Biomedical subjects
Publications and source records attributed to J Quackenbush.
The scientific process, and scientific progress, require a critical examination of all published reports. Recent publications detailing errors in the draft human genome sequence are an integral part of our quest to better understand nature and demonstrate the value of free access to scientific data.
Microarray analysis has become a widely used tool for the generation of gene expression data on a genomic scale. Although many significant results have been derived from microarray studies, one limitation has been the lack of standards for presenting and exchanging such data. Here we present a proposal, the Minimum Information About a Microarray Experiment (MIAME), that describes the minimum information required to ensure that microarray data can be easily interpreted and that results derived from its analysis can be independently verified. The ultimate goal of this work is to establish a standard for recording and reporting microarray-based gene expression data, which will in turn facilitate the establishment of databases and public repositories and enable the development of data analysis tools. With respect to MIAME, we concentrate on defining the content and structure of the necessary information rather than the technical format for capturing it.
An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained. Target tissues included those with highest likelihood to have impact on production parameters of animal health, growth, reproductive efficiency, and carcass merit. Success of normalization and inter- and intralibrary redundancy were assessed by collecting 6000-23,000 sequences from each of the libraries (68,520 total sequences deposited in GenBank). Sequence comparison and assembly of these sequences was performed in combination with 56,500 other bovine EST sequences present in the GenBank dbEST database to construct a cattle Gene Index (available from The Institute for Genomic Research at http://www.tigr.org/tdb/tgi.shtml). The 124,381 bovine ESTs present in GenBank at the time of the analysis form 16,740 assemblies that are listed and annotated on the Web site. Analysis of individual library sequence data indicates that the pooled-tissue approach was highly effective in preparing libraries for efficient deep sequencing.
Rice (Oryza sativa) is a model species for monocotyledonous plants, especially for members in the grass family. Several attributes such as small genome size, diploid nature, transformability, and establishment of genetic and molecular resources make it a tractable organism for plant biologists. With an estimated genome size of 430 Mb (Arumuganathan and Earle, 1991), it is feasible to obtain the complete genome sequence of rice using current technologies. An international effort has been established and is in the process of sequencing O. sativa spp. japonica var "Nipponbare" using a bacterial artificial chromosome/P1 artificial chromosome shotgun sequencing strategy. Annotation of the rice genome is performed using prediction-based and homology-based searches to identify genes. Annotation tools such as optimized gene prediction programs are being developed for rice to improve the quality of annotation. Resources are also being developed to leverage the rice genome sequence to partial genome projects such as expressed sequence tag projects, thereby maximizing the output from the rice genome project. To provide a low level of annotation for rice genomic sequences, we have aligned all rice bacterial artificial chromosome/P1 artificial chromosome sequences with The Institute of Genomic Research Gene Indices that are a set of nonredundant transcripts that are generated from nine public plant expressed sequence tag projects (rice, wheat, sorghum, maize, barley, Arabidopsis, tomato, potato, and barrel medic). In addition, we have used data from The Institute of Genomic Research Gene Indices and the Arabidopsis and Rice Genome Projects to identify putative orthologues and paralogues among these nine genomes.
Microarray expression analysis is providing unprecedented data on gene expression in humans and mammalian model systems. Although such studies provide a tremendous resource for understanding human disease states, one of the significant challenges is cross-referencing the data derived from different species, across diverse expression analysis platforms, in order to properly derive inferences regarding gene expression and disease state. To address this problem, we have developed RESOURCERER, a microarray-resource annotation and cross-reference database built using the analysis of expressed sequence tags (ESTs) and gene sequences provided by the TIGR Gene Index (TGI) and TIGR Orthologous Gene Alignment (TOGA) databases [now called Eukaryotic Gene Orthologs (EGO)].
Complementary DNA microarrays containing 3000 different rat genes were used to study the consequences of severe hormonal deficiency (hypophysectomy) on the gene expression patterns in heart, liver, and kidney. Hybridization signals were seen from a majority of the arrayed complementary DNAs; nonetheless, tissue-specific expression patterns could be delineated. Hypophysectomy affected the expression of genes involved in a variety of cellular functions. Between 16-29% of the detected transcripts from each tissue changed expression level as a reaction to this condition. Chronic treatment of hypophysectomized animals with human GH also caused significant changes in gene expression patterns. The study confirms previous knowledge concerning certain gene expression changes in the above-mentioned situations and provides new information regarding hypophysectomy and chronic human GH effects in the rat. Furthermore, we have identified several new genes that respond to GH treatment. Our results represent a first step toward a more global understanding of gene expression changes in states of hormonal deficiency.
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A wealth of molecular resources have been developed for rice genomics, including dense genetic maps, expressed sequence tags (ESTs), yeast artificial chromosome maps, bacterial artificial chromosome (BAC) libraries and BAC end sequence databases. Integration of genetic and physical maps involves labor-intensive empirical experiments. To accelerate the integration of the bacterial clone resources with the genetic map for the International Rice Genome Sequencing Project, we cleaned and filtered the available EST and BAC end sequences for repetitive sequences and then searched all available rice genetic markers with our filtered databases. We identified 418 genetic markers that aligned with at least one BAC end sequence with >95% sequence identity, providing a set of large insert clones with an average separation of 1 Mb that can serve as nucleation points for the sequencing phase of the International Rice Genome Sequencing Project.
The vast body of Expressed Sequence Tag (EST) data in the public databases provide an important resource for comparative and functional genomics studies and an invaluable tool for the annotation of genomic sequences. We have developed a rigorous protocol for reconstructing the sequences of transcribed genes from EST and gene sequence fragments. A key element in developing this protocol has been the evaluation of a number of sequence assembly programs to determine which most faithfully reproduce transcript sequences from EST data. The TIGR Gene Indices constructed using this protocol for human, mouse, rat and a variety of other plant and animal models have demonstrated their utility in a variety of applications and are freely available to the scientific research community.
The haploid nuclear genome of the African trypanosome, Trypanosoma brucei, is about 35 Mb and varies in size among different trypanosome isolates by as much as 25%. The nuclear DNA of this diploid organism is distributed among three size classes of chromosomes: the megabase chromosomes of which there are at least 11 pairs ranging from 1 Mb to more than 6 Mb (numbered I-XI from smallest to largest); several intermediate chromosomes of 200-900 kb and uncertain ploidy; and about 100 linear minichromosomes of 50-150 kb. Size differences of as much as four-fold can occur, both between the two homologues of a megabase chromosome pair in a specific trypanosome isolate and among chromosome pairs in different isolates. The genomic DNA sequences determined to date indicated that about 50% of the genome is coding sequence. The chromosomal telomeres possess TTAGGG repeats and many, if not all, of the telomeres of the megabase and intermediate chromosomes are linked to expression sites for genes encoding variant surface glycoproteins (VSGs). The minichromosomes serve as repositories for VSG genes since some but not all of their telomeres are linked to unexpressed VSG genes. A gene discovery program, based on sequencing the ends of cloned genomic DNA fragments, has generated more than 20 Mb of discontinuous single-pass genomic sequence data during the past year, and the complete sequences of chromosomes I and II (about 1 Mb each) in T. brucei GUTat 10.1 are currently being determined. It is anticipated that the entire genomic sequence of this organism will be known in a few years. Analysis of a test microarray of 400 cDNAs and small random genomic DNA fragments probed with RNAs from two developmental stages of T. brucei demonstrates that the microarray technology can be used to identify batteries of genes differentially expressed during the various life cycle stages of this parasite.
Expressed sequence tags (ESTs) have provided a first glimpse of the collection of transcribed sequences in a variety of organisms. However, a careful analysis of this sequence data can provide significant additional functional, structural and evolutionary information. Our analysis of the public EST sequences, available through the TIGR Gene Indices (TGI; http://www.tigr.org/tdb/tdb.html ), is an attempt to identify the genes represented by that data and to provide additional information regarding those genes. Gene Indices are constructed for selected organisms by first clustering, then assembling EST and annotated gene sequences from GenBank. This process produces a set of unique, high-fidelity virtual transcripts, or tentative consensus (TC) sequences. The TC sequences can be used to provide putative genes with functional annotation, to link the transcripts to mapping and genomic sequence data, and to provide links between orthologous and paralogous genes.
Although sequencing of the human genome will soon be completed, gene identification and annotation remains a challenge. Early estimates suggested that there might be 60,000-100,000 (ref. 1) human genes, but recent analyses of the available data from EST sequencing projects have estimated as few as 45,000 (ref. 2) or as many as 140, 000 (ref. 3) distinct genes. The Chromosome 22 Sequencing Consortium estimated a minimum of 45,000 genes based on their annotation of the complete chromosome, although their data suggests there may be additional genes. The nearly 2,000,000 human ESTs in dbEST provide an important resource for gene identification and genome annotation, but these single-pass sequences must be carefully analysed to remove contaminating sequences, including those from genomic DNA, spurious transcription, and vector and bacterial sequences. We have developed a highly refined and rigorously tested protocol for cleaning, clustering and assembling EST sequences to produce high-fidelity consensus sequences for the represented genes (F.L. et al., manuscript submitted) and used this to create the TIGR Gene Indices-databases of expressed genes for human, mouse, rat and other species (http://www.tigr.org/tdb/tgi.html). Using highly refined and tested algorithms for EST analysis, we have arrived at two independent estimates indicating the human genome contains approximately 120,000 genes.
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Microarray expression analysis has become one of the most widely used functional genomics tools. Efficient application of this technique requires the development of robust and reproducible protocols. We have optimized all aspects of the process, including PCR amplification of target cDNA clones, microarray printing, probe labeling and hybridization, and have developed strategies for data normalization and analysis.
The genome of the bacterium Borrelia burgdorferi B31, the aetiologic agent of Lyme disease, contains a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs. The chromosome contains 853 genes encoding a basic set of proteins for DNA replication, transcription, translation, solute transport and energy metabolism, but, like Mycoplasma genitalium, it contains no genes for cellular biosynthetic reactions. Because B. burgdorferi and M. genitalium are distantly related eubacteria, we suggest that their limited metabolic capacities reflect convergent evolution by gene loss from more metabolically competent progenitors. Of 430 genes on 11 plasmids, most have no known biological function; 39% of plasmid genes are paralogues that form 47 gene families. The biological significance of the multiple plasmid-encoded genes is not clear, although they may be involved in antigenic variation or immune evasion.
Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.
Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.
In earlier studies, we provided statistical evidence that individual differences in the angiotensinogen gene, the precursor of the vasoactive hormone angiotensin II, constitute inherited predispositions to essential hypertension in humans. We have now identified a common variant in the proximal promoter, the presence of an adenine, instead of a guanine, 6 bp upstream from the initiation site of transcription, in significant association with the disorder. Tests of promoter activity and DNA binding studies with nuclear proteins suggest that this nucleotide substitution affects the basal transcription rate of the gene. These observations provide some biological insight about the possible mechanism of a genetic predisposition to essential hypertension; they may also have important evolutionary implications.