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HTML
766 lines
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HTML
<!doctype html public "-//IETF//DTD HTML//EN">
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<html>
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<head>
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<title>Spring 1999</title>
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<meta name="GENERATOR" content="Microsoft FrontPage 3.0">
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<meta name="AUTHOR" content="RJohnson">
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</head>
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<body bgcolor="#FFFFFF" text="#000000" vlink="#0000FF" alink="#0000FF">
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<p align="left"><img src="newslogo.gif"> </p>
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<p> </p>
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<hr style="margin-top: -2in; margin-bottom: -2in; padding-top: ; padding-bottom:">
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<a name="toc">
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<p>Spring 1999</a></p>
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<hr style="margin-top: -3in; margin-bottom: ; padding-top: -9 in; padding-bottom: 0">
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<h3>In This Issue</h3>
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<p><a href="#New Human Genome Web Resource: A Nexus for Genome Data">New Human Genome Web
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Resource: A Nexus for Genome Data</a><br>
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<a href="#DNA Sequences">DNA Sequences from Times Past in GenBank</a><br>
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<a href="#Sequin 2.90">Sequin 2.90 Offers Simplified Network Access</a><br>
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<a href="#SAGEmap">SAGEmap Offers a Versatile Interface to Gene Expression Data</a><br>
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<a href="#Profile">Profile: PSI-BLAST's Impact is "High Profile"</a><br>
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<a href="#Lab">BLAST Lab</a><br>
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<a href="#EST Submissions">Mandatory Protocol for EST Submissions to Take Effect Soon</a><br>
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<a href="#FAQ">Frequently Asked Questions</a><br>
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<a href="#Recent Pubs">Selected Recent Publications by NCBI Staff</a><br>
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<a href="#Masthead">Masthead</a></p>
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<hr>
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<h3><a name="New Human Genome Web Resource: A Nexus for Genome Data"><font SIZE="5">New
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Human Genome Web Resource: A Nexus for Genome Data</font></a></h3>
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<p><font SIZE="5">C</font><font SIZE="3">ompletion of the sequencing and analysis of the
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human genome promises to be a complex task that will involve cooperation among researchers
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applying diverse tools to the problem. The data generated will be reported in a variety of
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forms reflecting this diverse set of tools. Genetic and physical maps, markers, nucleotide
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polymorphisms, disease phenotypes, expression profiles, and sequence data must be
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integrated and made accessible for analysis. A repository of sequence data represents a
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natural site for construction of a nexus into which data in many forms can flow and from
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which these data can be accessed. NCBI’s <a
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href="http://www.ncbi.nlm.nih.gov/genome/guide/">Human Genome Resources</a> page, designed
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to serve as such a nexus, is closely connected to the GenBank sequence database and also
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provides centralized access to a full range of human genome resources available within
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NCBI and elsewhere. </p>
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<p ALIGN="left">From the NCBI home page, the Human Genome Resources link leads to an
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organized set of links to human genome data in many forms. A screen clip of the upper half
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of the page is shown below. The first collection of links, called The Genome at a Glance,
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is an array of 24 chromosome ideograms serving as links to GeneMap ’98 (GeneMap
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’99 coming soon). Clicking on one of these chromosomes leads to radiation hybrid (RH)
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mapping data as well as information on gene distributions and gene-disease associations. </p>
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<p ALIGN="left"><img src="hgen3_html.jpg" alt="hgen3_html.jpg (54557 bytes)" border="1" width="660"
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height="705"><br>
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<small><em><font face="Helvetica">Partial display of recently introduced Human Genome
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Resources page on NCBI's Web site.</font></em></small></p>
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<p ALIGN="JUSTIFY"> </p>
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<p align="left"><strong>A Search Box for LocusLink</strong></p>
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<p ALIGN="left">A query box spanning the top of the page can be used to conduct a text
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search of any of six major NCBI resources. The default target is <a
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href="http://www.ncbi.nlm.nih.gov/LocusLink">LocusLink</a>, a new nomenclature
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cross-referencing tool developed by Donna Maglott. LocusLink allows searches that begin
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with queries as diverse as official gene names, aliases, sequence accession numbers,
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protein names, phenotypes, EC numbers, MIM numbers, other database identifiers, UniGene
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clusters, or mapping information to converge upon the same data. The target database can
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be changed from LocusLink to MEDLINE, OMIM, GenBank, GeneMap ’98, or UniGene.</p>
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<p align="left"><strong>A Column of Links to the Left</strong></p>
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<p ALIGN="left">A blue column along the left of the page contains an array of links to a
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variety of human genome data. A link to OMIM (Online Mendelian Inheritance in Man)
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provides access to over 10,000 descriptions of genetic diseases and genes, stressing
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genotype-phenotype correlations. A pointer to GeneMap ’98 provides access to mapping
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data on over 35,000 human genes. Gateways to UniGene, dbEST, and the Davis Human/Mouse
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Homology Map follow. There are also links for dbSNP, leading to a database of single
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nucleotide polymorphisms, and Mutation DBs, pointing to 27 disease-specific databases.</p>
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<p ALIGN="left">Of particular interest are links to Human Genome Sequencing, Reference
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mRNA Sequences, and SAGEmap. The first two of these resources are described below.
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The third, SAGEmap, is a new NCBI database of quantitative gene expression and is the
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subject of a separate <a href="#SAGEmap">article</a> in this issue of the <em>NCBI News.</em></p>
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<p align="left"><strong>A Look at the Genome Sequencing Page</strong></p>
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<p ALIGN="left">The Genome Sequencing site, developed by Greg Schuler, is an important new
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resource supporting the human sequencing effort. From the Human Genome Resources page,
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follow the <a
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href="http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsHome.html&ORG=Hs">Human
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Genome Sequencing</a> link to reach a colorful graphical display of genome sequencing
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progress. Finished sequence is indicated by hot red and orange bands on a set of
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chromosome ideograms. These data are also available in numerical form. A link labeled More
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Statistics leads to a table giving sequencing progress by chromosome. The table is
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extended with more links to the individual contigs involved. A query box at the head of
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the page provides access to a database of contigs plus six other NCBI databases. Down the
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left side of the page is a column of links pointing to genome sequencing centers, a contig
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browser, chromosome-specific BLAST searches, and information on downloading sequences.</p>
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<p align="left"><strong>A Quick Look at RefSeq</strong></p>
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<p ALIGN="left">RefSeq, a project developed by Kim Pruitt that provides reference
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sequences for chromosomes, mRNAs, and proteins, is reached via the <a
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href="http://www.ncbi.nlm.nih.gov/LocusLink/refseq.html">Reference mRNA Sequences</a>
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link. RefSeq standards provide a foundation for functional annotation of the genome. They
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provide a stable reference point for mutation analysis, studies of gene expression, and
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polymorphism discovery. In addition, RefSeq-to-LocusLink associations anchor UniGene
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clusters and support annotation of genomic contig sequence data generated by the Human
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Genome Project. RefSeq records are available through BLAST, Entrez, and LocusLink.</p>
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<p align="left"><strong>More Links to the Right</strong></p>
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<p ALIGN="left">The right side of the Human Genome Resources page is also lined with many
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links. The first is a pointer to the NCBI Genes and Disease page, which gives synopses of
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over 60 diseases of genetic origin and provides links to the literature and sequence
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databases. Below this is a block of links to resources of the National Human Genome
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Research Institute (NHGRI), including its home page, the Human Genome Project page, and a
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glossary of genetic terms.</p>
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<p ALIGN="left">A National Cancer Institute (NCI) block offers gateways to cGAP, cCAP,
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CancerNet, and the NCI home page. The path to cGAP allows access to gene expression data
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for normal, precancerous, and cancerous cells derived from the 121 sequence libraries of
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the Cancer Genome Anatomy Project. The database currently contains expression data for
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over 20,000 human genes. For data on physical chromosomal defects associated with cancer,
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follow the cCAP link to the Cancer Chromosomal Aberration Project.</p>
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<p align="left"><strong>News Items</strong></p>
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<p align="left">The Human Genome Resources page also features a central block of short
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newspaper-style announcements. Currently grouped under the headings What’s New and
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Human Genome Meetings, these items run the gamut from synopses of new human genome
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resources at NCBI to notices of symposia and workshops to recent news bulletins of
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relevance to the Human Genome Project. </p>
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<p><a href="#toc">Return to Table of Contents</a></p>
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<hr>
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<h3><font SIZE="5"><a name="DNA Sequences"><i>On the lighter side . . .<br>
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</i>DNA Sequences from Times Past in GenBank</a></font></h3>
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<p ALIGN="left"><font SIZE="5">A</font><font SIZE="3">lthough GenBank lacks dinosaur DNA,
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fragments of genomes past can be found here. A practical limit of about 100,000 years
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currently applies to the age of recoverable DNA samples. Beyond this limit, hydrolysis of
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the phosphate backbone of the DNA and oxidative damage to the bases that make up the DNA
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sequence become too great to allow for efficient PCR amplification. This is why deposition
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of significant amounts of dinosaur sequence (age </font><font FACE="Symbol"><EFBFBD></font><font
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SIZE="3"> 65 million years) in GenBank is unlikely to occur in the near future. However,
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many DNA sequences arising from extinct organisms and ancient genomes are in the database
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today, and the number is expected to grow as technology for the extraction and
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amplification of aged DNA progresses. A selection of sequences from the past now available
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in GenBank is given below.</font><b><font FACE="Helvetica" SIZE="1"></p>
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</font></b>
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<table border="0" width="84%" cellpadding="2">
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<tr>
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<td width="100%" bgcolor="#C0C0C0"><font face="Helvetica" size="2"><b>AF011222:</b><i>
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Mitochondrial DNA from a Neanderthal specimen discovered in 1856 near Dusseldorf, Germany.
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Source: Bone (3.5 g of right humerus). Age of Source: 30,000 to 100,000 years. Length: 379
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bp.</i><b><p>S69989: </b><i>Mitochondrial DNA from the Late Neolithic "Iceman"
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found mummified in the Tyrolean Alps. Source: Soft tissue. Age of Source: 5,000 years.
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Length: 354 bp.</i><b></p>
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<p>X73306:</b><i> Mitochondrial DNA from Egyptian mummy. Source: Tarsus bone. Age of
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Source: 2,000 years. Length: 122 bp.</i><b></p>
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<p>K02137: </b><i>Alu-repeat family DNA sequence from Egyptian mummy. Source: Mummified
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soft tissue. Age of Source: 2,400 years. Length: 919 bp.</i><b></p>
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<p>X88771:</b><i> Ribosomal RNA gene from an Iceman fungal clone. Source: Grass clothing
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of the Iceman (cloak, boots). Age of Source: 5,000 years. Length: 495 bp. </i></p>
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<p><strong>Z48945:</strong> <i>Ribosomal RNA gene from an extinct giant ground sloth.
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Source: Bone, teeth, and coprolites. Age of Source: 13,000 years. Length: 571 bp.</i><b></p>
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<p>L08480:</b> <i>Mitochondrial DNA from an extinct legume. Source: Leaf embedded in
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Dominican amber. Length: 348 bp.</i><b></p>
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<p>D50842:</b><i> Mitochondrial DNA from an extinct woolly mammoth (the baby Magadan
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mammoth known as Dima). Source: 1 g of muscle. Age of Source: 40,000 years. Length: 1,137
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bp.</i><b></p>
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<p>S72502: </b><i>Mitochondrial DNA from the extinct Siberian woolly mammoth. Source:
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Humerus cortical bone. Length: 242 bp.</i><b></p>
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<p>D83049: </b><i>Mitochondrial DNA from the extinct Stellar’s sea cow, a relative of
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the manatee. Source: Bone (2 g of bone from a scapula collected on Bering Island,
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Kamchatka). Length: 1,005 bp.</i><b></p>
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<p>X64307:</b><i> Mitochondrial DNA from a quagga (an extinct relative of the zebra from
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southern Africa). Length: 117 bp.</i><b></p>
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<p>S46659:</b><i> Mitochondrial ribosomal RNA from </i>Smilodon fatalis,<i> the
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saber-toothed tiger found in the Rancho La Brea Tar Pits in Los Angeles, California.
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Source: Bone from three specimens. Age of Source: 14,000 years. Length: 132 bp.</i><b></p>
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<p>X67636:</b><i> Mitochondrial ribosomal RNA of the Moa, an extinct bird. Source: Bones
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and soft tissues of four species of Moa. Length: 386 bp.</i><b></p>
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<p>S78028: </b><i>Mitochondrial DNA from Medieval French rabbits. Source: 1 to 4 g of
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bone. Age of Source: 400 to 600 years. Length: 233 bp.</i></font></td>
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</tr>
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</table>
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<p><a href="#toc">Return to Table of Contents</a></p>
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<hr>
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<h3><a name="Sequin 2.90"><font SIZE="5">Sequin 2.90 Offers Simplified Network Access </font></a></h3>
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<p align="left"><font SIZE="5">S</font>equin can function in either a stand-alone mode or
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a “network- aware” mode. The stand-alone mode provides the functions needed to
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prepare most sequence submissions. In its network-aware mode, however, Sequin acquires
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additional functionality through online access to GenBank and NCBI’s sequence
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analysis tools. For instance, network-aware Sequin can download sequences from GenBank in
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order to facilitate submitting multiple sequence alignments that include existing GenBank
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sequences. Network-aware Sequin can also conduct PowerBLAST searches, perform Entrez
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queries, and screen for the presence of contaminating vector sequences or repeat elements
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within a sequence submission. Setting up Sequin to communicate over the network has been
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simplified in version 2.90, which is now available at <a
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href="ftp://ncbi.nlm.nih.gov/sequin/">ftp://ncbi.nlm.nih.gov/sequin/</a>. </p>
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<p align="left">Sequin’s <strong>Network Configuration</strong> option, available on
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the initial Welcome to Sequin page as well as in the record viewer, is located under the
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Misc menu. To configure Sequin to use the network, most users need only select the <strong>Normal</strong>
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connection and click on <strong>Accept</strong> to begin the configuration. Users who are
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behind a firewall may need to contact their system administrator in order to fill in the
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Proxy and Port fields. Users outside the United States or with an unreliable Internet
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connection may need to increase the “timeout” value, which is the length of time
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Sequin will wait for a response from the network. Sequin must be restarted in order for
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the network configuration changes to take effect. </p>
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<p><a href="#toc">Return to Table of Contents</a></p>
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<hr>
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<h3 align="left"><a name="SAGEmap"><font SIZE="5">SAGEmap Offers a Versatile Interface to
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Gene Expression Data</font></a> </h3>
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<p ALIGN="left"><font SIZE="5">S</font><font SIZE="3">erial Analysis of Gene Expression
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(SAGE) refers to a technique for taking a snapshot of the messenger RNA population of a
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cell. The elements of the film, in this case, are oligonucleotides, each consisting of a
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concatenated array of short, 20- to 24-bp sequence tag pairs, or ditags. Each 10- to 12-bp
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tag of the ditag pair represents one parent messenger RNA. Many of these concatenated
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arrays are combined within a SAGE "library." The number of times a particular
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tag is detected in a library gives a digital measure of the abundance of its associated
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mRNA and, hence, provides a quantitative measure of gene expression. By using the SAGE
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technique, coupled with high-throughput sequencing technology, it is possible to obtain
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accurate expression data for thousands of genes within a cell. A major application of SAGE
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is in the identification of abnormal gene expression leading to, or diagnostic of, various
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disease states, such as cancer. NCBI’s <a href="http://www.ncbi.nlm.nih.gov/SAGE/">SAGEmap</a>
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site, developed by Alex Lash and introduced in March 1999, implements many functions
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useful in the analysis of SAGE data.</p>
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<b>
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<p align="left">Mapping Tools</b></p>
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<p align="left">SAGEmap provides tag-to-gene as well as gene-to-tag mappings. Both
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mappings are updated weekly, immediately following the updating of UniGene.</p>
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<p ALIGN="left">The tag-to-gene function maps a SAGE tag to one or more UniGene clusters.
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It also produces a table listing the SAGE libraries in which the gene tag occurs, the
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number of occurrences per library, and the total number of tags in the library. Following
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a link to a SAGE library leads to more information about the library and allows one to
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download the SAGE tag data. The full tag-to-gene mapping data for all the SAGE libraries,
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or a "reliably mapped" subset of these data, may also be downloaded as a single
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file.</p>
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<p ALIGN="left">The inverse function, gene-to-tag mapping, maps a UniGene cluster ID to
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the SAGE tags found within the cluster. For each SAGE tag found, library information is
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given. A link to the UniGene cluster used as the query leads to the UniGene page for this
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cluster.</p>
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<b>
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<p align="left">SAGE Data Analysis</b></p>
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<p ALIGN="left">SAGEmap can construct a user-configurable table of data comparing one
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group of SAGE libraries with another. Several CGAP SAGE libraries are currently available
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and may be included in the table. Libraries included in the table are assigned to one of
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two groups, A or B, between which a comparison can be made. For each group, the user
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specifies which tags within the designated libraries should be included in the table. A
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logical AND specifies that tags be included only if they are expressed in all libraries
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making up the group. A logical OR specifies that tags be included regardless of groupwide
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expression. Tags having wide intragroup variations in expression may be excluded from the
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comparison.</p>
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<p ALIGN="left">Tags may also be included in the table on the basis of intergroup
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expression differences. In this case, three schemes are available: OR, to include all
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tags; XOR, to include tags expressed in one group but not in the second group; AND, to
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include only tags expressed in both groups. A minimum difference threshold for use between
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groups may be defined. When the tabular display parameters are set, a click on the <b>Results
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</b>button displays the SAGE expression table.</p>
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<p ALIGN="left">For each SAGE tag, the expression table includes columns giving the
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associated UniGene cluster ID and the cluster description. A column summarizing the
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expression of each tag in groups A and B is colored red if the level is higher in group A
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and green if the level is higher in group B. The definition of "higher" depends
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on the difference threshold specified by the user.</p>
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<p align="left">SAGEmap may be reached from the NCBI home page, the Human Genome Resources
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page, or directly at <a href="http://www.ncbi.nlm.nih.gov/SAGE">http://www.ncbi.nlm.nih.gov/SAGE</a>.
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</font></p>
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<p ALIGN="left"><a href="#toc">Return to Table of Contents</a></p>
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<hr>
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||
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<h3><a name="EST Submissions"><font SIZE="5">Mandatory Protocol for EST Submissions to
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Take Effect Soon</font></a></h3>
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<p ALIGN="left"><font SIZE="5">T</font><font SIZE="3">o facilitate the submission of EST
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sequences, which are usually submitted in large batches, GenBank has offered a streamlined
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EST submission procedure using a specialized data format. Beginning May 31, 1999, the use
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of this specialized format will become mandatory for all EST submissions. EST submissions
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made with either BankIt or Sequin will no longer be accepted.</p>
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<p ALIGN="left">Expressed Sequence Tags (ESTs) are short (300 to 500 bp) single reads from
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cDNA complementary to mRNA, which are usually produced in large numbers. ESTs are useful
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in providing a snapshot of the mRNA population characteristic of a given tissue or of a
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given tissue at a particular developmental stage. EST sequences now represent
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approximately 70% of the sequences in GenBank and constitute the most rapidly growing
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GenBank division.</p>
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<p ALIGN="left">For instructions on using GenBank’s specialized EST submission
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format, see <a href="http://www.ncbi.nlm.nih.gov/dbEST/how_to_submit.html">http://www.ncbi.nlm.nih.gov/dbEST/how_to_submit.html</a>.
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</p>
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<p align="left">Completed EST submissions should be mailed to batch-sub@ncbi.nlm.nih.gov.
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After May 31, GenBank will no longer accept EST submissions at the gb-sub@ncbi.nlm.nih.gov
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address.</font></p>
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<p><a href="#toc">Return to Table of Contents</a></p>
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<hr>
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<table border="1" width="100%" bgcolor="#0080C0"
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style="border-left: medium none rgb(255,255,255); border-right: medium none; border-top: medium none; border-bottom: medium none"
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height="54">
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<tr>
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<td width="100%" height="48"><p align="center"><font SIZE="5"><font color="#FFFFFF"><a
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name="Profile"><strong>PROFILE</strong></a></font> </font></td>
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</tr>
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</table>
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|
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<h3 align="left"></font><a name="New Human Genome Web Resource: A Nexus for Genome Data"><font
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||
SIZE="5">PSI-BLAST's Impact Is "High Profile"</font></a><font SIZE="3"></h3>
|
||
|
||
<p align="left"><font SIZE="5">C</font><font SIZE="3">omparison, whether of morphological
|
||
features or protein sequences, lies at the heart of biology. The introduction of BLAST in
|
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1990 made it easier to rapidly scan huge sequence databases for overt homologies and
|
||
statistically evaluate the resulting matches. With more than 8,700 citations to date, the
|
||
paper describing the original algorithm</font><a href="#Altschul"><font face="Times"
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size="1"><sup>1</sup></font></a><font SIZE="3"> has since become the most heavily cited of
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the decade.</font><a href="#Russo"><sup><font SIZE="1">2</font></sup></a></p>
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<font SIZE="3">
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<p ALIGN="left">Not all significant homologies are overt, however. Some of the most
|
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interesting are subtle and do not rise to statistical significance during a standard BLAST
|
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search. NCBI’s Stephen Altschul has extended BLAST and its statistical methodology to
|
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address the problem of detecting weak but significant sequence similarities. With a small
|
||
group of other NCBI researchers, he has developed <a
|
||
href="http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-psi_blast">Position-Specific Iterated
|
||
BLAST</a> (PSI-BLAST), which searches sequence databases with a profile constructed from
|
||
BLAST alignments. </p>
|
||
|
||
<p ALIGN="left">As Altschul notes, "PSI-BLAST is by no means the first program for
|
||
searching sequence databases with protein profiles. However, it is the first program fully
|
||
to automate the construction of these profiles from the output of a standard database
|
||
search, and the first to apply fast heuristic search techniques to profile comparison. As
|
||
such, it has for the first time rendered the powerful profile-search methodology
|
||
accessible to the non-expert." </p>
|
||
|
||
<p ALIGN="left">Indeed, PSI-BLAST has proven to be popular. Among scientific articles
|
||
published in the past 2 years, the 1997 paper describing PSI-BLAST</font><a
|
||
href="#Altschul2"><font SIZE="1"><sup>3</sup></font></a><font SIZE="3"> has been the most
|
||
heavily cited for the fourth 2-month interval in a row, with over 700 citations to date,
|
||
according to statistics compiled by the Institute for Scientific Information. Due to this
|
||
strong citation record, PSI-BLAST and its development team were featured in the April 12,
|
||
1999, issue of <i>The Scientist</i>.</font><a href="#Russo"><sup><font SIZE="1">2</font></sup></a><font
|
||
SIZE="3"></p>
|
||
|
||
<p ALIGN="left">Just as users of PSI-BLAST are a diverse group, so are its developers,
|
||
with backgrounds spanning genetics, physics, and medicine.</p>
|
||
|
||
<p ALIGN="left">Stephen Altschul, Ph.D., leads the PSI-BLAST development team. He received
|
||
his Ph.D. in mathematics from MIT in 1987 and joined the Computational Biology Branch of
|
||
NCBI in 1989, shortly after its creation. His research continues to center on measures,
|
||
algorithms, and statistics for the comparison of DNA and protein sequences.</p>
|
||
|
||
<p ALIGN="left">Alejandro Sch<63>ffer, Ph.D., wrote most of the PSI-BLAST source code. His
|
||
current interests include development of software for genetics, including genetic linkage
|
||
analysis, sequence analysis, and modeling genetic changes in tumor progression.</p>
|
||
|
||
<p ALIGN="left">Tom Madden, Ph.D., is the main BLAST programmer at NCBI. He received a
|
||
doctorate in physics from the University of California at Santa Cruz. After performing
|
||
postdoctoral work in biophysics at Brandeis University, he joined NCBI in 1993, and has
|
||
since been involved in all BLAST development.</p>
|
||
|
||
<p ALIGN="left">David Lipman, M.D., has been a driving force behind improved database
|
||
searches for more than a decade. In 1988, he and William Pearson authored a seminal paper
|
||
describing a predecessor to the BLAST search, the well-known FASTA search.</font><a
|
||
href="#Pearson"><font SIZE="1"><sup>4</sup></font></a><font SIZE="3"> Since his
|
||
appointment as Director of NCBI in 1989, he has continued to take a lead role in
|
||
development of BLAST and its specialized variants.</p>
|
||
|
||
<p ALIGN="left">A tutorial based on a recent PSI-BLAST introductory article has been
|
||
prepared by Altschul.</font><a href="#Altschul3"><font SIZE="1"><sup>5</sup></font></a><font
|
||
SIZE="3"> It can be accessed via the <a
|
||
href="http://www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html">BLAST Course</a><b> </b>link
|
||
on the main BLAST page. </p>
|
||
|
||
<p ALIGN="left"></font><img src="profile.jpg" alt="NCBI05.jpg (136288 bytes)" width="294"
|
||
height="320" vspace="5"><br>
|
||
<font size="2" face="Helvetica"><i>(clockwise from left) Tom Madden, David Lipman, <br>
|
||
Stephen Altschul, and Alejandro Sch<63>ffer.</i></font><font SIZE="3"></p>
|
||
<b>
|
||
|
||
<p align="left">Notes</b></font><font SIZE="2"></p>
|
||
|
||
<p ALIGN="left">1. <a name="Altschul">Altschul</a>, SF, et al. <a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=2231712&form=6&db=m&Dopt=b">Basic
|
||
local alignment search tool</a>. <i>J Mol Biol</i> 215(3):403–10, 1990.</p>
|
||
|
||
<p ALIGN="left">2. <a name="Russo">Russo</a>, E, and S Bunk, eds. Hot papers. <i>The
|
||
Scientist </i>13(8):15, 1999.</p>
|
||
|
||
<p>3. <a name="Altschul2">Altschul</a>, SF, et al. <a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9254694&form=6&db=m&Dopt=b">Gapped
|
||
BLAST and PSI-BLAST: A new generation of protein database search programs</a>. <i>Nucleic
|
||
Acids Res</i> 25(17):3389–402, 1997.</p>
|
||
|
||
<p ALIGN="left">4. <a name="Pearson">Pearson</a>, WR, and DJ Lipman. <a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=3162770&form=6&db=m&Dopt=b">Improved
|
||
tools for biological sequence comparison</a>. <i>Proc Natl Acad Sci USA</i>
|
||
85(8):2444–8, 1988.</p>
|
||
|
||
<p align="left">5. <a name="Altschul3">Altschul</a>, SF, and EV Koonin. <a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9852764&form=6&db=m&Dopt=b">Iterated
|
||
profile searches with PSI-BLAST—a tool for discovery in protein databases</a>. <i>Trends
|
||
Biochem Sci</i> 23(11):444–7, 1998. </font></p>
|
||
|
||
<p align="left"><a href="#toc">Return to Table of Contents</a></p>
|
||
|
||
<hr>
|
||
|
||
<p ALIGN="left"> <img src="blast_lab.gif" alt="blast_lab.gif (22614 bytes)"
|
||
width="700" height="61"></p>
|
||
|
||
<h3><a name="Lab"><font color="#000000">How to Write and Load PSI-BLAST Checkpoint Files
|
||
and Inspect the PSSMs</font></a></h3>
|
||
<b>
|
||
|
||
<p ALIGN="left"></b><font size="2" face="Helvetica"><a
|
||
href="http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-psi_blast">PSI-BLAST</a> uses the
|
||
pairwise alignments it creates between query and database sequences during a preliminary
|
||
BLAST search to construct a Position-Specific Scoring Matrix, or PSSM.<a href="#Altschul4"><sup>1</sup></a>
|
||
This PSSM is then used in place of the usual combination of query and scoring matrix
|
||
during the first PSI-BLAST iteration. The PSSM is updated at the beginning of each
|
||
subsequent PSI-BLAST iteration using the data from all significant alignments generated
|
||
during the previous iteration. Stand-alone PSI-BLAST can write a "checkpoint"
|
||
file at the end of a search; this file contains the information necessary to regenerate
|
||
the last PSSM used. PSI-BLAST can also load a previously stored checkpoint file to conduct
|
||
a search using a previously computed PSSM. Because the PSSM encapsulates information
|
||
derived from PSI-BLAST alignments, the inspection of this matrix may provide insights into
|
||
the sequence patterns present within the query. </p>
|
||
|
||
<p align="left">This BLAST Lab describes how to write and load checkpoint files using the
|
||
stand-alone version of PSI-BLAST. Instructions are also given for producing an ASCII
|
||
version of the PSI-BLAST PSSM.</font></p>
|
||
|
||
<hr color="#0080C0" noshade>
|
||
|
||
<p><font face="Helvetica" size="2" color="#0080C0"><strong>Installing Stand-alone
|
||
PSI-BLAST </strong></font></p>
|
||
|
||
<p>Packages for installing stand-alone PSI-BLAST on a variety of computer platforms are
|
||
found at <a href="ftp://ncbi.nlm.nih.gov/blast/executables/">ftp://ncbi.nlm.nih.gov/blast/executables/</a>. The PC self-extracting archive is
|
||
"blastz.exe." Unix packages are those ending in ".Z".</p>
|
||
<font SIZE="3">
|
||
|
||
<p>See BLAST Lab in the <a
|
||
href="http://www.ncbi.nlm.nih.gov/Web/Newsltr/Winter99/winter99.htm">Winter 1999 <i>NCBI
|
||
News</i></a> for details on archive extraction.</font></p>
|
||
|
||
<hr color="#0080C0" noshade>
|
||
|
||
<p><font face="Helvetica" size="2" color="#0080C0"><strong>Writing a Checkpoint File</strong></font></p>
|
||
|
||
<p>The command line below will run PSI-BLAST using the sequence query contained in a file
|
||
named "tf1.fsa". </p>
|
||
<font SIZE="3">
|
||
|
||
<p ALIGN="JUSTIFY">blastpgp -i tf1.fsa -o tf1.out -d ecoli -j 2 -C tf1.chk</p>
|
||
|
||
<p>The output will be written to "tf1.out". The local database to be searched is
|
||
named "ecoli". The switch "-j 2" instructs PSI-BLAST to run one
|
||
regular BLAST round followed by one PSI-BLAST iteration. The syntax "-C tf1.chk"
|
||
("C" stands for "checkpoint") specifies that a checkpoint file called
|
||
"tf1.chk" be saved after the search. This checkpoint file can be loaded later to
|
||
perform a search of a different database as described below.</font></p>
|
||
|
||
<hr color="#0080C0" noshade>
|
||
|
||
<p><font face="Helvetica" size="2" color="#0080C0"><strong>Loading a Checkpoint File</strong></font><font
|
||
SIZE="3"></p>
|
||
|
||
<p>PSI-BLAST can use a previously stored checkpoint file to reconstruct a PSSM fabricated
|
||
by PSI-BLAST during a previous search. This feature enables you to search two separate
|
||
databases with the same query and PSSM. In the example above, a checkpoint file,
|
||
"tft1.chk", was saved after a PSI-BLAST search of the local database
|
||
"ecoli" using the query contained in "tf1.fsa". A search of a second
|
||
local database, named "saureusi", can be conducted with the same query and PSSM
|
||
by using the command line below. </p>
|
||
|
||
<p>blastpgp -i tf1.fsa -o tf1.out -d saureusi -j 2 -R tf1.chk</p>
|
||
|
||
<p>In this case, the checkpoint file, "tf1.chk", is read using the
|
||
"-R" switch ("R" stands for "restart"). When using the
|
||
"-R" switch, the query sequence must be identical to that which was used to
|
||
generate the checkpoint file to be read.</font></p>
|
||
|
||
<hr color="#0080C0" noshade>
|
||
|
||
<p><font face="Helvetica" size="2" color="#0080C0"><strong>Inspecting a Stored PSSM</strong></font></p>
|
||
|
||
<p>ASCII versions of the PSI-BLAST PSSM may be written to a file using the new command
|
||
line switch "-Q" followed by the name to be given to the file. A syntax such as<br>
|
||
<br>
|
||
blastpgp -i tf1.fsa -j 3 -Q pssm.dat<br>
|
||
<br>
|
||
will save the PSSM used in the last PSI-BLAST iteration in an ASCII file named
|
||
"pssm.dat".</p>
|
||
<font SIZE="3">
|
||
|
||
<p ALIGN="JUSTIFY">The syntax below will read a checkpoint file called
|
||
"tf1.chk", run one iteration of PSI-BLAST, and save an ASCII version of the PSSM
|
||
used in the file "pssm.dat".<br>
|
||
<br>
|
||
blastpgp -i tf1.fsa -R tf1chk -Q pssm.dat</p>
|
||
|
||
<p ALIGN="JUSTIFY">To use the new "-Q" switch with the blastpgp program,
|
||
download the latest stand-alone BLAST distribution (version 2.0.9).</p>
|
||
<b>
|
||
|
||
<p>Note</b></font><font SIZE="2"></p>
|
||
|
||
<p>1. <a name="Altschul4">Altschul</a> SF, TL Madden, AA Sch<63>ffer, J Zhang, Z Zhang, W
|
||
Miller, and DJ Lipman. <a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9254694&form=6&db=m&Dopt=b">Gapped
|
||
BLAST and PSI-BLAST: A new generation of protein database search programs</a>.<i> Nucleic
|
||
Acids Res</i> 25(17):3389–402, 1997.</font> </p>
|
||
|
||
<table border="1" width="50%" style="border: 1px solid rgb(0,0,255)" bordercolor="#2154CB"
|
||
cellpadding="0" cellspacing="0" bgcolor="#CCACD2">
|
||
<tr>
|
||
<td width="100%" bgcolor="#BBD2FD" style="border-bottom: medium none"><img
|
||
src="blast_standard.jpg" alt="blast_standard.jpg (46679 bytes)" width="494" height="42"></td>
|
||
</tr>
|
||
<tr>
|
||
<td width="100%" bgcolor="#BBD2FD"
|
||
style="border-left: 1px solid rgb(0,0,255); border-right: 1px solid rgb(0,0,255); border-top: 1px none rgb(0,0,255); border-bottom: 1px solid rgb(0,0,255); margin-left: 0; margin-right: 0; padding-left: 11px; padding-right: 11px"><i>The
|
||
BLAST Lab feature is intended to provide detailed technical information on some of the
|
||
more specialized uses of the BLAST family of programs. Topics are selected from the range
|
||
of questions received by the BLAST Help Group.</i></td>
|
||
</tr>
|
||
</table>
|
||
|
||
<p><a href="#toc">Return to Table of Contents</a></p>
|
||
|
||
<hr>
|
||
|
||
<table border="0" width="100%" cellpadding="12">
|
||
<tr>
|
||
<td width="29%" style="border:none"><img src="qanda.gif" alt="qanda.gif (1368 bytes)"
|
||
hspace="17" vspace width="117" height="156"></td>
|
||
<td width="71%" style="border: medium none"></font> <p> </p>
|
||
<h2><a name="FAQ"><font color="#0080C0" size="5">Frequently Asked Questions</font></a></h2>
|
||
</td>
|
||
</tr>
|
||
<tr>
|
||
<td width="100%" style="border: medium none" colspan="2"><hr color="#0080C0" noshade
|
||
size="3">
|
||
</td>
|
||
</tr>
|
||
<tr>
|
||
<td width="29%" style="border: medium none"><font SIZE="3"><i>Is there any way to
|
||
determine the total number of organisms currently represented in GenBank?</i></font></td>
|
||
<td width="71%" style="border: medium none"><font SIZE="3">Yes, you can obtain a monthly
|
||
updated count of the number of organisms represented in GenBank at <a
|
||
href="http://www.ncbi.nlm.nih.gov/Taxonomy/Taxresources/taxaJan0197.html">http://www.ncbi.nlm.nih.gov/Taxonomy/Taxresources/taxaJan0197.html</a>.
|
||
This page presents a table giving the total GenBank species count for each year since
|
||
1995. The table also gives yearly subtotals for Viruses, Bacteria, Archaea, and Eukaryota.</font></td>
|
||
</tr>
|
||
<tr>
|
||
<td width="29%" style="border: medium none"><font SIZE="3"><i>How can I determine which
|
||
portions of my nucleic acid sequence will be considered to be of low complexity during a
|
||
filtered blastn search?</i></font></td>
|
||
<td width="71%" style="border: medium none"><font SIZE="3">Blastn uses a program called
|
||
DUST to filter nucleic acid sequences for low complexity. DUST is available as source code
|
||
and in the form of Unix binaries at <a
|
||
href="ftp://ncbi.nlm.nih.gov/pub/tatusov/dust/version1/">ftp://ncbi.nlm.nih.gov/pub/tatusov/dust/version1/</a>.
|
||
To see the results of filtering for a FASTA-formatted nucleic acid sequence in a file
|
||
called, for example, "nuc.fsa", execute DUST as follows: dust nuc.fsa.</font></td>
|
||
</tr>
|
||
<tr>
|
||
<td width="29%" style="vertical-align: text-top; border: medium none"><font SIZE="3"><i>I
|
||
am looking for GeneMap ’98, but I no longer see this as a link on the NCBI home page.
|
||
Does NCBI have a search engine I can use to find it?</i></font></td>
|
||
<td width="71%" style="border: medium none"><font SIZE="3">GeneMap ’98 is now
|
||
accessible via a link from the new Human Genome Resources page. You can also use
|
||
NCBI’s new search engine to find it by entering GeneMap into the search box and
|
||
pressing the Search button. A link directly to GeneMap ’98 will appear as the first
|
||
hit. The NCBI search engine is reached through a link on the NCBI home page.</font></td>
|
||
</tr>
|
||
<tr>
|
||
<td width="29%" style="vertical-align: text-top; border: medium none"><font SIZE="3"><i>I
|
||
am interested in an old GenBank record entry that does not show a CDS feature. How can I
|
||
determine where a coding region might be?</i></font></td>
|
||
<td width="71%" style="border: medium none"><font SIZE="3" COLOR="#000000">You can do this
|
||
easily by pasting the sequence into NCBI’s ORF Finder or by simply specifying a
|
||
GenBank accession or gi number. ORF Finder will search for Open Reading Frames over the
|
||
entire sequence or over a range of nucleotides within the sequence using any of 15 genetic
|
||
codes. A link to ORF Finder is found on the NCBI home page.</font></td>
|
||
</tr>
|
||
<tr>
|
||
<td width="29%" style="vertical-align: text-top; border: medium none"><font SIZE="3"><i>On
|
||
your FTP server, I have only seen files containing the entire EST division. Can I download
|
||
the three subdivisions of human, murine, and other ESTs separately?</i></font></td>
|
||
<td width="71%" style="border: medium none"><font SIZE="3">Yes, you can download the raw
|
||
sequence data in this way. The EST data sets are available separated by organism, but only
|
||
in FASTA format and not as full GenBank records. These FASTA sequence files are available
|
||
at <a href="ftp://ncbi.nlm.nih.gov/blast/db/">ftp://ncbi.nlm.nih.gov/blast/db/</a>. From
|
||
the NCBI home page, select <b>Anonymous FTP,</b> then <b>BLAST,</b> and then the <b>db </b>subdirectory.
|
||
You will see est_human.z, est_mouse.z, and est_others.z. </font></td>
|
||
</tr>
|
||
</table>
|
||
|
||
<p><a href="#toc">Return to Table of Contents</a></p>
|
||
|
||
<hr>
|
||
<font FACE="Times" SIZE="5">
|
||
|
||
<p ALIGN="left"></font><font SIZE="5"><a name="Recent Pubs">Selected Recent Publications
|
||
by NCBI Staff</a></font><b></p>
|
||
|
||
<p ALIGN="left">Aravind, L, DR Walker,</b> and <b>EV Koonin.</b> <a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9973609&form=6&db=m&Dopt=b">Conserved
|
||
domains in DNA repair proteins and evolution of repair systems</a>. <i>Nucleic Acids Res</i>
|
||
27(5):1223–42, 1999.</p>
|
||
<b>
|
||
|
||
<p ALIGN="left">Aravind, L,</b> VM Dixit, and <b>EV Koonin. </b><a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=10098397&form=6&db=m&Dopt=b">The
|
||
domains of death: evolution of the apoptosis machinery</a>. <i>Trends Biochem Sci</i>
|
||
24(2):47–53, 1999.</p>
|
||
|
||
<p ALIGN="left">Desper, R, F Jiang, OP Kallioniemi, H Moch, CH Papadimitriou, and <b>AA
|
||
Sch<EFBFBD>ffer. </b><a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=10223663&form=6&db=m&Dopt=b">Inferring
|
||
tree models for oncogenesis from comparative genome hybridization data</a>. <i>J Comput
|
||
Biol</i> 6(1):37–51, 1999.</p>
|
||
<b>
|
||
|
||
<p ALIGN="left">Galperin, MY, </b>and D Frishman. Toward automated prediction of protein
|
||
function from microbial genomic sequences. <i>Methods of Microbiology </i>28:245–63.
|
||
London: Academic Press, 1999.</p>
|
||
<b>
|
||
|
||
<p ALIGN="left">Kuehl, PM,</b> <b>JM Weisemann,</b> JW Touchman, ED Green, and <b>MS
|
||
Boguski. </b><a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=10022984&form=6&db=m&Dopt=b">An
|
||
effective approach for analyzing "Prefinished" genomic sequence data</a>. <i>Genome
|
||
Res</i> 9(2):189–94, 1999.</p>
|
||
|
||
<p ALIGN="left">Menotti-Raymond, M, VA David, LA Lyons, <b>AA Sch<63>ffer, </b>JF Tomlin, MK
|
||
Hutton, and SJ O’Brien. <a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=10191079&form=6&db=m&Dopt=b">A
|
||
genetic linkage map of microsatellites in the domestic cat</a>. <i>Genomics </i>57(1):9–23,
|
||
1999. </p>
|
||
|
||
<p ALIGN="left">Pesole, G, S Liuni, G Grillo, M Ippedico, A Larizza, <b>W Makalowski, </b>and
|
||
C Saccone. <a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9847176&form=6&db=m&Dopt=b">UTRdb:
|
||
a specialized database of 5<font FACE="Symbol"><EFBFBD></font> and 3<font FACE="Symbol"><EFBFBD></font>
|
||
untranslated regions of eukaryotic mRNAs</a>. <i>Nucleic Acids Res</i> 27(1):188–91,
|
||
1999. </p>
|
||
<b>
|
||
|
||
<p ALIGN="left">Sch<EFBFBD>ffer, AA.</b> <a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=10030397&form=6&db=m&Dopt=b">Computing
|
||
probabilities of homozygosity by descent</a>. <i>Genet Epidemiol</i> 16(2):135–49,
|
||
1999.</p>
|
||
<b>
|
||
|
||
<p align="left">Wolf, YI,</b> SE Brenner, PA Bash, and <b>EV Koonin.</b> <a
|
||
href="http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9927481&form=6&db=m&Dopt=b">Distribution
|
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of protein folds in the three superkingdoms of life</a>. <i>Genome Res</i>
|
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9(1):17–26, 1999.</p>
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|
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<p><a href="#toc">Return to Table of Contents</a></p>
|
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|
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<hr>
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<h3 align="left"><font SIZE="5"><a name="Masthead">Masthead</a></font><font SIZE="3"></h3>
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<p></font><i>NCBI News</i> is distributed four times a year. Beginning in 1999, issues are
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E-mail: <a href="mailto:info@ncbi.nlm.nih.gov">info@ncbi.nlm.nih.gov</a></p>
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<i>
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<p></i><em>Editors</em><br>
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Barbara Rapp</p>
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<i>
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<p>Contributors</i><br>
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Stephen Altschul Donna Maglott<br>
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Jonathan Kans Kim Pruitt<br>
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Alex Lash
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Greg
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Schuler</p>
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<i>
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<p>Writer<br>
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</i>David Wheeler</p>
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<p>Editing, Graphics, and Production<br>
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<p>Photography</i><br>
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<p>Design Consultant<br>
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</i>Troy M. Hill</p>
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information systems for molecular biology and genetics data and perform research in
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computational molecular biology.</p>
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<p ALIGN="left">The contents of this newsletter may be reprinted without permission. The
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mention of trade names, commercial products, or organizations does not imply endorsement
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by NCBI, NIH, or the U.S. Government. </p>
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<p ALIGN="JUSTIFY">NIH Publication No. 99-3272<br>
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<br>
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ISSN 1060-8788 <br>
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ISSN 1098-8408 (Online Version)<font SIZE="3"></p>
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