On Graphical and Numerical Representation of Protein Sequences

Abstract

A new approach using a 3-D Cartesian coordinate system to represent protein sequences has been derived. By the 3-D Graphical representation we make a comparison of sequences belonging to nine different proteins.     

The Ha locus of wheat: identification of a polymorphic region for tracing grain hardness in crosses.

The grain softness proteins or friabilins are known to be composed of three main components: puroindoline a, puroindoline b, and GSP-1. cDNAs for GSP-1 have previously been mapped to group-5 chromosomes and their location on chromosome 5D is closely linked to the grain hardness (Ha) locus of hexaploid wheat. A genomic DNA clone containing the GSP-1 gene (wGSP1-A1) from hexaploid wheat has been identified by fluorescent in situ hybridization as having originated from the distal end of the short arm of chromosome 5A. A genomic clone containing the gene (wGSP1-D1) was also isolated from Aegilops tauschii, the donor of the D genome to bread wheat. There are no introns in the GSP-1 genes, and there is high sequence identity between wGSP1-A1 and wGSP1-D1 up to 1 kb 5' and 300 bp 3' to wGSP1-D1. However, regions further upstream and downstream of wGSP1-D1 share no significant sequence identity to corresponding sequences in wGSP1-A1. These regions therefore identified potentially valuable sequences for tracing the Ha locus through assaying polymorphic DNA sequences. The sequence from 300 to 500 bp 3' to wGSP1-D1 (wGSP1-D13) was mapped to the Ha locus in a mapping population. wGSP1-D13 was also tightly linked to genes for puroindoline a and puroindoline b which have been previously mapped to be at the Ha locus. In addition wGSP1-D13 was used to detect RFLPs between near isogenic soft and hard Falcon lines and in a random selection of soft and hard wheats.     

Structural study of irregular amino acid sequences in the heavy chain of Bombyx mori silk fibroin

Recently, genetic studies have revealed the entire amino acid sequence of Bombyx mori silk fibroin. It is known from X-ray diffraction studies that the beta-sheet crystalline structure (silk II) of fibroin is composed of hexaamino acid sequences of GAGAGS. However, in the heavy chain of B. mori silk fibroin, there are also present 11 irregular sequences, with about 31 amino acid residues (irregular GT approximately GT sequences). The structure and role of these irregular sequences have remained unknown. One of the most frequently appearing irregular sequences was synthesized and its 3-D solution structure was studied by high-resolution 2-D NMR techniques. The 3-D structure determined for this peptide shows that it makes a loop structure (distorted omega shape), which implies that the preceding backbone direction is changed by 180 degrees, i.e., reversed, by this sequence. This may facilitate the beta-sheet formation between the crystal-forming building blocks, GAGAGS/GY approximately GY sequences, in the fibroin heavy chain.     

2-D graphical representation for characteristic sequences of DNA and its application

DNA sequencing has resulted in an abundance of data on DNA sequences for various species. Hence, the characterization and comparison of sequences become more important but still difficult tasks. In this paper, we first give a 2-D ladderlike graphical representation for the characteristic sequences of a DNA sequence, and then construct a 3-component vector, in which the normalized ALE-indices extracted from such three 2-D graphs via D/D matrices are individual components, to characterize the DNA sequence. The examination of similarities/dissimilarities among sequences of the beta-globin genes of different species illustrates the utility of the approach.     

Noninvasive three-dimensional electrocardiographic imaging of ventricular activation sequence

Imaging the myocardial activation sequence is critical for improved diagnosis and treatment of life-threatening cardiac arrhythmias. It is desirable to reveal the underlying cardiac electrical activity throughout the three-dimensional (3-D) myocardium (rather than just the endocardial or epicardial surface) from noninvasive body surface potential measurements. A new 3-D electrocardiographic imaging technique (3-DEIT) based on the boundary element method (BEM) and multiobjective nonlinear optimization has been applied to reconstruct the cardiac activation sequences from body surface potential maps. Ultrafast computerized tomography scanning was performed for subsequent construction of the torso and heart models. Experimental studies were then conducted, during left and right ventricular pacing, in which noninvasive assessment of ventricular activation sequence by means of 3-DEIT was performed simultaneously with 3-D intracardiac mapping (up to 200 intramural sites) using specially designed plunge-needle electrodes in closed-chest rabbits. Estimated activation sequences from 3-DEIT were in good agreement with those constructed from simultaneously recorded intracardiac electrograms in the same animals. Averaged over 100 paced beats (from a total of 10 pacing sites), total activation times were comparable (53.3 +/- 8.1 vs. 49.8 +/- 5.2 ms), the localization error of site of initiation of activation was 5.73 +/- 1.77 mm, and the relative error between the estimated and measured activation sequences was 0.32 +/- 0.06. The present experimental results demonstrate that the 3-D paced ventricular activation sequence can be reconstructed by using noninvasive multisite body surface electrocardiographic measurements and imaging of heart-torso geometry. This new 3-D electrocardiographic imaging modality has the potential to guide catheter-based ablative interventions for the treatment of life-threatening cardiac arrhythmias.     

Phylogenetic Analysis of Ruminant Theileria spp. from China Based on 28S Ribosomal RNA Gene

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.     

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class ( Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue ( Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.     

The HSSP database of protein structure-sequence alignments.

HSSP is a derived database merging structural (3-D) and sequence (1-D) information. For each protein of known 3-D structure from the Protein Data Bank (PDB), the database has a multiple sequence alignment of all available homologues and a sequence profile characteristic of the family. The list of homologues is the result of a database search in SwissProt using a position-weighted dynamic programming method for sequence profile alignment (MaxHom). The database is updated frequently. The listed homologues are very likely to have the same 3-D structure as the PDB protein to which they have been aligned. As a result, the database is not only a database of aligned sequence families, but also a database of implied secondary and tertiary structures covering 29% of all SwissProt-stored sequences.     

The HSSP database of protein structure-sequence alignments.

HSSP is a derived database merging structural three dimensional (3-D) and sequence one dimensional(1-D) information. For each protein of known 3-D structure from the Protein Data Bank (PDB), the database has a multiple sequence alignment of all available homologues and a sequence profile characteristic of the family. The list of homologues is the result of a database search in Swissprot using a position-weighted dynamic programming method for sequence profile alignment (MaxHom). The database is updated frequently. The listed homologues are very likely to have the same 3-D structure as the PDB protein to which they have been aligned. As a result, the database is not only a database of aligned sequence families, but also a database of implied secondary and tertiary structures covering 27% of all Swissprot-stored sequences.     

Confocal-multilabeling, ultrasensitive TUNEL analysis of DNA breaks in individual cells

OBJECTIVE: To visualize and localize fragmented DNA strands within apoptotic cells by means of fluorescence using TdT-mediated dUTP-biotin nick end labeling (TUNEL) techniques, laser scanning confocal microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: For this experiment, lymphoid reverted cells were used as a model. Characteristic DNA breaks inside apoptotic cells were detected using TUNEL techniques by a reaction involving tetramethyl rhodamin isothyocyanate (TRITC). The DNA from cell nuclei was counterstained using chromomycin A3 (CA3). The tandem TRITC-CA3 in CLSM was applied to investigate the ability to detect DNA breaks in individual cells using TUNEL techniques and its amplified variants (TUNEL-CARD). FAMIS was applied on dynamic sequences of images of TUNEL preparations and on four-dimensional (4-D) sequences of images of TUNEL-CARD preparations. RESULTS: Distribution and amplitude of fluorescent structures were characterized on dynamic sequences of images. Characterization was improved when FAMIS was applied on 4-D sequences of images, taking into account differences in photobleaching and/or spectrum of TRITC and CA3. CONCLUSION: It is possible to discriminate targets from CA3. FAMIS and TUNEL methods can be used to visualize and localize multiple DNA breaks in lymphoid reverted cells in improved methods of experimentation.     

基于3-D图形表示的DNA序列的相似性分析

基于DNA序列的3D图形表示,通过L／L矩阵的规范化最大特征值组成的3维向量来刻画了DNA序列,并基于这种方法,用β-globin基因的第一个外显子分析了11个物种的相似性问题。     

Inverse sequence similarity of proteins does not imply structural similarity

There is a debate on the folding of proteins with inverted sequences. Theoretical approaches and experiments give contradictory results. Many proteins in the Protein Data Bank (PDB) show conspicuous inverse sequence similarity (ISS) to each other. Here we analyze whether this ISS is related to structural similarity. For the first time, we performed a large scale three-dimensional (3-D) superposition of corresponding Calpha atoms of forwardly and inversely aligned proteins and tested the degree of secondary structure identity between them. Comparing proteins of less than 50% pairwise sequence identity, only 0.5% of the inversely aligned pairs had similar folds (99 out of 19073), whereas about 9% of forwardly aligned proteins in the same score and length range show similar 3-D structures (1731 out of 19248). This observation strongly supports the view that the inversion of sequences in almost all cases leads to a different folding property of the protein. Inverted sequences are thus suitable as protein-like sequences for control purposes without relations to existing proteins.     

Genes encoding human killer-cell Ig-like receptors with D1 and D2 extracellular domains all contain untranslated pseudoexons encoding a third Ig-like domain

Human killer-cell immunoglobulin-like receptors (KIR) show three types of organization of their extracellular domains: D0-D1-D2 in KIR3D, D1-D2 in the majority of KIR2D, and D0-D2 in KIR2DL4 and the novel KIR2DL5. The gene for a KIR2DL3 variant, which has a D1-D2 structure, has been shown previously to have a nonexpressed region (pseudoexon 3) that is paralogous to the exon encoding the D0 domain of other KIR. This pseudoexon is not expressed because it is skipped during splicing of pre-mRNA. In this study, we demonstrate that all eight genes encoding human KIR with D1-D2 configuration (KIR2DL1-KIR2DL3, KIR2DS1-KIR2DS5) have similarly untranslated pseudoexons. Whereas the pseudoexons of four of these KIR genes bear nonsense mutations and/or altered splicing sites, the pseudoexons in the other four KIR genes have no major structural abnormalities, indicating that other mechanisms are responsible for inactivation of their exons 3. A comparison of the sequences on pseudoexons 3 with the paralogous expressed exons suggests that an exonic splicing enhancer may be necessary for the expression of exon 3 in KIR genes.     

Physical mapping of chromosome 3p25-p26 by flourescence in situ hybridisation (FISH)

As part of our effort to isolate and characterise the von Hippel-Lindau (VHL) disease gene, we constructed a physical map of chromosome 3p25-26 by fluorescence in situ hybridisation (FISH) studies on a panel of cytogenetic rearrangements involving this region. Biotinylated cosmid and lambda probes were hybridised to metaphase chromosome spreads and positioned with respect to each cytogenetic breakpoint. These studies unequivocally established the order of five loci linked to the VHL disease gene: cen-(RAF1,312)-D3S732-D3S1250-D3S601-D3S18-pter and determined the position of three other probes within this map. These results ordered RAF1 and D3S732 for the first time, confirmed the localisation of D3S1250 between RAF1 and D3S601 and determined the position of D3S651 with respect to other chromosome 3p25-p26 loci. The establishment of an ordered set of cytogenetic aberrations will enable the rapid assignment of polymorphic and nonpolymorphic cloned sequences within the chromosome region 3p25-p26.     

Mapping 3-D functional capillary geometry in rat skeletal muscle in vivo

We have developed a novel mapping software package to reconstruct microvascular networks in three dimensions (3-D) from in vivo video images for use in blood flow and O2 transport modeling. An intravital optical imaging system was used to collect video sequences of blood flow in microvessels at different depths in the tissue. Functional images of vessels were produced from the video sequences and were processed using automated edge tracking software to yield location and geometry data for construction of the 3-D network. The same video sequences were analyzed for hemodynamic and O2 saturation data from individual capillaries in the network. Simple user-driven commands allowed the connection of vessel segments at bifurcations, and semiautomated registration enabled the tracking of vessels across multiple focal planes and fields of view. The reconstructed networks can be rotated and manipulated in 3-D to verify vessel connections and continuity. Hemodynamic and O2 saturation measurements made in vivo can be indexed to corresponding vessels and visualized using colorized maps of the vascular geometry. Vessels in each reconstruction are saved as text-based files that can be easily imported into flow or O2 transport models with complete geometry, hemodynamic, and O2 transport conditions. The results of digital morphometric analysis of seven microvascular networks showed mean capillary diameters and overall capillary density consistent with previous findings using histology and corrosion cast techniques. The described mapping software is a valuable tool for the quantification of in vivo microvascular geometry, hemodynamics, and oxygenation, thus providing rich data sets for experiment-based computational models.     

Protein surface representation and analysis by dimension reduction

Background

Protein structures are better conserved than protein sequences, and consequently more functional information is available in structures than in sequences. However, proteins generally interact with other proteins and molecules via their surface regions and a backbone-only analysis of protein structures may miss many of the functional and evolutionary features. Surface information can help better elucidate proteins' functions and their interactions with other proteins. Computational analysis and comparison of protein surfaces is an important challenge to overcome to enable efficient and accurate functional characterization of proteins.

Methods

In this study we present a new method for representation and comparison of protein surface features. Our method is based on mapping the 3-D protein surfaces onto 2-D maps using various dimension reduction methods. We have proposed area and neighbor based metrics in order to evaluate the accuracy of this surface representation. In order to capture functionally relevant information, we encode geometric and biochemical features of the protein, such as hydrophobicity, electrostatic potential, and curvature, into separate color channels in the 2-D map. The resulting images can then be compared using efficient 2-D image registration methods to identify surface regions and features shared by proteins.

Results

We demonstrate the utility of our method and characterize its performance using both synthetic and real data. Among the dimension reduction methods investigated, SNE, LandmarkIsomap, Isomap, and Sammon's mapping provide the best performance in preserving the area and neighborhood properties of the original 3-D surface. The enriched 2-D representation is shown to be useful in characterizing the functional site of chymotrypsin and able to detect structural similarities in heat shock proteins. A texture mapping using the 2-D representation is also proposed as an interesting application to structure visualization.

     

Natural selection for 2,4,5-trichlorophenoxyacetic acid mineralizing bacteria in agent orange contaminated soil

Agent Orange contaminated soils were utilized in direct enrichment culture studies to isolate 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D) mineralizing bacteria. Two bacterial cultures able to grow at the expense of 2,4,5-T and/or 2,4-D were isolated. The 2,4,5-T degrading culture was a mixed culture containing two bacteria, Burkholderia species strain JR7B2 and Burkholderia species strain JR7B3. JR7B3 was able to metabolize 2,4,5-T as the sole source of carbon and energy, and demonstrated the ability to affect metabolism of 2,4-D to a lesser degree. Strain JR7B3 was able to mineralize 2,4,5-T in pure culture and utilized 2,4,5-T in the presence of 0.01 yeast extract. Subsequent characterization of the 2,4-D degrading culture showed that one bacterium, Burkholderiaspecies strain JRB1, was able to utilize 2,4-D as a sole carbon and energy source in pure culture. Polymerase chain reaction (PCR) experiments utilizing known genetic sequences from other 2,4-D and 2,4,5-T degrading bacteria demonstrated that these organisms contain gene sequences similar to tfdA, B, C, E, and R (Strain JRB1) and the tftA, C, and E genes (Strain JR7B3). Expression analysis confirmed that tftA, C, and E and tfdA, B, and C were transcribed during 2,4,5-T and 2,4-D dependent growth, respectively. The results indicate a strong selective pressure for 2,4,5-T utilizing strains under field condition.     

2,4-D impact on bacterial communities, and the activity and genetic potential of 2,4-D degrading communities in soil

The key role of telluric microorganisms in pesticide degradation is well recognized but the possible relationships between the biodiversity of soil microbial communities and their functions still remain poorly documented. If microorganisms influence the fate of pesticides, pesticide application may reciprocally affect soil microorganisms. The objective of our work was to estimate the impact of 2,4-D application on the genetic structure of bacterial communities and the 2,4-D-degrading genetic potential in relation to 2,4-D mineralization. Experiments combined isotope measurements with molecular analyses. The impact of 2,4-D on soil bacterial populations was followed with ribosomal intergenic spacer analysis. The 2,4-D degrading genetic potential was estimated by real-time PCR targeted on tfdA sequences coding an enzyme specifically involved in 2,4-D mineralization. The genetic structure of bacterial communities was significantly modified in response to 2,4-D application, but only during the intense phase of 2,4-D biodegradation. This effect disappeared 7 days after the treatment. The 2,4-D degrading genetic potential increased rapidly following 2,4-D application. There was a concomitant increase between the tfdA copy number and the 14C microbial biomass. The maximum of tfdA sequences corresponded to the maximum rate of 2,4-D mineralization. In this soil, 2,4-D degrading microbial communities seem preferentially to use the tfd pathway to degrade 2,4-D.     

Subregional mapping of 13 single-copy genes on the long arm of chromosome 12 by fluorescence in situ hybridization.

Subregional localization of 13 single-copy DNA sequences previously assigned to the long arm of chromosome 12 has been performed using the fluorescence in situ hybridization (FISH) technique. The following order is suggested for the 13 mapped genes: cen-->COL2A1-->(VDR-D12S15)-->(D12S17-D12S4++ +-D12S14-D12S6)-->D12S8-->(IAPP-MGF- D12S7-D12S12)-->IGF1-->qter. Eight of the mapped genes clustered at two regions, one at 12q13 (D12S17-D12S4-D12S14-D12S6) and the other at 12q22 (IAPP-MGF-D12S7-D12S12). Our results show that single-copy DNA sequences as small as 500 bp can be successfully mapped by FISH.