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At least 19 recordsLinked to original sources
PubMed1990

Folding and activity of hybrid sequence, disulfide-stabilized peptides.

Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a beta-turn and an alpha-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. We show that in S peptide the alpha-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure-function relationships for many biologically active sequences.

Amino Acid Sequence↗
PubMed1991

Scaffold-associated regions and repeated or cross-hybridizing sequences on an 800 kilobase DNA stretch of the Drosophila X chromosome.

DNA fragments binding to the scaffold (SARs) have been previously mapped on an 800-kb DNA fragment from the Drosophila X chromosome. Here we have examined whether these 86 DNA fragments bear sequences repeated in the Drosophila genome and/or cross-hybridizing sequences. Twenty-two middle-repeated sequences were localized by hybridizing Southern transfers of representative cloned DNA with total genomic DNA and, reciprocally, by hybridizing Southern transfers of total genomic DNA with this cloned DNA. Seventy-nine out of the 86 SAR-containing fragments appeared to be distinct from these 22 repeated motifs. Therefore, SARs, in their vast majority, are not members of middle-repeated sequence families. However, the 22 middle-repeated sequences were shown to be significantly located in the near vicinity of SARs. Hybridizations were also performed between SAR-containing DNA fragments, either at a high or at a low stringency. At a high stringency, the 86 SAR-containing DNA fragments did not cross-hybridize. However, at a low stringency, a complex hybridization network was evidenced. These nucleotide sequence similitudes support the idea that the various SARs may play common roles.

Animals↗
PubMed1995

Calculation of signal intensities in hybrid sequences for fast NMR imaging.

A number of techniques that recently have been used for fast NMR-imaging are based on a hybrid sequence of echo planar imaging (EPI) and FLASH imaging: after each NMR excitation several k-space lines are measured. The complete k-space is covered by performance of several excitations. It has been observed that there is usually an optimal hybrid sequence that maximizes the signal-to-noise ratio. In this work, a method is presented that allows a determination of the optimal sequence as a function of the relaxation times T1 and T2*.

Echo-Planar Imaging↗
PubMed1993

Likelihood DNA sequencing by hybridization.

Sequencing by hybridization (SBH) extracts local sequence information from a DNA fragment using hybridization with oligonucleotides and then reconstructs the sequence using the derived information. We describe an improvement to the SBH methodology which will allow it to work efficiently in the presence of hybridization errors. In particular, given a set of hybridizing probes, and the empirically derived rates of false positive and false negative hybridization, we can estimate the most likely DNA fragment to have produced the set of probes, and then estimate the probability that it generated the hybridization data. This methodology extends earlier results by identifying the most probable fragment to have generated the actual hybridization data. The methodology described will also generate longer unambiguous sequence fragments without the use of overlapping fragments.

Algorithms↗
PubMed1998

A hybrid sequence approach to the paracelsus challenge.

Inspired by the Paracelsus Challenge of Rose and Creamer (Proteins 19: 1-3, 1994), we have designed a protein sequence that is 50% identical to an all-helical protein but is intended to fold into a largely beta-sheet structure. Rather than attempt a de novo design, our strategy was to construct a hybrid sequence based on a helical "parent" protein (434 Cro) and a "target" protein with the desired fold (the B1 domain of protein G). The hybrid sequence (Crotein-G) is 50% identical to 434 Cro but is also 62% identical to the B1 domain of protein G. We also created a variant of Crotein-G (ZCrotein-G) that contains a potential His3Cys1 zinc binding site. At low protein concentrations and in the presence of 20% 2,2,2-trifluoroethanol (TFE) (v/v), the circular dichroism spectra of the designed proteins are distinct from that of 434 Cro and similar to that of the B1 domain of protein G. However, the proteins fail to denature in a cooperative manner. Furthermore, aggregation occurs at moderate protein concentrations or in the absence of TFE. Addition of zinc to ZCrotein-G does not promote structure formation. In summary, 434 Cro has been altered to something that may resemble the B1 domain of protein G, but the protein does not adopt a native structure.

Amino Acid Sequence↗
PubMed2003

Handling long targets and errors in sequencing by hybridization.

Sequencing by hybridization (SBH) is a DNA sequencing technique, in which the sequence is reconstructed using its k-mer content. This content, which is called the spectrum of the sequence, is obtained by hybridization to a universal DNA array. Standard universal arrays contain all k-mers for some fixed k, typically 8 to 10. Currently, in spite of its promise and elegance, SBH is not competitive with standard gel-based sequencing methods. This is due to two main reasons: lack of tools to handle realistic levels of hybridization errors and an inherent limitation on the length of uniquely reconstructible sequence by standard universal arrays. In this paper, we deal with both problems. We introduce a simple polynomial reconstruction algorithm which can be applied to spectra from standard arrays and has provable performance in the presence of both false negative and false positive errors. We also propose a novel design of chips containing universal bases that differs from the one proposed by Preparata et al. (1999). We give a simple algorithm that uses spectra from such chips to reconstruct with high probability random sequences of length lower only by a squared log factor compared to the information theoretic bound. Our algorithm is very robust to errors and has a provable performance even if there are both false negative and false positive errors. Simulations indicate that its sensitivity to errors is also very small in practice.

Computational Biology↗
PubMed1997

Software for DNA sequencing by hybridization.

Sequencing by hybridization (SBH) is a promising alternative approach to DNA sequencing and mutation detection. Analysis of the resolving power of SBH involves rather difficult combinatorial and probabilistic problems, and sometimes computer simulation is the only way to estimate the parameters and limitations of SBH experiments. This paper describes a software package, DNA-SPECTRUM, which allows one to analyze the resolving power and parameters of SBH. We also introduce the technique for visualizing multiple SBH reconstructions and describe applications of DNA-SPECTRUM to estimate various SBH parameters. DNA-SPECTRUM is available at http://www-hto.usc.edu/software/sbh/index. html.

Base Sequence↗
PubMed2002

Large scale sequencing by hybridization.

Sequencing by hybridization is a method for reconstructing a DNA sequence based on its k-mer content. This content, called the spectrum of the sequence, can be obtained from hybridization with a universal DNA chip. However, even with a sequencing chip containing all 4(9) 9-mers and assuming no hybridization errors, only about 400-bases-long sequences can be reconstructed unambiguously. Drmanac et al. (1989) suggested sequencing long DNA targets by obtaining spectra of many short overlapping fragments of the target, inferring their relative positions along the target, and then computing spectra of subfragments that are short enough to be uniquely recoverable. Drmanac et al. do not treat the realistic case of errors in the hybridization process. In this paper, we study the effect of such errors. We show that the probability of ambiguous reconstruction in the presence of (false negative) errors is close to the probability in the errorless case. More precisely, the ratio between these probabilities is 1 + O(p = (1 - p)(4). 1 = d) where d is the average length of subfragments, and p is the probability of a false negative. We also obtain lower and upper bounds for the probability of unambiguous reconstruction based on an errorless spectrum. For realistic chip sizes, these bounds are tighter than those given by Arratia et al. (1996). Finally, we report results on simulations with real DNA sequences, showing that even in the presence of 50% false negative errors, a target of cosmid length can be recovered with less than 0.1% miscalled bases.

Computational Biology↗
PubMed1996

Positional sequencing by hybridization.

Sequencing by hybridization (SBH) is a promising alternative to the classical DNA sequencing approaches. However, the resolving power of SBH is rather low: with 64kb sequencing chips, unknown DNA fragments only as long as 200 bp can be reconstructed in a single SBH experiment. To improve the resolving power of SBH, positional SBH (PSBH) has recently been suggested; this allows (with additional experimental work) approximate positions of every l-tuple in a target DNA fragment to be measured. We study the positional Eulerian path problem motivated by PSBH. The input to the positional eulerian path problem is an Eulerian graph G(V, E) in which every edge has an associated range of integers and the problem is to find an Eulerian path e1,...,e/E/ in G such that the range of ei contains i. We show that the positional Eulerian path problem is NP-complete even when the maximum out-degree (in-degree) of any vertex in the graph is 2. On a positive note we present polynomial algorithms to solve a special case of PSBH (bounded PSBH), where the range of the allowed positions for any edge is bounded by a constant (it corresponds to accurate experimental measurements of positions in PSBH). Moreover, if the positions of every l-tuple in an unknown DNA fragment of length n are measured with O(log n) error, then our algorithm runs in polynomial time. We also present an estimate of the resolving power of PSBH for a more realistic case when positions are measured with theta (n) error.

Algorithms↗
PubMed1997

Deletion of a single amino acid changes the folding of an apamin hybrid sequence peptide to that of endothelin.

The solution conformations of a hybrid sequence peptide related to the bee venom peptide apamin have been determined using two-dimensional 1H-nmr. Apamin is an 18 amino acid peptide containing a C-terminal helix that is stabilized by two disulfide bonds. The deletion of one residue (K4) of the N-terminal "scaffold" region of the apamin sequence results in a helical peptide, but with a change in the pairing of cysteines to form the disulfide cross links. The new disulfide arrangement is analogous to that of the vasoconstrictor peptide endothelin. Two sets of nmr resonances were observed for the apamin-deletion (AD) peptide, due to cistrans isomerism at the A4-P5 peptide bond. The cis isomer of the AD peptide contains a tight turn in residues 3-6, which is required for formation of the alpha-helix in residues 7-15. Nuclear Overhauser effects observed for the trans AD peptide are not consistent with any single unique fold, indicating the presence of conformational averaging when the peptide adopts the trans form. Distance geometry calculations on the cis AD peptide reveal an alpha-helical structure that appears to be more like that of apamin than the crystal structure of human endothelin, despite the reversal of the disulfide pattern in the AD peptide from that of apamin to that of endothelin.

Animals↗
PubMed1996

Poisson process approximation for sequence repeats, and sequencing by hybridization.

Sequencing by hybridization is a tool to determine a DNA sequence from the unordered list of all l-tuples contained in this sequence; typical numbers for l are l = 8, 10, 12. For theoretical purposes we assume that the multiset of all l-tuples is known. This multiset determines the DNA sequence uniquely if none of the so-called Ukkonen transformations are possible. These transformations require repeats of (l-1)-tuples in the sequence, with these repeats occurring in certain spatial patterns. We model DNA as an i.i.d. sequence. We first prove Poisson process approximations for the process of indicators of all leftmost long repeats allowing self-overlap and for the process of indicators of all left-most long repeats without self-overlap. Using the Chen-Stein method, we get bounds on the error of these approximations. As a corollary, we approximate the distribution of longest repeats. In the second step we analyze the spatial patterns of the repeats. Finally we combine these two steps to prove an approximation for the probability that a random sequence is uniquely recoverable from its list of l-tuples. For all our results we give some numerical examples including error bounds.

Algorithms↗
PubMed2001

Comparative expressed sequence hybridization to chromosomes for tumor classification and identification of genomic regions of differential gene expression.

Altered expression of genes can have phenotypic consequences in cancer development and treatment, developmental abnormalities, and differentiation processes. Here we describe a rapid approach, termed comparative expressed sequence hybridization (CESH), which gives a genome-wide view of relative expression patterns within tissues according to chromosomal location. No prior knowledge of genes or cloning is required, and minimal amounts of tissue can be used. Expression profiles are achieved in a manner similar to the identification of chromosomal imbalances by comparative genomic hybridization analysis. The approach is demonstrated to indicate a chromosomal region that harbors overexpressed genes that may be associated with a drug-resistant phenotype. In addition, known and new regions of differential gene expression in both normal tissues and tumor samples from the soft tissue sarcoma group of rhabdomyosarcoma (RMS) are indicated. These regions included 2p24; overexpression of MYCN at 2p24 was confirmed by quantitative reverse transcription-PCR for all of the alveolar RMS cases and did not necessarily correspond to genomic amplification. Evidence including region specific microarray analysis indicated that overexpression of several genes from a region may be required for detection by CESH. This evidence is consistent with clusters of functionally related genes and mechanisms that affect the expression of a number of genes at a particular genomic location. The distinctive CESH profiles demonstrated in different subtypes of RMS show potential for tumor classification.

Chromosomes, Artificial, Yeast↗
PubMed1980

Sequences hybridizing to mRNA, oligo(dT) and dsRNA from pre-mRNA are contiguous in the cloned mouse DNA fragments.

Fragments from the DNA of mouse embryos produced by restriction endonucleases HindIII were cloned in pBR322 plasmid and examined for the ability to hybridize in situ with [32P] labeled cDNA synthesized from the polysomal poly(A)+mRNA template. Several of the selected clones were examined for the presence of specific sequences inside the cloned mouse DNA fragments by the blotting procedure of southern [1]. The data obtained indicate that the majority of the cloned mouse DNA fragments contained sequences hybridizing with cDNA, oligo(dT) and double-stranded regions from pre-mRNA. The results of hybridization experiments and double digestion with HindIII+HaeIII endonucleases provide evidence that these sequences could be contiguous in the given restriction DNA fragments.

Animals↗
PubMed1990

Identification of human immunodeficiency virus hybridizing sequences in the peripheral blood of a patient with systemic lupus erythematosus.

Systemic lupus erythematosus, a multisystemic disorder, is considered a prototype of the autoimmune diseases. Although its cause remains unknown, a viral etiology has been proposed. We report that a rapid and sensitive messenger RNA in situ hybridization technique detected hybridizing sequences to the human immunodeficiency virus type in the peripheral blood cells of a woman with systemic lupus erythematosus in whom the presence of acquired immuno-deficiency syndrome was reasonably excluded.

Adult↗
PubMed1998

The stability of duplexes involving AT and/or G4EtC base pairs is not dependent on their AT/G4EtC ratio content. Implication for DNA sequencing by hybridization.

Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G4EtC base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51 degrees C). The 16 degrees C difference in thermal stability observed between the least stable and the most stable duplex built with AT and/or G4EtC base pairs is mainly due to the sequence effect and not to their AT/G4EtC ratio content. Thus N -4-ethyl-2'-deoxycytidine (d4EtC) hybridizes specifically with natural deoxyguanosine leading to a G4EtC base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d4EtC can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modified DNA targets were successfully amplified by random priming or PCR techniques using d4EtCTP, dATP, dGTP and dTTP in the presence of DNA polymerase. This new system might be very useful for DNA sequencing by hybridization.

Adenine↗
PubMed2001

Thermodynamics of the helix-coil transition: Binding of S15 and a hybrid sequence, disulfide stabilized peptide to the S-protein.

Pancreatic ribonuclease A may be cleaved to produce two fragments: the S-peptide (residues 1-20) and the S-protein (residues 21-124). The S-peptide, or a truncated version designated as the S15 peptide (residues 1-15), combines with the S-protein to produce catalytically active complexes. The conformation of these peptides and many of their analogues is predominantly random coil at room temperature; however, they populate a significant fraction of helical form at low temperature under certain solution conditions. Moreover, they adopt a helical conformation when bound to the S-protein. A hybrid sequence, disulfide-stabilized peptide (ApaS-25), designed to stabilize the helical structure of the S-peptide in solution, also combines with the S-protein to yield a catalytically active complex. We have performed high-precision titration microcalorimetric measurements to determine the free energy, enthalpy, entropy, and heat capacity changes for the binding of ApaS-25 to S-protein within the temperature range 5-25 degrees C. The thermodynamic parameters for both the complex formation reactions and the helix-to-coil transition also were calculated, using a structure-based approach, by calculating changes in accessible surface area and using published empirical parameters. A simple thermodynamic model is presented in an attempt to account for the differences between the binding of ApaS-25 and the S-peptide. From this model, the thermodynamic parameters of the helix-to-coil transition of S15 can be calculated.

Amino Acids↗
PubMed2001

Analysis of the constitution of the beer yeast genome by PCR, sequencing and subtelomeric sequence hybridization.

The lager brewing yeasts, Saccharomyces pastorianus (synonym Saccharomyces carlsbergensis), are allopolyploid, containing parts of two divergent genomes. Saccharomyces cerevisiae contributed to the formation of these hybrids, although the identity of the other species is still unclear. The presence of alleles specific to S. cerevisiae and S. pastorianus was tested for by PCR/RFLP in brewing yeasts of various origins and in members of the Saccharomyces sensu stricto complex. S. cerevisiae-type alleles of two genes, HIS4 and YCL008c, were identified in another brewing yeast, S. pastorianus CBS 1503 (Saccharomyces monacensis), thought to be the source of the other contributor to the lager hybrid. This is consistent with the hybridization of S. cerevisiae subtelomeric sequences X and Y' to the electrophoretic karyotype of this strain. S. pastorianus CBS 1503 (S. monacensis) is therefore probably not an ancestor of S. pastorianus, but a related hybrid. Saccharomyces bayanus, also thought to be one of the contributors to the lager yeast hybrid, is a heterogeneous taxon containing at least two subgroups, one close to the type strain, CBS 380T, the other close to CBS 395 (Saccharomyces uvarum). The partial sequences of several genes (HIS4, MET10, URA3) were shown to be identical or very similar (over 99%) in S. pastorianus CBS 1513 (S. carlsbergensis), S. bayanus CBS 380T and its close derivatives, showing that S. pastorianus and S. bayanus have a common ancestor. A distinction between two subgroups within S. bayanus was made on the basis of sequence analysis: the subgroup represented by S. bayanus CBS 395 (S. uvarum) has 6-8% sequence divergence within the genes HIS4, MET10 and MET2 from S. bayanus CBS 380T, indicating that the two S. bayanus subgroups diverged recently. The detection of specific alleles by PCR/RFLP and hybridization with S. cerevisiae subtelomeric sequences X and Y' to electrophoretic karyotypes of brewing yeasts and related species confirmed our findings and revealed substantial heterogeneity in the genome constitution of Czech brewing yeasts used in production.

Alleles↗