VIRS: A visual tool for identifying restriction sites in multiple DNA sequences

VIRS (A visual tool for identifying restriction sites in multiple DNA sequences) is an interactive web‐based program designed for restriction endonuclease cut sites prediction and visualization. It can afford to analyze multiple DNA sequences simultaneously and produce visual restriction maps with several useful options intended for users' customization. These options also perform in‐depth analysis of the restriction maps, such as providing virtual electrophoretic result for digested fragments. Different from other analytical tools, VIRS not only displays visual outputs but also provides the detailed properties of restriction endonucleases that are commercially available. All the information of these enzymes is stored in our internal database, which is updated monthly from the manufacturers' web pages. It is freely available online at http://bis.zju.edu.cn/virs/index.html . © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Delineating relative homogeneous G+C domains in DNA sequences

The concept of homogeneity of G+C content is always relative and subjective. This point is emphasized and quantified in this paper using a simple example of one sequence segmented into two subsequences. Whether the sequence is homogeneous or not can be answered by whether the two-subsequence model describes the DNA sequence better than the one-sequence model. There are at least three equivalent ways of looking at the 1-to-2 segmentation: Jensen-Shannon divergence measure, log likelihood ratio test, and model selection using Bayesian information criterion. Once a criterion is chosen, a DNA sequence can be recursively segmented into multiple domains. We use one subjective criterion called segmentation strength based on the Bayesian information criterion. Whether or not a sequence is homogeneous and how many domains it has depend on this criterion. We compare six different genome sequences (yeast S. cerevisiae chromosome III and IV, bacterium M. pneumoniae, human major histocompatibility complex sequence, longest contigs in human chromosome 21 and 22) by recursive segmentations at different strength criteria. Results by recursive segmentation confirm that yeast chromosome IV is more homogeneous than yeast chromosome III, human chromosome 21 is more homogeneous than human chromosome 22, and bacterial genomes may not be homogeneous due to short segments with distinct base compositions. The recursive segmentation also provides a quantitative criterion for identifying isochores in human sequences. Some features of our recursive segmentation, such as the possibility of delineating domain borders accurately, are superior to those of the moving-window approach commonly used in such analyses.     

The compositional distribution of coding sequences and DNA molecules in humans and murids

Summary The compositional distributions of coding sequences and DNA molecules (in the 50-100-kb range) are remarkably narrower in murids (rat and mouse) compared to humans (as well as to all other mammals explored so far). In murids, both distributions begin at higher and end at lower GC values. A comparison of homologous coding sequences from murids and humans revealed that their different compositional distributions are due to differences in GC levels in all three codon positions, particularly of genes located at both ends of the distribution. In turn, these differences are responsible for differences in both codon usage and amino acids. When GC levels at first+second codon positions and third codon positions, respectively, of murid genes are plotted against corresponding GC levels of homologous human genes, linear relationships (with very high correlation coefficients and slopes of about 0.78 and 0.60, respectively) are found. This indicates a conservation of the order of GC levels in homologous genes from humans and murids. (The same comparison for mouse and rat genes indicates a conservation of GC levels of homologous genes.) A similar linear relationship was observed when plotting GC levels of corresponding DNA fractions (as obtained by density gradient centrifugation in the presence of a sequence-specific ligand) from mouse and human. These findings indicate that orderly compositional changes affecting not only coding sequences but also noncoding sequences took place since the divergence of murids. Such directional fixations of mutations point to the existence of selective pressures affecting the genome as a whole.     

Retroviral integrase domains: DNA binding and the recognition of LTR sequences.

Integration of retroviral DNA into the host chromosome requires a virus-encoded integrase (IN). IN recognizes, cuts and then joins specific viral DNA sequences (LTR ends) to essentially random sites in host DNA. We have used computer-assisted protein alignments and mutagenesis in an attempt to localize these functions within the avian retroviral IN protein. A comparison of the deduced amino acid sequences for 80 retroviral/retrotransposon IN proteins reveals strong conservation of an HHCC N-terminal 'Zn finger'-like domain, and a central D(35)E region which exhibits striking similarities with sequences deduced for bacterial IS elements. We demonstrate that the HHCC region is not required for DNA binding, but contributes to specific recognition of viral LTRs in the cutting and joining reactions. Deletions which extend into the D(35)E region destroy the ability of IN to bind DNA. Thus, we propose that the D(35)E region may specify a DNA-binding/cutting domain that is conserved throughout evolution in enzymes with similar functions.     

Localization of DNA sequences tightly associated with synaptonemal complex in compositional fractions of golden hamster

The synaptonemal complex isolated from the spermatocyte nuclei by exhaustive hydrolysis of the latter by DNase II contains tightly associated DNA sequences (SCAR DNA). Here we studied the compositional properties of a cloned family of SCAR DNA of golden hamster, namely we performed the localization of 27 SCAR DNA clones on compositionally fractionated genomic DNA from golden hamster. We observed that sequences of the SCAR DNA family are mainly localized in the GC-poor isochore families L1 and L2, that showed 63% hybridization signals. This means that 37% of signals is referred to the GC-rich isochores, indicating the presence of SCAR DNA overall the genome, even if each isochore family presents differences in density and sequence type. Moreover, the SCAR DNA sequences containing regions of homology with LINE/SINE repeats were observed in all the isochore families. The compositional localization of SCAR DNA is in agreement with the hypothesis that SC and SCAR DNA participate in the chromatin organization during the meiosis prophase I, which should result in the attachment of chromatin loops to lateral elements of SC along the whole length of the latter.     

Codon preference and its use in identifying protein coding regions in long DNA sequences

This paper describes a computer method that uses codon preference to help find protein coding regions in long DNA sequences. The method can distinguish between introns and exons and can help to detect sequencing errors.     

A linear programming approach for identifying a consensus sequence on DNA sequences

MOTIVATION: Maximum-likelihood methods for solving the consensus sequence identification (CSI) problem on DNA sequences may only find a local optimum rather than the global optimum. Additionally, such methods do not allow logical constraints to be imposed on their models. This study develops a linear programming technique to solve CSI problems by finding an optimum consensus sequence. This method is computationally more efficient and is guaranteed to reach the global optimum. The developed method can also be extended to treat more complicated CSI problems with ambiguous conserved patterns. RESULTS: A CSI problem is first formulated as a non-linear mixed 0-1 optimization program, which is then converted into a linear mixed 0-1 program. The proposed method provides the following advantages over maximum-likelihood methods: (1) It is guaranteed to find the global optimum. (2) It can embed various logical constraints into the corresponding model. (3) It is applicable to problems with many long sequences. (4) It can find the second and the third best solutions. An extension of the proposed linear mixed 0-1 program is also designed to solve CSI problems with an unknown spacer length between conserved regions. Two examples of searching for CRP-binding sites and for FNR-binding sites in the Escherichia coli genome are used to illustrate and test the proposed method. AVAILABILITY: A software package, Global Site Seer for the Microsoft Windows operating system is available by http://www.iim.nctu.edu.tw/~cjfu/gss.htm     

Conjugative transposition: Tn916 integrase contains two independent DNA binding domains that recognize different DNA sequences.

Transposition of the conjugative transposon Tn916 requires the activity of a protein, called Int, which is related to members of the integrase family of site-specific recombinases. This family includes phage lambda integrase as well as the Cre, FLP and XerC/XerD recombinases. Different proteins, consisting of fragments of Tn916 Int protein fused to the C-terminal end of maltose binding protein (MBP) were purified from Escherichia coli. DNase I protection experiments showed that MBP-INT proteins containing the C-terminal end of Int bound to the ends of the transposon and adjacent plasmid DNA. MBP-INT proteins containing the N-terminal end of Int bound to sequences within the transposon close to each end. Competition binding experiments showed that the sites recognized by the C- and N-terminal regions of Int did not compete with each other for binding to MBP-INT. We suggest that Tn916 and related conjugative transposons are unique among members of the integrase family of site-specific recombination systems because the presence of two DNA binding domains in the Int protein might allow Int to bridge recombining sites, and this bridging seems to be the sole mechanism ensuring that only correctly aligned molecules undergo recombination.     

Limitations of compositional approach to identifying horizontally transferred genes

Genes with atypical G+C content and pattern of codon usage in a certain genome are possibly of exotic origin, and this idea has been applied to identify horizontal events. In this way, it was postulated that a total of 755 genes in the E. coli genome are relics of horizontal events after the divergence of E. coli from the Salmonella lineage 100 million years ago (Lawrence and Ochman, 1998). In this paper we propose a new way to study sequence composition more thoroughly. We found that although the 755 genes differ in composition from other genes in the E. coli genome, the difference is minor. If we accepted that these genes are horizontally transferred, then (1) it would be more likely that they were transferred from genomes evolutionarily closely related to E. coli; but (2) the dating method used by Lawrence and Ochman (1997, 1998) largely underestimated the average age of introduced sequences in the E. coli genome, in particular, most of the 755 genes should be introduced into E. coli before, instead of after, the divergence of E. coli from the Salmonella lineage. Our study reveals that atypical G+C content and pattern of codon usage are not reliable indicators of horizontal gene transfer events. Received: 27 September 2000 / Accepted: 9 April 2001     

Synthetic actin-binding domains reveal compositional constraints for function

The actin-binding domains of many proteins consist of a canonical type 1/type 2 arrangement of the structurally conserved calponin homology domain. Using the actin-binding domain of alpha-actinin-1 as a scaffold we have generated synthetic actin-binding domains by altering position and composition of the calponin homology domains. We show that the presence of two calponin homology domains alone and in the context of an actin-binding domain is not sufficient for actin-binding, and that both single and homotypic type 2 calponin homology domain tandems fail to bind to actin in vitro and in transfected cells. In contrast, single and tandem type 1 calponin homology domain arrays bind actin directly but result in defective turnover rates on actin filaments, and in aberrant actin bundling when introduced into the full-length alpha-actinin molecule. An actin-binding domain harboring the calponin homology domains in an inverted position, however, functions both in isolation and in the context of the dimeric alpha-actinin molecule. Our data demonstrate that the dynamics and specificity of actin-binding via actin-binding domains requires both the filament binding properties of the type 1, and regulation by type 2 calponin homology domains, and appear independent of their position.     

A method to assess compositional bias in biological sequences and its application to prion-like glutamine/asparagine-rich domains in eukaryotic proteomes

We have derived a novel method to assess compositional biases in biological sequences, which is based on finding the lowest-probability subsequences for a given residue-type set. As a case study, the distribution of prion-like glutamine/asparagine-rich ((Q+N)-rich) domains (which are linked to amyloidogenesis) was assessed for budding and fission yeasts and four other eukaryotes. We find more than 170 prion-like (Q+N)-rich regions in budding yeast, and, strikingly, many fewer in fission yeast. Also, some residues, such as tryptophan or isoleucine, are unlikely to form biased regions in any eukaryotic proteome.     

Visualizing DNA domains and sequences by microscopy: a fifty-year history of molecular cytogenetics.

This short review presents a historical perspective of chromosome research during the last 50 years. It shows how molecular knowledge and technology of DNA entered cytogenetics step by step making it now daily practice in almost every modern chromosome lab. A crucial milestone in these decades has been the development of in situ protocols by Pardue and Gall, among others, initially only with isotopic labels, and without fluorescence microscopy and sophisticated detection systems. But these very first in situ hybridizations played a decisive role in the discovery of chromosome banding profiles, which were obtained under specific chemical, physical, or enzymatic conditions, thus effecting stainability of specific chromosome regions. In the decades thereafter, numerous technical improvements were achieved leading to complex multi-colour fluorescence in situ hybridization (FISH) protocols for mammals, plants, and insects. Highly improved detection systems of the FISH signals further allowed detection of DNA targets of up to 50 bp, whereas other protocols, which were developed to stretch chromatin fibres to the full length of native DNA, improved spatial resolution of adjacent targets in the light microscope to 1 kb.     

Quantitative multicolor compositional imaging resolves molecular domains in cell-matrix adhesions

Background

Cellular processes occur within dynamic and multi-molecular compartments whose characterization requires analysis at high spatio-temporal resolution. Notable examples for such complexes are cell-matrix adhesion sites, consisting of numerous cytoskeletal and signaling proteins. These adhesions are highly variable in their morphology, dynamics, and apparent function, yet their molecular diversity is poorly defined.

Methodology/Principal Findings

We present here a compositional imaging approach for the analysis and display of multi-component compositions. This methodology is based on microscopy-acquired multicolor data, multi-dimensional clustering of pixels according to their composition similarity and display of the cellular distribution of these composition clusters. We apply this approach for resolving the molecular complexes associated with focal-adhesions, and the time-dependent effects of Rho-kinase inhibition. We show here compositional variations between adhesion sites, as well as ordered variations along the axis of individual focal-adhesions. The multicolor clustering approach also reveals distinct sensitivities of different focal-adhesion-associated complexes to Rho-kinase inhibition.

Conclusions/Significance

Multicolor compositional imaging resolves “molecular signatures” characteristic to focal-adhesions and related structures, as well as sub-domains within these adhesion sites. This analysis enhances the spatial information with additional “contents-resolved” dimensions. We propose that compositional imaging can serve as a powerful tool for studying complex multi-molecular assemblies in cells and for mapping their distribution at sub-micron resolution.     

Satellite DNA sequences flank amplified DHFR domains in marker chromosomes of mouse fibrosarcoma cells

This study centers on marker chromosomes carrying expanded chromosomal regions which were observed in two independent derivatives of the AA12 murine fibrosarcoma line, the 10^–3 M MTX-res H2 and the 5×10^–7 M MTX-res E. Previous characterization of the marker chromosomes of MTX-res variants showed their common derivation from a marker chromosome (m) of the parental line, endowed with two interstitial C-bands. Cytogenetic evidence pointed to one C-band ofm as the site involved in the chromosomal rearrangements leading to the HSR/ASR chromosomes. ISH of a³H-labeled satellite DNA probe allowed satellite sequences flanking the HSR/ASR in the marker chromosomes, where the C-band was no longer visible, to be detected. FISH experiments using biotinylated DHFR and satellite DNA probes showed that the respective target sequences are contiguous in new marker chromosomes. They also allowed inter- and intrachromosomal rearrangements to be seen at DHFR amplicons and satellite sequences. Double-color FISH using digoxygenated satellite DNA and biotinylated pDHFR7 showed that in a marker chromosome from the H2 cell line the two target sequences are not only adjacent, but closer than 3 Mb, as indicated by overlapping of the different fluorescence signals given by the two probes. Another marker chromosome in the E variant was shown to display a mixed ladder structure consisting of a head-to-head tandem of irregularly-sized satellite DNA blocks, with two symmetrical interspersed DHFR clusters.Abbreviations DHFR dihydrofolate reductase - MTX Methotrexate - HSR Homogeneously Staining Region - ASR Abnormally Staining Region - DM Double Minute - ISH In Situ Hybridization - FISH FluorescenceIn Situ Hybridization     

Prion domains: sequences, structures and interactions

Mammalian and most fungal infectious proteins (also known as prions) are self-propagating amyloid, a filamentous beta-sheet structure. A prion domain determines the infectious properties of a protein by forming the core of the amyloid. We compare the properties of known prion domains and their interactions with the remainder of the protein and with chaperones. Ure2p and Sup35p, two yeast prion proteins, can still form prions when the prion domains are shuffled, indicating a parallel in-register beta-sheet structure.     

Alignment statistic for identifying related protein sequences

Summary Closely related proteins show an obvious kinship by having numerous matching amino acids in their aligned sequences. Kinship between anciently separated proteins requires a statistical evaluation to rule out fortuitous similarities. A simple statistic is developed which assumes equal probability for all codon pairs, and a table of critical values for amino acid sequence alignments of length 200 or less is presented. Applying this statistic toV andC regions of immunoglobulin chains, aligned on the basis of shared features of three-dimensional structure, provides evidence that theV andC sequences descended from a common ancestor. Similarly the distant evolutionary relationship of dehydrogenases, flavdoxin, and subtilisin, suggested by structural alignments, is verified. On the other hand, the statistic does not verify a common evolutionary origin for the heme binding pocket in globins and cytochromeb ₅. Empirical evidence from the distribution of MMD values of amino acid pairs in comparisons of misaligned polypeptide chains and from Monte Carlo trials of sequences aligned with arbitrary gaps supports the validity of the statistic.     

Chromosome localization-dependent compositional bias of point mutations in Alu repetitive sequences

The Alu repetitive sequence family originated from a common ancestor. Its members, apparently free from functional constraints, are interspersed throughout primate genomes. We have found that base substitutions occurring during the evolution of primates caused a decrease in the average G + C content of those members of the family that are located in an A + T-rich region of the genome. The family members that are located in a G + C-rich genomic region have not changed their, already high, G + C content. This suggests that the regional differences in G + C content, which are responsible for chromosomal banding, are caused by an accumulation of mutations that, although selectively neutral in the majority, show different compositional bias in different regions of the vertebrate chromosome.