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At least 1,531 records · Page 85Linked to original sources
PubMed1996

An algorithm to analyse the hydrolysis pathway of peptides and proteins by sequence analyses of unfractionated digestion mixtures.

We have designed and implemented on a personal computer a program for identifying and quantifying the fragments present in a peptide mixture obtained by hydrolysing a polypeptide of known sequence using digesting agents. The qualitative data utilized by the main algorithm consist of the target sequence of the intact molecule and the amino acid residues identified at each step of the automatic sequence analysis of the unfractionated digestion mixture. In this way, the sequence of each fragment present in the mixture is quickly reconstructed. Furthermore, if the quantitative data of the amino acid residues identified at each step of the sequence analysis are utilized, the program will correlate the sequence of each fragment to its amount. We furnish an example of the application intended for the rapid identification and characterization of the extracellular proteinases produced by a basidiomycete fungus, utilizing the bovine insulin beta-chain as target substrate. A variety of uses for the method are discussed.

Algorithms↗
PubMed1999

Design of modified oligodeoxyribonucleotide probes to detect telomere repeat sequences in FISH assays.

A series of dye-labeled oligonucleotide probes containing base and sugar modifications were tested for the ability to detect telomeric repeat sequences in FISH assays. These modified oligonucleotides, all 18 nt in length, were complementary to either the cytidine-rich (C(3)TA(2))(n)or guanosine-rich (T(2)AG(3))(n)telomere target sequences. Oligonucleotides were modified to either increase target affinity by enhancing duplex stability [2'-OMe ribose sugars and 5-(1-propynyl)pyrimidine residues] or inhibit the formation of inter- or intramolecular structures (7-deazaguanosine and 6-thioguanosine residues), which might interfere with binding to the target. Several dye-labeled oligonucleotide probes were found that could effectively stain the telomeric repeat sequences of either cytidine- or guanosine-rich strands in a specific manner. Such probes could be used as an alternative to peptide nucleic acids for investigating the dynamics of telomere length and maintenance. In principle, these relatively inexpensive and readily synthesized modified oligonucleotides could be used for other FISH-related assays.

Base Sequence↗
PubMed1991

Sequence-specific alkylation of dsDNA with derivatives of pyrimidine oligonucleotides conjugated to 2-chloroethylamine groups.

Reaction of homopyrimidine oligonucleotides bearing a 5'-terminal alkylating aromatic 2-chloroethyl-amino group with a bovine papilloma vector expressing human interferon-gamma was investigated. The oligonucleotide derivatives bound to corresponding homopurine-homopyrimidine sequences in dsDNA and alkylated guanosine residues at these sites in the purine strand of the target. The alkylated DNA can be cleaved at the modified residues. At pH 5.4, the reaction was highly specific to the target sequences; at pH less than 5, some nonspecific reactions were observed at the sequences partially complementary to the oligonucleotides. Elongation of the linker between the alkylating group and the oligonucleotide phosphate increased the alkylation efficiency. Repeated treatment of the DNA with gradually increased concentrations of the reagent resulted in quantitative modification of the target guanosines.

Alkylation↗
PubMed2005

Issues in high-throughput comparative modelling: a case study using the ubiquitin E2 conjugating enzymes.

Sequences of the ubiquitin-conjugating enzyme (UBC or E2) family were used as a test set to investigate issues associated with the high-throughput comparative modelling of protein structures. A semi-automatic method was initially developed with particular emphasis on producing models of a quality suitable for structural comparison. Structural and sequence features of the E2 family were used to improve the sequence alignment and the quality of the structural templates. Initially, failure to correct for subtle structural inconsistencies between templates lead to problems in the comparative analysis of the UBC electrostatic potentials. Modelling of known UBC structures using Modeller 4.0 showed that multiple templates produced, on average, no better models than the use of just one template, as judged by the root-mean-squared deviation between the comparative model and crystal structure backbones. Using four different quality-checking methods, for a given target sequence, it was not possible to distinguish the model most similar to the experimental structure. The UBC models were thus finally modelled using only the crystal structure template with the highest sequence identity to the target to be modelled, and producing only one model solution. Quality checking was used to reject models with obvious structural anomalies (e.g., bad side-chain packing). The resulting models have been used for a comparison of UBC structural features and of their electrostatic potentials. The work was extended through the development of a fully automated pipeline that identifies E2 sequences in the sequence databases, aligns and models them, and calculates the associated electrostatic potential.

Amino Acid Motifs↗
PubMed2004

Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA).

Nucleic acid sequence based amplification (NASBA) is an isothermal nucleic acid amplification procedure based on target-specific primers and probes, and the co-ordinated activity of 3 enzymes: AMV reverse transcriptase, RNase H, and T7 RNA polymerase. We have developed a real-time NASBA procedure for detection of piscine nodaviruses, which have emerged as major pathogens of marine fish. Viral RNA was isolated by guanidine thiocyanate lysis followed by purification on silica particles. Primers were designed to target sequences in the nodavirus capsid protein gene, yielding an amplification product of 120 nucleotides. Amplification products were detected in real-time with a molecular beacon (FAM labelled/methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. Based on the detection of cell culture-derived nodavirus, and a synthetic RNA target, the real-time NASBA procedure was approximately 100-fold more sensitive than single-tube RT-PCR. When used to test a panel of 37 clinical samples (negative, n = 18; positive, n = 19), the real-time NASBA assay correctly identified all 18 negative and 19 positive samples. In comparison, the RT-PCR procedure identified all 18 negative samples, but only 16 of the positive samples. These results suggest that real-time NASBA may represent a sensitive and specific diagnostic procedure for piscine nodaviruses.

Animals↗
PubMed2003

Reduced expression of aconitase results in an enhanced rate of photosynthesis and marked shifts in carbon partitioning in illuminated leaves of wild species tomato.

Wild species tomato (Lycopersicon pennellii) plants bearing a genetic lesion in the gene encoding aconitase (Aco-1; aconitate hydratase EC 4.2.1.3) were characterized at molecular and biochemical levels. The genetic basis of this lesion was revealed by cloning the wild-type and mutant alleles. The mutation resulted in lowered expression of the Aco-1 transcript and lowered levels of both cytosolic and mitochondrial aconitase protein and activity. After in silico analysis, we concluded that in the absence of a recognizable target sequence, the best explanation for the dual location of this protein is inefficient targeting. Biochemical analysis of leaves of the Aco-1 accession suggested that they exhibited a restricted flux through the Krebs cycle and reduced levels of Krebs cycle intermediates but were characterized by elevated adenylate levels and an enhanced rate of CO2 assimilation. Furthermore, the analysis of both steady-state metabolite levels and metabolic fluxes revealed that this accession also exhibited elevated rates of photosynthetic Suc synthesis and a corresponding increase in fruit yield. Therefore, we conclude that the Krebs cycle normally competes with the Suc synthetic pathway for carbon but is not essential for the supply of energy to fuel the operation of this pathway.

Aconitate Hydratase↗
PubMed2001

Recognition of ATGA sequences by the unfused aromatic dication DB293 forming stacked dimers in the DNA minor groove.

Furamidine and related diamidines represent a promising series of drugs active against widespread parasites, in particular the Pneumocystic carinii pathogen. In this series, the phenylfuranbenzimidazole diamidine derivative DB293 was recently identified as the first unfused aromatic dication capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Here we present a detailed biochemical and biophysical characterization of the DNA sequence recognition properties of DB293. Three complementary footprinting techniques using DNase I, Fe(II)-EDTA, and an anthraquinone photonuclease were employed to locate binding sites for DB293 in different DNA restriction fragments. Two categories of sites were identified by DNase I footprinting: (i) 4/5 bp sequences containing contiguous A.T pairs, such as 5'-AAAA and 5'-ATTA; and (ii) sequences including the motif 5'-ATGA.5'-TCAT. In particular, a 13-bp sequence including two contiguous ATGA motifs provided a highly preferential recognition site for DB293. Quantitative footprinting analysis revealed better occupancy of the 5'-ATGA site compared to the AT-rich sites. Preferential binding of DB293 to ATGA sites was also observed with other DNA fragments and was confirmed independently by means of hydroxyl radical footprinting generated by the Fe(II)-EDTA system, as well as by a photofootprinting approach using the probe anthraquinone-2-sulfonate (AQS). In addition, this photosensitive reagent revealed the presence of sites of enhanced cutting specific to DB293. This molecule, but not other minor groove binders such as netropsin, induces specific local structural changes in DNA near certain binding sites, as independently shown by DNase I and the AQS probe. Recognition of the ATGA sequence by DB293 was investigated further using melting temperature experiments and surface plasmon resonance (SPR). The use of different hairpin oligonucleotides showed that DB293 can interact with AT sites via the formation of 1:1 drug-DNA complexes but binds much more strongly, and cooperatively, to ATGA-containing sequences to form 2:1 drug-DNA complexes. DB293 binds strongly to ATGA sequences with no significant context dependence but is highly sensitive to the orientation of the target sequence. The formation of 2:1 DB293/DNA complexes is abolished by reversing the sequence 5'-ATGA-->3'-ATGA, indicating that directionality plays an important role in the drug-DNA recognition process. Similarly, a single mutation in the A[T-->G]GA sequence is very detrimental to the dimer interactions of DB293. From the complementary footprinting and SPR data, the 5'-ATGA sequence is identified as being a highly favored dimer binding site for DB293. The data provide clues for delineating a recognition code for diamidine-type minor groove binding agents, and ultimately to guide the rational design of gene regulatory molecules targeted to specific sites of the genetic material.

Antifungal Agents↗
PubMed1993

Use of polymerase chain reaction and electroporation of Escherichia coli to monitor the persistence of extracellular plasmid DNA introduced into natural soils.

A modified protocol for DNA amplification by polymerase chain reaction (PCR) coupled with laser densitometric determination of the amount of PCR products, which allowed quantitation of target sequence numbers in soil extracts, was developed. The method was applied to monitor target loss during incubation of purified plasmid DNA in natural nonsterile soils. It revealed soil-specific kinetics of target loss. After 60 days, 0.2, 0.05, and 0.01% of the initially added nahA genes on plasmids were detectable by PCR in a loamy sand soil, a clay soil, and a silty clay soil, respectively. Electroporation of Escherichia coli was used in parallel to quantitate plasmid molecules in soil extracts by their transforming activity. It was found that transformation by electroporation was about 20 times more efficient and much less inhibited by constituents of soil extracts than transformation of Ca(2+)-treated cells (G. Romanowski, M.G. Lorenz, G. Sayler, and W. Wackernagel, Appl. Environ. Microbiol. 58:3012-3019, 1992). By electroporation, greater than 10,000-fold plasmid loss was monitored in nonsterile soils. Transforming activity was found up to 60 days after inoculation of the soils. The studies indicate that PCR and electroporation are sensitive methods for monitoring the persistence of extracellular plasmid DNA in soil. It is proposed that plasmid transformation by electroporation can be used for the monitoring in soil and other environments of genetically engineered organisms with recombinant plasmids. The data suggest that genetic material may persist in soil for weeks and even for months after its release from cells.

Base Sequence↗
PubMed1989

Transcriptional occlusion of transposon targets.

In Salmonella typhimurium, insertion of transposons Tn5, Tn10 and bacteriophage Mu is inhibited by transcription of some target sequences. The transcription effects on Tn5 are large when the lac operon is a target but are limited to a slight effect on the hisG gene of the his operon. The Tn10 element shows target occlusion in both operons. Phage Mu has been shown previously to be inhibited for insertion into the lac operon. In the his operon Mu is only inhibited for insertion into the hisG gene. The variability of the inhibition effect from one sequence to another suggests site or regional specificity for transcription effects. Reducing the probability of insertion into transcribed sequences may be of selective importance to transposons since it reduces the risk of killing the host while maintaining the ability to transpose.

DNA Transposable Elements↗
PubMed1992

In vitro resolution of poxvirus replicative intermediates into linear minichromosomes with hairpin termini by a virally induced Holliday junction endonuclease.

Available evidence suggests that one or more late viral gene products are involved in processing poxvirus replicative intermediates into mature progeny hairpin-terminated genomes. Cloned versions of the Shope fibroma virus (SFV) replicated telomere in the inverted repeat configuration were used as substrates to assay lysates from poxvirus-infected cells for protein fractions that participate in the resolution of the circular substrate plasmid into a linear minichromosome with viral hairpin termini. An activity in a crude protein fraction obtained from vaccinia virus-infected cells at late times during the replicative cycle was capable of accurately resolving all poxviral inverted repeat replicative intermediates tested. The resolved linear products are identical to the products of in vivo resolution and possessed symmetrical nicks which mapped at the borders of the inverted repeat sequence. Strand-specific nicks were also identified, which mapped within the telomere resolution target sequence known to be required for telomere resolution in vivo. The resolving activity that we have identified is specific to virus-infected cells at late times during replication and cleaves cloned poxviral telomeric substrates in a fashion expected of a classic Holliday junction-resolving enzyme in addition to possessing a telomere resolution target-specific nicking activity. Although a Holliday junction-resolving activity would also be expected to play a role in the recombination induced by poxvirus infection, the appearance of the activity described here only after the commencement of viral late protein synthesis suggests that it functions strictly at late times. Other non-viral Holliday junction analogs can also be cleaved by this extract, suggesting that this component of the resolution activity may also play a role in other viral processes that require cleavage of a branched DNA structure. Thus, we have identified a poxviral activity that may be a part of a protein complex which resolves concatemeric replicative intermediates of viral DNA as well as participate in general recombination late during infection.

Animals↗
PubMed2001

HyBeacon probes: a new tool for DNA sequence detection and allele discrimination.

Technologies that permit rapid investigation of DNA sequences, such as those containing single nucleotide polymorphisms (SNPs), are of great consequence to many sectors that perform molecular diagnostic analyses. We have developed a novel fluorescent oligonucleotide probe technology, termed HyBeacons, which provides a new homogeneous method for fluorescence-based sequence detection, allele discrimination and DNA quantification. Hybridization of HyBeacons to complementary DNA target sequences results in a measurable elevation of probe fluorescence emission. HyBeacon probes may be incorporated into real-time polymerase chain reaction (PCR) assays to detect the presence and monitor the accumulation of specific DNA sequences. Furthermore, closely related sequences differing by as little as a single nucleotide may be discriminated by measuring the melting temperatures (T(m)) of various probe/target duplexes and exploiting the differences in T(m) that exist between different duplexes. We demonstrate here that HyBeacon probes are efficient tools for rapid sequence analysis and that a single probe may be employed to reliably identify homozygous and heterozygous samples. Additional benefits exhibited by the HyBeacon technology derive from their simple mode of action, ease of design, relatively inexpensive synthesis and potential for multiplex analysis.

Alleles↗
PubMed1991

Characterization of cDNA clones for the beta subunit of pyruvate dehydrogenase from Ascaris suum.

The pyruvate dehydrogenase complex occupies a unique position in the anaerobic mitochondrial metabolism of the parasitic nematode Ascaris suum. This paper describes cDNA clones for the beta subunit of the pyruvate dehydrogenase component of this complex. A cDNA library has been constructed in lambda gt11 from poly(A)+ RNA isolated from adult ascarid body wall muscle. The library was screened with antiserum prepared against the beta subunit of pyruvate dehydrogenase. Full-length clones of 1.2 kb have been characterized. The first 15 amino acids determined from the purified protein match exactly those predicted from the cDNA sequence. The deduced protein sequence contains a 26-amino-acid presequence that has characteristics of mitochondrial targeting sequences. The mature protein is predicted to contain 334 amino acids and is 62% identical to the predicted sequence of the corresponding human subunit. Full-length in vitro transcripts have been translated in vitro to yield a 39-kDa polypeptide consistent with the open reading frame present in the cDNA sequence. The 90 nucleotides at the 3' end of the cDNA sequence have the potential to form a cruciform structure that may play a role in the synthesis of the enzyme.

Amino Acid Sequence↗
PubMed1994

Cloning cassettes containing the reporter gene xylE.

Two pUC-derived vectors containing the promoterless xylE gene (encoding catechol 2,3-dioxygenase) of Pseudomonas putida mt-2 were constructed. The t(o) transcriptional terminator of phage lambda was placed downstream from the stop codon of xylE. The new vectors, pXT1 and pXT2, contain xylE and the t(o) terminator within a cloning cassette which can be excised with several endonucleases. When inserted into a transcribed sequence, this xylE cassette reports promoter activity and interrupts downstream transcription of the target sequence.

Bacteriophage lambda↗
PubMed2003

Nuclear large subunit rDNA group I intron distribution in a population of Beauveria bassiana strains: phylogenetic implications.

Four group I introns, designated Bb1, Bb2, Bb3 and Bb4, were identified in the entomopathogenic hyphomycete Beauveria bassiana. Sequence analyses of these introns verified that they were invariably inserted at specific target sequences after conserved positions Ec2563, Ec2449, Ec2066 and Ec1921 of the large nuclear subunit (LSU) rDNA 3'-end. Secondary structure modelling confirmed that Bb1 and Bb3 belonged to subgroup IE while Bb2 and Bb4 belonged to subgroup IC1. Intron presence, distribution and size-variation were studied in a population of 125 B. bassiana strains using site-specific primers. Nucleotide sequences and secondary structures were compared and showed considerable variations usually at P1, P6 and P9 helices, but concomitantly, high homology between members of the same site-specific group. Intron distribution studies revealed that few (7.2%) strains were intron-less, most contained one (28%), two (48%) or three (16%) introns, while only one strain contained all four introns. Bb4-like introns (Ec1921) were the most abundant (86.4%), whereas the other three introns were evenly represented (ca 30%) in the B. bassiana population. Analysis of intron genotype distribution indicated a tenuous dependence upon geographic origin or insect host species. Phylogenetic analysis of all B. bassiana LSU introns and their close relatives from other entomopathogenic fungi showed a strong correlation between specific insertion sites and intron subgroups, fully supported by corresponding clades, suggesting common ancestry of the site specific LSU introns.

Animals↗
PubMed1998

Gene expression induced by physical impedance in maize roots.

Two cDNA clones, pIIG1 and pIIG2, corresponding to mRNAs that accumulate in maize root tips subjected to 10 min of physical impedance, were isolated by differential screening of a cDNA library. The deduced proteins, based on DNA sequence analysis, have molecular masses of 13 and 23 kDa for pIIG1 and pIIG2, respectively. pIIG1 showed 97% similarity at the nucleic acid level to a maize root cortical cell delineating protein (pZRP3) and was also similar to some bimodular proteins that are developmentally or stress regulated in other plant species. In situ localization of pIIG1 showed some expression in cortical cells of control maize roots; however, after a 10 min physical impedance treatment, pIIG1 accumulation increased greatly in cortical cells and extended to include the procambial region. pIIG2 did not show sequence similarity with any identified gene of known function, but a bipartite nuclear targeting sequence occurs in its deduced amino acid sequence which indicates it may function in the nucleus. Thus, rapid accumulation of specific mRNAs occurs in maize roots in response to impedance stress, and these mRNAs may be responsible for some responses of the roots to physical impedance.

Amino Acid Sequence↗
PubMed2003

LINE-mediated retrotransposition of marked Alu sequences.

Alu elements are the most successful transposons in humans. They are 300-bp non-coding sequences transcribed by RNA polymerase III (Pol III) and are expected to retrotranspose with the aid of reverse transcriptases of cellular origin. We previously showed that human LINEs can generate cDNA copies of any mRNA transcript by means of a retroposition process involving reverse transcription and integration by the LINE-encoded endonuclease and reverse transcriptase. Here we show mobility of marked Alu sequences in human HeLa cells with the canonical features of a retrotransposition process, including splicing out of an autocatalytic intron introduced into the marked sequence, target site duplications of varying lengths and integrations into consensus A-rich sequences. We further show that the poly-A stretch at the Alu 3' end is essential for mobility, that LINEs are required for transposition and that the rate of retroposition is 100-1,000 times higher for Alu transcripts than for control mRNAs, thus accounting for the high mutational activity of these elements observed in humans.

Alu Elements↗
PubMed1999

CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in gram-positive bacteria.

clpP and clpC of Bacillus subtillis encode subunits of the Clp ATP-dependent protease and are required for stress survival, including growth at high temperature. They play essential roles in stationary phase adaptive responses such as the competence and sporulation developmental pathways, and belong to the so-called class III group of heat shock genes, whose mode of regulation is unknown and whose expression is induced by heat shock or general stress conditions. The product of ctsR, the first gene of the clpC operon, has now been shown to act as a repressor of both clpP and clpC, as well as clpE, which encodes a novel member of the Hsp100 Clp ATPase family. The CtsR protein was purified and shown to bind specifically to the promoter regions of all three clp genes. Random mutagenesis, DNasel footprinting and DNA sequence deletions and comparisons were used to define a consensus CtsR recognition sequence as a directly repeated heptad upstream from the three clp genes. This target sequence was also found upstream from clp and other heat shock genes of several Gram-positive bacteria, including Listeria monocytogenes, Streptococcus salivarius, S. pneumoniae, S. pyogenes, S. thermophilus, Enterococcus faecalis, Staphylococcus aureus, Leuconostoc oenos, Lactobacillus sake, Lactococcus lactis and Clostridium acetobutylicum. CtsR homologues were also identified in several of these bacteria, indicating that heat shock regulation by CtsR is highly conserved in Gram-positive bacteria.

Adenosine Triphosphatases↗
PubMed1989

A set of Macintosh computer programs for the design and analysis of synthetic genes.

Computer programs that can be used for the design of synthetic genes and that are run on an Apple Macintosh computer are described. These programs determine nucleic acid sequences encoding amino acid sequences. They select DNA sequences based on codon usage as specified by the user, and determine the placement of base changes that can be used to create restriction enzyme sites without altering the amino acid sequence. A new algorithm for finding restriction sites by translating the restriction endonuclease target sequence in all three reading frames and then searching the given peptide or protein amino acid sequence with these short restriction enzyme peptide sequences is described. Examples are given for the creation of synthetic DNA sequences for the bovine prethrombin-2 and ribonuclease A genes.

Algorithms↗