several cleavage sites not present in the ori- circles, including the rare site for the very efficient I-SceI enzyme, that are used to inactivate the ori+ circles and any unresolved plasmid DNA. We have used this system to insert cloned DNA into the host genome at (i) the attB site, by Int-mediated integration and (ii) at any predetermined sequence, as mediated by the Rec system(s) of the host. The genomes of the resulting transformants were analyzed by NotI digestion of the chromosomal DNA, embedded in agarose microbeads, followed by pulsed-field gel electrophoresis. A system for the retrieval of DNA fragments inserted at the attB site was also developed.

Base Sequence↗
PubMed1994

Assembly of 18-nucleotide primers by ligation of three hexamers: sequencing of large genomes by primer walking.

A novel method (SPEL-6) for sequencing large genomes permits 10-min synthesis of 18-mer primers and their immediate use in DNA sequencing by primer walking. Primers are ligated from three contiguous hexamers complementary to the single-stranded or denatured DNA (150:1 ratio), which is used both as the template for ligation and as the DNA to be sequenced. The complete library consists of 4096 hexamers, but sequencing remains very efficient with a fourfold smaller library composed of 1024 degenerate hexamers containing all four nucleotides at their position 3. The SSB protein (which binds to single-stranded DNA) greatly enhances the quality of sequencing ladders. The SPEL-6 method eliminates the need for subcloning, permits direct sequencing of large DNA fragments (of 50 kb or larger), is ideally suited for automation, and should accelerate the sequencing of large genomes by more than one order of magnitude.

Base Sequence↗
PubMed1994

In vivo excision and amplification of large segments of the Escherichia coli genome.

In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E. coli. Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E. coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E. coli MG1655 genome.

Antiporters↗
PubMed1993

From the double-helix to novel approaches to the sequencing of large genomes.

Elucidation of the structure of DNA by Watson and Crick [Nature 171 (1953) 737-738] has led to many crucial molecular experiments, including studies on DNA replication, transcription, physical mapping, and most recently to serious attempts directed toward the sequencing of large genomes [Watson, Science 248 (1990) 44-49]. I am totally convinced of the great importance of the Human Genome Project, and toward achieving this goal I strongly favor 'top-down' approaches consisting of the physical mapping and preparation of contiguous 50-100-kb fragments directly from the genome, followed by their automated sequencing based on the rapid assembly of primers by hexamer ligation together with primer walking. Our 'top-down' procedures totally avoids conventional cloning, subcloning and random sequencing, which are the elements of the present 'bottom-up' procedures. Fragments of 50-100 kb are prepared in sufficient quantities either by in vitro excision with rare-cutting restriction systems (including Achilles' heel cleavage [AC] or the RecA-AC procedures of Koob et al. [Nucleic Acids Res. 20 (1992) 5831-5836]) or by in vivo excision and amplification using the yeast FRT/Flp system or the phage lambda att/Int system. Such fragments, when derived directly from the Escherichia coli genome, are arranged in consecutive order, so that 50 specially constructed strains of E. coli would supply 50 end-to-end arranged approx. 100-kb fragments, which will cover the entire approx. 5-Mb E. coli genome. For the 150-Mb Drosophila melanogaster genome, 1500 of such consecutive 100-kb fragments (supplied by 1500 strains) are required to cover the entire genome. The fragments will be sequenced by the SPEL-6 method involving hexamer ligation [Szybalski, Gene 90 (1990) 177-178; Fresenius J. Anal. Chem. 4 (1992) 343] and primer walking. The 18-mer primers are synthesized in only a few minutes from three contiguous hexamers annealed to the DNA strand to be sequenced when using an over 100-fold excess of hexamers and T4 DNA ligase at room temperature, preferably in the presence of the single-strand-binding (SSB) protein of E. coli. These 18-nt primers are immediately extended by the DNA polymerase, Sequenase 2.0, in the dideoxy sequencing reaction. Very high quality sequencing ladders are obtained for single-stranded DNA or denatured double-stranded approx. 50-kb fragments, as exemplified by phage lambda DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗