Induction and isolation of mutants in a specific region of gene A of bacteriophage ∅X174

Borrias, Wilhelmina E.; Wilschut, Ina J. C.; Vereijken, J.M.; Weisbeek, Peter; Arkel, G.A. van

doi:10.1016/0042-6822(76)90251-8

Cited by 21 publications

(10 citation statements)

References 4 publications

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“…Weissmann and his colleagues (26) described a site-directed mutagenesis procedure that uses coliphage Qfl replicase to incorporate nucleotide analogs at specific sites in Q(3 RNA, and Borrias et al (27) isolated temperature-sensitive mutants of coliphage 4X174 after transfection with a partial heteroduplex consisting of singlestranded phage DNA and a mutagenized fragment. With suitable modifications, each of these approaches might have more general applicability.…”

Section: Methodsmentioning

confidence: 99%

Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome.

Shortle¹,

Nathans²

1978

Proc. Natl. Acad. Sci. U.S.A.

249

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DNA from simian virus 40 (SV40) was prepared for local mutagenesis by nicking the molecule at a specific site with a restriction endonuclease that recognizes one site in SV40 DNA and then extending the nick enzymatically to expose a short, single-stranded segment of DNA. The "gapped" DNA was treated with a single-strand-specific mutagen, sodium bisulfite, which converts cytosine to uracil. After mutagenesis, the gap was repaired with DNA polymerase, generating molecules resistant to the restriction enzyme used to make the initial nick. From cells infected with DNA thus modified, SV40 mutants were isolated that had enzyme-resistant genomes. In some cases, precise positions of G.C to A.T transitions could be inferred from the patterns of susceptibility of mutant DNA to other restriction endonucleases whose recognition sequences were altered by the mutagenesis procedure. One of the restriction endonuclease sites mutagenized (Bgl I) maps at the origin of SV40 DNA replication and near sequences corresponding to the 5' ends of viral mRNAs. Many of the resulting Bgl I-resistant mutants yielded small plaques, suggesting partial defectiveness in DNA replication or transcription.

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Section: Methodsmentioning

confidence: 99%

Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome.

Shortle¹,

Nathans²

1978

Proc. Natl. Acad. Sci. U.S.A.

249

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“…In this approach, deletions or insertions can be introduced rather easily. However, methods to generate point mutations at predetermined sites in a DNA molecule by base-specific chemical treatment (2)(3)(4)(5) or by incorporation of nucleotide analogs (1) give rise to only a few mutants among a large background ofnonmutants. Because this yield is too low to permit saturation of a genetic element with point mutations, we developed an improved method for sodium bisulfite mutagenesis in vitro.…”

mentioning

confidence: 99%

Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element.

Weiher¹,

Schaller²

1982

Proc. Natl. Acad. Sci. U.S.A.

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A method for highly efficient segment-specific mutagenesis is described. The method uses as target for sodium bisulfite mutagenesis the DNA single strands ofa DNA restriction fragment that had been separated by cloning into base-complementary regions of a pair of phage fd vectors. After repair synthesis in vitro, the mutagenized DNA fragment is recovered by cloning into a nonmutated plasmid vector and analyzed for sequence and by functional tests. By using this method, the nucleotide sequence of a 109-base pair restriction fragment containing the lac promoter/operator from Escherichia coli was extensively modified. More than 90% of the 235 isolates obtained showed a change in phenotype; all of 22 analyzed for their nucleotide sequence were found to carry multiple C -* T point mutations in up to 60% of the possible target positions. Nevertheless, few isolates showed major changes in promoter activity relative to the nonmutated promoter element, which indicates a high degree of flexibility in the promoter sequence.Molecular cloning methods have been used to develop a type of genetic analysis in which a cloned genetic element is first altered at predetermined sites and then assayed for changes in phenotype in the appropriate biological environment. This "reversed genetics" (1) allows construction and study of silent mutations that are often essential to correlate structure and function of a genetic element. In this approach, deletions or insertions can be introduced rather easily. However, methods to generate point mutations at predetermined sites in a DNA molecule by base-specific chemical treatment (2)(3)(4)(5) or by incorporation of nucleotide analogs (1) give rise to only a few mutants among a large background ofnonmutants. Because this yield is too low to permit saturation of a genetic element with point mutations, we developed an improved method for sodium bisulfite mutagenesis in vitro. This reagent has been used before for site-specific mutagenesis of sequences next to a unique restriction site in which a DNA sequence could be rendered single stranded to become a selective target for the mutagen (2). We have overcome these limitations by using a singlestranded phage vector system to clone either strand of the desired target DNA separately. Mutagenesis of the single-stranded recombinant DNA enabled us to introduce any number of C -* T base changes into one strand of a defined restriction fragment. The present study exemplifies the scope of the method by an extensive variation of the nucleotide sequence of the lac promoter/operator, a regulatory signal that controls the expression of the lac operon in Escherichia coli.MATERIALS AND METHODS E. coli C600 rm+ and KB35 and phages fdlO9 and fdlO9-2 were obtained from R. Herrmann (6). E. coli C600 galK (gal E'T+K-) and pKOl were obtained from K. McKenney (7). Construction of plasmids and recombinant phages, including transformation and cultivation of bacteria, and isolation of recombinant DNA was done essentially as described by Herrmann et al. (6). DNA se...

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“…The local mutagenesis methods as well as other methods for site-directed mutagenesis (Borrias et al 1976;Weissmann et al, this volume)should be increasingly useful for modifying specific nucleotides in genes and regulatory signals of viral or other cloned DNA segments. The availability of restriction maps and extensive nucleotide sequences should make it possible to select functionally interesting sites for systematic perturbation, whether in coding or in regulatory sequences.…”

Section: Discussionmentioning

confidence: 98%

Mutants of Simian Virus 40 with Base Substitutions at the Origin of DNA Replication

Shortle¹,

Nathans

1979

Cold Spring Harbor Symposia on Quantitative Biology

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Induction and isolation of mutants in a specific region of gene A of bacteriophage ∅X174

Cited by 21 publications

References 4 publications

Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome.

Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome.

Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element.

Mutants of Simian Virus 40 with Base Substitutions at the Origin of DNA Replication

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