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The complete coding sequence of the gene for bacteriophage T7 RNA polymerase (T7 gene 1) has been cloned in the plasmid pBR322. Large amounts of active enzyme can be accumulated in Escherichia coli when the cloned gene is transcribed from the lac UV5 promoter. A protease activity that apparently can nick the protein without causing it to fall apart can be a problem during purification, but a procedure is described that gives good yields of essentially homogeneous, highly active enzyme suitable for biochemical and physical studies. T7 RNA polymerase has a stringent specificity for its own promoters and will selectively transcribe DNA that has been linked to such a promoter. This specificity makes the enzyme useful both for producing specific RNAs in vitro and for directing the expression of selected genes inside the cell. Having the cloned gene also makes possible a detailed mutational analysis of the functioning of T7 RNA polymerase.Bacteriophage T7 RNA polymerase (EC 2.7.7.6) is produced early in T7 infection and plays a central role in regulating gene expression (for a review, see ref. 1). A single-chain enzyme with a molecular weight close to 100,000, T7 RNA polymerase has a stringent specificity for its own promoters, which contain a highly conserved sequence of 23 continuous base pairs including the start site for the RNA. Seventeen such promoters are found in T7 DNA, but few if any such promoters are found in host DNA or in any other DNAs unrelated to T7. Once T7 RNA polymerase has been produced during infection, other T7 gene products inactivate the host RNA polymerase, leaving all transcription in the cell directed to T7 DNA.The great specificity of T7 RNA polymerase for its own promoters is not only central to the strategy of T7 infection but is also interesting and potentially useful to biochemists. Understanding the basis for this specificity is a challenge in its own right. Furthermore, the purified enzyme can be used to produce large amounts of specific RNAs simply by transcribing DNA that has been joined to a promoter for T7 RNA polymerase. Such RNAs could be useful as hybridization probes, mRNAs for in vitro translation, substrates for analyzing processing reactions or RNA splicing, or for any purpose requiring a specific RNA. T7 RNA polymerase, together with a suitably positioned promoter, can also be used to direct the transcription of selected genes inside the cell. Target genes potentially could be expressed at very high levels, using the same strategy employed by T7 itself: once T7 RNA polymerase had been made, the host RNA polymerase could be inactivated, thereby eliminating the synthesis of competing mRNAs. The T7 RNA polymerase made during T7 infection has already been shown to be capable of directing the transcription of genes cloned in plasmids (2-4).The yield of purified T7 RNA polymerase from infected cells is not particularly good, because the enzyme is synthesized for only a few minutes during infection and does not accumulate to high levels. Nor does T7 infection provide an ...

Human platelets are anucleate blood cells that retain cytoplasmic mRNA and maintain functionally intact protein translational capabilities. We have adapted complementary techniques of microarray and serial analysis of gene expression (SAGE) for genetic profiling of highly purified human blood platelets. Microarray analysis using the Affymetrix HGU95Av2 approximately 12 600-probe set maximally identified the expression of 2147 (range, 13%-17%) platelet-expressed transcripts, with approximately 22% collectively involved in metabolism and receptor/signaling, and an overrepresentation of genes with unassigned function (32%). In contrast, a modified SAGE protocol using the Type IIS restriction enzyme