A single amino acid substitution in B subunit of Escherichia coli enterotoxin affects its oligomer formation

We isolated a mutant strain of enterotoxigenic Escherichia coli by nitrosoguanidine mutagenesis, which produces an immunologically altered B subunit of heat-labile enterotoxin. This mutant B subunit was detected as a monomer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis even without prior heating, suggesting a problem in oligomer formation. Furthermore, this mutant B subunit could not form holotoxin with the native A subunit, and the affinity to GM1-ganglioside receptor was 10-fold lower than that of the native B subunit. The amino acid sequence analysis of this mutant B subunit revealed only one amino acid substitution compared with the native B subunit, at the 64th position from the N terminus (valine instead of alanine). These data suggest that the alanine at position 64 from the N terminus is important for the native B subunit to form an oligomer structure and express its functions.     

Site-directed mutagenesis: Generation of an extracistronic mutation in bacteriophage Qβ RNA

The preparation in vitro and chemical characterization of bacteriophage Qβ RNA with an extracistronic mutation, a G → A transition in the 16th position from the 3′-terminus, is described. The 5′-terminal region of the Qβ minus strand was synthesized in vitro up to position 14 (inclusive) by using ATP and GTP as the only substrates. The mutagenic nucleotide analog N⁴-hydroxyCMP was then incorporated into position 15 instead of CMP. The minus strand was completed with the four standard ribonucleoside triphosphates, purified and used as a template for the synthesis of plus strands. Of the plus strand product, 33% had a G → A transition in the 16th position from the 3′-end (which corresponds to position 15 of the minus strand), as shown by nucleotide sequence analysis of the terminal T₁ oligonucleotide. The modified RNA was efficiently replicated by Qβ replicase and a preparation containing 55% of the mutant RNA was obtained.The general approach to directed mutagenesis outlined above should allow the introduction of mutations into the 5′ and 3′-terminal regions of Qβ RNA as well as into the intercistronic sequences.     

Quantifying DNA-protein interactions by double-stranded DNA arrays.

We have created double-stranded oligonucleotide arrays to perform highly parallel investigations of DNA-protein interactions. Arrays of single-stranded DNA oligonucleotides, synthesized by a combination of photolithography and solid-state chemistry, have been used for a variety of applications, including large-scale mRNA expression monitoring, genotyping, and sequence-variation analysis. We converted a single-stranded to a double-stranded array by synthesizing a constant sequence at every position on an array and then annealing and enzymatically extending a complementary primer. The efficiency of second-strand synthesis was demonstrated by incorporation of fluorescently labeled dNTPs (2'-deoxyribonucleoside 5'-triphosphates) and by terminal transferase addition of a fluorescently labeled ddNTP. The accuracy of second-strand synthesis was demonstrated by digestion of the arrayed double-stranded DNA (dsDNA) on the array with sequence-specific restriction enzymes. We showed dam methylation of dsDNA arrays by digestion with DpnI, which cleaves when its recognition site is methylated. This digestion demonstrated that the dsDNA arrays can be further biochemically modified and that the DNA is accessible for interaction with DNA-binding proteins. This dsDNA array approach could be extended to explore the spectrum of sequence-specific protein binding sites in genomes.     

DNA detection system using molecularly imprinted polymer as the gel matrix in electrophoresis

To develop a simple and inexpensive method for DNA detection, we prepared a molecularly imprinted polymer (MIP) for recognizing a specific double-stranded DNA (dsDNA) sequence and used it in an electrophoretic gel matrix. The MIP gel has many binding sites that are complementary in size, shape, and arrangement of functional groups of the target dsDNA sequence. During MIP gel electrophoresis (MIPGE), migration of the target dsDNA should be hindered by the capture effect of the binding sites in the MIP gel. This was confirmed by observation of deviations from the linear relationship between the migration distances of the DNA standard size markers in the polyacrylamide gel and those in the MIP gel. The migration distances of nontarget dsDNA maintained a linear relationship, however. In addition, the sequence selectivity of dsDNA in this method was investigated by using the Ha-ras gene and its point mutants. Except for A.T to T.A base pair substitution, mutant dsDNA (for example, substitution from A.T to C.G and from G.C to T.A) could be distinguished from the target (wild-type) dsDNA. Although some improvement in A.T (T.A) base pair distinction is still needed, this study is the first to demonstrate detection of a specific dsDNA sequence with MIPs and, as such, opens up a new realm for practical applications of MIPs.     

FIMO: scanning for occurrences of a given motif

A motif is a short DNA or protein sequence that contributes to the biological function of the sequence in which it resides. Over the past several decades, many computational methods have been described for identifying, characterizing and searching with sequence motifs. Critical to nearly any motif-based sequence analysis pipeline is the ability to scan a sequence database for occurrences of a given motif described by a position-specific frequency matrix. RESULTS: We describe Find Individual Motif Occurrences (FIMO), a software tool for scanning DNA or protein sequences with motifs described as position-specific scoring matrices. The program computes a log-likelihood ratio score for each position in a given sequence database, uses established dynamic programming methods to convert this score to a P-value and then applies false discovery rate analysis to estimate a q-value for each position in the given sequence. FIMO provides output in a variety of formats, including HTML, XML and several Santa Cruz Genome Browser formats. The program is efficient, allowing for the scanning of DNA sequences at a rate of 3.5 Mb/s on a single CPU. Availability and Implementation: FIMO is part of the MEME Suite software toolkit. A web server and source code are available at http://meme.sdsc.edu.     

Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon

Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.     

Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level

Fluorescent labeling of a short sequence of double-stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem–loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand-induced triple helix formation, its extremities hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non-repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 ± 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules.     

Mutational analysis of amiloride sensitivity of the NhaA Na+/H+ antiporter from Vibrio parahaemolyticus.

The activity of the NhaA Na+/H+ antiporter of Vibrio parahaemolyticus is inhibited by amiloride. We found an amino acid sequence in the NhaA that was identical to a putative amiloride binding domain of the Na+/H+ exchanger in mammalian cells. We constructed mutant NhaAs that had amino acid substitutions in the putative amiloride binding domain by site-directed mutagenesis. These include V62L (Val62 replaced by Leu), F63Y, F64Y, and L65F. Most mutant NhaAs showed decreased sensitivity for amiloride. Among these, the F64Y mutant NhaA showed the least amiloride sensitivity, with a Ki value 7 to 10 times greater than that in the wild type. Thus, the sequence between residues V62 and L65 in NhaA, especially F64, is very important for the inhibitory effect of amiloride on the antiporter.     

Bacteriophage ST64B, a genetic mosaic of genes from diverse sources isolated from Salmonella enterica serovar typhimurium DT 64

The complete sequence of the double-stranded DNA (dsDNA) genome of the Salmonella enterica serovar Typhimurium ST64B bacteriophage was determined. The 40,149-bp genomic sequence of ST64B has an overall G+C content of 51.3% and is distinct from that of P22. The genome architecture is similar to that of the lambdoid phages, particularly that of coliphage lambda. Most of the putative tail genes showed sequence similarity to tail genes of Mu, a nonlambdoid phage. In addition, it is likely that these tail genes are not expressed due to insertions of fragments of genes related to virulence within some of the open reading frames. This, together with the inability of ST64B to produce plaques on a wide range of isolates, suggests that ST64B is a defective phage. In contrast to the tail genes, most of the head genes showed similarity to those of the lambdoid phages HK97 and HK022, but these head genes also have significant sequence similarities to those of several other dsDNA phages infecting diverse bacterial hosts, including Escherichia, Pseudomonas, Agrobacterium, Caulobacter, Mesorhizobium, and Streptomyces: This suggests that ST64B is a genetic mosaic that has acquired significant portions of its genome from sources outside the genus Salmonella.     

A strategy for single site PCR amplification of dsDNA: priming digested cloned or genomic DNA from an anchor-modified restriction site and a short internal sequence

Amplification of dsDNA by polymerase chain reaction (PCR) has been limited to those instances in which segments of known sequence flank the fragment to be amplified. A strategy for the PCR amplification of cloned or genomic dsDNA that necessitates sequence information from only a single short segment (single site PCR) has been devised. The region of known sequence may be located at any position within or adjacent to the segment to be amplified. The basic procedure for amplification consists of 1) digestion of dsDNA with one or more restriction enzymes, 2) ligation with a universal anchor adaptor and 3) PCR amplification using an anchor primer and the primer for the single site of known sequence. The anchor adaptor is designed in such a way as to facilitate the amplification of only those fragments containing the sequence of interest. We have demonstrated the utility of this technique by specifically amplifying and directly sequencing antibody variable region genes from cloned dsDNA and from genomic DNA.     

PSIC: profile extraction from sequence alignments with position-specific counts of independent observations.

Sequence weighting techniques are aimed at balancing redundant observed information from subsets of similar sequences in multiple alignments. Traditional approaches apply the same weight to all positions of a given sequence, hence equal efficiency of phylogenetic changes is assumed along the whole sequence. This restrictive assumption is not required for the new method PSIC (position-specific independent counts) described in this paper. The number of independent observations (counts) of an amino acid type at a given alignment position is calculated from the overall similarity of the sequences that share the amino acid type at this position with the help of statistical concepts. This approach allows the fast computation of position-specific sequence weights even for alignments containing hundreds of sequences. The PSIC approach has been applied to profile extraction and to the fold family assignment of protein sequences with known structures. Our method was shown to be very productive in finding distantly related sequences and more powerful than Hidden Markov Models or the profile methods in WiseTools and PSI-BLAST in many cases. The profile extraction routine is available on the WWW (http://www.bork.embl-heidelberg. de/PSIC or http://www.imb.ac.ru/PSIC).     

Homology-extended sequence alignment

We present a profile–profile multiple alignment strategy that uses database searching to collect homologues for each sequence in a given set, in order to enrich their available evolutionary information for the alignment. For each of the alignment sequences, the putative homologous sequences that score above a pre-defined threshold are incorporated into a position-specific pre-alignment profile. The enriched position-specific profile is used for standard progressive alignment, thereby more accurately describing the characteristic features of the given sequence set. We show that owing to the incorporation of the pre-alignment information into a standard progressive multiple alignment routine, the alignment quality between distant sequences increases significantly and outperforms state-of-the-art methods, such as T-COFFEE and MUSCLE. We also show that although entirely sequence-based, our novel strategy is better at aligning distant sequences when compared with a recent contact-based alignment method. Therefore, our pre-alignment profile strategy should be advantageous for applications that rely on high alignment accuracy such as local structure prediction, comparative modelling and threading.     

Site-specific cleavage of double-strand DNA by hydroperoxide of linoleic acid

The breakage of double-strand (ds) DNA by 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid (LAHPO) was investigated by agarose gel electrophoresis of supercoiled pBR322 DNA and the site of cleavage on the DNA molecule was determined by the method of DNA sequence analysis using 3'-end and 5'-end-labeled DNA fragments as substrates. LAHPO caused cleavage at the position of guanine nucleotide in dsDNA. LAHPO caused dsDNA breaks at specific sites, but linoleic acid (LA) and 13-L-hydroxy-cis-9,trans-11-octadecadienoic acid (LAHO) have no such effects on dsDNA. The active oxygen atom of the hydroperoxy group of LAHPO was perhaps responsible for the site-specific cleavage of dsDNA.     

cDNA cloning and sequencing of tobacco chloroplast ribosomal protein L12

Tobacco chloroplast ribosomal protein L12 was isolated as a ssDNA-cellulose-binding protein from a chloroplast soluble protein fraction. Based on the N-terminal amino acid sequence of chloroplast L12, a cDNA clone was isolated and characterized. The precursor protein deduced from the DNA sequence consists of a transit peptide of 53 amino acid residues and a mature L12 protein of 133 amino acid residues. The chloroplast L12 protein was synthesized with a reticulocyte lysate and subjected to nucleic acid-binding assays. L12 synthesized in vitro does not bind to ssDNA, dsDNA nor ribonucleotide homopolymers, but it binds to cellulose matrix.     

A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe.

A small and highly fluorescent non-natural amino acid that contains an anthraniloyl group (atnDap) was incorporated into various positions of streptavidin. The positions were directed by a CGGG/CCCG four-base codon/anticodon pair. The non-natural mutants were obtained in excellent yields and some of them retained strong biotin-binding activity. The fluorescence wavelength as well as the intensity of the anthraniloyl group at position 120 were sensitive to biotin binding. These unique properties indicate that the atnDap is the most suitable non-natural amino acid for a position-specific fluorescent labeling of proteins that is highly sensitive to microenvironmental changes.     

Cloning of soybean leghemoglobin structural gene sequences synthesized in vitro.

Double-stranded soybean leghemoglobin DNA was synthesized from leghemoglobin mRNA isolated from soybean nodules. The dsDNA was inserted into the Bam H1 site of plasmid pBR322 using the poly-dAT-joiner method. A cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The restriction cleavage map and the DNA sequence of a selected part of the inserted DNA are in complete accordance with the amino-acid sequence of soybean leghemoglobin.     

Novel biosensor system model based on fluorescence quenching by a fluorescent streptavidin and carbazole‐labeled biotin

In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole‐labeled biotin. BODIPY‐FL‐aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position‐specifically incorporated into Trp120 position of streptavidin by four‐base codon method. On the other hand, carbazole‐labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole‐labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole‐labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole‐labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd.     

Dimerization-based homogeneous fluorosensor proteins for the detection of specific dsDNA

While there are many hybridization-based DNA sensors, none of them can detect native double-stranded DNA (dsDNA), which is most commonly found in physiological conditions. Here we made novel fluorosensor proteins comprised of a pair of two zinc fingers with an N-terminal dimerization motif and a C-terminal GFP variant to detect specific dsDNA sequence in a homogeneous solution. When a pair of purified zinc finger-GFP color variant proteins (Zif12-eCFP, Zif12-eYFP) were mixed and added with specific dsDNA with 12 bp inverted repeat (IR), fluorescence spectra of the solution showed significant concentration-dependent enhancement of fluorescence resonance energy transfer (FRET), with the detection limit of approximately 10nM. No significant change in FRET was observed if nonspecific DNA was added, indicating dsDNA-dependent dimerization of the two proteins. This dimerization-based dsDNA sensors will have a range of applications where conventional hybridization-based assay is difficult.