1. Introduction Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes acquaintance with the principles and practice of PCR, as outlined in, for example, refs. 1–4. Primer3 is a computer program that suggests PCR primers for a variety of applications, for example to create STSs (sequence tagged sites) for radiation hybrid mapping (5), or to amplify sequences for single nucleotide polymor-phism discovery (6). Primer3 can also select single primers for sequencing reactions and can design oligonucleotide hybridization probes. In selecting oligos for primers or hybridization probes, Primer3 can consider many factors. These include oligo melting temperature, length, GC content , 3′ stability, estimated secondary structure, the likelihood of annealing to or amplifying undesirable sequences (for example interspersed repeats), the likelihood of primer–dimer formation between two copies of the same primer, and the accuracy of the source sequence. In the design of primer pairs Primer3 can consider product size and melting temperature, the likelihood of primer– dimer formation between the two primers in the pair, the difference between primer melting temperatures, and primer location relative to particular regions of interest or to be avoided.

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Primer3 on the WWW for general users and for biologist programmers.

In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's website. NCBI resources include Entrez, PubMed, PubMed Central, LocusLink, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, SARS Coronavirus Resource, SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheritance in Man (OMIM), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD) and the Conserved Domain Architecture Retrieval Tool (CDART). 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.

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Database resources of the National Center for Biotechnology Information

The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. The discovery of extensive transcription of large RNA transcripts that do not code for proteins, termed long noncoding RNAs (lncRNAs), provides an important new perspective on the centrality of RNA in gene regulation. Here, we discuss genome-scale strategies to discover and characterize lncRNAs. An emerging theme from multiple model systems is that lncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators and then target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, lncRNAs can function as modular scaffolds to specify higher-order organization in RNP complexes and in chromatin states. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs for proper gene control across all kingdoms of life.

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Genome Regulation by Long Noncoding RNAs

The widely expressed protein Fas is a member of the tumour necrosis factor receptor family which can trigger apoptosis1 However, Fas surface expression does not necessarily render cells susceptible to Fas ligand-induced death signals1,2, indicating that inhibitors of the apoptosis-signalling pathway must exist Here we report the characterization of an inhibitor of apoptosis, designated FLIP (for FLICE-inhibitory protein), which is predominantly expressed in muscle and lymphoid tissues The short form, FLIPS, contains two death effector domains and is structurally related to the viral FLIP inhibitors of apoptosis3, whereas the long form, FLIPL, contains in addition a caspase-like domain in which the active-centre cysteine residue is substituted by a tyrosine residue FLIPS and FLIPL interact with the adaptor protein FADD4,5 and the protease FLICE6,7, and potently inhibit apoptosis induced by all known human death receptors1 FLIPL is expressed during the early stage of T-cell activation, but disappears when T cells become susceptible to Fas ligand-mediated apoptosis High levels of FLIPL protein are also detectable in melanoma cell lines and malignant melanoma tumours Thus FLIP may be implicated in tissue homeostasis as an important regulator of apoptosis

/pdf/inhibition-of-death-receptor-signals-by-cellular-flip-1848gtju9y.pdf

Inhibition of death receptor signals by cellular FLIP

The development and progression of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. Microarrays of cDNA provide a powerful tool for studying these complex phenomena. The tumorigenic properties of a human melanoma cell line, UACC-903, can be suppressed by introduction of a normal human chromosome 6, resulting in a reduction of growth rate, restoration of contact inhibition, and suppression of both soft agar clonogenicity and tumorigenicity in nude mice. We used a high density microarray of 1,161 DNA elements to search for differences in gene expression associated with tumour suppression in this system. Fluorescent probes for hybridization were derived from two sources of cellular mRNA [UACC-903 and UACC-903(+6)] which were labelled with different fluors to provide a direct and internally controlled comparison of the mRNA levels corresponding to each arrayed gene. The fluorescence signals representing hybridization to each arrayed gene were analysed to determine the relative abundance in the two samples of mRNAs corresponding to each gene. Previously unrecognized alterations in the expression of specific genes provide leads for further investigation of the genetic basis of the tumorigenic phenotype of these cells.

Use of a cDNA microarray to analyse gene expression patterns in human cancer.

The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

https://science.sciencemag.org/content/sci/274/5287/540.full.pdf

K. Swanson

Papers

A gene map of the human genome