Synthetic lacoperator DNA is functional in vivo

150

The inducible protein X of Escherichia coli has been compared to the recA+ protein made by specialized recA transducing phages. The molecular weights and isoelectric points of these proteins are identical. Two In Escherichia cohl a complex set of responses is observed after treatments that disrupt DNA synthesis or damage DNA. UV irradiation of cells produces mutations, induces prophage A in lysogens, and stimulates reactivation and mutagenesis of phage containing DNA damage (Weigle or W-reactivation and Wmutagenesis) (see ref. 1 for review). Witkin (2) and Radman (3) have presented evidence that expression of these and other diverse processes (SOS functions) results from their activation by a common regulatory signal. SOS functions are not expressed constitutively in cells but are induced by treatments that inhibit DNA synthesis, such as UV or X irradiation, thymine starvation, and nalidixic acid or mitomycin C treatment (see ref. 1).The principal evidence that mutagenesis, prophage induction, and W-reactivation share a common pathway is the isolation of pleiotropic mutations that alter the cells' response to inducing treatments. In recA -or lexA -strains, expression of SOS functions is not observed. These mutants display no UVinduced mutagenesis, W-reactivation, or prophage induction (3). Another mutation, tif-1, is closely linked to the recA gene (refs. 4 and 5; unpublished data) and shows conditional induction of SOS functions in the absence of DNA damage. All of the processes observed after exposure of wild-type cells to UV light or nalidixic acid are observed after shift of the tif-1 mutant to high temperature (4). Furthermore, tif-1-mediated induction of SOS functions is abolished by recA -or lexAmutations (6). The inducibility of SOS functions is demonstrated by kinetic studies of tif-l-or UV-mediated bacterial mutagenesis and phage A reactivation (7,8) as well as the demonstration that de novo protein synthesis is required after inducingThe costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked "advertIsement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5275 treatments for expression of these functions (2, 8, 9).At least one protein that is induced in E. coli after treatments eliciting expression of SOS functions has been studied. Inouye and Pardee (10) and Gudas and Pardee (11) (12). The tsl-mutation is tightly linked to the lexA locus and appears to alter expression of SOS functions (13). Although the induction of protein X is usually correlated with expression of SOS functions, the precise relationship between protein X and the genes controlling these inducible functions has not been determined.The recent isolation of A transducing derivatives carrying the recA gene has made possible the identification of the recA protein (14). A preliminary characterization of this protein suggested that it might be related to the inducible protein X (14). In this paper, biochemical and genetic evidence i...

Section: Discussionmentioning

confidence: 99%

Protein X is the product of the recA gene of Escherichia coli.

McEntee¹

1977

150

“…The oligodeoxyribonucleotides were synthesized by the modified triester method (9)(10)(11), which leaves free 5'-OH ends. The oligonucleotides were phosphorylated at the 5' end by phage T4 polynucleotide kinase (Miles Laboratories) and ["y-32P]ATP as described (12 Spheroplast Infection by Synthetic DNA. Spheroplasts were prepared from E. coli W6 cells as described by Guthrie and Sinsheimer (13).…”

Section: Methodsmentioning

confidence: 99%

“…The single-burst experiments of Baas and Jansz (5) 12 hr at 12°C to a synthetic 5'-32P-labeled 6mer with am3 (+) strands serving as templates. Then polymerase I and dNTP mix were added and incubation was continued for 6 hr at 12°C.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Efficient correction of a mutation by use of chemically synthesized DNA.

Razin¹,

Hirose²,

Itakura³

et al. 1978

The mutated base in the am3 lysis-defective mutant of the bacteriophage OX174 has been corrected by a combined in vitro enzymatic DNA synthesis and in vivo replication of the heteroduplex product. Chemically synthesized oligodeoxyribonucleotides carrying the wild-type sequence have been used to prime DNA synthesis with am3 OX174 DNA serving as a template. The resultant semisynthetic heteroduplex composed of an am3 (+) strand and a wild-type (-) strand, with one mismatched base pair at position 587 on the 4X174 DNA sequence, was used to infect spheroplasts. The progeny phage were analyzed by a parallel plaque assay on wild-type host, Escherichia coli C, to screen for wild-type phenotype, and on E. coli HF4714, an amber suppressor strain, to determine the total progeny phage. When a 23-base-long synthetic primer was used, about one-third of total progeny were found to be wild type. Shorter primers yielded lower percentages of wild type; they also had poorer priming activity.The ability to specifically and efficiently substitute one or a few bases in a DNA sequence should greatly aid numerous genetic manipulations. Such a procedure would allow, for example: repair or creation of mutations; conversion of a gene of one species to the same gene of another species; and creation or elimination of special sequences such as restriction sites. In the present study, the am3 lysis-defective mutant of the bacteriophage 4X174 has been used to test the feasibility of specifically changing one base in a DNA sequence. The am3 mutation was chosen because large amounts of DNA carrying it can be prepared easily, the complete DNA sequence of 4X174 is known (1), the precise location of the mutation has been established (2), and the mutant progeny can be distinguished easily from wild type.The strategy used in the present experiments involved the chemical synthesis of an oligodeoxyribonucleotide complementary to the wild-type DNA at the mutation region (2) (see Fig. 1) and its annealing to am3 OX174 single-stranded circular DNA. This partial duplex with one mismatched base pair was used for primed repair synthesis in vitro by Escherichia coli DNA polymerase I followed by ligation of the product to form a covalently closed circular duplex (RFI). These duplex molecules are infective to spheroplasts and genetically heterozygous, composed of an am3 mutant (+) strand and a wild-type (-) strand.We report here that a substantial fraction of the progeny produced after infection of spheroplasts was found to bear the wild-type marker derived from the chemically synthesized oligonucleotide. The "salvage of mutations" of 4X174 by hybridizing wild-type DNA with mutant DNA has been reported before (3-6). Results similar to those presented here were achieved with enzymatically synthesized primers (7). MATERIALS AND METHODSThe bacteria and phage strains used in this study were: E. coli C (wild type), HF4714 (carrying a suppressor), E. coli W6; bacteriophage 4X174 wild type and am3cs70 (a gene E mutant). All were obtained from R. L. Sinsheimer...

“…Foreign DNA can be inserted by blunt end ligation. The same ligation technique has been used for fusing "linker" molecules to synthetic lac DNA (24) and for the ring closure of recombinant plasmid DNA (25).…”

Section: Discussionmentioning

confidence: 99%

Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro.

Messing¹,

Gronenborn²,

Müller‐Hill³

et al. 1977

602

138

A HindII restriction fragment comprising the Escherichia coli lac regulatory region and the genetic information for the a peptide of ,-galactosidase (A-D-galactoside galactohydrolase, EC 3.2.1.23) has been inserted into 1 of the 10 Bsu I cleavage sites of M13 by blunt end ligation. A stable hybrid phage was isolated and identified by its ability to complement the lac a function. Further characterization of the h brid phage includes retransformation studies, agarose gel electrophoresis, DNADNA hybridization, and heteroduplex mapping. The insertion point has been localized at 0.083 map unit on the wild-type circular map-i.e., within the intergenic region. The results prove that part of the intergenic region is nonessential and that the phage can be used as a cloning vehicle.Two vector systems, plasmids of the ColEl family or pSC 101 and the bacteriophage X genome (1-8), have been developed to clone prokaryotic and eukaryotic DNA in Escherichia coli. Vectors generated from filamentous phages like M13 containing single-stranded circular DNA may offer some special advantages. Upon infection, phage DNA is converted into a doublestranded supercoiled replicative form (RF I) and amplified to about 300 copies per cell, and the phage-producing cells do not lyse but continue to replicate (9). In addition to the doublestranded DNA from the infected bacteria, single-stranded DNA can easily be obtained from the progeny phages, thus exerting a pronounced advantage over the isolation procedure of plasmid DNA from transformed bacteria. Because bacterial growth is slightly retarded by M13 infection, infected bacteria can be detected by "turbid-plaque" formation (9) and no selective pressure, as for instance for drug resistance factors, would be necessary to detect bacteria transfected by M13 DNA. Finally, due to the filamentous shape of the phage, it can be expected that additional DNA could be packed over a broad range of size.