Mutational analysis of an ssi region carried by plasmid pACYC184

To investigate the functional contribution of some structural components of the signal that directs single-stranded initiation of DNA replication (ssi signal) carried by a 119-nt segment of plasmid pACYC184 (Bahk et al., 1988), we constructed mutants carrying one-base substitutions and insertions using oligodeoxyribonucleotide (oligo) directed mutagenesis. Two one-base substitution mutants were obtained. The mutants, M13 delta lac 184/Sp and M13 delta lac 184/Ev, carried an SplI site and an EcoRV site, respectively, created by base substitution. Three kinds of synthetic oligos, that is, a 10-bp EcoRI linker, an 8-bp ScaI linker and an 8-bp SmaI linker, were inserted into the SplI site of M13 delta lac 184/Sp, and into the EcoRV site of M13 delta lac 184/Ev. The SSI activity of each mutant examined indicated that the one-base substitutions had different effects on the SSI functions of the altered ssi signals. This fact suggests that some structural components within the 119-bp region make distinct contributions to the SSI function. Moreover, when the three kinds of synthetic linkers were inserted into the mutants M13 delta lac 184/Sp and M13 delta lac 184/Ev, each of the insertion mutations affected the rate of conversion of ss DNA to RFI in vivo and the growth of the recombinant phages in a distinct manner. Judging from the above results, the base composition and the length of a certain specific site were crucial for maintenance of the SSI functional activity, and structural components of the ssi signal contributed distinctly to the SSI function.     

The isolation and characterization of plaque-forming arabinose transducing bacteriophage lambda.

Non-defective arabinose transducing phage, λpara, were isolated in two steps: first, Escherichia coli strains containing rare insertions of λ DNA into the arabinose C or B genes were selected; and second, these lysogens were induced and transducing phage were selected from the resulting lysates. The approximate location of the bacterial substitution on the phage and the ara gene content of the substitution were determined genetically. The precise location of the substitution was determined by electron microscopy of DNA heteroduplexes.Transducing phage, derived from the strain possessing λ inserted into the araC gene, carried part of the araC gene, the ara regulatory region, and all of the araB gene. Transducing phage, derived from eight independent strains possessing λ inserted in the same orientation and in the same position in the araB gene, carried a portion of the araB gene, the ara regulatory region and all of the araC gene. In these nine cases the ara DNA on the phage was immediately adjacent to the normal phage attachment site, indicating that the transducing phage were formed by the same type of abnormal excision which produces gal or bio transducing λ phage. The relative orientations of ara and phage genes were deduced from the topology of such excisions. One anomalous transducing phage was also characterized.     

Site-localized mutagenesis directed by phosphotriester analogs of oligonucleotides

17- and 20-mer oligodeoxyribonucleotides and their analogues, containing one to four phosphate groups esterified with ethyl alcohol in different positions of oligonucleotide chain, were synthesized by modified triester method. Ethylated di- and trinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. The structures of the oligonucleotides were confirmed by Maxam-Gilbert sequencing method. Oligonucleotides were not totally complementary to the N-terminal region of lac Z'gene (coding for N-terminal fragment of beta-galactosidase) of phage M13mpB DNA and induced the formation of the proposed deletion mutant DNA M13mp1 delta T. Phosphotriester analogues were more effective mutagens as compared to phosphodiester oligonucleotides due to their stability to nucleases. The use of E. coli DNA-polymerase I provided the increase in the mutant yields in case of the phosphotriester analogues. The stability of the analogues to 5'----3'----5'-endonuclease action, the specificity of oligonucleotide: DNA binding and the structure of mutant DNA were studied by the Sanger sequencing method.     

Construction of pBR322-ara hybrid plasmids by in vivo recombination

Summary In vivo recombination was used to clone deletions of the araBAD-araC genes of Escherichia coli onto a hybrid pBR322-ara plasmid. Genetic and physical analysis demonstrated that the desired deletions had been recombined onto the plasmid. In addition to permitting a detailed physical analysis of various ara deletions, this procedure has generated a series of plasmid cloning vehicles that can be used to clone, by in vivo recombination, any ara point mutation located within the region covered by the deletions. Hybrid plasmids containing the cloned point mutation can be distinguished from the original cloning vehicle by genetic complementation. The desired recombinant plasmid can be easily obtained because the frequency of recombination between the plasmid ara region and the chromosomal ara region is 0.025%–3%. A plasmid containing a deletion which removes the ara controlling site region and the araC gene was used to clone two types of araBAD promoter mutations and an araCmutation by in vivo recombination. Genetic and physical analysis of these plasmids established that the mutations in question had been recombined on to the ara deletion plasmid. The application of this procedure to the ara genes and to other genetic systems is discussed.     

The regulatory region of the L-arabinose operon: a physical, genetic and physiological study.

A fine-structure physical and genetic map was constructed of a 1000 base-pair region of the l-arabinose operon of Escherichia coli. This region consists of the ara regulatory sequences contained between the araC and araB genes and portions of these flanking genes. Point mutations, Mu phage insertions, and bacterial deletions as well as arabinose-induced and basal enzyme levels in the strains were used in constructing a genetic map of the region. These ordered positions were then located more accurately by mapping the point mutations against physically located endpoints of deletions isolated on the two non-defective transducing phage λparaB114 and λparaC116. Phage possessing deletions ending in the arabinose regulatory region were isolated from indicating-plates on which deletions removing none, part, or all of either the araC or araB genes carried on the phage could be distinguished. Phage stocks were enriched for such deletions prior to plating by treatment with chelating agents and heat (Parkinson &; Huskey, 1971). Deletions into the ara region on either phage shorten the ara DNA homology region formed from heteroduplexes between λparaB114 and λparaB116. Therefore the physical locations of these deletion endpoints were determined by electron microscopy of the appropriate heteroduplexes and/or by gel electrophoresis of the central duplex following S₁ nuclease digestion (Lis &; Schleif, 1975b). 18 of the 32 deletions isolated and mapped in this region were measured physically. The space between araC and araB, containing the regulatory elements of the operon, is estimated to be about 300 base-pairs.     

Salmonella typhimurium metC operator-constitutive mutations

Abstract We used an Escherichia coli lac deletion strain lysogenized with a metC-lacZ fusion phage (λClac) to select operator-constitutive mutations in the Salmonella typhimurium metC gene control region. The mutations were located in a region containing 2 tandemly repeated 8 bp palindromes previously proposed to be the MetJ repressor binding site. Lysogens carrying λClac mutant phage exhibit high β-galactosidase levels that are only partially repressible by methionine. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product.     

The regulatory region of the L-arabinose operon: its isolation on a 1000 base-pair fragment from DNA heteroduplexes.

A DNA fragment containing the l-arabinose operon regulatory region of Escherichia coli was purified from DNA heteroduplexes formed between opposite strands of two non-defective ara transducing phage. The phage and arabinose gene orientation is such that the heteroduplex contains two single-stranded “bubbles”. The ara regulatory region and short portions of the flanking araB and araC genes are in the short duplex between the “bubbles”. Extensive regions of homology between the phage genomes allowed nearly half of the DNA renatured from a mixture of the two phage DNAs to be in the form of heteroduplexes. Digestion of the reannealed DNA containing heteroduplexes and homoduplexes with the easily purified, single-strand specific nuclease S₁ yielded the 1000 (1017 ± 20, n = 36) base-pair ara DNA duplex plus half and whole phage-length duplexes. The larger DNA duplexes were selectively precipitated by polyethylene glycol before the final purification by preparative electrophoresis on polyacryl-amide gels. By these methods 10 to 20 μg of the 1000 base-pair DNA fragment were purified.     

Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: II. In vitro selection of mutant DNA

A method for the in vitro selection of mutant DNA has been devised as an adjunct to the recently developed method for the use of short enzymatically-synthesized oligodeoxyribonucleotides of defined sequence as sitespecific mutagens for circular DNA. The selection method uses the mutating oligodeoxyribonucleotide as a primer for Escherichia coli DNA polymerase I (large fragment) under conditions where there is preferential interaction with mutant DNA template. After ligation using T4 DNA ligase, endonuclease Sl is used to degrade single-stranded non-mutant DNA leaving the desired mutant as closed circular duplex DNA. This paper describes the development of the method using mutants in ØX174 DNA as the model system. Studies on the changes A → G and G → A at position 587 of ØX174 viral DNA (am 3 to wild-type and its reversal) show that one or two cycles of selection can lead to a population of phage consisting of close to 100% mutants.     

Salmonella typhimurium metC operator-constitutive mutations

We used an Escherichia coli lac deletion strain lysogenized with a metC-lacZ fusion phage (lambda Clac) to select operator-constitutive mutations in the Salmonella typhimurium metC gene control region. The mutations were located in a region containing 2 tandemly repeated 8 bp palindromes previously proposed to be the MetJ repressor binding site. Lysogens carrying lambda Clac mutant phage exhibit high beta-galactosidase levels that are only partially repressible by methionine. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product.     

Selective ‘stencil’-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS

The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS alleles are cleaved prior to PCR. We took advantage of an AluI recognition site located immediately upstream of the K-RAS codon 12. The site was reconstituted upon DNA denaturation followed by annealing with a ‘stencil’, a 16-bp synthetic oligonucleotide complementary to the wild-type sequence. As opposed to normal K-RAS, the mutant allele forms, upon annealing with the stencil, a mismatch at the codon 12 which lies within the AluI enzyme binding site and partially inhibits its activity. The mismatch also lowers the melting temperature of the stencil-mutant K-RAS double helix as compared to stencil–wild-type duplex, so that only the latter is double stranded and selectively digested by AluI at elevated temperatures. The proposed method of stencil-aided mutation analysis (SAMA) based on selective pre-PCR elimination of wild-type sequences can be highly advantageous for detection of mutant K-RAS due to: (i) an enhanced sensitivity because of reduced competition with a great excess of normal K-RAS, and (ii) a decrease in a number of false-positive results from Taq polymerase errors. Application of SAMA for generalized detection of DNA mutations is discussed.     

Molecular cloning and characterization of a complete DNA copy of potato spindle tuber viroid RNA.

Double-stranded cDNA has been synthesized from RNA of a severe strain of potato spindle tuber viroid using a synthetic oligodeoxyribonucleotide as a primer. Upon cloning in bacteriophage M13mp9, two recombinant phages were selected to construct a pBR322-derived plasmid containing a complete viroid DNA copy. Elucidation of the nucleotide sequence revealed four differences with the previously established sequence of another PSTV strain, three of which were base exchanges and one a deletion.     

Applications of synthetic oligodeoxyribonucleotides to problems in molecular biology

The applications of synthetic oligodeoxyribonucleotides to problems in molecular biology described in this article are those where the oligodeoxyribonucleotide is a probe for a specific region of a nucleic acid. This includes the isolation of the iso-1-cytochrome c gene of yeast; the sequence determination of RNAs and DNAs including regions of double-stranded DNA; the introduction of defined site-specific point mutations into bacteriophage OX174 and in the in vitro selection of mutant DNA from a mixture with wild-type DNA.     

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA^- strain were G:C → T:A transversions, occurring within the sequence which in recA⁺ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.     

Whole-genome synthesis and characterization of viable S13-like bacteriophages

Background

Unprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template.

Methodology/Principal Findings

The sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl₂, the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition.

Conclusions/Significance

The bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.     

UNG-dependent correction of molecular heteroduplexes of M13 phage DNA in Escherichia coli cells

Correction of heteroduplex DNA obtained by hybridization of uracil-containing single-stranded M13mp18 phage DNA and "mutant" synthetic oligonucleotide with deletion of cytosine in SalGI site was studied in ung+ and ung- E. coli strains. Uracil-containing DNA was prepared after growth of phage in an E. coli strain dut- ung-. The DNA was hybridized with "mutant" oligonucleotide then complementary DNA chain was synthesized by T4 DNA polymerase. Ung+ and ung- E. coli cells were transformed by DNA. In all experiments mutation frequency in ung+ was higher than in ung- cells (approximately 6-fold) and reached 11-50%. Absolute number of mutants was higher in ung+ cells. The results indicate that high level of mutagenesis depends on uracil repair system polarizing the correction of heteroduplex DNA.     

Analysis of internal (n-1)mer deletion sequences in synthetic oligodeoxyribonucleotides by hybridization to an immobilized probe array.

The purity of a drug substance can influence its toxicity and potency, so impurities must be specifically determined. In the case of synthetic oligodeoxyribonucleotide drugs, however, product complexity makes complete impurity speciation difficult. The goal of the present work was to develop a new analytical method for speciation of individual internal (n-1)mer impurities arising from formal nucleotide deletion in synthetic oligodeoxyribonucleotides. A complete series of oligodeoxyribonucleotide probes were designed, each complementary to an (n-1)mer deletion sequence of the drug in question. Glass plates were used as a solid support for individually immobilizing the entire probe array. The total mixture of internal (n-1) length impurities was isolated from a synthetic oligodeoxyribonucleotide by PAGE and labeled with 35S. Under stringently optimized conditions, only the perfectly sequence-matched oligodeoxyribonucleotide hybridized to each probe, while all other deletion sequences were removed by washing with buffer. The 35S signal intensity of the bound oligodeoxyribonucleotide was proportional to the concentration of each (n-1)mer deletion sequence in the analyte solution. This method has been applied to a number of synthetic phosphorothioate oligodeoxy-ribonucleotide lots and shown to be reliable for speciation and relative quantitation of the internal (n -1)mer deletion sequences present.     

Mutagenesis directed by phosphotriester analogs of oligonucleotides

20-mer oligodeoxyribonucleotides d-ACGACGG (R') CCAG (R') TGATCCGTA, where R' = R' = H (20), F' = Et, R' = H (20-Et), or R' = R' = Et (20-Et2) were synthesized by modified triester method. Ethylated dinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. Structures of oligonucleotides were confirmed by Maxam - Gilbert method. Mutagenesis induced by oligonucleotides was studied on DNA of M13mpB phage. Oligonucleotides were not totally complementary to this DNA in the region of 4-11 codons of Z'-gene. They all were shown to direct the formation of the designed deletion mutants, phosphotriester analogues (20-Et) and (20-Et2) being more effective mutagens. The specificity of oligonucleotides: DNA binding and mutant DNA structure were shown by Sanger method.     

Binding of complement by complexes between antibodies to nucleic acid constituents and short oligodeoxyribonucleotides

Oligodeoxyribonucleotides act as inhibitors of the complement fixation caused by complexes between antibodies to defined oligodeoxyribonucleotides and denatured DNA. At concentrations higher than 50 micrograms oligodeoxyribonucleotide/ml complement fixation occurred in the absence of antigen. The extent of complement binding depends on the specificity of the antibodies as well as on the composition of the oligodeoxyribonucleotides. Complement fixation is observed most strongly with antisera to oligodeoxyribonucleotides and to denatured DNA, which belong predominantly to the IgM class. With two LE-sera, containing antibodies to denatured and to native DNA, no complement fixation was found. It is supposed that specific interactions of the oligodeoxyribonucleotides with amino acid residues closely neighbored to the antibody combining site lead to conformational changes in the antibody molecules and to an activation of the complement binding site.     

Distal CCAAT box deletion in the A gamma globin gene of two black adolescents with elevated fetal A gamma globin.

Point mutations in G gamma and A gamma globin gene promoters are associated with increased production of G gamma and A gamma globin, respectively. To determine whether an upstream promoter mutation could account for elevated A gamma in a Black adolescent with A gamma-beta+-HPFH and sickle cell trait, we cloned the 13 kb BglII fragment containing both gamma genes into phage lambda vector EMBL3. For one clone, the A gamma upstream promoter showed no hybridization to a 19 bp oligonucleotide whose sequence centered at -117. A gamma promoter sequence data for this mutant clone revealed a 13 bp deletion which eliminated the A gamma distal CCAAT box. Amplified A gamma genomic DNA of this and a similar case showed hybridization to both deletion-mutant and normal oligonucleotide probes. We propose that this 13 bp deletion removes part of the binding site for a repressor protein which is abundant in adult erythroid cells.