Construction of an M13 histidine-transducing phage: a single-stranded cloning vehicle with one EcoRI site.

In order to create a ready source of single-stranded DNA for DNA sequence determination by the dideoxy chain-termination method, the promoter-proximal part of the histidine operon, the hisOGD region of Salmonella typhimurium, was cloned onto the single-stranded phage M13. Both orientations of the his DNA were cloned to supply DNA template for sequencing of each strand. Insertion was achieved at an HaeIII site in the intergenic region (IR) of M13, and a single EcoRI site was purposely regenerated at one boundary of the his DNA insert. Infected colonies, not plaques, were selected using the hisD gene as a selective marker. The single RI site and the hisD marker for auxotrophic selection represent improvements on the wild type M13 as a single-stranded vector for cloning other DNA.     

Isolation of Specialized Transducing Bacteriophages for Gluconate 6-Phosphate Dehydrogenase (gnd) of Escherichia coli

Specialized transducing phages for gluconate 6-phosphate dehydrogenase (gnd), a constitutive enzyme in Escherichia coli, have been isolated using a method previously described for other genes. The gnd-his region, carried on an F' episome, was first transposed to tonB. Rare phages carrying gnd were selected, by transduction, from phi80 lysogens of these strains; one phage also carried his (phi80gndhis). From the transductants, high-frequency transducing lysates were obtained; low multiplicity of infection then yielded defective lysogens. tonB deletion analysis of the phi80dgndhis lysogen shows the order of genes in the prophage to be imm80...hisOGD...gnd; according to a marker rescue experiment most phage late genes have been replaced by bacterial deoxyribonucleic acid. A heat-inducible, lysis-defective lambda-phi80 hybrid derivative of phi80dgndhis has been prepared.     

The transposable element Tn3 promotes general recombination at the neighboring regions

Transposon Tn3 was inserted into a tRNA operon of the amber suppressor Su+2 on a transducing phage (lambda hcI857nin5pSu+2) by selecting phages with ampicillin resistance and Su- phenotypes. In a strain thus obtained, Tn3 was inserted between the promoter and the first tRNA gene of the operon, which was determined by DNA sequencing. The Su+2 tRNA operon on the transducing phage consisted of two tRNA genes for tRNA(Met) and Su+2 tRNA(2Gln), which was a deletion derivative of the supB-E tRNA operon of E. coli containing seven tRNA genes in the order of promoter-Met-Leu-Gln1-Gln1-Met-Gln2-Gln2. Proliferating the lambda hcI857nin5pSu+2::Tn3 in E. coli cells, a number of phages which had lost Tn3 were isolated, and their tRNA gene compositions as well as the DNA structures of the tRNA operon were analyzed. In many cases the tRNA genes which had been deleted from the original transducing phage were regained from the chromosomal supB-E operon. Thus the loss of Tn3 from the phages was not due to excision of the transposon but due to the replacement of a portion of the tRNA operon, including Tn3, with the host homologous region that did not contain Tn3. This type of replacement takes place rather efficiently as a consequence of Tn3 insertion, owing to the general recombination occurring between homologous tRNA genes of phage and host chromosomes in the presence of either host recA or phage red. No such enhanced recombination in a similar cross between phage and host chromosomes was observed with the Tn3 present in the trans position on an independent plasmid. We conclude that inserting Tn3 in cis promotes general recombination in the neighboring regions. Possible mechanisms for this new type of genetic effect of Tn3 are discussed. During the course of this study, a natural defective mutation (T11) was also detected in one of the duplicated tRNA(2Gln) genes in an E. coli K12 strain we used.     

Deletion analysis of the expression of rRNA genes and associated tRNA genes carried by a lambda transducing bacteriophage.

Transducing phage lambdailv5 carries genes for rRNA's, spacer tRNA's (tRNA1 Ile and tRNA1B Ala), and two other tRNA's (TRNA1 Asp and tRNA Trp). We have isolated a mutant of lambdailv5, lambdailv5su7, which carries an amber suppressor mutation in the tRNA Trp gene. A series of deletion mutants were isolated from the lambdailv5su7 phage. Genetic and biochemical analyses of these deletion mutants have confirmed our previous conclusion (E. A. Morgan, T. Ikemura, L. Lindahl, A. M. Fallon, and M. Nomura, Cell 13:335--344, 1978) that the genes for tRNA1 Asp and tRNA Trp located at the distal end of the rRNA operon (rrnC) are cotranscribed with other rRNA genes in that operon. In addition, these deletions were used to define roughly the physical location of the promoter(s) of the rRNA operon carried by the lambdailv5su7 transducing phage.     

Deoxyhexanucleotide containing a vinyl chloride induced DNA lesion, 1,N6-ethenoadenine: synthesis, physical characterization, and incorporation into a duplex bacteriophage M13 genome as part of an amber codon

Organic synthesis and recombinant DNA techniques have been used to situate a single 1,N6-ethenoadenine (epsilon Ade) DNA adduct at an amber codon in the genome of an M13mp19 phage derivative. The deoxyhexanucleotide d[GCT(epsilon A)GC] was chemically synthesized by the phosphotriester method. Mild nonaqueous conditions were employed for deprotection because of the unstable nature of the epsilon Ade adduct in aqueous basic milieu. Physical studies involving fluorescence, circular dichroism, and 1H NMR indicated epsilon Ade to be very efficiently stacked in the hexamer, especially with the 5'-thymine. Melting profile and circular dichroism studies provided evidence of the loss of base-pairing capabilities attendant with formation of the etheno ring. The modified hexanucleotide was incorporated into a six-base gap formed in the genome of an M13mp19 insertion mutant; the latter was constructed by blunt-end ligation of d(GCTAGC) in the center of the unique SmaI site of M13mp19. Phage of the insertion mutant, M13mp19-NheI, produced light blue plaques on SupE strains because of the introduced amber codon. Formation of a hybrid between the single-strand DNA (plus strand) of M13mp19-NheI with SmaI-linearized M13mp19 replicative form produced a heteroduplex with a six-base gap in the minus strand. The modified hexamer [5'-32P]d-[GCT(epsilon A)GC], after 5'-phosphorylation, was ligated into this gap by using bacteriophage T4 DNA ligase to generate a singly adducted genome with epsilon Ade at minus strand position 6274. Introduction of the radiolabel provided a useful marker for characterization of the singly adducted genome, and indeed the label appeared in the anticipated fragments when digested by several restriction endonucleases. Evidence that ligation occurred on both 5' and 3' sides of the oligonucleotide also was obtained. The adduct was introduced into a unique NheI site, and it was observed that this restriction endonuclease was able to cleave the adducted genome, albeit at a lower rate compared to unmodified DNA. The M13mp19-NheI genome containing epsilon Ade will be used as a probe for studying mutagenesis and repair of this DNA adduct in Escherichia coli.     

A genetic enrichment for mutations constructed by oligodeoxynucleotide-directed mutagenesis

A genetic enrichment procedure for mutations constructed by oligodeoxynucleotide(oligo)-directed mutagenesis of DNA cloned in M13mp vectors is described. The procedure uses an M13 vector that contains the cloned target DNA and amber (am) mutations within the phage genes I and II. This vector cannot replicate in a suppressor-free (sup degrees) bacterial strain. A gapped heteroduplex is formed by annealing portions of a complementary (-)strand containing wild-type copies of genes I and II to the am-containing template (+)strand. The oligo is annealed to the single-stranded (ss) region and the remaining gaps and nicks are repaired enzymatically to form a closed circular heteroduplex structure. By transfecting the DNA into a sup degrees host we promote the propagation of heteroduplexes with the oligo-containing (-)strand since only this construction contains the wild-type copies of genes I and II. This procedure eliminates the need for any physical separation of the covalently closed circular DNA that contains the oligo from the ss template. Using this technique we have constructed 17 point mutations with mutation frequencies ranging from 2-20% for single base changes and from 0.3-9% for multiple base changes. In addition, we found that the mutation frequencies were affected by the state of DNA methylation in the (+) and (-)strands.     

Polarity of amber mutations in ribosomal protein genes of Escherichia coli.

Two amber mutations have been mapped inside the spcA-strA region (now called rpsE-rpsL) on the bacterial genome. Derivatives of the transducing phage lambda fus3 carrying each mutation were constructed and assayed in ultraviolet-irradiated bacteria to identify the mutated genes and measure the polarity of the mutations. The data indicated that both mutations, 3162(Am) and 3161(Am), affect genes coding for ribosomal proteins: rplC (L3) and rpsN (S14), respectively. It was shown also that each mutation exerts, inside of its respective operon (S10 and spc units), a relatively strong polar effect on genes distal to the mutated locus.     

Gene replacement and retrieval with recombinant M13mp bacteriophages.

We have developed an allele exchange system for shuttling sequences of DNA to and from their original chromosomal loci. Cloned segments of the histidine operon of Salmonella typhimurium and the lactose operon of Escherichia coli served as target sequences and were used to develop the system. Replacement and retrieval of target sequences used the phage M13mp vectors and proceeded through an M13 lysogen intermediate. The intermediates and products of allele exchange were characterized by genetic and hybridization analyses. Several unique properties of M13 lysogens were exploited to devise positive selections to detect integration and excision. These positive selections were used to manipulate phenotypically silent alleles.     

Nucleotide sequence and expression in E. coli of a human interferon-alpha gene selected from a genomic library using synthetic oligonucleotides

The complete nucleotide sequence of a human interferon-alpha gene is reported. The gene, designated IFN-alpha M1, was isolated from a human genomic library in phage lambda Charon 4A using synthetic oligonucleotides as hybridization probes. Based on a comparison of nucleotide sequence data obtained from this recombinant phage with published interferon-alpha gene sequences, a region of DNA capable of coding for a pre-interferon of 189 amino acids was identified. An AluI fragment containing the coding region for the mature interferon was inserted into the HincII site of the phage M13mp11, resulting in a fusion of portions of the IFN-alpha M1 and the beta-galactosidase genes. Antiviral activity was detected in extracts from E. coli infected with the recombinant M13 phage carrying the fused gene. The antiviral activity was completely neutralized by antibodies to human interferon-alpha.     

Genes 55, alpha gt, 47 and 46 of bacteriophage T4: the genomic organization as deduced by sequence analysis

The nucleotide sequence of T4 genes 55, alpha gt, 47 and 46 was determined by a combination of 'classical' procedures and a shotgun approach. Small DNA fragments generated by frequent cleavage with restriction enzymes or by sonication of restriction fragments were cloned in phage M13 vectors and sequenced by the dideoxy method. The positions of the genes were determined by marker rescue between the corresponding T4 amber mutants and the cloned T4 DNA fragments used in the sequencing experiments. The sequence gives an insight into the organization of this 7.1-kb early region of the T4 genome and shows that genetically 'silent' portions within this region are not void of genetic information.     

Genetic analysis of attTn7, the transposon Tn7 attachment site in Escherichia coli, using a novel M13-based transduction assay

The large (14 kb; kb = 10(3) bases) bacterial transposon, Tn7 (encoding resistance to trimethoprim and streptomycin/spectinomycin), has unusual properties. Like other elements, Tn7 transposes with low efficiency and low target-site specificity, but Tn7 also transposes, with high frequency in a unique orientation, to a preferred "attachment" site, called attTn7, in the Escherichia coli chromosome and similarly into plasmids containing attTn7. We developed a novel bacteriophage M13-based assay system to measure the transposition frequency of Tn7 to M13mp phage vectors containing attTn7 on a cloned 1 kb fragment of chromosomal DNA. Phage harvested from a Tn7 donor strain were used to infect recipient bacteria with selection for trimethoprim resistance. Transposition frequency, expressed as the number of trimethoprim-resistant colonies per plaque-forming unit, was found to be approximately 10(-4) to M13mp::attTn7, in contrast to 10(-10) to M13mp recombinants with approximately 1 kb insertions of other, "generic brand", DNA. By deletion analysis of M13mp::attTn7, we show that attTn7 is contained within a 64 base-pair region; sequences adjacent to the actual insertion site and encoding the carboxy terminus of the glmS gene are required. This assay also provided evidence for transposition immunity conferred by the right end of Tn7.     

Analysis of promoter mutations in the histidine transport operon of Salmonella typhimurium: use of hybrid M13 bacteriophages for cloning, transformation, and sequencing.

Mutations that cause an increased level of expression of the histidine transport operon were isolated and characterized genetically. Five independently isolated promoter-up mutations were transferred to an M13 hybrid phage that carries the histidine transport operon, and their nucleotide sequences were determined. For all five mutations, the change was the same as the one previously determined for promoter-up mutation dhuA1: a C-to-T change in the Pribnow box rendered this region more homologous to the consensus sequence. Methods for enabling Salmonella typhimurium to support growth of M13 phage effectively and for easy transfer of chromosomal mutations onto the hybrid phage are presented.     

A versatile primer for DNA sequencing in the M13mp2 cloning system

A primer for DNA sequencing by the chain-termination method in the M13mp2 cloning system was constructed and amplified. The primer was isolated as an EcoRI/AluI restriction fragment. After conversion of the AluI end into an EcoRI end the fragment was cloned in pBR325 from which it can be recovered by cleavage with EcoRI. The primer hybridizes to the single-stranded DNA of the mature M13mp2 phage next to the site of insertion thereby directing DNA synthesis along the inserted DNA.     

Improved oligonucleotide site-directed mutagenesis using M13 vectors.

An improved method is described for the construction of mutations in M13 vectors using synthetic oligonucleotides. The DNA is first cloned into a novel M13 vector (based upon M13mp18 or M13mp19), which carries a genetic marker that can be selected against, such as an EcoK or EcoB site, or an amber mutation in an essential phage gene. In this "coupled priming" technique, one primer is used to construct the silent mutation of interest, and a second primer is used to eliminate the selectable marker on the minus strand. After primer extension and ligation, the heteroduplex DNA is transfected into a strain of E. coli which is repair deficient and selects against the plus strand marker. Over 50 mutants have been constructed with this approach, and the yields can be excellent (up to 70%). For the stepwise construction of mutations using separate rounds of mutagenesis, the EcoK and EcoB markers offer a particular advantage over the amber marker. They permit selection in each round, as it is possible to cycle between the two markers. However for construction of multiple mutations over a short region, long synthetic oligonucleotides with multiple mismatches to the template can offer an alternative strategy.     

Physical map of the Salmonella typhimurium histidine transport operon: correlation with the genetic map.

A detailed restriction map of a 12.4-kilobase EcoRI fragment of Salmonella typhimurium deoxyribonucleic acid (DNA) containing the entire histidine transport operon and the argT gene is presented. Subclones of specific regions of the transport operon of S. typhimurium were constructed in plasmid vectors. An accurate correlation between the restriction map and the location of genetically defined deletions was obtained by hybridizing restriction digests of chromosomal DNA from strains carrying each deletion with cloned transport operon DNA as a probe. These data were used to position the histidine transport genes on the cloned 12.4-kilobase fragment of DNA.     

A system for shotgun DNA sequencing.

A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.