Structure of a region in the genome of bacteriophage N15, necessary for formation of hairpins at ends of the linear plasmid prophage]

A fragment containing telRL site of bacteriophage N15 has been cloned in the vector plasmid pUC19. The nucleotide sequence of a small region from EcoRV-PstI fragment has been defined by Maxam-Gilbert technique. The analysis of the obtained sequence has shown the telRL site to be a nonideal palindrome (the size of 56 nucleotide ops) in which two nucleotide pairs differ in the positions 12 and 14 on both sides of the palindrome centre. The DNA region with alteration of purines and pyrimidines (GC) surrounded by AT-rich regions: 5'-ATTATACGCGCGTATAAT-3'--in the symmetry centre of palindrome is characteristic of the telRL site structure. This characteristic of the region may play a key role in recognition of the site by the specific enzyme at formation of linear prophage-plasmid during lysogenization.     

Sequence specificity of the deoxyribonuclease activity of 1,10-phenanthroline-copper ion

A statistical analysis of a data set composed of over 1600 scission events of DNA produced by the 2:1 1,10-phenanthroline-copper complex (OP-Cu) has demonstrated that the nucleotide 5' to the site of phosphodiester bond scission is a primary influence in the kinetics of cleavage at any sequence position. The scission was less affected by the 3' neighbor. For each of the sixteen possible dinucleotides, a kinetic parameter can be computed reflecting scission at the 3' nucleotide. When used to predict the scission pattern of a DNA sequence not part of the present data set, correlation coefficients of about 0.6 between predicted and observed patterns were obtained.     

Determination of the primary structure of oligodeoxyribonucleotides containing P-S-C(5') bonds

Analogues of oligodeoxynucleotides with P-S-C(5') bonds, which, due to their unusual substrate properties, may find interesting applications in molecular biology, can not be structurally analysed by the Maxam-Gilbert or Sanger (fingerprinting) methods. We, therefore, devised a modification of the fingerprinting technique making possible the sequence determination of these analogues.     

Defining the sequence recognized with BmFTZ-F1, a sequence specific DNA binding factor in the silkworm, Bombyx mori, as revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis.

BmFTZ-F1 is a Bombyx mori homologue of FTZ-F1, a positive regulator of the fushi tarazu gene of Drosophila melanogaster. In order to determine the sequence recognized with this factor, we made three sets of oligonucleotide mixture which contain 4 possible nucleotides at different positions within the previously proposed 12-bp binding consensus sequence. Oligonucleotides which bound to purified BmFTZ-F1 were separated by a gel mobility shift procedure and a binding sequence was determined by direct sequencing through Maxam-Gilbert method. By this analysis, 7 positions showed clear sequence preference and 5 positions showed weak or no sequence preference. The importance of each nucleotide at each position was confirmed by a gel mobility shift competition analysis and results were presented as a quantitative difference in the binding affinity. From these analyses, we conclude that the best binding sequence of BmFTZ-F1 is 5'-PyCAAGGPyCPu-3'. This method may be useful for the determination of a binding sequence of other sequence specific DNA binding factor.     

Enzymic detection of uracil in a cloned and sequenced deoxyribonucleic acid segment

Uracil can occur in DNA either as a result of utilization of dUTP by DNA polymerases or from in situ deamination of cytosine. The latter results in transition mutations following the next round of replication. We describe a technique for the detection of uracil in DNA by a modification of the Maxam-Gilbert sequencing procedure. Reaction of end-labeled DNA with uracil-DNA glycosylase followed by 1 M piperidine results in scission products corresponding to locations of uracils. These are detected by autoradiography following electrophoresis through a sequencing gel. Comparison of these scission products with the DNA sequences elucidates the mechanism of origin of the DNA uracils. The technique was tested with a cloned human DNA sequence grown in a dut-,ung-strain of Escherichia coli, which incorporates uracil in place of thymine in its DNA, and by chemical deamination of cytosines in that sequence. The technique was expanded by use of alkaline and enzymic probes to investigate possible accumulation of uracil, base losses, and other modifications in human liver and brain DNA. No damaged DNA moieties were detected. This method is applicable to the study of any recoverable reiterated sequence by any enzyme preparation that can recognize modifications in DNA.     

Primary structure of fragment 7 of the influenza virus A/USSR/90/77(H1N1) RNA

The double-stranded DNA copy of the matrix protein (M) gene of the influenza virus A/USSR/90/77 (H1N1) has been inserted in PstI site of plasmid pBR322 and cloned in E. coli. The full-length DNA copy of the M gene has been sequenced using Maxam-Gilbert method. Analysis of nucleotide sequence of segment 7 RNA of influenza virus A/USSR/90/77 and nucleotide substitutions, as compared with known primary structures of segment 7 RNA for other strains, is presented. A hypothetical model of secondary structure of segment 7 RNA of influenza virus and repeating sequences at nucleotide and amino acid levels, revealed in the central region of M1 protein, are discussed.     

1-Tuple DNA sequencing: computer analysis

A new method of DNA reading was proposed at the end of 1988 by Lysov et al. According to the authors' claims it has certain advantages as compared to the Maxam-Gilbert and Sanger methods, which are revealed by automation and rapidity of DNA sequencing. Nevertheless its employment is hampered by a number of biological and mathematical problems. The present study proposes an algorithm that allows to overcome the computational difficulties occurring in the course of the method during reconstruction of the DNA sequence by its l-tuple composition. It is shown also that the biochemical problems connected with the loss of information about the l-tuple DNA composition during hybridization are not crucial and can be overcome by finding the maximal flow of minimal cost in the special graph.     

Specific binding of o-phenanthroline at a DNA structural lesion.

DNA intercalators are found to recognize a DNA lesion as a high affinity receptor site. This lesion-specific binding is observed when one strand of a DNA double helix contains an extra, unpaired nucleotide. Our assay for binding controls for the effects of sequence with a series of oligodeoxynucleotide duplexes which are identical except for the location of the lesion, an extra cytidine. Scission of the series of oligodeoxynucleotides by the cuprous complex of ortho-phenanthroline (OP-Cu) indicates that OP-Cu binds at the lesion-specific stable intercalation site, suggesting that OP-Cu intercalates into DNA. The dispersion of OP-Cu scission sites over three residues is consistent with scission via a diffusible intermediate. The location of the scission sites, directly on the 3' side of the lesion, is consistent with minor groove binding in B DNA.     

Sequence-specific spin labeling of DNA

Several DNA fragments deriving from plasmid pBR322 were used to determine the modification sites caused by the reaction with alkylating spin-labeling probes. At a high spin-label concentration, all guanines became alkylated, causing the cleavage of the phosphodiester bonds upon the treatment with piperidine. The lengths of the breakage products of 5'-end labeled DNA treated with spin labels were compared with the length of DNA scission products generated by Maxam-Gilbert procedure for DNA sequence analysis. The distribution of the guanine modifications is dependent on the amount of the reagent used for the alkylation and the ionic conditions of the reaction. The frequency of alkylation by spin labels was greatly enhanced within continuous runs of guanines in DNA. The stabilization of the DNA structure by magnesium or spermine directs the spin-label binding specifically to the most exposed region of DNA fragment containing GGTGG sequence. The sequence-dependent interaction of spin labels with DNA enables the development of the method for the selective spin labeling of DNA molecule.     

Site-specific analysis of drug interactions and damage in DNA using sequencing techniques

DNA-sequencing techniques can be adapted to provide powerful analytical tools for pinpointing the sites at which DNA is modified by either radiations or chemicals. Base modifications, covalent adducts, crosslinks, and noncovalent binding can all be detected. This review outlines the adaptions of the Maxam-Gilbert and Sanger dideoxy sequencing techniques which make such studies possible. Practical aspects and limitations of the various methods are given. Assays which test the ability of DNA polymerase to bypass damage and misincorporate bases are also discussed. It is concluded that these techniques constitute the most powerful method currently available for the study of sequence-specific DNA interactions.     

High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley

An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

APEX disease gene resequencing: mutations in exon 7 of the p53 tumor suppressor gene.

Detection of mutations in disease genes will be a significant application of genomic research. Methods for detecting mutations at the single nucleotide level are required in highly mutated genes such as the tumor suppressor p53. Resequencing of an individual patient's DNA by conventional Sanger methods is impractical, calling for novel methods for sequence analysis. Toward this end, an arrayed primer extension (APEX) method for identifying sequence alterations in primary DNA structure was developed. A two-dimensional array of immobilized primers (DNA chip) was fabricated to scan p53 exon 7 by single bases. Primers were immobilized with 200 microm spacing on a glass support. Oligonucleotide templates of length 72 were used to study individual APEX resequencing reactions. A template-dependent DNA polymerase extension was performed on the chip using fluorescein-labeled dideoxynucleotides (ddNTPs). Labeled primers were evanescently excited and the induced fluorescence was imaged by CCD. The average signal-to-noise ratio (S/N) observed was 30:1. Software was developed to analyze high-density DNA chips for sequence alterations. Deletion, insertion, and substitution mutations were detected. APEX can be used to scan for any mutation (up to two-base insertions) in a known region of DNA by fabricating a DNA chip comprising complementary primers addressing each nucleotide in the wild-type sequence. Since APEX is a parallel method for determining DNA sequence, the time required to assay a region is independent of its length. APEX has a high level of accuracy, is sequence-based, and can be miniaturized to analyze a large DNA region with minimal reagents.     

Nucleotide sequence of the genes for β and ε subunits of proton-translocating ATPase from Escherichia coli

The 1855-nucleotide long DNA sequence of part of the gene cluster for the proton-translocating ATPase from $\text{E. coli}$ was determined by the method of Maxam-Gilbert. The sequence covers the genes for the β and ε subunits of F₁ along with the flanking region. The amino acid sequence of these subunits deduced from the nucleotide sequence indicates that the β and ε subunits have 459 and 138 amino acids, respectively. The possible secondary structure of the both subunits was estimated from the deduced primary structures. A possible nucleotide binding site in the β subunit is also discussed on the basis of the primary and secondary structures. The codons used in the genes for all the components of F₁F₀ were different in different genes, suggesting that the amount of each subunit in the F₁F₀ is determined to some extent on a translational level.     

DNAse footprinting: a simple method for the detection of protein-DNA binding specificity.

A method for studying the sequence-specific binding of proteins to DBA is described. The technique is a simple conjoining of the Maxam-Gilbert DNA-sequencing method and the technique of DNAase-protected fragment isolation. Fragments of a 5' end-labelled, double-stranded DNA segment, partially degraded by DNAase in the presence and absence of the binding protein, are visualized by electrophoresis and autoradiography alongside the base-specific reaction products of the Maxam-Gilbert sequencing method. It is then possible to see the protective "footprint" of the binding protein on the DNA sequence. The binding of lac repressor to lac operator is visualized by "footprinting" as an example. Equillibrium estimates indicate that 10-fold sequence-specificity (differential binding constant) could be studied easily using this technique.     

The nucleotide sequence of bacteriophage φX174

The complete nucleotide sequence of the DNA of bacteriophage φX174 has been determined. The provisional sequence (Sanger et al., 1977a) deduced largely by the plus and minus method, has been completed and confirmed, predominantly using the terminator method (Sanger et al., 1977b). About 30 revisions were found to be necessary in the 5386-nucleotide sequence. The amino acid sequences of the ten proteins for which the DNA codes have also been deduced.     

Investigation of restriction-modification enzymes from M. varians RFL19 with a new type of specificity toward modification of substrate.

The characterization of MvaI restriction-modification enzymes, isolated from Micrococcus varians RFL19, is reported. Both enzymes recognize the 5'CC decreases (A/T)GG nucleotide sequence. The endonuclease cleaves the sequence at the position indicated by the arrow, whereas the methylase modifies the internal cytosine, yielding N4-methylcytosine. This type of modification protects the substrate from R.MvaI cleavage. 5-Methylcytosine in the same position of the recognition sequence does not protect the substrate from R.MvaI cleavage. R.MvaI proved to be the first example of a restriction endonuclease differentiating the position of the methyl group in the heterocyclic ring of cytosine, located in the same site of the recognition sequence. M.MvaI modifies DNA dcm+ in vitro yielding N4,5-dimethylcytosine. N4-methylcytosine cannot be differentiated from cytosine using the Maxam-Gilbert DNA sequencing procedure.     

Specific-primer-directed DNA sequencing

A simple and rapid strategy for DNA sequence analysis based on the Sanger chain-termination method is described. This procedure utilizes full-sized inserts of 1 to 4 kb of DNA cloned into M13 bacteriophage vectors. After the sequence of the first 600-650 bp of the insert DNA has been determined with the commercially available universal vector primer, a specific oligonucleotide is synthesized utilizing the sequence data obtained from the 3' end of the sequence and used as a primer to extend the sequence analysis for another 600-650 nucleotides. Additional primers are synthesized in a similar manner until the nucleotide sequence of the entire insert DNA has been determined. General guidelines for the selection of oligonucleotide length and composition and the use of unpurified primers are discussed. The use of the specific-primer-directed approach to dideoxynucleotide sequence analysis, in association with highly purified single-stranded template DNA, reduces considerably the time required for the analysis of large segments of DNA.