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W Szybalski

Publications and source records attributed to W Szybalski.

At least 19 recordsLinked to original sources
PubMed1992

RecA-AC: single-site cleavage of plasmids and chromosomes at any predetermined restriction site.

We have developed a novel version of the Achilles' Cleavage (AC) reaction in which virtually any restriction site on DNA of any size can be converted to a unique cleavage site. We first polymerized RecA protein on a synthetic oligodeoxyribonucleotide (oligo) in the presence of a nonhydrolyzable ATP analogue to generate oligo:RecA nucleoprotein filaments. These filament were then incubated with plasmid or intact chromosomal DNA from Saccharomyces cerevisiae to form stable complexes in the yeast LEU2 gene at the target sequence identical (or complementary) to that of the oligo. When HhaII (HinfI) methyltransferase (M.HhaII) was added, all of the recognition sites for HhaII with the exception of the one protected by the RecA filament were methylated and thus no longer cleaved by the cognate restriction endonuclease (HinfI). After inactivation of the RecA and the M.HhaII, HinfI was used to efficiently cleave the plasmid or chromosome specifically at the targeted restriction site. Since oligos specific for any sequence can be easily synthesized and the other reagents necessary to perform RecA-mediated AC (RecA-AC) reactions on both plasmids and intact chromosomes are readily available, this procedure can be applied immediately to the precise dissection and analysis of genomic DNA from any source and to any other research problem requiring efficient, highly specific cleavage of DNA at predetermined sites.

Base Sequence↗
PubMed1992

Integration host factor (IHF) binds to many sites in the A + T-rich b2 region of phage lambda DNA.

Computer analysis of almost the entire b2 region of lambda phage (nt 22346-27475) revealed 23 consensus-like ihf sites, with eleven pointing in one direction and twelve in the opposite direction [27 bp; Kur et al., Gene 81 (1989) 1-15]. To confirm the significance of this finding experimentally, the region was subdivided into 21 fragments and examined for integration host factor (IHF) binding by gel retardation and a variety of footprinting methods. Out of 21 fragments examined 13 were found to be retarded on gels in the presence of IHF and to contain one to three ihf sites each. All sites which differ by up to 2 bp from our 27-bp consensus ihf sequence can bind IHF in vitro. However, three of the computer-predicted sites overlap with sites of opposite orientation; therefore we could not determine at the present time which of the two antiparallel sequences binds IHF. We have compared the predictive values of various kinds of consensus sequences and show that our 27-bp consensus ihf sequence agrees best with the experimental data. It was demonstrated by Kur et al. [Virology 168 (1989) 236-244] that the IHF protein represses transcription from promoters located close to the right terminus of the b2 region, within the phage lambda attachment site. We discuss the possibility that some of the IHF-binding sites could be instrumental in repressing in vivo transcription from the A + T-rich b2 region during the lambda prophage state [Rosenvold et al., Virology 107 (1980) 476-487].

Amino Acid Sequence↗
PubMed1992

A novel method for converting common restriction enzymes into rare cutters: integration host factor-mediated Achilles' cleavage (IHF-AC).

Integration host factor (IHF)-mediated protection against enzymatic methylation at ihf-overlapping sites provides the basis for this novel application of the Achilles' cleavage (AC) technique [Koob et al., Science 241 (1988) 1084-1086] for generating rare natural cleavage sites. When applying IHF-AC to plasmid, phage lambda, Escherichia coli and yeast genomes, only a few of the EcoRI, HinfI, and MboI sites (which overlapped the ihf sites) remained cleavable after prior methylation with the cognate M.EcoRI, M.HinfI, or Dam methyltransferases in the presence of IHF. Thus, IHF-AC essentially converted these enzymes into very rare cutters. The extent of cleavage could be controlled by varying the IHF:DNA ratio and temperature. Moreover, the method permits the genomic location and strength of the ihf sites to be determined.

Bacterial Proteins↗
PubMed1992

Use of the HPRT gene and the HAT selection technique in DNA-mediated transformation of mammalian cells: first steps toward developing hybridoma techniques and gene therapy.

In 1956, I decided to apply my experience in microbial genetics to developing analogous systems for human cell lines, including the selection of mutants with either a loss or gain of a biochemical function. For instance, mutants resistant to azahypoxanthine showed a loss of the HPRT enzyme (hypoxanthine phosphoribosyl transferase), whereas gain of the same enzyme was accomplished by blocking de novo purine biosynthesis with aminopterin, while supplying hypoxanthine and thymine (HAT selection). Using HAT selection, we: (i) genetically transformed HPRT- mutant cells to HPRT+ wild type by using DNA extracted from HPRT+ cells, and (ii) selected HPRT+ hybrid cells by fusing HPRT- D98/AH2 cells with skin cells. These approaches, which we dubbed in 1962 as a 'first step toward gene therapy', contributed to the later development of (i) cell fusion techniques, (ii) the development of monoclonal antibodies, (iii) routine transformation of mammalian cells with cloned genes, and (iv) methods for creating transgenic organisms.

Animals↗
PubMed1991

Class-IIS restriction enzymes--a review.

Class-IIS restriction enzymes (ENases-IIS) interact with two discrete sites on double-stranded DNA: the recognition site, which is 4-7 bp long, and the cleavage site, usually 1-20 bp away from the recognition site. The recognition sequences of ENases-IIS are totally (or partially) asymmetric and all of the characterized ENases-IIS are monomeric. A total of 35 ENases-IIS are described (80, if all isoschizomers are taken into consideration) together with ten related ENases (class IIT), and 15 cognate methyltransferases (MTases-IIS). The physical, chemical, and molecular properties of the ENases-IIS and MTases-IIS are reviewed and many unique applications of this class of enzymes are described, including: precise trimming of DNA; retrieval of cloned fragments; gene assembly; use as a universal restriction enzyme; cleavage of single-stranded DNA; detection of point mutations; tandem amplification; printing-amplification reaction; and localization of methylated bases.

Base Sequence↗
PubMed1991

A novel gene-fusing vector: construction of a 5'-GGmCC-specific chimeric methyltransferase, M.BspRI/M.BsuRI.

A vector was designed to allow predetermined and precise fusion between two cloned genes by constructing a cassette with two unique class-IIS restriction sites, 5'-ACCTGC-3' (BspMI) and 5'-CCGGATG-3' (FokI overlapping with MspI), arranged back-to-back in a divergent manner and inserted at the HincII site of a multiple cloning site (MCS) in plasmid pUC18 or analogous vehicle. Two DNA fragments or genes to be precisely fused are cloned into the MCS parts located on each side of the cassette containing the two unique class-IIS restriction sites. The BspMI and MspI/FokI sites are used to generate unidirectional deletions of the genes as previously described [Hasan et al., Gene 50 (1986) 55-62; Pósfai and Szybalski, Nucleic Acids Res. 16 (1988) 6245]. The precisely trimmed genes are ligated after the cassette containing the unique class-IIS restriction sites are excised with BspMI + FokI and the termini were blunted with mung-bean nuclease. This method was used to construct a hybrid methyltransferase (MTase) from the M.BspRI and M.BsuRI MTases, which share a high degree of overall homology (about 65%) and have the identical sequence specificity (5'-GGmCC-3'). A hybrid MTase composed of the N-terminal part of M.BspRI and the C-terminal part of M.BsuRI was constructed and found to be fully functional.

Base Sequence↗
PubMed1990

Cleaving yeast and Escherichia coli genomes at a single site.

The 15-megabase pair Saccharomyces cerevisiae and the 4.7-megabase pair Escherichia coli genomes were completely cleaved at a single predetermined site by means of the Achilles' heel cleavage (AC) procedure. The symmetric lac operator (lacOs) was introduced into the circular Escherichia coli genome and into one of the 16 yeast chromosomes. Intact chromosomes from the resulting strains were prepared in agarose microbeads and methylated with Hha I (5'-GCGC) methyltransferase (M.Hha I) in the presence of lac repressor (LacI). All Hae II sites (5'-[sequence: see text]) with the exception of the one in lacOs, which was protected by LacI, were modified and thus no longer recognized by Hae II. After inactivation of M.Hha I and LacI, Hae II was used to completely cleave the chromosomes specifically at the inserted lacOs. These experiments demonstrate the feasibility of using the AC approach to efficiently extend the specificity of naturally occurring restriction enzymes and create new tools for the mapping and precise molecular dissection of multimegabase genomes.

Base Sequence↗
PubMed1990

Achilles' heel cleavage: creation of rare restriction sites in lambda phage genomes and evaluation of additional operators, repressors and restriction/modification systems.

A novel technique for the creation of rare restriction sites was described by Koob et al. [Science 241 (1988) 1084-1086]. This technique, Achilles' heel cleavage (AC), relies on the use of a bound repressor molecule to protect only one of many identical restriction sites from a modification methyltransferase that inactivates all other restriction sites. The technique was applied to a small plasmid and shown to work efficiently with two repressor/operator systems: lac repressor/lacO operator and lambda repressor/lambda oL1 operator. Here, we have extended these results to a lac operator carried by a much larger vector, namely a 44-kb phage lambda construct. In addition, we have evaluated the effect of altering the stability of the lac repressor/lac operator complex by varying both the operator and the repressor. We have also evaluated several more restriction/modification systems (MboI, Dam, MspI and AluI) in addition to HhaI and HaeII used earlier. Finally, we extended the AC technique to a third system, that of the phage 434 repressor and a synthetic 434 operator. From our results we conclude that the AC method should be applicable to the mapping of large genomes and to measuring the strength of operator-repressor interactions. AC could also be applied to identifying and evaluating many different DNA-binding proteins and their sites of action.

Bacteriophage lambda↗
PubMed1990

Proposal for sequencing DNA using ligation of hexamers to generate sequential elongation primers (SPEL-6).

The efficiency of the directed-priming proposal of Studier [Proc. Natl. Acad. Sci. USA 86 (1989) 6917-1921] can be improved upon by employing a library of less than 4094 hexamers, allowing for the easy construction of all required primers using template-directed automated ligation. This 'top-down' sequencing procedure should be several times more efficient than the present 'bottom-up' procedures [see Watson, Science 248 (1990) 44-49].

Base Sequence↗
PubMed1990

Alterations in the p'R promoter of coliphage lambda modify both its activity and interaction with the integration host factor (IHF).

A limited number of deletion/insertions and a point mutation in the -35 region of the p'R promoter of phage lambda were examined and found to influence both transcription and its repression by the integration host factor (IHF). Positive effects on transcription (in the absence of IHF) are small (up to 1.4-fold) and are caused by a deletion-substitution upstream of the -35/ihf site. Up to three base changes in the -35 promoter element seem to be tolerated, with only a small negative effect on transcription. In some cases, effective transcription requires supercoiling of such mutant template. Since an ihf sequence overlaps the -35 region of p'R, IHF represses transcription. Repression is correlated with IHF binding and consequent DNA bending, as assessed by gel retardation experiments. Nine p'R mutants were tested for their IHF binding and repression; the results confirm the consensus sequence, 5'-W2WWWWN7WWWWCARNWN2TTR derived from the hydroxyl radical footprinting, where the bold letters indicate the IHF-protected bases and W is A or T, R is A or G and N represents A, T, G or C. Perhaps surprisingly, some mutations just upstream or downstream of this ihf sequence also affect IHF binding, as observed not only for the pR'/ihf but also for the att H' site of lambda. Supercoiling in some cases permits the IHF-mediated repression to be overcome, probably by increasing the RNA polymerase binding and/or decreasing the interaction with IHF. All our data are consistent with a model which assumes that IHF initially binds to one or two ihf contact points depending on preexisting DNA topology, bends DNA, and completes the remaining contacts while finally adjusting the DNA conformation to establish the best fit within the minor groove of the double helix. Effective IHF repression of transcription would thus depend on several factors, including: (1) the sequence, and (2) the initial conformation of the ihf site, together with (3) the capacity of IHF to compete with RNA polymerase for the overlapping binding sites.

Bacterial Proteins↗
PubMed1990

The role of the direct repeat in qut-controlled antitermination in phage lambda.

For antitermination of transcription from the late p'R promoter of phage lambda, a cis-acting qut sequence, which overlaps with p'R, is required, together with the product of lambda gene Q. Using our BspMI-mediated multicycle technique for generation of precise deletions, we have confirmed that deletions removing DNA downstream of +18 bp (counted from the p'R-controlled transcriptional start point s'R = +1) do not affect the efficiency of qut antitermination; at the same time we found that deleting one more bp (shifting the right-hand boundary to bp +17) reduces antitermination by only 20%. Deleting another 5 or 6 bp (+11 or +12 bp right-hand qut boundary), decreases antitermination by about 80%. These deletions reduce the 9/10-bp-direct repeat (5'-TGGGT(A or T)AATT)2 in qut to only the five italicized bp. Similar strong reduction in antitermination (by about 68%) was obtained with +16 bp qut boundaries, in constructs which also contained 2- or 3-bp insertions between bp +11 and +12 or between +12 and +13. Since the latter deletions retain only 5/10 bp of the direct repeat, it appears that antitermination is dependent on the length and intactness of the direct repeat.

Bacteriophage lambda↗
PubMed1989

An MboII/FokI trimming plasmid allowing consecutive cycles of precise 1- to 12-base-pair deletions in cloned DNA.

A novel trimming plasmid has been designed which allows, in a preprogrammed fashion, the precise deletion of up to 12 bp per cleavage cycle, from one end of a cloned fragment. The plasmid, which carries the dhfr gene, contains unique recognition sites for two class-IIS restriction enzymes, MboII and FokI, which are arranged in the form of a cassette, so that consecutive cleavages with these endonucleases, followed by blunting with mung bean nuclease (MB), will precisely delete 12 bp of adjacent cloned DNA. When either MboII or FokI is used alone (followed by MB), 1 or 4 bp are removed, respectively. The final step in the trimming cycle is religation of the plasmid with T4 ligase. After required number of cycles, plasmids were transformed into Escherichia coli C600, and transformants selected by resistance to trimethoprim. Since the MboII/FokI cassette remains intact during these operations, one can repeat the cycle, consisting of cleaving, MB blunting and religation, several times, each time removing up to 12 bp from the cloned target DNA. Examples are provided of one-, two- and three-cycle trimmings.

Base Sequence↗
PubMed1989

Physical and biological consequences of interactions between integration host factor (IHF) and coliphage lambda late p'R promoter and its mutants.

The integration host factor (IHF) binds to a site (ihf) that overlaps the -35 region of the phage lambda late rightward promoter (p'R). This interaction represses p'R-promoted transcription, both in vivo and in vitro. In vivo repression was observed when a plasmid carrying both p'R and the galK reporter gene was transfected into IHF+ or IHF- hosts. In vitro repression of transcription by IHF was observed only with linear, but not with supercoiled wild-type p'R templates. When binding to ihf, IHF imposes a strong bend on the DNA and protects this site from cleavage by neocarzinostatin, pancreatic DNase I, and hydroxyl radicals, as assessed by footprinting experiments. Both the functional and nonfunctional p'R mutants, in which the upstream part of the -35 region was replaced by an EcoRI linker, show modified behavior toward IHF. Some are more sensitive to IHF-mediated repression, even in the supercoiled form, while others have lost their affinity for IHF. We conclude that IHF binding depends not only on the consensus ihf sequence, but also on a suitable combination of the sequences of both ihf and neighboring regions, together with the DNA conformation, which includes both natural and imposed bends in DNA and the degree of supercoiling. Based on most of the present data, it is difficult to predict the relationship between the ihf sequence and IHF interaction, since two very different sequences (less than 50% homology) show strong IHF binding, whereas very similar sequences (80-87% homology) show a very different behavior. However, the hydroxylradical footprinting data show that three A + T-rich sequences are protected by IHF: the central sequence, which overlaps the -35 region of p'R, and two flanking sequences removed by one helix turn. All three sequences are located on the same face of the helix, and the amino acid side chains of IHF seem to occupy the narrow minor groove. A novel consensus sequence is proposed.

Bacterial Proteins↗
PubMed1989

Repression of transcription from the b2-att region of coliphage lambda by integration host factor.

The central b2-att region of coliphage lambda is known to be transcriptionally active in vitro, but silent in vivo in lambda lysogens. To explain such in vivo repression of transcription originating in the b2-att region, we explored the effect of the Escherichia coli integration host factor (IHF), the product of E. coli genes himA and himD, especially since the att region contains several IHF-binding sites. Using various lambda DNA templates, we mapped the transcripts which are initiated in vitro in the attP region by the RNA polymerase and found that there are three rightward (RI, RII, and RIII) and one leftward (LI) transcripts. All four of them are repressed by a factor of about 10 by 10 micrograms IHF/ml. Moreover, in in vivo experiments we found that plasmids carrying the attP fragment cannot be established and maintained in IHF-hosts. These results indicate that IHF may play a significant auxiliary role in repressing transcription in the prophage state.

Attachment Sites, Microbiological↗