Abstract: Accurate and comprehensive sequence coverage for large genomes has been restricted to only a few species of specific interest. Lower sequence coverage (survey sequencing) of related species can yield a wealth of information about gene content and putative regulatory elements. But survey sequences lack long-range continuity and provide only a fragmented view of a genome. Here we show the usefulness of combining survey sequencing with dense radiation-hybrid (RH) maps for extracting maximum comparative genome information from model organisms. Based on results from the canine system, we propose that from now on all low-pass sequencing projects should be accompanied by a dense, gene-based RH map-construction effort to extract maximum information from the genome with a marginal extra cost.
Abstract: BACKGROUND: The 156 breeds of dog recognized by the American Kennel Club offer a unique opportunity to map genes important in genetic variation. Each breed features a defining constellation of morphological and behavioral traits, often generated by deliberate crossing of closely related individuals, leading to a high rate of genetic disease in many breeds. Understanding the genetic basis of both phenotypic variation and disease susceptibility in the dog provides new ways in which to dissect the genetics of human health and biology. RESULTS: To facilitate both genetic mapping and cloning efforts, we have constructed an integrated canine genome map that is both dense and accurate. The resulting resource encompasses 4249 markers, and was constructed using the RHDF5000-2 whole genome radiation hybrid panel. The radiation hybrid (RH) map features a density of one marker every 900 Kb and contains 1760 bacterial artificial chromosome clones (BACs) localized to 1423 unique positions, 851 of which have also been mapped by fluorescence in situ hybridization (FISH). The two data sets show excellent concordance. Excluding the Y chromosome, the map features an RH/FISH mapped BAC every 3.5 Mb and an RH mapped BAC-end, on average, every 2 Mb. For 2233 markers, the orthologous human genes have been established, allowing the identification of 79 conserved segments (CS) between the dog and human genomes, dramatically extending the length of most previously described CS. CONCLUSIONS: These results provide a necessary resource for the canine genome mapping community to undertake positional cloning experiments and provide new insights into the comparative canine-human genome maps.
Abstract: The purebred dog population consists of >300 partially inbred genetic isolates or breeds. Restriction of gene flow between breeds, together with strong selection for traits, has led to the establishment of a unique resource for dissecting the genetic basis of simple and complex mammalian traits. Toward this end, we present a comprehensive radiation hybrid map of the canine genome composed of 3,270 markers including 1,596 microsatellite-based markers, 900 cloned gene sequences and ESTs, 668 canine-specific bacterial artificial chromosome (BAC) ends, and 106 sequence-tagged sites. The map was constructed by using the RHDF5000-2 whole-genome radiation hybrid panel and computed by using MULTIMAP and TSP/CONCORDE. The 3,270 markers map to 3,021 unique positions and define an average intermarker distance corresponding to 1 Mb. We also define a minimal screening set of 325 highly informative well spaced markers, to be used in the initiation of genome-wide scans. The well defined synteny between the dog and human genomes, established in part as a function of this work by the identification of 85 conserved fragments, will allow follow-up of initial findings of linkage by selection of candidate genes from the human genome sequence. This work continues to define the canine system as the method of choice in the pursuit of the genes causing mammalian variation and disease.
Abstract: Radiation hybrid (RH) map construction allows investigators to locate both type I and type II markers on a given genome map. The process is composed of two steps. The first consists of determining the pattern distribution of a set of markers within the different cell lines of an RH panel. This is mainly done by polymerase chain reaction (PCR) amplification and gel electrophoresis, and results in a series of numbers indicating the presence or the absence of each marker in each cell line. The second step consists of a comparison of these numbers, using various algorithms, to group and then order markers. Because different algorithms may provide (slightly) different orders, we have compared the merits of the MultiMap and TSP/CONCORDE packages using a data set of information currently under analysis for construction of the canine genome RH map.
Abstract: BACKGROUND: Olfactory receptors (ORs), the first dedicated molecules with which odorants physically interact to arouse an olfactory sensation, constitute the largest gene family in vertebrates, including around 900 genes in human and 1,500 in the mouse. Whereas dogs, like many other mammals, have a much keener olfactory potential than humans, only 21 canine OR genes have been described to date. RESULTS: In this study, 817 novel canine OR sequences were identified, and 640 have been characterized. Of the 661 characterized OR sequences, representing half of the canine repertoire, 18% are predicted to be pseudogenes, compared with 63% in human and 20% in mouse. Phylogenetic analysis of 403 canine OR sequences identified 51 families, and radiation-hybrid mapping of 562 showed that they are distributed on 24 dog chromosomes, in 37 distinct regions. Most of these regions constitute clusters of 2 to 124 closely linked genes. The two largest clusters (124 and 109 OR genes) are located on canine chromosomes 18 and 21. They are orthologous to human clusters located on human chromosomes 11q11-q13 and HSA11p15, containing 174 and 115 ORs respectively. CONCLUSIONS: This study shows a strongly conserved genomic distribution of OR genes between dog and human, suggesting that OR genes evolved from a common mammalian ancestral repertoire by successive duplications. In addition, the dog repertoire appears to have expanded relative to that of humans, leading to the emergence of specific canine OR genes.
Abstract: The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.
Abstract: We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have significant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.
Abstract: We present the sequence of a contiguous 2.63 Mb of DNA extending from the tip of the X chromosome of Drosophila melanogaster. Within this sequence, we predict 277 protein coding genes, of which 94 had been sequenced already in the course of studying the biology of their gene products, and examples of 12 different transposable elements. We show that an interval between bands 3A2 and 3C2, believed in the 1970s to show a correlation between the number of bands on the polytene chromosomes and the 20 genes identified by conventional genetics, is predicted to contain 45 genes from its DNA sequence. We have determined the insertion sites of P-elements from 111 mutant lines, about half of which are in a position likely to affect the expression of novel predicted genes, thus representing a resource for subsequent functional genomic analysis. We compare the European Drosophila Genome Project sequence with the corresponding part of the independently assembled and annotated Joint Sequence determined through "shotgun" sequencing. Discounting differences in the distribution of known transposable elements between the strains sequenced in the two projects, we detected three major sequence differences, two of which are probably explained by errors in assembly; the origin of the third major difference is unclear. In addition there are eight sequence gaps within the Joint Sequence. At least six of these eight gaps are likely to be sites of transposable elements; the other two are complex. Of the 275 genes in common to both projects, 60% are identical within 1% of their predicted amino-acid sequence and 31% show minor differences such as in choice of translation initiation or termination codons; the remaining 9% show major differences in interpretation.
Abstract: The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.
Abstract: Sinorhizobium meliloti is an alpha-proteobacterium that forms agronomically important N(2)-fixing root nodules in legumes. We report here the complete sequence of the largest constituent of its genome, a 62.7% GC-rich 3,654,135-bp circular chromosome. Annotation allowed assignment of a function to 59% of the 3,341 predicted protein-coding ORFs, the rest exhibiting partial, weak, or no similarity with any known sequence. Unexpectedly, the level of reiteration within this replicon is low, with only two genes duplicated with more than 90% nucleotide sequence identity, transposon elements accounting for 2.2% of the sequence, and a few hundred short repeated palindromic motifs (RIME1, RIME2, and C) widespread over the chromosome. Three regions with a significantly lower GC content are most likely of external origin. Detailed annotation revealed that this replicon contains all housekeeping genes except two essential genes that are located on pSymB. Amino acid/peptide transport and degradation and sugar metabolism appear as two major features of the S. meliloti chromosome. The presence in this replicon of a large number of nucleotide cyclases with a peculiar structure, as well as of genes homologous to virulence determinants of animal and plant pathogens, opens perspectives in the study of this bacterium both as a free-living soil microorganism and as a plant symbiont.
Abstract: One of the rewards of having a Drosophila melanogaster whole-genome sequence will be the potential to understand the molecular bases for structural features of chromosomes that have been a long-standing puzzle. Analysis of 2.6 megabases of sequence from the tip of the X chromosome of Drosophila identifies 273 genes. Cloned DNAs from the characteristic bulbous structure at the tip of the X chromosome in the region of the broad complex display an unusual pattern of in situ hybridization. Sequence analysis revealed that this region comprises 154 kilobases of DNA flanked by 1.2-kilobases of inverted repeats, each composed of a 350-base pair satellite related element. Thus, some aspects of chromosome structure appear to be revealed directly within the DNA sequence itself.
Abstract: The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.
Abstract: We describe here the construction of six deletion mutants and their basic phenotypic analysis. Six open reading frames (ORFs) from chromosome X, YJR039w, YJR041c, YJR043c, YJR046w, YJR053w and YJR065c, were disrupted by deletion cassettes with long (LFH) or short (SFH) flanking regions homologous to the target locus. The LFH deletion cassette was made by introducing into the kanMX4 marker module two polymerase chain reaction (PCR) fragments several hundred base pairs (bp) in size homologous to the promoter and terminator regions of a given ORF. The SFH gene disruption construct was obtained by PCR amplification of the kanMX4 marker with primers providing homology to the target gene. The region of homology to mediate homologous recombination was about 70 bp. Sporulation and tetrad analysis revealed that ORFs YJR041c, YJR046w and YJR065c are essential genes. Complementation tests by corresponding cognate gene clones confirmed this observation. The non-growing haploid segregants were observed under the microscope. The yjr041c delta haploid cells gave rise to microcolonies comprising about 20 to 50 cells. Most yjr046w delta cells were blocked after one or two cell cycles with heterogeneous bud sizes. The yjr065c delta cells displayed an unbudded spore or were arrested before completion of the first cell division cycle with a bud of variable size. The deduced protein of ORF YJR065c, that we named Act4, belongs to the Arp3 family of actin-related proteins. Three other ORFs, YJR039w, YJR043c and YJR053w are non-essential genes. The yjr043c delta cells hardly grew at 15 degrees C, indicating that this gene is required for growth at low temperature. Complementation tests confirmed that the disruption of YJR043c is responsible for this growth defect. In addition, the mating efficiency of yjr043c delta and yjr053w delta cells appear to be moderately affected.
