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PubMed2004

A first generation physical map of the medaka genome in BACs essential for positional cloning and clone-by-clone based genomic sequencing.

In order to realize the full potential of the medaka as a model system for developmental biology and genetics, characterized genomic resources need to be established, culminating in the sequence of the medaka genome. To facilitate the map-based cloning of genes underlying induced mutations and to provide templates for clone-based genomic sequencing, we have created a first-generation physical map of the medaka genome in bacterial artificial chromosome (BAC) clones. In particular, we exploited the synteny to the closely related genome of the pufferfish, Takifugu rubripes, by marker content mapping. As a first step, we clustered 103,144 public medaka EST sequences to obtain a set of 21,121 non-redundant sequence entities. Avoiding oversampling of gene-dense regions, 11,254 of EST clusters were successfully matched against the draft sequence of the fugu genome, and 2363 genes were selected for the BAC map project. We designed 35mer oligonucleotide probes from the selected genes and hybridized them against 64,500 BAC clones of strains Cab and Hd-rR, representing 14-fold coverage of the medaka genome. Our data set is further supplemented with 437 results generated from PCR-amplified inserts of medaka cDNA clones and BAC end-fragment markers. Our current, edited, first generation medaka BAC map consists of 902 map segments that cover about 74% of the medaka genome. The map contains 2721 markers. Of these, 2534 are from expressed sequences, equivalent to a non-redundant set of 2328 loci. The 934 markers (724 different) are anchored to the medaka genetic map. Thus, genetic map assignments provide immediate access to underlying clones and contigs, simplifying molecular access to candidate gene regions and their characterization.

Animals↗

PubMed2006

Genome-wide analysis of ATP-binding cassette (ABC) proteins in a model legume plant, Lotus japonicus: comparison with Arabidopsis ABC protein family.

ATP-binding cassette (ABC) proteins constitute a large family in plants with more than 120 members each in Arabidopsis and rice, and have various functions including the transport of auxin and alkaloid, as well as the regulation of stomata movement. In this report, we carried out genome-wide analysis of ABC protein genes in a model legume plant, Lotus japonicus. For analysis of the Lotus genome sequence, we devised a new method 'domain-based clustering analysis', where domain structures like the nucleotide-binding domain (NBD) and transmembrane domain (TMD), instead of full-length amino acid sequences, are used to compare phylogenetically each other. This method enabled us to characterize fragments of ABC proteins, which frequently appear in a draft sequence of the Lotus genome. We identified 91 putative ABC proteins in L. japonicus, i.e. 43 'full-size', 40 'half-size' and 18 'soluble' putative ABC proteins. The characteristic feature of the composition is that Lotus has extraordinarily many paralogs similar to AtMRP14 and AtPDR12, which are at least six and five members, respectively. Expression analysis of the latter genes performed with real-time quantitative reverse transcription-PCR revealed their putative involvement in the nodulation process.

ATP-Binding Cassette Transporters↗

PubMed2002

Characterization of tissue expression and full-length coding sequence of a novel human gene mapping at 3q12.1 and transcribed in oligodendrocytes.

Macro-array differential hybridization of a collection of 5058 human gene transcripts represented in an IMAGE infant brain cDNA library has led to the identification of transcripts displaying preferential or specific expression in brain (Genome Res. 9 (1999) 195; http://idefix.upr420.vjf.cnrs.fr/IMAGE). Most of these genes correspond to as yet undescribed functions. Detailed characterization of the expression, sequence, and genome assignment of one of these genes named C3orf4, is reported here. The full-length sequence of the transcript was obtained by 5' extension RT-PCR. The gene transcript (2.8 kb) encodes a 253 amino acid long protein, with four transmembrane domains. The position of the C3orf4 gene was determined at 3q12.1 thanks to the draft sequence of the human genome. It is composed of five exons spanning more than 7 kb. No TATAA box but a CpG island was found upstream of the beginning of the gene. Northern blot analysis and in situ hybridization revealed a predominant expression in myelinated structures such as corpus callosum and spinal cord. RT-PCR showed expression of the C3orf4 gene in rat optic nerve and cultured oligodendrocytes, the myelinating cells of the central nervous system, but not in astrocytes. This work supports further investigations aimed at determining the role of the C3orf4 gene in myelinating cells.

Adult↗

PubMed2003

Comprehensive analysis of two Alu Yd subfamilies.

Alu elements have inserted in the human genome throughout primate evolution. A small number of Alu insertions have occurred after the divergence of humans from nonhuman primates and therefore should not be present in nonhuman primate genomes. Most of these recently integrated Alu elements are contained with a series of discrete Alu subfamilies that are related to each other based upon diagnostic nucleotide substitutions. We have extracted members of the Alu Yd subfamily that are derivatives of the Alu Y subfamily that share a common 12-bp deletion that defines the Yd lineage from the draft sequence of the human genome. Analysis of the Yd Alu elements resulted in the recovery of two new Alu subfamilies, Yd3 and Yd6, which contain a total of 295 members (198 Yd3 and 97 Yd6). DNA sequence analysis of each of the Alu Yd subfamilies yielded age estimates of 8.02 and 1.20 million years old for the Alu Yd3 and Yd6 subfamilies, respectively. Two hundred Alu Yd3 and Yd6 loci were screened using polymerase chain reaction (PCR) assays to determine their phylogenetic origin and associated levels of human genomic diversity. The Alu Yd3 subfamily appears to have started amplifying relatively early in primate evolution and continued propagating albeit at a low level as many of its members are found in a variety of hominoid (humans, greater and lesser ape) genomes. Only two of the elements are polymorphic in the human genome and absent from the genomes of nonhuman primates. By contrast all of the members of the Alu Yd6 subfamily are restricted to the human genome, with 12% of the elements representing insertion polymorphisms in human populations. A single Alu Yd6 locus contained an independent parallel forward insertion of a paralogous Alu Sq sequence in the owl monkey. These Alu subfamilies are a source of genomic fossil relics for the study of primate phylogenetics and human population genetics.

Alu Elements↗

PubMed2005

Genome-wide prediction of human VNTRs.

Polymorphic minisatellites, also known as variable number of tandem repeats (VNTRs), are tandem repeat regions that show variation in the number of repeat units among chromosomes in a population. Currently, there are no general methods for predicting which minisatellites have a high probability of being polymorphic, given their sequence characteristics. An earlier approach has focused on potentially highly polymorphic and hypervariable minisatellites, which make up only a small fraction of all minisatellites in the human genome. We have developed a model, based on available minisatellite and VNTR sequence data, that predicts the probability that a minisatellite (unit size > or = 6 bp) identified by the computer program Tandem Repeats Finder is polymorphic (VNTR). According to the model, minisatellites with high copy number and high degree of sequence similarity are most likely to be VNTRs. This approach was used to scan the draft sequence of the human genome for VNTRs. A total of 157,549 minisatellite repeats were found, of which 29,224 are predicted to be VNTRs. Contrary to previous results, VNTRs appear to be widespread and abundant throughout the human genome, with an estimated density of 9.1 VNTRs/Mb.

Genome, Human↗

PubMed2003

Mobilization of a transposon in the rice genome.

Rice (Oryza sativa L.) is an important crop worldwide and, with the availability of the draft sequence, a useful model for analysing the genome structure of grasses. To practice efficient rice breeding through genetic engineering techniques, it is important to identify the economically important genes in this crop. The use of mobile transposons as gene tags in intact plants is a powerful tool for functional analysis because transposon insertions often inactivate genes. Here we identify an active rice transposon named miniature Ping (mPing) through analysis of the mutability of a slender mutation of the glume-the seed structure that encloses and determines the shape of the grain. The mPing transposon is inserted in the slender glume (slg) mutant allele but not in the wild-type allele. Search of the O. sativa variety Nipponbare genome identified 34 sequences with high nucleotide similarity to mPing, indicating that mPing constitutes a family of transposon elements. Excision of mPing from slg plants results in reversion to a wild-type phenotype. The mobility of the transposon mPing in intact rice plants represents a useful alternative tool for the functional analysis of rice genes.

Alleles↗

PubMed2001

The human genome: an immuno-centric view of evolutionary strategies.

A hallmark of modern biology is the realization of the fundamental unity of biological processes in all life forms. Consequently, the complete genome sequencing of various bacteria, yeast (Saccharomyces cerevisiae), fly (Drosophila melanogaster) and worm (Caenorhabditis elegans) over the past five years has already had an impact on all of biology. "Model organisms" have contributed a great deal to immunology; for example, the Toll receptors of the fly provided the impetus for the investigation of Toll-like receptors, which proved to be fundamental elements in the mammalian innate immune system. The recent release of a draft sequence of the human genome provides the first panoramic view of the 30000-35000 human genes in the human genetic blueprint and provides a plethora of new details, the significance of which will take some time to appreciate. The over-riding concepts that emerge from these studies relate primarily to general evolutionary processes that are equally as relevant to immunology as they are to other disciplines of biology.

Amino Acid Sequence↗

PubMed2006

A comprehensive catalog of human KRAB-associated zinc finger genes: insights into the evolutionary history of a large family of transcriptional repressors.

Krüppel-type zinc finger (ZNF) motifs are prevalent components of transcription factor proteins in all eukaryotes. KRAB-ZNF proteins, in which a potent repressor domain is attached to a tandem array of DNA-binding zinc-finger motifs, are specific to tetrapod vertebrates and represent the largest class of ZNF proteins in mammals. To define the full repertoire of human KRAB-ZNF proteins, we searched the genome sequence for key motifs and then constructed and manually curated gene models incorporating those sequences. The resulting gene catalog contains 423 KRAB-ZNF protein-coding loci, yielding alternative transcripts that altogether predict at least 742 structurally distinct proteins. Active rounds of segmental duplication, involving single genes or larger regions and including both tandem and distributed duplication events, have driven the expansion of this mammalian gene family. Comparisons between the human genes and ZNF loci mined from the draft mouse, dog, and chimpanzee genomes not only identified 103 KRAB-ZNF genes that are conserved in mammals but also highlighted a substantial level of lineage-specific change; at least 136 KRAB-ZNF coding genes are primate specific, including many recent duplicates. KRAB-ZNF genes are widely expressed and clustered genes are typically not coregulated, indicating that paralogs have evolved to fill roles in many different biological processes. To facilitate further study, we have developed a Web-based public resource with access to gene models, sequences, and other data, including visualization tools to provide genomic context and interaction with other public data sets.

Computational Biology↗

PubMed2003

The genome sequence of the filamentous fungus Neurospora crassa.

Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes--more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca2+ signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes.

Calcium Signaling↗

PubMed2004

Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78.

White rot fungi efficiently degrade lignin, a complex aromatic polymer in wood that is among the most abundant natural materials on earth. These fungi use extracellular oxidative enzymes that are also able to transform related aromatic compounds found in explosive contaminants, pesticides and toxic waste. We have sequenced the 30-million base-pair genome of Phanerochaete chrysosporium strain RP78 using a whole genome shotgun approach. The P. chrysosporium genome reveals an impressive array of genes encoding secreted oxidases, peroxidases and hydrolytic enzymes that cooperate in wood decay. Analysis of the genome data will enhance our understanding of lignocellulose degradation, a pivotal process in the global carbon cycle, and provide a framework for further development of bioprocesses for biomass utilization, organopollutant degradation and fiber bleaching. This genome provides a high quality draft sequence of a basidiomycete, a major fungal phylum that includes important plant and animal pathogens.

Base Composition↗

PubMed2001

Genomic organisation of the approximately 1.5 Mb Smith-Magenis syndrome critical interval: transcription map, genomic contig, and candidate gene analysis.

Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation syndrome associated with an interstitial deletion of chromosome 17 involving band p11.2. SMS is hypothesised to be a contiguous gene syndrome in which the phenotype arises from the haploinsufficiency of multiple, functionally-unrelated genes in close physical proximity, although the true molecular basis of SMS is not yet known. In this study, we have generated the first overlapping and contiguous transcription map of the SMS critical interval, linking the proximal 17p11.2 region near the SMS-REPM and the distal region near D17S740 in a minimum tiling path of 16 BACs and two PACs. Additional clones provide greater coverage throughout the critical region. Not including the repetitive sequences that flank the critical interval, the map is comprised of 13 known genes, 14 ESTs, and six genomic markers, and is a synthesis of Southern hybridisation and polymerase chain reaction data from gene and marker localisation to BACs and PACs and database sequence analysis from the human genome project high-throughput draft sequence. In order to identify possible candidate genes, we performed sequence analysis and determined the tissue expression pattern analysis of 10 novel ESTs that are deleted in all SMS patients. We also present a detailed review of six promising candidate genes that map to the SMS critical region.

Abnormalities, Multiple↗

PubMed2002

Entire sequence of a mouse chromosomal segment containing the gene Rhced and a comparative analysis of the homologous human sequence.

The mouse genomic sequence of the region containing the gene Rhced, the orthologue to the human gene RH30, was determined to elucidate the structure of Rhced and its flanking regions and to compare these with the corresponding human genomic region. Two genes, Smp1 and AK003528 (an orthologue of FLJ10747), flank Rhced. Neither sequences homologous to the characteristic nucleotide elements flanking the RHD gene in humans (rhesus boxes) nor an additional Rh gene were found within the mouse region sequenced. This result and that of a previous report demonstrate that this chromosomal region of the mouse comprises five genes (FLJ10747-RHCE-SMP1-NPD014-P29) that exhibit syntenic homology with the corresponding human region, which suggests that the RHD gene and rhesus boxes were inserted later. Evaluations of tissue distribution and subcellular localization of these genes indicate that the SMP1 orthologue has a ubiquitous tissue distribution and cytoplasmic localization, whereas AK003528 is expressed slightly higher in testis with a strong subcellular localization in the nucleus. Despite the steady improvements in the draft sequence of the human genome, this study demonstrates the continuing benefits of comparative genetic analyses in increasing our understanding of human genomic structure.

3' Flanking Region↗

PubMed2005

Draft versus finished sequence data for DNA and protein diagnostic signature development.

Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10(-3)-10(-5) (approximately 8x coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of approximately 1% (3x to 6x coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures.

Computational Biology↗

PubMed2001

High-resolution mapping of the human 4q21 and the mouse 5E3 SCYB chemokine cluster by fiber-fluorescence in situ hybridization.

The CXC chemokine or small inducible cytokine B (SCYB) subfamily includes the T-cell chemoattractants MIG (CXCL9, SCYB9), IP-10 (CXCL10, SCYB10), and I-TAC (CXCL11, SCYB11). These three highly homologous chemokines lack the glutamic acid-leucine-arginine (ELR) motif and signal via the CXCR3 receptor. Previous work showed that the genes encoding these chemokines are localized in an individual mini-cluster on human Chromosome (Chr) 4 at position 4q21.2. Recently, we identified mouse Scyb11 and mapped this gene by fluorescence in situ hybridization (FISH) to mouse Chr 5E3, the orthologous locus to human 4q21 where the other two homologous mouse genes, Scyb9 and Scyb10, have also been localized. Since SCYB10 and SCYB11 are not represented in the recently published draft sequence of the human genome, we wanted to clarify exactly the order and distances of the three chemokine genes using two-color FISH on stretched DNA fiber preparations. Here, we report the simultaneous localization of all three genes and provide high-resolution visual maps of this chemokine cluster from both mouse and human. The three chemokine genes were found within a range of 32 kb on mouse and 29 kb on human DNA fiber targets. The precise physical distances were defined, and an almost identical arrangement of the human and mouse homologues was identified, indicating that this CXC chemokine mini-cluster has been completely conserved evolutionarily since the divergence of mouse and human. Our results refine previous maps of the three genes, support the hypothesis that they resulted from gene duplication that took place in a common ancestor of mouse and human, and provide complementary information on a region of the draft sequence of human Chr 4 that is not yet covered.

Animals↗

PubMed2003

Genomic analysis and initial characterization of the chitinolytic system of Microbulbifer degradans strain 2-40.

The marine bacterium Microbulbifer degradans strain 2-40 produces at least 10 enzyme systems for degrading insoluble complex polysaccharides (ICP). The draft sequence of the 2-40 genome allowed a genome-wide analysis of the chitinolytic system of strain 2-40. The chitinolytic system includes three secreted chitin depolymerases (ChiA, ChiB, and ChiC), a secreted chitin-binding protein (CbpA), periplasmic chitooligosaccharide-modifying enzymes, putative sugar transporters, and a cluster of genes encoding cytoplasmic proteins involved in N-acetyl-D-glucosamine (GlcNAc) metabolism. Each chitin depolymerase was detected in culture supernatants of chitin-grown strain 2-40 and was active against chitin and glycol chitin. The chitin depolymerases also had a specific pattern of activity toward the chitin analogs 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside (MUF-diNAG) and 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside (MUF-triNAG). The depolymerases were modular in nature and contained glycosyl hydrolase family 18 domains, chitin-binding domains, and polycystic kidney disease domains. ChiA and ChiB each possessed polyserine linkers of up to 32 consecutive serine residues. In addition, ChiB and CbpA contained glutamic acid-rich domains. At 1,271 amino acids, ChiB is the largest bacterial chitinase reported to date. A chitodextrinase (CdxA) with activity against chitooligosaccharides (degree of polymerization of 5 to 7) was identified. The activities of two apparent periplasmic (HexA and HexB) N-acetyl-beta-D-glucosaminidases and one cytoplasmic (HexC) N-acetyl-beta-D-glucosaminidase were demonstrated. Genes involved in GlcNAc metabolism, similar to those of the Escherichia coli K-12 NAG utilization operon, were identified. NagA from strain 2-40, a GlcNAc deacetylase, was shown to complement a nagA mutation in E. coli K-12. Except for the GlcNAc utilization cluster, genes for all other components of the chitinolytic system were dispersed throughout the genome. Further examination of this system may provide additional insight into the mechanisms by which marine bacteria degrade chitin and provide a basis for future research on the ICP-degrading systems of strain 2-40.

Alteromonadaceae↗

PubMed2006

Identification and characterization of novel polymorphic LINE-1 insertions through comparison of two human genome sequence assemblies.

Mobile elements represent a relatively new class of markers for the study of human evolution. Long interspersed elements (LINEs) belong to a group of retrotransposons comprising approximately 21% of the human genome. Young LINE-1 (L1) elements that have integrated recently into the human genome can be polymorphic for insertion presence/absence in different human populations at particular chromosomal locations. To identify putative novel L1 insertion polymorphisms, we computationally compared two draft assemblies of the whole human genome (Public and Celera Human Genome assemblies). We identified a total of 148 potential polymorphic L1 insertion loci, among which 73 were candidates for novel polymorphic loci. Based on additional analyses we selected 34 loci for further experimental studies. PCR-based assays and DNA sequence analysis were performed for these 34 loci in 80 unrelated individuals from four diverse human populations: African-American, Asian, Caucasian, and South American. All but two of the selected loci were confirmed as polymorphic in our human population panel. Approximately 47% of the analyzed loci integrated into other repetitive elements, most commonly older L1s. One of the insertions was accompanied by a BC200 sequence. Collectively, these mobile elements represent a valuable source of genomic polymorphism for the study of human population genetics. Our results also suggest that the exhaustive identification of L1 insertion polymorphisms is far from complete, and new whole genome sequences are valuable sources for finding novel retrotransposon insertion polymorphisms.

Alleles↗

PubMed2005

GC composition of the human genome: in search of isochores.

The isochore theory, proposed nearly three decades ago, depicts the mammalian genome as a mosaic of long, fairly homogeneous genomic regions that are characterized by their guanine and cytosine (GC) content. The human genome, for instance, was claimed to consist of five distinct isochore families: L1, L2, H1, H2, and H3, with GC contents of <37%, 37%-42%, 42%-47%, 47%-52%, and >52%, respectively. In this paper, we address the question of the validity of the isochore theory through a rigorous sequence-based analysis of the human genome. Toward this end, we adopt a set of six attributes that are generally claimed to characterize isochores and statistically test their veracity against the available draft sequence of the complete human genome. By the selection criteria used in this study: distinctiveness, homogeneity, and minimal length of 300 kb, we identify 1,857 genomic segments that warrant the label "isochore." These putative isochores are nonuniformly scattered throughout the genome and cover about 41% of the human genome. We found that a four-family model of putative isochores is the most parsimonious multi-Gaussian model that can be fitted to the empirical data. These families, however, are GC poor, with mean GC contents of 35%, 38%, 41%, and 48% and do not resemble the five isochore families in the literature. Moreover, due to large overlaps among the families, it is impossible to classify genomic segments into isochore families reliably, according to compositional properties alone. These findings undermine the utility of the isochore theory and seem to indicate that the theory may have reached the limits of its usefulness as a description of genomic compositional structures.

Base Composition↗

PubMed2005

The genome sequence of the rice blast fungus Magnaporthe grisea.

Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.

Fungal Proteins↗