Search PubMed⌕ Search

Biomedical subjects

S Pongor

Publications and source records attributed to S Pongor.

At least 37 records · Page 2Linked to original sources
PubMed1999

Sequence-dependent modelling of local DNA bending phenomena: curvature prediction and vibrational analysis.

Bending is a local conformational micropolymorphism of DNA in which the original B-DNA structure is only distorted but not extensively modified. Bending can be predicted by simple static geometry models as well as by a recently developed elastic model that incorporate sequence dependent anisotropic bendability (SDAB). The SDAB model qualitatively explains phenomena including affinity of protein binding, kinking, as well as sequence-dependent vibrational properties of DNA. The vibrational properties of DNA segments can be studied by finite element analysis of a model subjected to an initial bending moment. The frequency spectrum is obtained by applying Fourier analysis to the displacement values in the time domain. This analysis shows that the spectrum of the bending vibrations quite sensitively depends on the sequence, for example the spectrum of a curved sequence is characteristically different from the spectrum of straight sequence motifs of identical basepair composition. Curvature distributions are genome-specific, and pronounced differences are found between protein-coding and regulatory regions, respectively, that is, sites of extreme curvature and/or bendability are less frequent in protein-coding regions. A WWW server is set up for the prediction of curvature and generation of 3D models from DNA sequences (http:@www.icgeb.trieste.it/dna).

Base Sequence↗
PubMed1999

Single-chain 434 repressors with altered DNA-binding specificities. Isolation of mutant single-chain repressors by phenotypic screening of combinatorial mutant libraries.

Combinatorial mutant libraries of the single-chain 434 repressor were used to discover novel DNA-binding specificities. Members of the library contain one wild type domain and one mutant domain which are connected by a recombinant peptide linker. The mutant domain contains randomized amino acids in place of the DNA-contacting residues. The single-chain derivatives are expected to recognize artificial operators containing the DNA sequence of ACAA--6 base-pairs--NNNN, where ACAA is bound by the wild-type and NNNN by the mutant domain. An in vivo library screening method was used to isolate mutant DNA-binding domains which recognize the TTAA site of an asymmetric operator. Several mutants showed high affinity binding to the selection target and also strong (up to 80 fold) preference for TTAA over the wild type TTGT sequence. Some of the isolated mutants bound with very high affinities (10-50 pM) to operators containing the TTAC sequence, a close homologue of the TTAA selection target.

Base Sequence↗
PubMed1999

A thermodynamic study of the 434-repressor N-terminal domain and of its covalently linked dimers.

The isolated N-terminal 1-69 domain of the 434-phage repressor, R69, and its covalently linked (head-to-tail and tail-to-tail) dimers have been studied by differential scanning microcalorimetry (DSC) and CD. At neutral solvent conditions the R69 domain maintains its native structure, both in isolated form and within the dimers. The stability of the domain depends highly upon pH within the acidic range, thus at pH 2 and low ionic strength R69 is already partially unfolded at room temperature. The thermodynamic parameters of unfolding calculated from the DSC data are typical for small globular proteins. At neutral pH and moderate ionic strength, the domains of the dimers behave as two independent units with unfolding parameters similar to those of the isolated domain, which means that linking two R69 domains, either by a long peptide linker or by a designed C-terminal disulfide bridge, does not induce any cooperation between them.

Calorimetry, Differential Scanning↗
PubMed1999

The domain-server: direct prediction of protein domain-homologies from BLAST search.

RESULTS: A WWW server for protein domain homology prediction, based on BLAST search and a simple data-mining algorithm (Hegyi,H. and Pongor,S. (1993) Comput. Appl. Biosci., 9, 371-372), was constructed providing a tabulated list and a graphic plot of similarities. AVAILABILITY: http://www.icgeb.trieste.it/domain. Mirror site is available at http://sbase.abc.hu/domain. A standalone programme will be available on request. SUPPLEMENTARY INFORMATION: A series of help files is available at the above addresses.

Algorithms↗
PubMed1998

A somatostatin analogue induces translocation of Ku 86 autoantigen from the cytosol to the nucleus in colon tumour cells.

Flow cytometric and electron microscopic immunocytochemical studies have been performed in HT-29 human colon tumour cells in vitro, to determine and localise p86 Ku protein, which is a regulatory subunit of DNA-dependent kinase and a specific binding site for somatostatin. We have demonstrated that HT-29 cells contain p86 Ku and that the distribution between the cytoplasm and the nucleus is even. After administration of the somatostatin analogues Sandostatin and TT-232 to HT-29 cells, the p86 Ku content of the cytoplasmic compartment decreased in the first 4 h. An increase in the content of this protein in the nuclear compartment was observed at hour 1 followed by a decrease at hour 4 after treatment. Quantitative differences between the two analogues have been observed in this respect. The practical significance of these findings is discussed.

Antigens, Nuclear↗
PubMed1998

Rod models of DNA: sequence-dependent anisotropic elastic modelling of local bending phenomena.

Local bending phenomena can be predicted by elastic models that incorporate sequence-dependent anisotropic-bendability (SDAB). SDAB models consider DNA to be an initially straight, segmented, elastic rod, in which the flexibility of each segment is greater towards the major groove than it is in other directions. While local bending can be predicted by static-geometry models as well, SDAB models, in addition, qualitatively explain such phenomena as the affinity of protein binding and kinking. A set of prediction tools is available at http:/(/)www.icgeb.trieste.it/dna.

Animals↗
PubMed1998

Immunolocalization of a novel annexin-like protein encoded by a stress and abscisic acid responsive gene in alfalfa.

We report here on the isolation and characterization of a full-length cDNA clone from alfalfa termed AnnMs2 encoding a 333 amino acid long polypeptide that shows 32-37% sequence identity with both mammalian and plant annexins, and has four tandem repeats. While other plant annexins exhibit a high level of sequence similarity to each other (up to 77% identity at amino acid level), AnnMs2 appears to be a distinct type of plant annexins. All the four endonexin folds contain the conserved eukaryotic motif within this alfalfa protein, but this element is considerably different in the second repeat. The AnnMs2 gene is expressed in various tissues of alfalfa with elevated mRNA accumulation in root and flower. This gene is activated in cells or tissues exposed to osmotic stress, abscisic acid (ABA) or water deficiency. The recombinant AnnMs2 protein is able to bind to phospholipid in the presence of Ca2+. Indirect immunofluorescence studies using affinity purified rabbit anti-AnnMs2 peptide antibody show mainly nucleolar localization, but the protein sequence lacks the usual nuclear localization signal. The potential role of this novel annexin-like protein in the basic and stress-induced cellular functions is discussed.

Abscisic Acid↗
PubMed1997

The role of DNA bending in Cro protein-DNA interactions.

Binding energy of DNA-Cro protein complexes is analyzed in terms of DNA elasticity, using a sequence-dependent anisotropic bendability (SDAB) model of DNA, developed recently [M.M. Gromiha, M.G. Munteanu, A. Gabrielian and S. Pongor, J. Biol. Phys. 22(1996) 227-243.]. The protein is considered to bind aspecifically to DNA that reduces the freedom of movement in the DNA molecule. In cognate DNA, the Cro protein moves on to form specific interactions and bends DNA. A comparison of the experimental data [Y. Takeda, A. Sarai and V.M. Rivera, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 439-443.] with the calculated DNA stiffness data shows that delta G of the complex formation increases with stiffness of the ligand when the interactions are nonspecific ones, while an opposite trend is observed for specific binding. Both of these trends are in agreement with our approach using the SDAB model. A decomposition of the energy terms suggests that binding energy in the nonspecific case is used maily to compensate the free energy changes due to entropy lost by DNA, while the energy of specific interactions provide enough energy both to bend the DNA molecule and to change the conformation of the Cro protein upon ligand binding.

Base Sequence↗
PubMed1997

Recognition of DNA by single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non-contacted base pairs.

Single-chain derivatives of the phage 434 repressor, termed single-chain repressors, contain covalently dimerized DNA-binding domains (DBD) which are connected with a peptide linker in a head-to-tail arrangement. The prototype RR69 contains two wild-type DBDs, while RR*69 contains a wild-type and an engineered DBD. In this latter domain, the DNA- contacting amino acids of thealpha3 helix of the 434 repressor are replaced by the corresponding residues of the related P22 repressor. We have used binding site selection, targeted mutagenesis and binding affinity studies to define the optimum DNA recognition sequence for these single-chain proteins. It is shown that RR69 recognizes DNA sequences containing the consensus boxes of the 434 operators in a palindromic arrangement, and that RR*69 optimally binds to non-palindromic sequences containing a 434 operator box and a TTAA box of which the latter is present in most P22 operators. The spacing of these boxes, as in the 434 operators, is 6 bp. The DNA-binding of both single-chain repressors, similar to that of the 434 repressor, is influenced indirectly by the sequence of the non-contacted, spacer region. Thus, high affinity binding is dependent on both direct and indirect recognition. Nonetheless, the single-chain framework can accommodate certain substitutions to obtain altered DNA-binding specificity and RR*69 represents an example for the combination of altered direct and unchanged indirect readout mechanisms.

Bacteriophage lambda↗
PubMed1997

Distribution of sequence-dependent curvature in genomic DNA sequences.

The distribution of inherent, sequence-dependent curvature was calculated for a number of prokaryotic (M. genitalium, H. influenzae, M. jannaschii), viral (adenovirus 2, equine herpes virus 1), phage (M13, lambda), eukaryotic (S. cerevisiae) and mitochondrial genomes as well as E. coli and human genomic fragments. The genomic averages are in the range of 6-8 degrees/helical turn and only about 20% of DNA is curved less than 3 degrees/helical turn. The prokaryotes and phages appear to have a consistently higher frequency of curved DNA in their genomes than the other genomes tested. Long, highly curved segments, similar to artificially designed curved DNA, are apparently absent from the genomes. Short, curved segments, differing in G+C content may provide environmentally modulated conformational signals for gene regulation. A WWW-server was constructed for the prediction of curved sites from DNA sequences (http://icgeb.trieste.it/dna/curve_it.html/)..

Animals↗
PubMed1997

Single-chain repressors containing engineered DNA-binding domains of the phage 434 repressor recognize symmetric or asymmetric DNA operators.

Single-chain (sc) DNA-binding proteins containing covalently dimerized N-terminal domains of the bacteriophage 434 repressor cI have been constructed. The DNA-binding domains (amino acid residues 1 to 69) were connected in a head-to-tail arrangement with a part of the natural linker sequence that connects the N and C-terminal domains of the intact repressor. Compared to the isolated N-terminal DNA-binding domain, the sc molecule showed at least 100-fold higher binding affinity in vitro and a slightly stronger repression in vivo. The recognition of the symmetric O(R)1 operator sequence by this sc homodimer was indistinguishable from that of the naturally dimerized repressor in terms of binding affinity, DNase I protection pattern and in vivo repressor function. Using the new, sc framework, mutant proteins with altered DNA-binding specificity have also been constructed. Substitution of the DNA-contacting amino acid residues of the recognition helix in one of the domains with the corresponding residues of the Salmonella phage P22 repressor c2 resulted in a sc heterodimer of altered specificity. This new heterodimeric molecule recognized an asymmetric, artificial 434-P22 chimeric operator with high affinity. Similar substitutions in both 434 domains have led to a new sc homodimer which showed high affinity binding to a natural, symmetric P22 operator. These findings, supported by both in vitro and in vivo experiments, show that the sc architecture allows for the introduction of independent changes in the binding domains and suggest that this new protein framework could be used to generate new specificities in protein-DNA interaction.

Amino Acid Sequence↗
PubMed1997

The SBASE protein domain library, release 5.0: a collection of annotated protein sequence segments.

SBASE 5.0 is the fifth release of SBASE, a collection of annotated protein domain sequences that represent various structural, functional, ligand-binding and topogenic segments of proteins. SBASE was designed to facilitate the detection of functional homologies and can be searched with standard database-search programs. The present release contains over 79863 entries provided with standardized names and is cross-referenced to all major sequence databases and sequence pattern collections. The information is assigned to individual domains rather than to entire protein sequences, thus SBASE contains substantially more cross-references and links than do the protein sequence databases. The entries are clustered into >16 000 groups in order to facilitate the detection of distant similarities. SBASE 5.0 is freely available by anonymous 'ftp' file transfer from . Automated searching of SBASE with BLAST can be carried out with the WWW-server . and with the electronic mail server which now also provides a graphic representation of the homologies. A related WWW-server and e-mail server predicts SBASE domain homologies on the basis of SWISS-PROT searches.

Amino Acid Sequence↗
PubMed1997

Cell cycle phase specificity of putative cyclin-dependent kinase variants in synchronized alfalfa cells.

The eukaryotic cell division cycle is coordinated by cyclin-dependent kinases (CDKs), represented by a single major serine/threonine kinase in yeasts (Cdc2/CDC28) and a family of kinases (CDK1 to CDK8) in human cells. Previously, two cdc2 homologs, cdc2MsA and cdc2MsB, have been identified in alfalfa (Medicago sativa). By isolating cDNAs using a cdc2MsA probe, we demonstrate here that at least four additional cdc2 homologous genes are expressed in the tetraploid alfalfa. Proteins encoded by the new cdc2MsC to cdc2MsF cDNAs share the characteristic functional domains of CDKs with the conserved and plant-specific sequence elements. Transcripts from cdc2MsA, cdc2MsB, cdc2MsC, and cdc2MsE genes are synthesized throughout the cell cycle, whereas the amounts of cdc2MsD and cdc2MsF mRNAs peak during G2-to-M phases. The translation of Cdc2MsA/B, Cdc2MsD, and Cdc2MsF proteins follows the pattern of transcript accumulation. The multiplicity of kinase complexes with cell cycle phase-dependent activities was revealed by in vitro phosphorylation experiments. Proteins bound to p13suc1-Sepharose or immunoprecipitated with Cdc2MsA/B antibodies from cells at G1-to-S and G2-to-M phase boundaries showed elevated kinase activities. the Cdc2MsF antibodies separated a G2-to-M phase-related kinase complex. Detection of histone H1 phosphorylation activities in fractions immunoprecipitated with antimitotic cyclin (CyclinMs2) antibodies from G2-to-M phase cells indicates the complex formation between this cyclin and a kinase partner in alfalfa. The observed fluctuation of transcript levels, amounts, and activities of kinases in different cell cycle phases reflects a multilevel regulatory system during cell cycle progression in plants.

Amino Acid Sequence↗
PubMed1997

Synthesis and cystine/cysteine-catalyzed oxidative folding of the amaranth alpha-amylase inhibitor.

We report here the total synthesis of the alpha-amylase inhibitor (AAI), a 32-residue-long peptide with three disulfide bridges, isolated from amaranth seeds (Chagolla-Lopez, A., Blanco-Labra, A., Patthy, A., Sanchez, R. & Pongor S. (1994) J. Biol. Chem. 269, 23675-23680). The synthesis was carried out using a stepwise solid-phase approach based on the Fmoc/t-Bu chemistry, combined with the S-acetamidomethyl protection for cysteines. The linear, reduced peptide was obtained after two reduction steps, using 1,4-dithio-DL-threitol and tri(2-carboxyethyl)phosphine hydrochloride in basic and acidic conditions, respectively. Disulfide bridges were formed by oxidative folding in a cystine/cysteine redox buffer, these conditions were found superior to air oxidation and to glutathione-catalyzed oxidative folding. The physiochemical and enzyme inhibitory properties of synthetic AAI were found identical with those of natural product. Several orthogonal protection schemes proved unsuccessful in obtaining a biologically active product.

Amino Acid Sequence↗
PubMed1996

Primary structure and specificity of a serine proteinase inhibitor from paprika (Capsicum annuum) seeds.

Several fractions demonstrating trypsin inhibitory activity were isolated from the seeds of the paprika plant (Capsicum annuum). One of the inhibitors, PSI-1.1, was purified to homogeneity and characterised. The mature form of PSI-1.1 has a molecular mass of 6053 Da and consists of 55 amino acids in a sequence showing over 80% identity with members of the inhibitors of potato-2 family. PSI-1.1 is a potent inhibitor of trypsin (Ki = 4.8 x 10(-10) M) and a somewhat weaker inhibitor of chymotrypsin (Ki = 4.7 x 10(-8) M) and pronase E (Ki = 5.9 x 10(-8) M). PSI-1.1 is resistant to heat up to 85 degrees C, to acidic conditions (down to pH 2.0) and to pepsin digestion, presumably due to its four disulfide bridges.

Amino Acid Sequence↗
PubMed1996

Distribution of bending propensity in DNA sequences.

Local bending propensity and curvature of DNA can be characterized using a vector description of DNA bendability, based on a set of parameters derived from deoxyribonuclease I (DNase I) cleavage experiments. Two characteristics-arithmetic and vector averages of bendability-were successfully used to predict experimentally known bendable, rigid and curved segments in DNA. A characteristic distribution of bendability is conserved in evolutionarily related kinetoplast sequences. An analysis of the M. genitalium and H. influenzae genomes as well as fragments of human and yeast genomes shows, on the other hand, that highly curved segments--similar to artificially designed curved oligonucleotides--are extremely rare in natural DNA.

Animals↗