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At least 613 records · Page 34Linked to original sources
PubMed2000

Structural basis for the diversity of DNA recognition by bZIP transcription factors.

The basic region leucine zipper (bZIP) proteins form one of the largest families of transcription factors in eukaryotic cells. Despite relatively high homology between the amino acid sequences of the bZIP motifs, these proteins recognize diverse DNA sequences. Here we report the 2.0 A resolution crystal structure of the bZIP motif of one such transcription factor, PAP1, a fission yeast AP-1-like transcription factor that binds DNA containing the novel consensus sequence TTACGTAA. The structure reveals how the Pap1-specific residues of the bZIP basic region recognize the target sequence and shows that the side chain of the invariant Asn in the bZIP motif adopts an alternative conformation in Pap1. This conformation, which is stabilized by a Pap1-specific residue and its associated water molecule, recognizes a different base in the target sequence from that in other bZIP subfamilies.

Amino Acid Sequence↗
PubMed2004

Enhancement of p53 sequence-specific binding by DNA supercoiling.

Using a new competition assay, we investigated the effect of DNA negative supercoiling on the DNA sequence-specific binding (SSDB) of human wild-type (wt) p53 protein. We found that supercoiled (sc) pBluescript DNAs with different inserted p53 target sequences were stronger competitors than a mixture of scDNA pBluescript with the given 20-mer target oligodeoxynucleotide. ScDNAs were always better competitors than their linearized or relaxed forms. Two DNAs with extruded cruciforms within the target sequence were the best competitors; removal of the cruciforms resulted in a decrease of competitor strength. In contrast to the full-length wt p53, the deletion mutant p53CDelta30 and the p53 core domain (93-312 aa) showed no enhancement of p53 SSDB to scDNA, suggesting that, in addition to the p53 core domain, the C-terminal was involved in this binding. We conclude that cruciforms and DNA bends contribute to the enhancement of p53 SSDB to scDNA and that the DNA supercoiling is an important determinant in the p53 sequence-specific binding. Supercoiling may thus play a significant role in the complex p53-regulatory network.

Amino Acid Sequence↗
PubMed2001

Chrysanthemyl diphosphate synthase: isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium.

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1'-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium lambda cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni(2+) chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1'-2-3 cyclopropanation reactions similar to the CPPase reaction.

Amino Acid Sequence↗
PubMed1995

[Leu5]enkephalin-encoding sequences are targets for a specific DNA-binding factor.

A DNA-binding factor with high affinity and specificity for the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has been characterized. The factor has the highest affinity for the [Leu5]-enkephalin-encoding sequence in the dynorphin B-encoding region of the prodynorphin gene, has relatively high affinity for other [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes, but has no apparent affinity for similar DNA sequences coding for [Met5]-enkephalin in the prodynorphin or proopiomelanocortin genes. The factor has been named [Leu5]enkephalin-encoding sequence DNA-binding factor (LEF). LEF has a nuclear localization and is composed of three subunits of about 60, 70, and 95 kDa, respectively. The highest levels were observed in rat testis, cerebellum, and spleen and were generally higher in late embryonal compared to newborn or adult animals. LEF activity was also recorded in human clonal tumor cell lines. LEF inhibited the transcription of reporter genes in artificial gene constructs where a [Leu5]enkephalin-encoding DNA fragment had been inserted between the transcription initiation site and the coding region of the reporter genes. These observations suggest that the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes also have regulatory functions realized through interaction with a specific DNA-binding factor.

Aging↗
PubMed1988

Peptide sequences that target proteins for enhanced degradation during serum withdrawal.

Fibroblasts increase the catabolism of certain intracellular proteins in response to serum withdrawal, and these proteins contain specific peptide regions that may be required for their increased degradation. We show that the increased degradation of microinjected ribonuclease A during serum withdrawal can be blocked by co-injection of a pentapeptide corresponding to residues 7-11 of ribonuclease A, Lys-Phe-Glu-Arg-Gln. Furthermore, similar peptide sequences appear to play a widespread role in targeting proteins for enhanced degradation. Affinity-purified antibodies raised against the pentapeptide are able to precipitate 20-35% of radiolabeled cytosolic proteins from fibroblasts. Such proteins are preferentially degraded when cells are deprived of serum while nonimmunoprecipitable proteins are degraded at the same rate in the presence and absence of serum. Immunoreactive cytosolic proteins also exist in rat liver and kidney, and these proteins are depleted when protein degradation rates are enhanced due to starvation. Several types of evidence suggest that the peptides recognized in cellular proteins are similar to Lys-Phe-Glu-Arg-Gln but are not this exact sequence. Analyses of amino acid sequences for four proteins whose degradative rates are enhanced in response to serum withdrawal and for four proteins that are degraded in a serum-independent manner indicate two possible peptide motifs related to Lys-Phe-Glu-Arg-Gln that may target cellular proteins for enhanced degradation. These results, combined with previous studies (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993), suggest that these peptide regions target specific proteins to a lysosomal pathway of degradation during serum withdrawal.

Amino Acid Sequence↗
PubMed1999

Folding protein models with a simple hydrophobic energy function: the fundamental importance of monomer inside/outside segregation.

The present study explores a "hydrophobic" energy function for folding simulations of the protein lattice model. The contribution of each monomer to conformational energy is the product of its "hydrophobicity" and the number of contacts it makes, i.e., E(h,c) = -Sigma N/i=1 c(i)h(i) = -(h.c) is the negative scalar product between two vectors in N-dimensional cartesian space: h = (h1,., hN), which represents monomer hydrophobicities and is sequence-dependent; and c = (c(1),., c(N)), which represents the number of contacts made by each monomer and is conformation-dependent. A simple theoretical analysis shows that restrictions are imposed concomitantly on both sequences and native structures if the stability criterion for protein-like behavior is to be satisfied. Given a conformation with vector c, the best sequence is a vector h on the direction upon which the projection of c - c is maximal, where c is the diagonal vector with components equal to c, the average number of contacts per monomer in the unfolded state. Best native conformations are suggested to be not maximally compact, as assumed in many studies, but the ones with largest variance of contacts among its monomers, i.e., with monomers tending to occupy completely buried or completely exposed positions. This inside/outside segregation is reflected on an apolar/polar distribution on the corresponding sequence. Monte Carlo simulations in two dimensions corroborate this general scheme. Sequences targeted to conformations with large contact variances folded cooperatively with thermodynamics of a two-state transition. Sequences targeted to maximally compact conformations, which have lower contact variance, were either found to have degenerate ground state or to fold with much lower cooperativity.

Models, Chemical↗
PubMed1991

The tdh and serA operons of Escherichia coli: mutational analysis of the regulatory elements of leucine-responsive genes.

The tdh promoter of Escherichia coli is induced seven- to eightfold when cells are grown in the presence of exogenous leucine. A scheme was devised to select mutants that exhibited high constitutive expression of the tdh promoter. The mutations in these strains were shown to lie within a previously identified gene (lrp) that encodes Lrp (leucine-responsive regulatory protein). By deletion analysis, the site of action of Lrp was localized to a 25-bp region between coordinates -69 and -44 of the tdh promoter. Disruption of a 12-bp presumptive target sequence found in this region of tdh resulted in constitutively derepressed expression from the tdh promoter. Similar DNA segments (consensus, TTTATTCtNaAT) were also identified in a number of other promoters, including each of the Lrp-regulated promoters whose nucleotide sequence is known. The sequence of the promoter region of serA, an Lrp-regulated gene, was determined. No Lrp consensus target sequence was present upstream of serA, suggesting that Lrp acts indirectly on the serA promoter. A previously described mutation in a leucine-responsive trans-acting factor, LivR (J. J. Anderson, S. C. Quay, and D. L. Oxender, J. Bacteriol. 126:80-90, 1976), resulted in constitutively repressed expression from the tdh promoter and constitutively induced expression from the serA promoter. The possibility that LivR and Lrp are allelic is discussed.

Alcohol Oxidoreductases↗
PubMed1997

Multivariant confirmation of sickle cell disease using a non-radioactive minisequencing reaction.

A non-radioactive solid-phase minisequencing method for confirmation of abnormal hemoglobin variants causing sickle cell disease has been developed. In this method amplified 5'-biotinylated target sequences containing normal and mutation sites are immobilized onto streptavidin-coated microplates. Detection primers corresponding to target sequences are annealed immediately adjacent to the mutation site and single-step, hapten-labeled nucleotide primer extension reactions are performed. The incorporation of the labeled nucleotide is detected through immunological reaction with an enzyme-labeled anti-hapten conjugate and a substrate. The method enables confirmation of mutations of the beta-globin gene variants (Hbs S, C, E D-Punjab, O-Arab) and the alpha-globin gene variant (Hb G-Philadelphia). The test was evaluated using characterized dried blood spot specimens (n = 100) The advantages of the procedure are easy performance and objectiveness. The non-radioactive minisequencing assay will prove helpful for genotyping in neonatal screening for hemoglobinopathies and in prenatal and pre-implantational diagnostics.

Anemia, Sickle Cell↗
PubMed1998

Role of endoproteolytic dibasic proprotein processing in maturation of secretory proteins in Trichoderma reesei.

Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T. reesei. Secretion of xylanase I (proprotein processing sequence -R-R- downward arrow-R- downward arrow-A-) and xylanase II (-K-R- downward arrow-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R- downward arrow-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A- downward arrow-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from Streptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different endoproteolytic proprotein processing enzymes in T. reesei and demonstrate that dibasic processing is obligatory for the secretion of the proproteins containing this target.

Amino Acid Sequence↗
PubMed1996

Identification of cis-acting sequences in the promoter of the herpes simplex virus type 1 latency-associated transcripts required for activation by nerve growth factor and sodium butyrate in PC12 cells.

In the absence of detectable viral proteins, expression of the latency-associated transcripts (LATs) is likely regulated by cellular factors during latent infection of neurons with herpes simplex virus type 1. The amounts and activation states of these factors may in turn be regulated by extracellular regulatory factors. Consistent with this hypothesis, we have recently demonstrated that LAT expression is significantly enhanced by nerve growth factor (NGF) and sodium butyrate (NaB) in neurally derived PC12 cells. With the ultimate goal of identifying trans-acting cellular factors involved in regulating LAT expression during latency, we have attempted to identify the cis-acting elements to which these putative cellular factors bind by characterizing the LAT promoter and a series of 5' promoter deletion mutants in PC12 cells following treatment with the LAT-enhancing agents NGF and NaB. Transient expression assays demonstrated that distinct cis-acting sequences mediate basal and induced LAT promoter expression. Basal activity in PC12 cells is mediated by two elements: a negative regulatory element between -435 and -270 and a positive element between -240 and -204. The positive element contains binding sites for the transactivator Sp-1, whereas the negative element bears some resemblance to known neuron-specific silencer elements. In contrast to basal expression, maximum induction of the LAT promoter by NGF and NaB requires sequences between -159 and -81. Using gel mobility shift assays, we have identified three sets of protein-DNA complexes that bind to this 78-bp region and shown by competition analysis that binding is specific. The abundance and mobility of these complexes were altered by treatment with NGF or NaB. The nucleotide sequences to which these complexes bind were fine mapped by competition analysis with oligonucleotide probes containing substitution mutations. The target sequences identified exhibit no homology to binding sites of known transcription factors. These regions were critical for complex formation in vitro and for maximum induction of the LAT promoter by NGF and NaB in transient expression assays. The protein complexes that form with target sequences likely participate in the regulation of LAT expression in response to physiological stimuli in neurons in vivo.

Animals↗
PubMed1995

MATS: a rapid and efficient method for the development of microsatellite markers from YACs.

In this report, we describe the successful application of a rapid and efficient procedure, based on subtractive hybridization and PCR amplification, for generating microsatellite-based markers directly from yeast artificial chromosomes (YACs). This strategy, termed MATS (marker addition through subtraction), exploits the fact that the only difference between a yeast host strain harboring a YAC and the host strain alone is the artificial chromosome. Given the low complexity of the yeast genome and relatively large target size presented by a YAC, only a single round of subtraction is required before amplification of the target sequences (YAC) and cloning into a plasmid vector for further analysis. Several key steps have been designed to achieve optimal subtraction and to obtain preferential amplification and recovery of the target sequences. Methods for efficient construction of small insert libraries and rapid, nonradioactive screening have also been integrated into the protocol. Using a 750-kb YAC as a target, we identified a minimum of 14 unique microsatellite containing clones, leading to the development of 12 polymorphic STSs (sequence-tagged sites). These new markers will facilitate the genetic localization of targeted locus and allow the accurate ordering by STS content mapping of a cloned contig spanning the interval. In addition to the utility of this approach in positional cloning, this strategy may provide an approach for filling gaps in the emerging genetic maps.

Base Sequence↗
PubMed1994

Noncanonical Oct-sequences are targets for mouse Oct-2B transcription factor.

We have suggested a random modification method for determining preferable binding sites of a DNA-binding protein and applied this method to the Oct-2B transcription factor. Our results indicate that the Oct-2B protein interacts with canonical oct sequence ATGC/TAAAT and degenerated sequences which contain TAAT motif in the binding site. We have determined nucleotides in the binding sites, involved in the DNA-protein interaction, and the equilibrium dissociation constants Kd for these sequences. These data show that a much greater number of potential targets for Oct proteins exist on DNA and changed our view on the gene expression regulation by this protein factor.

Animals↗
PubMed2001

Sequence-specific targeting of duplex DNA by peptide nucleic acids via triplex strand invasion.

Because of a set of exceptional chemical, physical, and biological properties, polyamide or peptide nucleic acids (PNAs) hold a distinctive position among various synthetic ligands designed for DNA-targeting purposes. Cationic pyrimidine PNAs (cpyPNAs) represent a special group of PNAs, which effectively form strand invasion triplexes with double-stranded DNA (dsDNA) also known as P-loops. Extraordinary stability of the invasion triplexes and high sequence specificity of their formation combined with local opening of the DNA double helix within the P-loops make these complexes very attractive for sequence-specific manipulation with dsDNA. Important for applications is the fact that the discrimination between correct and mismatched binding sites in dsDNA by cpyPNAs is a nonequilibrium, kinetically controlled process. Therefore, a careful choice of experimental conditions that are optimal for the kinetic discrimination of correct versus mismatched cpyPNA binding is crucial for sequence-specific recognition of dsDNA by cpyPNAs. The experimental and theoretical data presented make it possible to select those solution parameters and cpyPNA constructions that are most favorable for sequence specificity without compromising the affinity of dsDNA targeting.

Base Pair Mismatch↗
PubMed2003

The Flp double cross system a simple efficient procedure for cloning DNA fragments.

BACKGROUND: While conventional cloning methods using restriction enzymes and polynucleotide ligase are adequate for most DNAs, fragments made by the polymerase chain reaction are difficult to clone because the amplifying DNA polymerase tends to add untemplated nucleotides to the 3'-termini of the amplified strands. Conservative site-specific recombinases offer an efficient alternative to conventional cloning methods. RESULTS: In this paper I describe the use of the Flp site-specific recombinase for cloning PCR-amplified fragments. A DNA fragment is amplified with primers that contain at their ends inverted target sequences for Flp. Flp readily recombines these fragments in vitro into a vector that also contains two inverted Flp target sequences surrounding the alpha-complementing region of the lacZ gene of E. coli. The recombinants are conveniently detected as white colonies by the familiar blue/white screening test for lacZ activity. A useful feature of the system is that both orientations of the inserted DNA are usually obtained. If the recipient vector is cut between the two inverted Flp targets, Flp "heals" the double-strand break by inserting a linear fragment flanked by Flp targets. CONCLUSION: This system ("The Flp Double Cross System") should be useful for cloning multiple PCR fragments into many sites in several vectors. It has certain advantages over other available recombinase-based cloning procedures.

Bacterial Outer Membrane Proteins↗
PubMed2000

Plant mercaptopyruvate sulfurtransferases: molecular cloning, subcellular localization and enzymatic activities.

Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) and thiosulfate sulfurtransferase (TST, rhodanese, EC 2.8.1.1) are evolutionarily related enzymes that catalyze the transfer of sulfur ions from mercaptopyruvate and thiosulfate, respectively, to cyanide ions. We have isolated and characterized two cDNAs, AtMST1 and AtMST2, that are Arabidopsis homologs of TST and MST from other organisms. Deduced amino-acid sequences showed similarity to each other, although MST1 has a N-terminal extension of 57 amino acids containing a targeting sequence. MST1 and MST2 are located in mitochondria and cytoplasm, respectively, as shown by immunoblot analysis of subcellular fractions and by green fluorescent protein (GFP) analysis. However, some regions of MST1 fused to GFP were found to target not only mitochondria, but also chloroplasts, suggesting that the regions on the targeting sequence recognized by protein import systems of mitochondria and chloroplasts are not identical. Recombinant proteins, expressed in Escherichia coli, exhibited MST/TST activity ratios determined from kcat/Km values of 11 and 26 for MST1 and MST2, respectively. This indicates that the proteins encoded by both AtMST1 and AtMST2 are MST rather than TST type. One of the hypotheses proposed so far for the physiological function of MST and TST concerns iron-sulfur cluster assembly. In order to address this possibility, a T-DNA insertion Arabidopsis mutant, in which the AtMST1 was disrupted, was isolated by PCR screening of T-DNA mutant libraries. However, the mutation had no effect on levels of iron-sulfur enzyme activities, suggesting that MST1 is not directly involved in iron-sulfur cluster assembly.

Amino Acid Sequence↗
PubMed2000

Spectrum alignment: efficient resequencing by hybridization.

Recent high-density microarray technologies allow, in principle, the determination of all k-mers that appear along a DNA sequence, for k = 8 - 10 in a single experiment on a standard chip. The k-mer contents, also called the spectrum of the sequence, is not sufficient to uniquely reconstruct a sequence longer than a few hundred bases. We have devised a polynomial algorithm that reconstructs the sequence, given the spectrum and a homologous sequence. This situation occurs, for example, in the identification of single nucleotide polymorphisms (SNPs), and whenever a homologue of the target sequence is known. The algorithm is robust, can handle errors in the spectrum and assumes no knowledge of the k-mer multiplicities. Our simulations show that with realistic levels of SNPs, the algorithm correctly reconstructs a target sequence of length up to 2,000 nucleotides when a polymorphic sequence is known. The technique is generalized to handle profiles and HMMs as input instead of a single homologous sequence.

Algorithms↗
PubMed1989

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.

Chromosome Deletion↗
PubMed1998

ISD1, an insertion element from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough: structure, transposition, and distribution.

Insertion element ISD1, discovered when its transposition caused the insertional inactivation of an introduced sacB gene, is present in two copies in the genome of Desulfovibrio vulgaris Hildenborough. Southern blot analysis indicated at least two insertion sites in the sacB gene. Cloning and sequencing of a transposed copy of ISD1 indicated a length of 1,200 bp with a pair of 44-bp imperfect inverted repeats at the ends, flanked by a direct repeat of the 4-bp target sequence. AAGG and AATT were found to function as target sequences. ISD1 encodes a transposase from two overlapping open reading frames by programmed translational frameshifting at an A6G shifty codon motif. Sequence comparison showed that ISD1 belongs to the IS3 family. Isolation and analysis of the chromosomal copies, ISD1-A and ISD1-B, by PCR and sequencing indicated that these are not flanked by direct repeats. ISD1-A is inserted in a region of the chromosome containing the gapdh-pgk genes (encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase). Active transposition to other loci in the genome was demonstrated, offering the potential of a new tool for gene cloning and mutagenesis. ISD1 is the first transposable element described for the sulfate reducers, a large and environmentally important group of bacteria. The distribution of ISD1 in genomes of sulfate-reducing bacteria is limited. A single copy is present in the genome of D. desulfuricans Norway.

Amino Acid Sequence↗