High speed DNA sequencing by capillary electrophoresis.

A major challenge of the Human Genome Initiative is the development of a rapid, accurate, and efficient DNA sequencing technology. A major limitation of current technology is the relatively long time required to perform the gel electrophoretic separations of DNA fragments produced in the sequencing reactions. We demonstrate here that it is possible to increase the speed of sequence analysis by over an order of magnitude by performing the electrophoresis and detection in ultra thin capillary gels. An instrument which utilizes these high speed separations to simultaneously analyze many samples will constitute a second generation automated DNA sequencer suitable for large-scale sequence analysis.     

Molecular studies of human genetic disease.

A wide variety of techniques are available for detecting disease-causing mutations within human genes; this report provides a brief review of such procedures. Good communication and exchange of materials between the clinical genetics field and the Human Genome Initiative will benefit both.     

Sequence errors described in GenBank: a means to determine the accuracy of DNA sequence interpretation.

The accuracy of nucleic acid sequence data interpretation was determined by assessing and quantifying the discrepancies reported in the GenBank database. This permitted the calculation of an Error Rate (ER) for nucleic acid sequence determination. If one assumes that most entries (TB, Total Bases) were independently verified or those without reported discrepancies were correct, the ER is 0.368 errors per 1000 bases. However, if one assumes that only those sequences with reported discrepancies (TBIQ, Total Bases from entries In Question) were verified and are thus correct, the ER is 2.887 errors per 1000 bases. This establishes the first set of limit boundaries of the ER for sequence interpretation and sequence errors within the GenBank database and provides the foundation for future assessments and the monitoring of sequence data accumulation. In addition, the ER measure provides a basis to evaluate the efficiency and merit of present and future automated nucleic acid sequencing technologies which will have a direct impact upon the final outcome of the "Human Genome Initiative".     

Molecular and genetic characterization and physical mapping of 11 new markers detecting multiallele restriction fragment length polymorphisms on the short arm of human chromosome 3

Summary Genetic markers with high degrees of polymorphisms are of vital importance in the construction of high resolution (2–4 cM) linkage maps of human chromosomes as specified in the short-term goals of the Human Genome Initiative. In this paper, we report on molecular and genetic characterization and physical localization of 11 new multiallele restriction fragment length polymorphism markers on human chromosome 3p. Ten of these represent three- and four-allele polymorphisms of the base substitution type probably at two adjacent restriction sites. One has been identified as a novel minisatellite sequence comprising a variable copy number tandem repeat array of a G/T-rich 79-bp sequence. This collection of multiallele polymorphic (PIC values: 0.40–0.60) markers should prove valuable and increase the resolution power of the available chromosome 3p genetic markers.     

Large-scale sequencing of plant genomes

The large number of ESTs generated for Arabidopsis and rice in recent years now act as an important complement to whole genome sequencing projects. The Arabidopsis Genome Initiative has begun a coordinated effort to sequence the entire genome and, as a result, increasing numbers of large sequence entries can be found in the public databases. In addition, the mitochondrial genome of Arabidopsis has been completely sequenced. Genome sequencing studies and the public sequence databases have begun to influence the direction of diverse areas of research from physiology to evolution.     

A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni

To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (http://biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 x coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.     

Globalizing Genomics: The Origins of the International Nucleotide Sequence Database Collaboration

Genomics is increasingly considered a global enterprise – the fact that biological information can flow rapidly around the planet is taken to be important to what genomics is and what it can achieve. However, the large-scale international circulation of nucleotide sequence information did not begin with the Human Genome Project. Efforts to formalize and institutionalize the circulation of sequence information emerged concurrently with the development of centralized facilities for collecting that information. That is, the very first databases build for collecting and sharing DNA sequence information were, from their outset, international collaborative enterprises. This paper describes the origins of the International Nucleotide Sequence Database Collaboration between GenBank in the United States, the European Molecular Biology Laboratory Databank, and the DNA Database of Japan. The technical and social groundwork for the international exchange of nucleotide sequences created the conditions of possibility for imagining nucleotide sequences (and subsequently genomes) as a “global” objects. The “transnationalism” of nucleotide sequence was critical to their ontology – what DNA sequences came to be during the Human Genome Project was deeply influenced by international exchange.     

Positional cloning in Arabidopsis. Why it feels good to have a genome initiative working for you

Positional (or map-based) cloning techniques are widely used to identify the protein products of genes defined by mutation. In Arabidopsis the information generated by the Genome Initiative is giving this approach a decisive boost. A wealth of sequence polymorphisms and molecular markers is now available and can be exploited for fine mapping with technically simple and robust polymerase chain reaction-based methods. As a result it has become possible to complete positional cloning projects in a short time and with relatively little effort.     

一种高效的功能基因分离解析法

人类基因组计划（Human Genome Project）的实施揭开了各种生物基因组解析的序幕［1~3］。随着各种生物的基因组解析的顺利进行,遗传基因的功能研究以及寻找新的功能基因变得越来越重要。本文介绍的MegacloneTM技术、MegasortTM技术［4］以及MPSS技术［5］可以高效地分离解析各种功能基因。 Abstract:The implementation of the Human Genome Project preludes the analyzing of biologic genomes［1~3］.Following the successful analysis of diverse biologic genomes,it becomes more and more important to research the functions of genes and to find new functional genes.In this article,we use the techniques of MegacloneTM,MegasortTM［4］ and MPSS［5］ to sort and sequence effectively different functional genes.     

Genome-wide characterization of tetrahymena thermophila chromosome breakage sites. I. Cloning and identification of functional sites

The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific and directed by a conserved 15-bp chromosome breakage sequence (Cbs element). This article reports the construction of a library enriched for chromosome breakage junctions and the development of a successful scheme for the genome-wide isolation and characterization of functional Cbs junctions. Twenty-three new Cbs junctions were characterized and each was assigned to a specific micronuclear chromosome or chromosome arm. Two distinct previously unreported variant chromosome breakage sequences were found, each in two or more functional Cbs elements. Analysis of natural Cbs junctions confirmed that microheterogeneity in the macronuclear telomere addition site is associated with chromosome fragmentation. The physical and genetic characterization of these functional chromosome breakage junctions is reported in the accompanying article in this issue. The whole-genome shotgun sequencing and auto-assembly phase of the Tetrahymena Genome Initiative has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from the natural ends of macronuclear chromosomes, Cbs junctions characterized in the work reported here will serve as useful sequence tags for relating macro- and micronuclear genetic, physical, and sequence maps.     

Plagiarized bacterial genes in the human book of life

The initial analysis of the human genome draft sequence reveals that our 'book of life' is multi-authored. A small but significant proportion of our genes owes their heritage not to antecedent eukaryotes but instead to bacteria. The publicly funded Human Genome Project study indicates that about 0.5% of all human genes were copied into the genome from bacterial sources. Detailed sequence analyses point to these 'horizontal gene transfer' events having occurred relatively recently. So how did the human 'book of life' evolve to be a chimaera, part animal and part bacterium? And what was the probable evolutionary impact of such gene plagiarism?     

Societal and Medical Consequences of the Human Genome Project

The Human Genome Project is a US-based molecular biological project, the results of which are likely to be implemented on humans. The sociopolitical dimension of this is highly neglected. The aim of the conference was to fill this gap by drawing together scientists of natural and political sciences to discuss the consequences of the Human Genome Project across the disciplines.     

人类变异组计划及其进展

基因组学构建了人类的基因组图谱,后基因组时代的主要任务是解释基因组如何影响生命活动,由此产生了各种新类型的组学:结构基因组学,功能基因组学,蛋白质组学,代谢组学等。人类基因组突变学会于2006年6月在澳大利亚的墨尔本会议上正式启动了人类变异组计划。该计划旨在全球范围内广泛收集所有基因和蛋白质序列变异和多态性的数据,采用全基因组级别的基因型与表型关联等方法,系统地搜索并确定与人类疾病相关的变异,以指导临床应用。鉴于该计划对人类健康领域将产生的潜在影响,文章较为全面地介绍了该计划的起源和主要内容,并对其意义和前景进行了讨论。     

Mammalian cAMP-dependent protein kinase functionally replaces its homolog in yeast

The cDNA encoding the catalytic subunit (C alpha) from mouse cAMP-dependent protein kinase (PK) was expressed in Saccharomyces cerevisiae. By a plasmid swap procedure, we demonstrated that the mammalian C alpha subunit can functionally replace its yeast homolog to maintain the viability of a yeast strain containing genetic disruptions of the three TPK genes encoding the yeast C subunits. C alpha subunit produced in yeast was purified and its biochemical properties were determined. The protein isolated from yeast appears to be myristylated, as has been found for C subunits from higher eukaryotic cells. This system would be useful for studying the biochemistry of the mammalian enzyme in vitro and its biological role in a model in vivo system. These studies demonstrate that the PK substrate(s) required for viability are recognized by the mammalian enzyme. In general terms, these results demonstrate that heterologous proteins with only 50% sequence conservation with their yeast counterparts can be functional in yeast. This is an important result because it validates the use of yeast to identify the biological role of newly cloned genes from heterologous systems, a key tenet of the Human Genome Initiative.     

Isochores Merit the Prefix 'Iso'

The isochore concept in human genome sequence was challenged in an analysis by the International Human Genome Sequencing Consortium (IHGSC). We argue here that a statement in IGHSC analysis concerning the existence of isochore is incorrect, because it had applied an inappropriate statistical test. To test the existence of isochores should be equivalent to a test of homogeneity of windowed GC%. The statistical test applied in the IHGSC's analysis, the binomial test, is however a test of a sequence being random on the base level. For testing the existence of isochore, or homogeneity in GC%, we propose to use another statistical test: the analysis of variance (ANOVA). It can be shown that DNA sequences that are rejected by binomial test may not be rejected by the ANOVA test.     

Perspectives on functional genomics

As the first assembly of the human genome was announced on June 26, 2000, we have entered post genome era. The genome sequence represents a new starting point for science and medicine with possible impact on research across the life sciences. In this review I tried to offer brief summaries of history and progress of the Human Genome Project and two major challenges ahead, functional genomics and DNA sequence variation research.     

Database resources of the National Center for Biotechnology Information

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources that operate on the data in GenBank and a variety of other biological data made available through NCBI's Web site. NCBI data retrieval resources include Entrez, PubMed, LocusLink and the Taxonomy Browser. Data analysis resources include BLAST, Electronic PCR, OrfFinder, RefSeq, UniGene, HomoloGene, Database of Single Nucleotide Polymorphisms (dbSNP), Human Genome Sequencing, Human MapViewer, GeneMap'99, Human-Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, Cancer Genome Anatomy Project (CGAP), SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheri-tance in Man (OMIM), the Molecular Modeling Database (MMDB) and the Conserved Domain Database (CDD). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih. gov.     

Full-speed mammalian genetics: in vivo target validation in the drug discovery process.

The completion of the Human Genome Project has signaled the beginning of the post-genome era, with a corresponding shift in focus from the sequencing and identification of genes to the exploration of gene function. A rate-limiting step in deriving value from this gene sequence information is determining the potential pharmaceutical applications of genes and their encoded proteins. This validation step is crucial for focusing efforts and resources on only the most promising targets. Strategies using reverse mouse genetics provide excellent methods for validating potential targets and therapeutic proteins in vivo in a mammalian model system.     

The Human Genome Project: a paradigm for information management in the life sciences

The major product of the Human Genome Project will be a series of linked data sets containing the genetic and physical location of all genes on each chromosome, plus the complete nucleotide sequence of the genome for humans and several model organisms. Here we summarize the current status of attempts to collect, analyze, and distribute this information in an electronically accessible form. Although formidable problems remain to be solved in the acquisition and adequate representation of the genetic, physical, and biological data, this project is a model for the rapid dissemination of genome and related information in biology and medicine.