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PubMed2004

DNA mapping using microfluidic stretching and single-molecule detection of fluorescent site-specific tags.

We have developed a rapid molecular mapping technology--Direct Linear Analysis (DLA)--on the basis of the analysis of individual DNA molecules bound with sequence-specific fluorescent tags. The apparatus includes a microfluidic device for stretching DNA molecules in elongational flow that is coupled to a multicolor detection system capable of single-fluorophore sensitivity. Double-stranded DNA molecules were tagged at sequence-specific motif sites with fluorescent bisPNA (Peptide Nucleic Acid) tags. The DNA molecules were then stretched in the microfluidic device and driven in a flow stream past confocal fluorescence detectors. DLA provided the spatial locations of multiple specific sequence motifs along individual DNA molecules, and thousands of individual molecules could be analyzed per minute. We validated this technology using the 48.5 kb lambda phage genome with different 8-base and 7-base sequence motif tags. The distance between the sequence motifs was determined with an accuracy of +/-0.8 kb, and these tags could be localized on the DNA with an accuracy of +/-2 kb. Thus, DLA is a rapid mapping technology, suitable for analysis of long DNA molecules.

Bacteriophage lambda↗
PubMed2005

A configuration space of homologous proteins conserving mutual information and allowing a phylogeny inference based on pair-wise Z-score probabilities.

BACKGROUND: Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pair-wise sequence alignments, respect probabilistic properties of Z-scores (Monte Carlo methods applied to pair-wise comparisons) and be the basis for a novel method of consistent and stable phylogenetic reconstruction. RESULTS: We have built up a spatial representation of protein sequences using concepts from particle physics (configuration space) and respecting a frame of constraints deduced from pair-wise alignment score properties in information theory. The obtained configuration space of homologous proteins (CSHP) allows the representation of real and shuffled sequences, and thereupon an expression of the TULIP theorem for Z-score probabilities. Based on the CSHP, we propose a phylogeny reconstruction using Z-scores. Deduced trees, called TULIP trees, are consistent with multiple-alignment based trees. Furthermore, the TULIP tree reconstruction method provides a solution for some previously reported incongruent results, such as the apicomplexan enolase phylogeny. CONCLUSION: The CSHP is a unified model that conserves mutual information between proteins in the way physical models conserve energy. Applications include the reconstruction of evolutionary consistent and robust trees, the topology of which is based on a spatial representation that is not reordered after addition or removal of sequences. The CSHP and its assigned phylogenetic topology, provide a powerful and easily updated representation for massive pair-wise genome comparisons based on Z-score computations.

Algorithms↗
PubMed2026

MX1+ effector T cells hyperactivation at the maternal-fetal interface in unexplained recurrent pregnancy loss.

BACKGROUND: Immune tolerance breakdown at the maternal-fetal interface is implicated in unexplained recurrent pregnancy loss (URPL), but the interplay between T cell hyperactivation and dendritic cells (DCs)-mediated signaling remains poorly defined. METHODS: First-trimester decidual tissues from 5 healthy controls and 6 URPL patients underwent single-cell RNA sequencing (scRNA-seq, 10× Genomics). Computational analyses included clustering (Seurat), trajectory inference (scTour), intercellular communication (CellChat) and metabolic pathway enrichment (Gene Ontology and scMetabolism). Flow cytometry was performed from 11 patients and 11 healthy controls. Spatial validation was performed via multiplex immunohistochemistry and immunohistochemistry on 12 additional controls and 12 URPL cases. Statistical significance was assessed using Student’s t-test. RESULTS: URPL decidua exhibited marked CD3+ T cells and MX1+effector T (Tem) cells infiltration and activation. Flow cytometry analysis confirmed a significant decidua-specific upregulation of T cell activation markers CD25 and CD69 specifically on the MX1+Tem subset in URPL patients compared to controls. MX1+Tem cell subset demonstrated interferon hyperactivation, proliferative hyperactivity and lipid-biased immunometabolism. Pseudotemporal analysis positioned MX1+ Tem cells between classical Tem and exhausted T cell states, suggesting progressive differentiation. CellChat identified DCs as key regulators of MX1+ Tem expansion via aberrant ICOSL signaling, validated by spatial co-localization of ICOSL+ DCs and MX1+ Tem cells in URPL tissues. CONCLUSION: Our findings demonstrate that the aberrant activation and proliferation of MX1+Tem cells as a key immunological feature associated with URPL patients.

Humans↗
PubMed2006

The tortuous route from genes to behavior: A neuroconstructivist approach.

In their excitement at using the human genome project to uncover the functions of specific genes, researchers have often ignored one fundamental factor: the gradual process of ontogenetic development. The view that there might be a gene for spatial cognition or language has emanated from a focus on the structure of the adult brain in neuropsychological patients whose brains were fully and normally developed until their brain insult. The developing brain is very different. It starts out highly interconnected across regions and is neither localized nor specialized at birth, allowing interaction with the environment to play an important role in gene expression and the ultimate cognitive phenotype. This article takes a neuroconstructivist perspective, arguing that domain-specific end states can stem from more domain-general start states, that associations may turn out to be as informative as dissociations, and that genetic mutations that alter the trajectory of ontogenetic development can inform nature/nurture debates.

Animals↗
PubMed2000

Differential expression of the Arabidopsis genes coding for Em-like proteins.

Late embryogenesis abundant (lea) genes are a large and diverse group of genes highly expressed during late stages of seed development. Five major groups of LEA proteins have been described. Two Em genes (group I lea genes) are present in the genome of Arabidopsis thaliana L., AtEm1 and AtEm6. Both genes encode for very similar proteins which differ basically in the number of repetitions of a highly hydrophilic amino acid motif. The spatial patterns of expression of the two Arabidopsis Em genes have been studied using in situ hybridization and transgenic plants transformed with the promoters of the genes fused to the beta-glucuronidase reporter gene (uidA). In the embryo, AtEm1 is preferentially expressed in the pro-vascular tissues and in meristems. In contrast, AtEm6 is expressed throughout the embryo. The activity of both promoters disappears rapidly after germination, but is ABA-inducible in roots of young seedlings, although in different cells: the AtEm1 promoter is active in the internal tissues (vasculature and pericycle) whereas the AtEm6 promoter is active in the external tissues (cortex, epidermis and root hairs). The AtEm1 promoter, but not AtEm6, is also active in mature pollen grains and collapsed nectaries of young siliques. These data indicate that the two Em proteins could carry out at least slightly different functions and that the expression of AtEm1 and AtEm6 is controlled at, at least, three different levels: temporal, spatial and hormonal (ABA).

Amino Acid Motifs↗
PubMed2005

Identification of a new family of putative PD-(D/E)XK nucleases with unusual phylogenomic distribution and a new type of the active site.

BACKGROUND: Prediction of structure and function for uncharacterized protein families by identification of evolutionary links to characterized families and known structures is one of the cornerstones of genomics. Theoretical assignment of three-dimensional folds and prediction of protein function even at a very general level can facilitate the experimental determination of the molecular mechanism of action and the role that members of a given protein family fulfill in the cell. Here, we predict the three-dimensional fold and study the phylogenomic distribution of members of a large family of uncharacterized proteins classified in the Clusters of Orthologous Groups database as COG4636. RESULTS: Using protein fold-recognition we found that members of COG4636 are remotely related to Holliday junction resolvases and other nucleases from the PD-(D/E)XK superfamily. Structure modeling and sequence analyses suggest that most members of COG4636 exhibit a new, unusual variant of the putative active site, in which the catalytic Lys residue migrated in the sequence, but retained similar spatial position with respect to other functionally important residues. Sequence analyses revealed that members of COG4636 and their homologs are found mainly in Cyanobacteria, but also in other bacterial phyla. They undergo horizontal transfer and extensive proliferation in the colonized genomes; for instance in Gloeobacter violaceus PCC 7421 they comprise over 2% of all protein-encoding genes. Thus, members of COG4636 appear to be a new type of selfish genetic elements, which may fulfill an important role in the genome dynamics of Cyanobacteria and other species they invaded. Our analyses provide a platform for experimental determination of the molecular and cellular function of members of this large protein family. CONCLUSION: After submission of this manuscript, a crystal structure of one of the COG4636 members was released in the Protein Data Bank (code 1wdj; Idaka, M., Wada, T., Murayama, K., Terada, T., Kuramitsu, S., Shirouzu, M., Yokoyama, S.: Crystal structure of Tt1808 from Thermus thermophilus Hb8, to be published). Our analysis of the Tt1808 structure reveals that we correctly predicted all functionally important features of the COG4636 family, including the membership in the PD-(D/E)xK superfamily of nucleases, the three-dimensional fold, the putative catalytic residues, and the unusual configuration of the active site.

Amino Acid Sequence↗
PubMed2006

Predicting transcription factor binding sites using local over-representation and comparative genomics.

BACKGROUND: Identifying cis-regulatory elements is crucial to understanding gene expression, which highlights the importance of the computational detection of overrepresented transcription factor binding sites (TFBSs) in coexpressed or coregulated genes. However, this is a challenging problem, especially when considering higher eukaryotic organisms. RESULTS: We have developed a method, named TFM-Explorer, that searches for locally overrepresented TFBSs in a set of coregulated genes, which are modeled by profiles provided by a database of position weight matrices. The novelty of the method is that it takes advantage of spatial conservation in the sequence and supports multiple species. The efficiency of the underlying algorithm and its robustness to noise allow weak regulatory signals to be detected in large heterogeneous data sets. CONCLUSION: TFM-Explorer provides an efficient way to predict TFBS overrepresentation in related sequences. Promising results were obtained in a variety of examples in human, mouse, and rat genomes. The software is publicly available at http://bioinfo.lifl.fr/TFM-Explorer.

Algorithms↗
PubMed2004

Hox cluster disintegration with persistent anteroposterior order of expression in Oikopleura dioica.

Tunicate embryos and larvae have small cell numbers and simple anatomical features in comparison with other chordates, including vertebrates. Although they branch near the base of chordate phylogenetic trees, their degree of divergence from the common chordate ancestor remains difficult to evaluate. Here we show that the tunicate Oikopleura dioica has a complement of nine Hox genes in which all central genes are lacking but a full vertebrate-like set of posterior genes is present. In contrast to all bilaterians studied so far, Hox genes are not clustered in the Oikopleura genome. Their expression occurs mostly in the tail, with some tissue preference, and a strong partition of expression domains in the nerve cord, in the notochord and in the muscle. In each tissue of the tail, the anteroposterior order of Hox gene expression evokes spatial collinearity, with several alterations. We propose a relationship between the Hox cluster breakdown, the separation of Hox expression domains, and a transition to a determinative mode of development.

Animals↗
PubMed2026

Characterizing the ecological niche of insertion sequences within prokaryotic genomes.

Insertion sequences (ISs) are widespread prokaryotic transposable elements, often regarded as genomic parasites that primarily cause deleterious mutations. However, they can also promote adaptive changes. These antagonistic properties make their overall impact on prokaryotic evolution difficult to grasp. Here, we address this challenge by leveraging the framework of transposon ecology to analyze IS occurrences across and within 30 499 prokaryotic genomes. Combining phylogenomics with multi-scale genomic analysis, quantitative ecology, and mathematical modeling, we provide evidence that although genomes generally provide sufficient resources for IS coexistence, universal mechanisms shape their occurrence and chromosomal distribution across genomes. These include (i) the preferential localization of ISs within highly variable and GC-heterogeneous chromosomal regions of genomic plasticity, which act as the primary reservoir of IS niches; (ii) a linear scaling between IS abundance and niche size, with an average of $5.4$ additional accessible insertion sites per IS; (iii) a dependence of IS occurrence on the presence of other ISs, suggesting a form of group behavior; (iv) the accumulation of AT-rich sequences in both coding and noncoding regions up to 100 kb around ISs, indicative of ecological isolation; and (v) the spatial partitioning of mobile genetic elements around ISs, reminiscent of ecological niche differentiation. Besides these general principles, we also uncover niche specificities associated with particular IS families, hinting at regulatory mechanisms that modulate IS activity. Altogether, this comprehensive transposon ecology approach offers new insights and avenues for understanding IS-host interactions and genome evolution, moving beyond traditional host-centric perspectives.

DNA Transposable Elements↗
PubMed2005

TileMap: create chromosomal map of tiling array hybridizations.

MOTIVATION: Tiling array is a new type of microarray that can be used to survey genomic transcriptional activities and transcription factor binding sites at high resolution. The goal of this paper is to develop effective statistical tools to identify genomic loci that show transcriptional or protein binding patterns of interest. RESULTS: A two-step approach is proposed and is implemented in TileMap. In the first step, a test-statistic is computed for each probe based on a hierarchical empirical Bayes model. In the second step, the test-statistics of probes within a genomic region are used to infer whether the region is of interest or not. Hierarchical empirical Bayes model shrinks variance estimates and increases sensitivity of the analysis. It allows complex multiple sample comparisons that are essential for the study of temporal and spatial patterns of hybridization across different experimental conditions. Neighboring probes are combined through a moving average method (MA) or a hidden Markov model (HMM). Unbalanced mixture subtraction is proposed to provide approximate estimates of false discovery rate for MA and model parameters for HMM. AVAILABILITY: TileMap is freely available at http://biogibbs.stanford.edu/~jihk/TileMap/index.htm. SUPPLEMENTARY INFORMATION: http://biogibbs.stanford.edu/~jihk/TileMap/index.htm (includes coloured versions of all figures).

Algorithms↗
PubMed2004

Cloning and developmental expression of nonmuscle myosin IIA (Myh9) in the mammalian inner ear.

MYH9 encoding a nonmuscle myosin heavy chain has been linked to nonsyndromic and syndromic forms of autosomal dominant hereditary hearing loss, suggesting a critical biological role of this motor protein in the auditory organ. While Myh9 expression has been described in the adult mouse, critical parameters pertaining to its developmental expression remain to be characterized. The current study describes cloning of the mouse Myh9 cDNA and the temporal onset and spatial distribution of Myh9 expression in the inner ear of the developing fetus, the neonate, and the adult. The cloned Myh9 cDNA contained two single-base-pair differences from the published genomic sequence: T990C (G330G) and T5198A (L1733Q). Immunoblotting of embryonic (E15.5) and adult tissues from several organs, including the cochlea, identified a single 250-kDa anti-Myh9-immunoreactive band, supporting an absence of Myh9 splice variants in the fetus and the adult. In situ expression analysis identified Myh9 distributed within the epithelial layer of the otic vesicle at E10.5. Myh9 expression was found to persist within the epithelia surrounding the cochlear duct at E13.5 and E16.5. The sensory cells of the developing cochlea were positive for Myh9 expression at E16.5. Within the neonate and the adult cochlea, Myh9 expression was observed within the sensory hair cells and the supporting hair cells of the organ of Corti, the spiral ligament, and the spiral limbus, but not in the stria vascularis. Identification of Myh9 in the developing and mature inner ear suggests a role for this protein in the development and maintenance of auditory function.

Animals↗
PubMed1995

The MLC1f/3f gene is an early marker of somitic muscle differentiation in Xenopus laevis embryo.

cDNAs clones encoding the MLC1f and MLC3f proteins of Xenopus laevis have been isolated from a stage 42 cDNA library. Sequence analysis reveals that the amphibian MLC1f and MLC3f isoforms are similar to the mammalian and avian cognates. The two isoforms share a common 141-amino-acid carboxy-terminal regions. These are 49 and 9 residues long for the MLC1f and MLC3f isoforms, respectively. This suggests a genomic organization similar to the mammalian and avian genes, with two promoters and alternative splicing. The developmental expression of the MLC1f/3f mRNAs was studied by Northern blot and RNase protection and their spatial expression analyzed by in situ hybridization. Both the MLC1f and MLC3f mRNAs can be detected in the developing embryo from the end of gastrulation and accumulate rapidly in the somitic mesoderm. Expression of the MLC1f/3f gene can also be detected in animal cap explants which have been induced to form mesodermal derivatives by exposure to activin A or bFGF. However, unlike other muscle-specific markers, neither transcript from the MLC1f/3f gene can be detected in embryonic or adult cardiac muscle, their expression being restricted to somitic muscle. Together, these data demonstrate that expression of the MLC1f/3f gene provides a sensitive and specific marker for skeletal muscle differentiation. Ectopic expression of myogenic factors in animal caps induces the expression of the MLC1f/3f gene, suggesting that the amphibian gene, like its mammalian and avian counterparts, is a regulatory target for members of the MyoD family of transcription factors.

Amino Acid Sequence↗
PubMed1996

Molecular organization and alternative splicing in zipper, the gene that encodes the Drosophila non-muscle myosin II heavy chain.

Genomic sequence of the entire zipper gene, that encodes non-muscle myosin II heavy chain (MHC) in Drosophila melanogaster, reveals a new, differentially spliced exon in this essential locus and identifies a molecular lesion that is responsible for a severe embryonic lethal zipper allele. There are two alternative splices in the head domain. The first is present in the 5' untranslated sequence which, when employed, produces an N-terminal extension of 45 amino acids (aa). This splicoform produces a protein that is stable in flies but less prevalent than the isoform that lacks the extension. The second alternative exon (40 aa) is close to the nucleotide binding pocket. The position, size and sequence of this exon is conserved in D. simulans and putative alternative exons of different size (7 to 16 aa) but identical position have been reported for other myosins throughout phylogeny. The functional significance of neither alternative splice is clear. Sequence analysis of genomic DNA identifies the lesion responsible for zipIIF107, one of the original severe embryonic lethal zipper alleles. Our primary structural data confirm and correct our previous sequence of the cDNA, establish the spatial relationship between zipper and unzipped (the gene originally thought to have been disrupted in zipper mutations), and provide a high resolution template for the precise mapping of mutations.

Alleles↗
PubMed2004

Determination of infectious load of Mycoplasma genitalium in clinical samples of human vaginal cells.

Mycoplasma genitalium is a leading cause of chlamydia-negative, nongonoccocal urethritis and has been directly implicated in numerous other genitourinary as well as extragenitourinary tract pathologies. Detection of M. genitalium has relied almost entirely on PCR amplification of clinical specimens and evidence of seroconversion since these mycoplasmas are highly fastidious and culture isolation by microbiological techniques is very rare. We have established a combinatorial strategy using confocal immunoanalysis (CIA) and real-time PCR to qualitatively and quantitatively assess patterns of M. genitalium infection in women attending a sexually transmitted disease-related health clinic in San Antonio, Tex. CIA allows spatial examination of mycoplasmas on surfaces and inside human target cells, plus the ability to evaluate cell-to-cell patterns and variances within samples. Real-time PCR permits determination of genome copy numbers of mycoplasmas and human cells by multiplex amplification using mycoplasma gyrA and human RNase P gene sequences, which indicates overall levels of mycoplasma infection and degree of parasitism. These assays are strongly correlated and, in combination, permit detection and elucidation of heretofore-unrecognized patterns of M. genitalium infections in clinical and experimental samples.

Base Sequence↗
PubMed1993

The beta 1 integrin distal promoter is developmentally regulated in transgenic mice.

Transgenic mice harbouring 5' flanking sequences of the human beta 1 integrin gene linked to the Escherichia coli lacZ gene have been generated to examine spatial and temporal distribution of the promoter activity during development. Our previous data showed that this regulatory region is composed by two promoters, called distal and proximal, located closely on the human genome. To determine the role of each promoter region during development we generated transgenic mice using these two sequences linked to the lacZ reporter gene. Their analysis shows that these two sequences, as determined by in vitro studies, have different efficiencies in promoting transcription. Actually mice carrying the proximal promoter region exhibit a weak lacZ expression resulting in an undetectable beta-galactosidase activity in both embryonic and adult tissues. On the other hand, transgenic mice carrying the distal promoter express beta-galactosidase at high efficiency during embryonic development. The pattern of transgene expression is consistent with the localization of beta 1 protein on mouse embryos evidenced by immunohistochemistry. Moreover the distal promoter is subjected to a temporal modulation since in adult transgenic mice lacZ expression decreases to a level detected only by RT-PCR analysis. We have determined a similar down-regulation analysing by Northern blot beta 1 mRNA in adult and embryonic organs such as heart and gut.

Animals↗
PubMed2006

A Hidden Markov model web application for analysing bacterial genomotyping DNA microarray experiments.

Whole genome DNA microarray genomotyping experiments compare the gene content of different species or strains of bacteria. A statistical approach to analysing the results of these experiments was developed, based on a Hidden Markov model (HMM), which takes adjacency of genes along the genome into account when calling genes present or absent. The model was implemented in the statistical language R and applied to three datasets. The method is numerically stable with good convergence properties. Error rates are reduced compared with approaches that ignore spatial information. Moreover, the HMM circumvents a problem encountered in a conventional analysis: determining the cut-off value to use to classify a gene as absent. An Apache Struts web interface for the R script was created for the benefit of users unfamiliar with R. The application may be found at http://hmmgd.cryst.bbk.ac.uk/hmmgd. The source code illustrating how to run R scripts from an Apache Struts-based web application is available from the corresponding author on request. The application is also available for local installation if required.

Algorithms↗
PubMedDate not supplied

[Levels in the structural organization of Escherichia coli promoter DNA].

A number of additional structural elements were identified by statistic analysis of nucleotide sequences in promoters recognized by Escherichia coli RNA polymerase. Together with canonical hexanucleotides, these elements characterize different levels in the structural organization of promoter DNA. Sequence motifs exhibiting the highest statistical significance, which dominate in the contact regions with RNA polymerase alpha and sigma subunits, are considered as targets for specific interaction with RNA polymerase. A typical feature of these elements is the presence of easily deformable dinucleotides (TG, CA and TA) or tracts containing only A/T base pairs. Thus, we noticed that the frequency of occurrence of TA in the promoter DNA is essentially higher than the average value for the genome. Besides the regions of specific interaction with RNA polymerase, these dinucleotides are often located in the number of other sites periodically distributed along the promoter DNA. This preferred disposition suggests that deformable elements participate in the adaptive conformational transitions of the promoter DNA favoring optimal configuration of the transcription complex. Probably, the most important feature of promoter DNA revealed by statistic analysis is the presence of A/T-tracts regularly distributed in the wide range from -160 up to +75 relative to the transcription start point. Both of these spatially distributed elements (TA dinucleotides and A/T-tracts) are linked with canonical regions and, therefore, may contribute to the conformational or dynamic features of the transcription machinery. Having high statistic significance, these elements might be considered as additional factors discriminating the promoter DNA on the background of other nucleotide sequences in the genome.

Base Sequence↗
PubMed2004

High-throughput gene silencing using cell arrays.

A recently established transfected cell array (TCA) technology has opened new experimental dimensions in the field of functional genomics. Cell arrays allow for transfection of several thousands different DNA molecules in microarray format. The effects of overexpression of hundreds of proteins on cellular physiology can be observed in a single experiment. The TCA technique has also found its application in RNA interference (RNAi) research. Small interfering RNAs (siRNA) as well as plasmid expressing short hairpin RNAs can be transferred into the cells through the process of reverse transfection. The silencing of numerous genes in spatially separated manner can be thus monitored. This review will provide an overview on current concepts concerning combination of cell array and RNAi for high-throughput loss-of-function studies.

Gene Silencing↗