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Differential gene transcription is a fundamental regulatory mechanism of biological systems during development, body homeostasis, and disease. Comparative genomics is believed to be a rapid means for the identification of regulatory sequences in genomes. We tested this approach to identify regulatory sequences that control expression in the midline of the zebrafish embryo. We first isolated a set of genes that are coexpressed in the midline of the zebrafish embryo during somitogenesis stages by gene array analysis and subsequent rescreens by in situ hybridization. We subjected 45 of these genes to Compare and DotPlot analysis to detect conserved sequences in noncoding regions of orthologous loci in the zebrafish and Takifugu genomes. The regions of homology that were scored in nonconserved regions were inserted into expression vectors and tested for their regulatory activity by transient transgenesis in the zebrafish embryo. We identified one conserved region from the connective tissue growth factor gene (ctgf), which was able to drive expression in the midline of the embryo. This region shares sequence similarity with other floor plate/notochord-specific regulatory regions. Our results demonstrate that an unbiased comparative approach is a relevant method for the identification of tissue-specific cis-regulatory sequences in the zebrafish embryo.

Together with the hemichordates, sea urchins represent basal groups of nonchordate invertebrate deuterostomes that occupy a key position in bilaterian evolution. Because sea urchin embryos are also amenable to functional studies, the sea urchin system has emerged as one of the leading models for the analysis of the function of genomic regulatory networks that control development. We have analyzed a total of 107,283 cDNA clones of libraries that span the development of the sea urchin Strongylocentrotus purpuratus. Normalization by oligonucleotide fingerprinting, EST sequencing and sequence clustering resulted in an EST catalog comprised of 20,000 unique genes or gene fragments. Around 7000 of the unique EST consensus sequences were associated with molecular and developmental functions. Phylogenetic comparison of the identified genes to the genome of the urochordate Ciona intestinalis indicate that at least one quarter of the genes thought to be chordate specific were already present at the base of deuterostome evolution. Comparison of the number of gene copies in sea urchins to those in chordates and vertebrates indicates that the sea urchin genome has not undergone extensive gene or complete genome duplications. The established unique gene set represents an essential tool for the annotation and assembly of the forthcoming sea urchin genome sequence. All cDNA clones and filters of all analyzed libraries are available from the resource center of the German genome project at http://www.rzpd.de.

The 2R hypothesis predicting two genome duplications at the origin of vertebrates is highly controversial. Studies published so far include limited sequence data from organisms close to the hypothesized genome duplications. Through the comparison of a gene catalog from amphioxus, the closest living invertebrate relative of vertebrates, to 3453 single-copy genes orthologous between Caenorhabditis elegans (C), Drosophila melanogaster (D), and Saccharomyces cerevisiae (Y), and to Ciona intestinalis ESTs, mouse, and human genes, we show with a large number of genes that the gene duplication activity is significantly higher after the separation of amphioxus and the vertebrate lineages, which we estimate at 650 million years (Myr). The majority of human orthologs of 195 CDY groups that could be dated by the molecular clock appear to be duplicated between 300 and 680 Myr with a mean at 488 million years ago (Mya). We detected 485 duplicated chromosomal segments in the human genome containing CDY orthologs, 331 of which are found duplicated in the mouse genome and within regions syntenic between human and mouse, indicating that these were generated earlier than the human–mouse split. Model based calculations of the codon substitution rate of the human genes included in these segments agree with the molecular clock duplication time-scale prediction. Our results favor at least one large duplication event at the origin of vertebrates, followed by smaller scale duplication closer to the bird–mammalian split.

The zebrafish is a powerful system for understanding the vertebrate genome, allowing the combination of genetic, molecular, and embryological analysis. Expressed sequence tags (ESTs) provide a rapid means of identifying an organism's genes for further analysis, but any EST project is limited by the availability of suitable libraries. Such cDNA libraries must be of high quality and provide a high rate of gene discovery. However, commonly used normalization and subtraction procedures tend to select for shorter, truncated, and internally primed inserts, seriously affecting library quality. An alternative procedure is to use oligonucleotide fingerprinting (OFP) to precluster clones before EST sequencing, thereby reducing the re-sequencing of common transcripts. Here, we describe the use of OFP to normalize and subtract 75,000 clones from two cDNA libraries, to a minimal set of 25,102 clones. We generated 25,788 ESTs (11,380 3′ and 14,408 5′) from over 16,000 of these clones. Clustering of 10,654 high-quality 3′ ESTs from this set identified 7232 clusters (likely genes), corresponding to a 68% gene diversity rate, comparable to what has been reported for the best normalized human cDNA libraries, and indicating that the complete set of 25,102 clones contains as many as 17,000 genes. Yet, the library quality remains high. The complete set of 25,102 clones is available for researchers as glycerol stocks, filters sets, and as individual EST clones. These resources have been used for radiation hybrid, genetic, and physical mapping of the zebrafish genome, as well as positional cloning and candidate gene identification, molecular marker, and microarray development.[The sequence data described in this paper have been submitted to the dbEST/GenBank data library under accession nos. AA497144–AA497369, AA542435–AA542678,AA545709–AA545724, AI384176–AI384205, AI384761–AI384796,AI396646–AI396663, AI396733–AI396777, AI396895–AI396938,AI397015–AI397130, AI397219–AI397252, AI397388–AI397484,AI415743–AI416403, AI436854–AI437493, AI444118–AI444540,AI461280–AI461395, AI476823–AI478024, AI496677–AI497576,AI522337–AI522810, AI544445–AI546083, AI558267–AI558995,AI584192–AI585023, AI585025–AI585238, AI588088–AI588836,AI601277–AI601868, AI626134–AI626875, AI629052–AI629398,AI641018–AI641780, AI657549–AI658347, AI666929–AI667197,AI667264–AI667414, AI667488–AI667567, AI721460–AI721747,AI721839–AI721978, and AI722283–AI722483.]

Clustering is one of the main mathematical challenges in large-scale gene expression analysis. We describe a clustering procedure based on a sequential k-means algorithm with additional refinements that is able to handle high-throughput data in the order of hundreds of thousands of data items measured on hundreds of variables. The practical motivation for our algorithm is oligonucleotide fingerprinting—a method for simultaneous determination of expression level for every active gene of a specific tissue—although the algorithm can be applied as well to other large-scale projects like EST clustering and qualitative clustering of DNA-chip data. As a pairwise similarity measure between two p-dimensional data points,x and y, we introduce mutual information that can be interpreted as the amount of information about x iny, and vice versa. We show that for our purposes this measure is superior to commonly used metric distances, for example, Euclidean distance. We also introduce a modified version of mutual information as a novel method for validating clustering results when the true clustering is known. The performance of our algorithm with respect to experimental noise is shown by extensive simulation studies. The algorithm is tested on a subset of 2029 cDNA clones coming from 15 different genes from a cDNA library derived from human dendritic cells. Furthermore, the clustering of these 2029 cDNA clones is demonstrated when the entire set of 76,032 cDNA clones is processed.

The generation of genome-wide data derived from methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) has become a major tool for epigenetic studies in health and disease. The computational analysis of such data, however, still falls short on accuracy, sensitivity, and speed. We propose a time-efficient statistical method that is able to cope with the inherent complexity of MeDIP-seq data with similar performance compared with existing methods. In order to demonstrate the computational approach, we have analyzed alterations in DNA methylation during the differentiation of human embryonic stem cells (hESCs) to definitive endoderm. We show improved correlation of normalized MeDIP-seq data in comparison to available whole-genome bisulfite sequencing data, and investigated the effect of differential methylation on gene expression. Furthermore, we analyzed the interplay between DNA methylation, histone modifications, and transcription factor binding and show that in contrast to de novo methylation, demethylation is mainly associated with regions of low CpG densities.