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PubMed2002

Directed proteomic analysis of the human nucleolus.

BACKGROUND: The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized. RESULTS: We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions. CONCLUSIONS: This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.

Amino Acid Sequence↗

PubMed2002

Retroviral insertion sites and cancer: fountain of all knowledge?

Retroviral gene tagging is enjoying a renaissance as a gene discovery method since the completion of the draft mouse genome sequence. The potential of this approach to elucidate the genetic basis of cancer is reviewed in the light of a series of recent papers that report the results of high-throughput screens.

Animals↗

PubMed2001

Can sequencing shed light on cell cycling?

Every organism must have cells that can replicate indefinitely. Can the draft human genome sequence tell us how the cell cycle works and how it evolved? We studied two protein families--the cyclins and their partners the cyclin-dependent kinases (Cdks)--and a conserved regulatory circuit, the spindle checkpoint. Disappointingly, we discovered a few novel cyclins and no new Cdks or components of the spindle checkpoint, and could shed little light on the organization of the cell cycle.

Amino Acid Sequence↗

PubMed2001

Comparison of human genetic and sequence-based physical maps.

Recombination is the exchange of information between two homologous chromosomes during meiosis. The rate of recombination per nucleotide, which profoundly affects the evolution of chromosomal segments, is calculated by comparing genetic and physical maps. Human physical maps have been constructed using cytogenetics, overlapping DNA clones and radiation hybrids; but the ultimate and by far the most accurate physical map is the actual nucleotide sequence. The completion of the draft human genomic sequence provides us with the best opportunity yet to compare the genetic and physical maps. Here we describe our estimates of female, male and sex-average recombination rates for about 60% of the genome. Recombination rates varied greatly along each chromosome, from 0 to at least 9 centiMorgans per megabase (cM Mb(-1)). Among several sequence and marker parameters tested, only relative marker position along the metacentric chromosomes in males correlated strongly with recombination rate. We identified several chromosomal regions up to 6 Mb in length with particularly low (deserts) or high (jungles) recombination rates. Linkage disequilibrium was much more common and extended for greater distances in the deserts than in the jungles.

Female↗

PubMed2006

The DNA sequence, annotation and analysis of human chromosome 3.

After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion that occurred some time after the split of Homininae from Ponginae, and propose an evolutionary history of the inversion.

Animals↗

PubMed2002

Efficient discovery of single-nucleotide polymorphisms in coding regions of human genes.

Single nucleotide polymorphisms in protein coding regions (cSNPs) are of great interest for their effects on phenotype and potential for mapping disease genes. We have identified 5,400 novel exonic SNPs from alignments of public EST data to the draft human genome sequence, and approximately 12,000 more novel exonic SNPs from EST cluster alignments. We found 82% of the genomic-aligned SNPs and 63% of the EST-only SNPs to be detectably polymorphic in 20 Finnish DNA samples. 37% of the SNPs mapped to known protein coding regions, yielding 6,500 distinct, novel cSNPs from the two datasets. These data reveal selection against mutations that alter protein structure, and distinct classes of genes under strongly positive vs. negative pressure from natural selection for amino acid replacement (detected by K(A)/K(S)ratio). We have searched these cSNPs for compatibility with the amino acid profile at each site and structural impact on protein core stability.

Chromosome Mapping↗

PubMed2001

The genetics of type 2 diabetes.

Type 2 diabetes mellitus is not a single disease but a genetically heterogeneous group of metabolic disorders sharing glucose intolerance. The precise underlying biochemical defects are unknown and almost certainly include impairments of both insulin secretion and action. The rapidly increasing prevalence of T2D world wide makes it a major cause of morbidity and mortality. Understanding the genetic aetiology of T2D will facilitate its diagnosis, treatment and prevention. The results of linkage and association studies to date demonstrate that, as with other common diseases, multiple genes are involved in the susceptibility to T2D, each making a modest contribution to the overall risk. The completion of the draft human genome sequence and a brace of novel tools for genomic analysis promise to accelerate progress towards a more complete molecular description of T2D.

Animals↗

PubMed2005

Two CD1 genes map to the chicken MHC, indicating that CD1 genes are ancient and likely to have been present in the primordial MHC.

CD1 molecules play an important role in the immune system, presenting lipid-containing antigens to T and NKT cells. CD1 genes have long been thought to be as ancient as MHC class I and II genes, based on various arguments, but thus far they have been described only in mammals. Here we describe two CD1 genes in chickens, demonstrating that the CD1 system was present in the last common ancestor of mammals and birds at least 300 million years ago. In phylogenetic analysis, these sequences cluster with CD1 sequences from other species but are not obviously like any particular CD1 isotype. Sequence analysis suggests that the expressed proteins bind hydrophobic molecules and are recycled through intracellular vesicles. RNA expression is strong in lymphoid tissues but weaker to undetectable in some nonlymphoid tissues. Flow cytometry confirms expression from one gene on B cells. Based on Southern blotting and cloning, only two such CD1 genes are detected, located approximately 800 nucleotides apart and in the same transcriptional orientation. The sequence of one gene is nearly identical in six chicken lines. By mapping with a backcross family, this gene could not be separated from the chicken MHC on chromosome 16. Mining the draft chicken genome sequence shows that chicken has only these two CD1 genes located approximately 50 kb from the classical class I genes. The unexpected location of these genes in the chicken MHC suggests the CD1 system was present in the primordial MHC and is thus approximately 600 million years old.

Amino Acid Sequence↗

PubMed2006

A Sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes.

Since its introduction a decade ago, whole-genome shotgun sequencing (WGS) has been the main approach for producing cost-effective and high-quality genome sequence data. Until now, the Sanger sequencing technology that has served as a platform for WGS has not been truly challenged by emerging technologies. The recent introduction of the pyrosequencing-based 454 sequencing platform (454 Life Sciences, Branford, CT) offers a very promising sequencing technology alternative for incorporation in WGS. In this study, we evaluated the utility and cost-effectiveness of a hybrid sequencing approach using 3730xl Sanger data and 454 data to generate higher-quality lower-cost assemblies of microbial genomes compared to current Sanger sequencing strategies alone.

Biotechnology↗

PubMed2000

Shotgun sequencing of the human transcriptome with ORF expressed sequence tags.

Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription-PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome.

Animals↗

PubMed2002

Comprehensive proteomic analysis of breast cancer cell membranes reveals unique proteins with potential roles in clinical cancer.

Proteins associated with cancer cell plasma membranes are rich in known drug and antibody targets as well as other proteins known to play key roles in the abnormal signal transduction processes required for carcinogenesis. We describe here a proteomics process that comprehensively annotates the protein content of breast tumor cell membranes and defines the clinical relevance of such proteins. Tumor-derived cell lines were used to ensure an enrichment for cancer cell-specific plasma membrane proteins because it is difficult to purify cancer cells and then obtain good membrane preparations from clinical material. Multiple cell lines with different molecular pathologies were used to represent the clinical heterogeneity of breast cancer. Peptide tandem mass spectra were searched against a comprehensive data base containing known and conceptual proteins derived from many public data bases including the draft human genome sequences. This plasma membrane-enriched proteome analysis created a data base of more than 500 breast cancer cell line proteins, 27% of which were of unknown function. The value of our approach is demonstrated by further detailed analyses of three previously uncharacterized proteins whose clinical relevance has been defined by their unique cancer expression profiles and the identification of protein-binding partners that elucidate potential functionality in cancer.

Amino Acid Sequence↗

PubMed2001

Gene structure for adenosine kinase in Chinese hamster and human: high-frequency mutants of CHO cells involve deletions of several introns and exons.

The structure for the adenosine kinase (AK) gene has been determined from Chinese hamster (CH) and human cells. The AK gene in CH is comprised of 11 exons ranging in length from 36 to 765 nt, with the majority <100 nt. The exact lengths of the intervening introns have not been determined, but most of them are indicated to be very large (>15 kb). A 6.6-kb fragment from human cells was also sequenced, and it contained only a single exon corresponding to exon 10 in CH. The BLAST searches of the subsequently released draft human genome sequence have revealed that the AK gene structure in human is identical to that in CH. In the human genome, the AK exons are distributed over four genomic clones totaling 752 kb, providing direct evidence that the AK gene in mammalian species is unusually large. In contrast to CH and human, the AK genes from several other eukaryotic organisms whose complete genomes are now known are quite small (between 1.2 and 2.5 kb) and either contain no introns (Saccharomyces cerevisiae and Schizosaccharomyces pombe) or various numbers of introns (Drosophila melanogaster [2], Caenorhabditis elegans [4], Arabidopsis thaliana [10]). Some of the intron-exon junctions in these species are in the same positions as in mammals. The AK gene in CH and human, as well as mouse, is linked upstream in a head-to-head fashion with the gene for the clathrin adaptor mu3 protein (or beta 3A subunit of the AP-3 protein complex), which is affected in type 2 Hermansky-Pudlak syndrome. These two genes are separated by <200 nt, and it is possible that they have a common or overlapping promoter(s). We have also determined the nature of the genetic alterations in two of the class A AK(-) mutants of CHO cells, which are obtained at a very high spontaneous frequency (10(-3)-10(-4)) in this cell line. Both mutants contained large deletions within the AK gene and greatly shortened AK transcripts. The cloning and sequencing of the transcripts from these mutants showed that the deletion in one of them led to the loss of exons 5 through 8, whereas in the other, all exons from 2 through 8 are deleted. The endpoints of these deletions lie in the large introns within the AK gene.

Adenosine Kinase↗

PubMed2002

The Gene Resource Locator: gene locus maps for transcriptome analysis.

Since the advent of the draft human genome sequence there has been growing interest in transcriptome analysis based on genomic data. The Gene Resource Locator (GRL) assembles gene maps that include information on gene-expression patterns, cis-elements in regulatory regions and alternatively spliced transcripts. The database was constructed using customized software, and currently contains 2.2 million alignments (exon-intron structures). The alignments have been annotated and integrated into a system that encompasses approximately 90 000 EST loci sharing common exons, 8091 alternatively spliced transcript groups, 10 801 expression-profile groups, 8066 candidate regulatory regions in full-length cDNAs, and 1 million SNP loci. We have used Flash technology to build a dynamic web viewer that facilitates browsing through the millions of alignments. All of the information is available through the World Wide Web at the Gene Resource Locator web site (http://grl.gi.k.u-tokyo.ac.jp).

Alternative Splicing↗

PubMed2005

FlyBase: genes and gene models.

FlyBase (http://flybase.org) is the primary repository of genetic and molecular data of the insect family Drosophilidae. For the most extensively studied species, Drosophila melanogaster, a wide range of data are presented in integrated formats. Data types include mutant phenotypes, molecular characterization of mutant alleles and aberrations, cytological maps, wild-type expression patterns, anatomical images, transgenic constructs and insertions, sequence-level gene models and molecular classification of gene product functions. There is a growing body of data for other Drosophila species; this is expected to increase dramatically over the next year, with the completion of draft-quality genomic sequences of an additional 11 Drosophila species.

Animals↗

PubMed2002

EZ-Retrieve: a web-server for batch retrieval of coordinate-specified human DNA sequences and underscoring putative transcription factor-binding sites.

The availability of a draft human genome sequence and ability to monitor the transcription of thousands of genes with DNA microarrays has necessitated the need for new computational tools that can analyze cis-regulatory elements controlling genes that display similar expression patterns. We have developed a tool designated EZ-Retrieve that can: (i) retrieve any particular region of human genome sequence from the NCBI database and (ii) analyze retrieved sequences for putative transcription factor-binding sites (TFBSs) as they appear on the TRANSFAC database. The tool is web-based, user-friendly and offers both batch sequence retrieval and batch TFBS prediction. A major application of EZ-Retrieve is the analysis of co-expressed genes that are highlighted as expression clusters in DNA microarray experiments.

Animals↗

PubMed2003

The human transcriptome map reveals extremes in gene density, intron length, GC content, and repeat pattern for domains of highly and weakly expressed genes.

The chromosomal gene expression profiles established by the Human Transcriptome Map (HTM) revealed a clustering of highly expressed genes in about 30 domains, called ridges. To physically characterize ridges, we constructed a new HTM based on the draft human genome sequence (HTMseq). Expression of 25,003 genes can be analyzed online in a multitude of tissues (http://bioinfo.amc.uva.nl/HTMseq). Ridges are found to be very gene-dense domains with a high GC content, a high SINE repeat density, and a low LINE repeat density. Genes in ridges have significantly shorter introns than genes outside of ridges. The HTMseq also identifies a significant clustering of weakly expressed genes in domains with fully opposite characteristics (antiridges). Both types of domains are open to tissue-specific expression regulation, but the maximal expression levels in ridges are considerably higher than in antiridges. Ridges are therefore an integral part of a higher order structure in the genome related to transcriptional regulation.

Base Composition↗

PubMed2005

Fatty acid desaturases from the microalga Thalassiosira pseudonana.

Analysis of a draft nuclear genome sequence of the diatom Thalassiosira pseudonana revealed the presence of 11 open reading frames showing significant similarity to functionally characterized fatty acid front-end desaturases. The corresponding genes occupy discrete chromosomal locations as determined by comparison with the recently published genome sequence. Phylogenetic analysis showed that two of the T. pseudonana desaturase (Tpdes) sequences grouped with proteobacterial desaturases that lack a fused cytochrome b5 domain. Among the nine remaining gene sequences, temporal expression analysis revealed that seven were expressed in T. pseudonana cells. One of these, TpdesN, was previously characterized as encoding a Delta11-desaturase active on palmitic acid. From the six remaining putative desaturase genes, we report here that three, TpdesI, TpdesO and TpdesK, respectively encode Delta6-, Delta5- and Delta4-desaturases involved in production of the health beneficial polyunsaturated fatty acid DHA (docosahexaenoic acid). Furthermore, we show that one of the remaining genes, TpdesB, encodes a Delta8-sphingolipid desaturase with strong preference for dihydroxylated substrates.

Chromatography, Gas↗

PubMed2002

A draft sequence of the rice genome (Oryza sativa L. ssp. japonica).

The genome of the japonica subspecies of rice, an important cereal and model monocot, was sequenced and assembled by whole-genome shotgun sequencing. The assembled sequence covers 93% of the 420-megabase genome. Gene predictions on the assembled sequence suggest that the genome contains 32,000 to 50,000 genes. Homologs of 98% of the known maize, wheat, and barley proteins are found in rice. Synteny and gene homology between rice and the other cereal genomes are extensive, whereas synteny with Arabidopsis is limited. Assignment of candidate rice orthologs to Arabidopsis genes is possible in many cases. The rice genome sequence provides a foundation for the improvement of cereals, our most important crops.

Arabidopsis↗