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At least 685 records · Page 38Linked to original sources
PubMed2004

Stable expression of 1Dx5 and 1Dy10 high-molecular-weight glutenin subunit genes in transgenic rye drastically increases the polymeric glutelin fraction in rye flour.

We generated and characterized transgenic rye synthesizing substantial amounts of high-molecular-weight glutenin subunits (HMW-GS) from wheat. The unique bread-making characteristic of wheat flour is closely related to the elasticity and extensibility of the gluten proteins stored in the starchy endosperm, particularly the HMW-GS. Rye flour has poor bread-making quality, despite the extensive sequence and structure similarities of wheat and rye HMW-GS. The HMW-GS 1Dx5 and 1Dy10 genes from wheat, known to be associated with good bread-making quality were introduced into a homozygous rye inbred line by the biolistic gene transfer. The transgenic plants, regenerated from immature embryo derived callus cultures were normal, fertile, and transmitted the transgenes stably to the sexual progeny, as shown by Southern blot and SDS-PAGE analysis. Flour proteins were extracted by means of a modified Osborne fractionation from wildtype (L22) as well as transgenic rye expressing 1Dy10 (L26) or 1Dx5 and 1Dy10 (L8) and were quantified by RP-HPLC and GP-HPLC. The amount of transgenic HMW-GS in homozygous rye seeds represented 5.1% (L26) or 16.3% (L8) of the total extracted protein and 17% (L26) or 29% (L8) of the extracted glutelin fraction. The amount of polymerized glutelins was significantly increased in transgenic rye (L26) and more than tripled in transgenic rye (L8) compared to wildtype (L22). Gel permeation HPLC of the un-polymerized fractions revealed that the transgenic rye flours contained a significantly lower proportion of alcohol-soluble oligomeric proteins compared with the non-transgenic flour. The quantitative data indicate that the expression of wheat HMW-GS in rye leads to a high degree of polymerization of transgenic and native storage proteins, probably by formation of intermolecular disulfide bonds. Even gamma-40k secalins, which occur in non-transgenic rye as monomers, are incorporated into these polymeric structures. The combination 1Dx5 + 1Dy10 showed stronger effects than 1Dy10 alone. Our results are the first example of genetic engineering to significantly alter the polymerization and composition of storage proteins in rye. This may be an important step towards improving bread-making properties of rye whilst conserving its superior stress resistance.

Chromatography, High Pressure Liquid↗
PubMed2003

ASTRO-FOLD: a combinatorial and global optimization framework for Ab initio prediction of three-dimensional structures of proteins from the amino acid sequence.

The field of computational biology has been revolutionized by recent advances in genomics. The completion of a number of genome projects, including that of the human genome, has paved the way toward a variety of challenges and opportunities in bioinformatics and biological systems engineering. One of the first challenges has been the determination of the structures of proteins encoded by the individual genes. This problem, which represents the progression from sequence to structure (genomics to structural genomics), has been widely known as the structure-prediction-in-protein-folding problem. We present the development and application of ASTRO-FOLD, a novel and complete approach for the ab initio prediction of protein structures given only the amino acid sequences of the proteins. The approach exhibits many novel components and the merits of its application are examined for a suite of protein systems, including a number of targets from several critical-assessment-of-structure-prediction experiments.

Algorithms↗
PubMed1998

Application of a computer program for genetic typing of classical swine fever virus isolates from Germany.

The commercial software program HLA SequiTyper (Amersham Pharmacia Biotech), designed originally for human leukocyte antigen typing, was adapted for rapid typing of classical swine fever (CSF) virus isolates. The program compares new sequence data with those stored in a database file and calculates the most probable assignment. For generating the CSF virus sequence database, 150 bp of the 5' nontranslated genomic region (5'-NTR) from 88 German classical swine fever virus isolates from outbreaks between 1984 and 1997 were solid-phase sequenced directly after RT-PCR amplification. Sequence alignments showed that they all belonged to the previously defined genetic group 2. Within this group, six different subgroups could be distinguished, and were designated according to the geographic location where they are either still endemic or where they appeared most commonly. The advantage of using the HLA SequiTyper program is that it reads directly the sequence files as generated by the ALF sequencer (Amersham Pharmacia Biotech), making any manipulations unnecessary. In addition, a constant quality control of the raw sequence data can be achieved, as more than one sequence from the same isolate can be evaluated at once. Using this approach, new CSF isolates can be typed within 2 days.

Animals↗
PubMed2004

Algorithms for sequence analysis via mutagenesis.

MOTIVATION: Despite many successes of conventional DNA sequencing methods, some DNAs remain difficult or impossible to sequence. Unsequenceable regions occur in the genomes of many biologically important organisms, including the human genome. Such regions range in length from tens to millions of bases, and may contain valuable information such as the sequences of important genes. The authors have recently developed a technique that renders a wide range of problematic DNAs amenable to sequencing. The technique is known as sequence analysis via mutagenesis (SAM). This paper presents a number of algorithms for analysing and interpreting data generated by this technique. RESULTS: The essential idea of SAM is to infer the target sequence using the sequences of mutants derived from the target. We describe three algorithms used in this process. The first algorithm predicts the number of mutants that will be required to infer the target sequence with a desired level of accuracy. The second algorithm infers the target sequence itself, using the mutant sequences. The third algorithm assigns quality values to each inferred base. The algorithms are illustrated using mutant sequences generated in the laboratory.

Algorithms↗
PubMed2003

[The dual-index sequence analytical method of common peak ratio and variant ratio for analysing UV fingerprint spectra of Radix Glycyrrhizae].

OBJECTIVE: To set up the dual-index sequence analytical method for UV fingerprint spectra (FPS) in which the dual indices are common peak ratio and variant ration. METHODS: Three kinds of solvents chloroform, alcohol and water were used to extract different polarity components from Radix Glycyrrhizae, and then the UV FPS of the components were measured, which were applied to distinguish the samples. RESULTS: This method is effective for distinguishing Radix Glycyrrhizae from inner Mongolina, Gansu and Xinjiang. CONCLUSION: The UV-FPS dual-index sequence analytical method is feasible.

Glycyrrhiza↗
PubMed2000

Method enabling pyrosequencing on double-stranded DNA.

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlén, and P. Nyrén, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA. In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.

Alkaline Phosphatase↗
PubMed2006

Microarray quality control.

Physically separated groups of specific sequences (probes) provide useful high through put (HTP) measurements for the amount of selected DNA/RNA sequences in a biological target sample. Unfortunately, these measurements are impacted by various technical sources, such as platform production factors, target preparation processes, hybridization method/conditions, and signal-extraction devices and methods. Given the typically huge population of signals, statistical methods are especially effective at estimation and removal of such technical distortions (Churchill, 2002; Kerr et al., 2000; Yue et al., 2001), as well as providing metrics for computer-based quality control (QC), for example, autoQC (Minor et al., 2002a). This chapter reviews statistical procedures that have been validated by successful applications in both large-scale commercial ventures (Ganter et al., 2005) and individual research studies (Parisi et al., 2003, 2004) involving HTP projects. This chapter focuses on methods for spatially distributed probes on a flat medium surface such as glass, collectively known as a microarray.

Algorithms↗
PubMed2006

Evidence standards in experimental and inferential INSDC Third Party Annotation data.

The Third Party Annotation (TPA) project collects and presents high-quality annotation of nucleotide sequence. Annotation is submitted by researchers who have not themselves generated novel nucleotide sequence. In its first few years, the resource has proven to be popular with submitters from a range of biological research areas. Central to the project is the requirement for high-quality data, resulting from experimental and inferred analysis discussed in peer-reviewed publications. The data are divided into two tiers: those with experimental evidence and those with inferential evidence. Standards for TPA are detailed and illustrated with the aid of case studies.

Animals↗
PubMed2004

COACH: profile-profile alignment of protein families using hidden Markov models.

MOTIVATION: Alignments of two multiple-sequence alignments, or statistical models of such alignments (profiles), have important applications in computational biology. The increased amount of information in a profile versus a single sequence can lead to more accurate alignments and more sensitive homolog detection in database searches. Several profile-profile alignment methods have been proposed and have been shown to improve sensitivity and alignment quality compared with sequence-sequence methods (such as BLAST) and profile-sequence methods (e.g. PSI-BLAST). Here we present a new approach to profile-profile alignment we call Comparison of Alignments by Constructing Hidden Markov Models (HMMs) (COACH). COACH aligns two multiple sequence alignments by constructing a profile HMM from one alignment and aligning the other to that HMM. RESULTS: We compare the alignment accuracy of COACH with two recently published methods: Yona and Levitt's prof_sim and Sadreyev and Grishin's COMPASS. On two sets of reference alignments selected from the FSSP database, we find that COACH is able, on average, to produce alignments giving the best coverage or the fewest errors, depending on the chosen parameter settings. AVAILABILITY: COACH is freely available from www.drive5.com/lobster

Algorithms↗
PubMed2004

A graph based algorithm for generating EST consensus sequences.

MOTIVATION: EST sequences constitute an abundant, yet error prone resource for computational biology. Expressed sequences are important in gene discovery and identification, and they are also crucial for the discovery and classification of alternative splicing. An important challenge when processing EST sequences is the reconstruction of mRNA by assembling EST clusters into consensus sequences. RESULTS: In contrast to the more established assembly tools, we propose an algorithm that constructs a graph over sequence fragments of fixed size, and produces consensus sequences as traversals of this graph. We provide a tool implementing this algorithm, and perform an experiment where the consensus sequences produced by our implementation, as well as by currently available tools, are compared to mRNA. The results show that our proposed algorithm in a majority of the cases produces consensus of higher quality than the established sequence assemblers and at a competitive speed. AVAILABILITY: The source code for the implementation is available under a GPL license from http://www.ii.uib.no/~ketil/bioinformatics/ CONTACT: ketil@ii.uib.no.

Algorithms↗
PubMed2006

FeatureMap3D--a tool to map protein features and sequence conservation onto homologous structures in the PDB.

FeatureMap3D is a web-based tool that maps protein features onto 3D structures. The user provides sequences annotated with any feature of interest, such as post-translational modifications, protease cleavage sites or exonic structure and FeatureMap3D will then search the Protein Data Bank (PDB) for structures of homologous proteins. The results are displayed both as an annotated sequence alignment, where the user-provided annotations as well as the sequence conservation between the query and the target sequence are displayed, and also as a publication-quality image of the 3D protein structure with the selected features and sequence conservation enhanced. The results are also returned in a readily parsable text format as well as a PyMol (http://pymol.sourceforge.net/) script file, which allows the user to easily modify the protein structure image to suit a specific purpose. FeatureMap3D can also be used without sequence annotation, to evaluate the quality of the alignment of the input sequences to the most homologous structures in the PDB, through the sequence conservation colored 3D structure visualization tool. FeatureMap3D is available at: http://www.cbs.dtu.dk/services/FeatureMap3D/.

Amino Acid Sequence↗
PubMed1996

[A new application of MR tomography of the lung using ultra-short turbo spin echo sequences].

PURPOSE: Development of an improved MR sequence for examining the lung. METHOD: T2 weighted ultra-short turbo spin echo sequences were used in five individuals with variations in echo times, delayed triggering and echo intervals. To reduce movement artifacts all examinations were carried out with ECG and respiratory triggering. The sequences giving optimal image quality were then employed in 19 patients having various pulmonary abnormalities. Image resolutions, artifacts, image contrasts and diagnostic value were then judged by two observers and compared with CT. RESULTS: In the first study, a diastole-triggered UTSE sequence with the shortest echo proved optimal (TE = 90 ms, TR = 2-4 s, echo = 9 ms, turbo factor = 19). In the patient series studied, MRT was inferior to CT with regard to resolution and number of artifacts, but better in respect of contrast and diagnostic value. CONCLUSION: Using UTSE of the lung, MRT can produce images of good quality. Compared with CT, contrast is better with MRT, offering diagnostic advantages for MRT.

Diagnosis, Differential↗
PubMedDate not supplied

MR of focal liver lesions: comparison of breath-hold and non-breath-hold hybrid RARE and conventional spin-echo T2-weighted pulse sequences.

To compare liver lesion detection rates, tissue signal and noise data, and qualitative parameters for breath-hold (BH) and non-breath-hold (NBH) hybrid rapid acquisition with relaxation enhancement (RARE) and conventional spin-echo (CSE) T2-weighted (CSE-T2) MR sequences, 20 patients were imaged using all three sequences. Lesion detection rates were 73.5% for the CSE-T2 sequence and 81.1% and 88.6% for the BH-RARE and NBH-RARE sequences, respectively (P = .027). Mean lesion-to-liver signal-difference-to-noise ratio for the NBH-RARE sequence was 14.0 +/- 11.5, significantly greater than 9.8 +/- 7.8 obtained for the BH-RARE sequence (P = .050) and 9.0 +/- 6.2 obtained for the CSE-T2 sequence (P = .015). The NBH-RARE sequence demonstrated fewer artifacts and greater overall image quality compared to the CSE-T2 sequence. The NBH-RARE sequence is a useful alternative to the liver signal-difference-to-noise ratio and lesion detection rate and better overall image quality.

Adult↗
PubMed2002

Imaging articular cartilage defects in the ankle joint with 3D fat-suppressed echo planar imaging: comparison with conventional 3D fat-suppressed gradient echo imaging.

PURPOSE: To shorten the examination time for articular cartilage imaging, using a recently developed three-dimensional (3D) multishot echo planar imaging (EPI) sequence with fat saturated (FS), compared to a conventional 3D fat-saturated spoiled gradient echo sequence (3D FS GRE). MATERIAL AND METHODS: There were 32 consecutive patients with ankle joint disorders who underwent magnetic resonance imaging (MRI) in a 1.0-T unit. Hyaline cartilage was imaged with a 3D FS EPI sequence and a 3D FS GRE sequence. Image assessment criteria included lesion conspicuity, contrast between different types of normal tissue, and image artifacts. In addition, contrast-to-noise ratios (CNRs) of cartilage vs. joint fluid and bone marrow were measured. RESULTS: The 3D FS EPI sequence provided a high CNR between cartilage and subchondral bone, similar to that of the 3D FS GRE sequence. The CNR between cartilage and effusion was significantly lower on the 3D EPI sequence due to the higher signal intensity of fluid. Both sequences were equal in lesion detection ability. The image quality of the 3D FS GRE sequence was slightly higher than that of the 3D FS EPI, but the difference was not statistically significant. CONCLUSION: We conclude that the 3D FS EPI sequence is comparable to the 3D FS GRE sequence in the detection of cartilage lesions, with the additional advantage of reduction in scan time by a factor of 4.

Adolescent↗
PubMed2005

Quality assessment of microarray experiments.

OBJECTIVES: We describe a quality assurance procedure to maximize the value of oligonucleotide microarray expression profiles. DESIGN, METHODS, AND RESULTS: Background microarray noise was 82.2+/-54.5 and 51.8+/-12.4 units, respectively, before and after enacting the program (P<0.0001). We also noted improved concordance of microarray expression fold-changes for selected genes with results of RT-PCR validation. CONCLUSIONS: This multi-step procedure, including quantification of RNA sample degradation and detection of outlier data points, has increased data quality from our microarray facility.

Animals↗
PubMed1999

Establishing a method of vector contamination identification in database sequences.

MOTIVATION: The nucleotide sequence databases are invaluable tools both for the private and the academic research communities, from the retrieval of sequences to homology searching. Several issues related to data quality, such as the existence of sequencing artifacts and errors, are facing the databases. We investigated a major source of these errors, i.e. the presence of vector-contaminated sequences. RESULTS: Using a panel of 180 vector polylinker sequences, we found 0.36% or 3029 vector-matching sequences in GenBank Release 95-96, with an average vector-matching length of 72 nucleotides. The number of vector-contaminated sequences has been growing with the database; however, the percent contamination has remained approximately constant at an average of 0.28% from 1982 to 1996. AVAILABILITY: Access to the database of vector polylinker sequences via sequence similarity searching is available at http://seqsim.ncgr.org/vector/ CONTACT: gas@molinfo.com

Animals↗