Transcription in mammalian cells can be assessed at a genome-wide level, but it has been difficult to reliably determine whether individual transcripts are derived from the plus or minus strands of chromosomes. This distinction can be critical for understanding the relationship between known transcripts (sense) and the complementary antisense transcripts that may regulate them. Here, we describe a technique that can be used to (i) identify the DNA strand of origin for any particular RNA transcript, and (ii) quantify the number of sense and antisense transcripts from expressed genes at a global level. We examined five different human cell types and in each case found evidence for antisense transcripts in 2900 to 6400 human genes. The distribution of antisense transcripts was distinct from that of sense transcripts, was nonrandom across the genome, and differed among cell types. Antisense transcripts thus appear to be a pervasive feature of human cells, which suggests that they are a fundamental component of gene regulation.

Biological sensing of small molecules such as NO, O 2 , and CO is an important area of research; however, little is know about how CO is sensed biologically. The photosynthetic bacterium Rhodospirillum rubrum responds to CO by activating transcription of two operons that encode a CO-oxidizing system. A protein, CooA, has been identified as necessary for this response. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from Escherichia coli. In this study we report the purification of wild-type CooA from its native organism, R. rubrum, to greater than 95% purity. The purified protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. Gel filtration experiments reveal the protein to be a dimer in the absence of CO. Purified CooA contains 1.6 mol heme per mol of dimer. Upon interacting with CO, the electronic spectrum of CooA is perturbed, indicating the direct binding of CO to the heme of CooA. A hypothesis for the mechanism of the protein's response to CO is proposed.The sensing of small molecules such as NO, CO, and O 2 is a highly active area of research in biology. Although NO-sensing (soluble guanylyl cyclase) (1, 2) and O 2 -sensing (FixL) (3, 4) proteins have been studied, little data on CO-sensing proteins exists. The CO binding and CO inhibition of metalloproteins such as cytochromes has been extensively researched, but little is known of regulatory factors that have a clear biological role in the recognition of CO. In this paper, we report the isolation and characterization of a bacterial protein that binds CO and elicits a physiologically important response upon activation with CO.

In the photosynthetic bacterium Rhodospirillum rubrum, the presence of carbon monoxide (CO) induces expression of several proteins. These include carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. Together these enzymes catalyze the following conversion: CO ؉ H 2 O 3 CO 2 ؉ H 2 . This system enables R. rubrum to grow in the dark on CO as the sole energy source. Expression of this system has been shown previously to be regulated at the transcriptional level by CO. We have now identified the remainder of the CO-regulated genes encoded in a contiguous region of the R. rubrum genome. These genes, cooMKLXU, apparently encode proteins related to the function of the CO-induced hydrogenase. As seen before with the gene for the large subunit of the CO-induced hydrogenase (cooH), most of the proteins predicted by these additional genes show significant sequence similarity to subunits of Escherichia coli hydrogenase 3. In addition, all of the newly identified coo gene products show similarity to subunits of NADH-quinone oxidoreductase (energyconserving NADH dehydrogenase I) from various eukaryotic and prokaryotic organisms. We have found that dicyclohexylcarbodiimide, an inhibitor of mitochondrial NADH dehydrogenase I (also called complex I), inhibits the CO-induced hydrogenase as well. We also show that expression of the cooMKLXUH operon is regulated by CO and the transcriptional activator CooA in a manner similar to that of the cooFSCTJ operon that encodes the subunits of CODH and related proteins.In the presence of carbon monoxide (CO), Rhodospirillum rubrum induces synthesis of a CO-oxidizing system. This system catalyzes the net reaction CO ϩ H 2 O 3 CO 2 ϩ H 2 . The reaction is carried out by two enzymes: CO dehydrogenase (CODH) and a CO-tolerant hydrogenase. CODH is a wellcharacterized nickel-iron enzyme (10) that carries out the oxidation of CO to CO 2 , producing two reducing equivalents. Hydrogenase then consumes these reducing equivalents by the reduction of two protons to H 2 (9, 17). Intermediate electron carriers (including the ferredoxin-like small subunit of CODH [17]) may also be involved in the reaction.The hydrogenase is tightly membrane bound and has yet to be purified, although the sequence of its large subunit (CooH) and several of its properties have been described previously (22). A wide variety of hydrogenases from other organisms, however, have been purified and characterized. These hydrogenases have been found to fall into three distinct categories: Fe-only, Ni-Fe, and Ni-Fe-Se enzymes (for reviews, see references 1, 4, and 56). The CO-induced hydrogenase from R. rubrum apparently belongs to the Ni-Fe class, on the basis of its protein sequence and the requirement of Ni for activity (22).There seem to be two CO-regulated transcripts in R. rubrum. The first, cooFSCTJ, encodes CODH and related proteins (27,29,43) and has been shown to be regulated by the product of the cooA gene (43). CooA appears to bind to a specific region of DNA upstream of the cooF promoter in a CO-dependent m...