Deamination of cytosine to uracil is the most common promutagenic change in DNA, and it is greatly increased at the elevated growth temperatures of hyperthermophilic archaea. If not repaired to cytosine prior to replication, uracil in a template strand directs incorporation of adenine, generating a G⅐C 3 A⅐U transition mutation in half the progeny. Surprisingly, genomic analysis of archaea has so far failed to reveal any homologues of either of the known families of uracil-DNA glycosylases responsible for initiating the base-excision repair of uracil in DNA, which is otherwise universal. Here we show that DNA polymerases from several hyperthermophilic archaea (including Vent and Pfu) specifically recognize the presence of uracil in a template strand and stall DNA synthesis before mutagenic misincorporation of adenine. A specific template-checking function in a DNA polymerase has not been observed previously, and it may represent the first step in a pathway for the repair of cytosine deamination in archaea.

The dsRNA-activated protein kinase PKR is involved in signal transduction pathways that mediate cellular processes as diverse as cell growth and differentiation, the stress response, and apoptosis. PKR was originally described as an interferon-inducible elF2alpha kinase involved in the antiviral defense mechanism of the cell. The interaction of the kinase with specific viral RNAs has been studied in much detail, but information about cellular mRNAs, which are able to bind and activate PKR, is scarce. In search for such cellular mRNAs, we developed a cloning strategy to identify individual mRNA species from the dsRNA-rich fraction of Daudi cell poly(A)+ RNA. Two out of five cDNA clones we obtained contained sequences derived from the mRNA of the translationally controlled tumor protein P23/TCTP, indicating that this mRNA is present in the dsRNA-rich fraction. Secondary structure predictions and gel electrophoretic mobility investigations on P23/TCTP transcripts confirmed the potential of this mRNA to form extensive secondary structure. A full-length P23 transcript, but not a truncated version thereof, was able to bind to PKR in vitro and in vivo. Transient transfection experiments in human 293 cells showed that coexpression of full-length P23 mRNA leads to partial inhibition of the expression of a beta-galactosidase reporter gene in trans. Additional coexpression of a dominant negative mutant of PKR or of adenovirus VA1 RNA suppressed this inhibition, indicating that it is mediated by PKR. Studies on P23/TCTP expression in cells from PKR-knockout mice suggest that P23/TCTP mRNA translation is regulated by PKR. Hence, our results demonstrate that the mRNA of P23/TCTP may both activate PKR and be subject to translational regulation by this kinase.

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Interactive regulation of ERK and JNK signaling pathways by the ' 551 M3-muscarinic receptor and B2-adrenoceptor co-expressed in CHO cells Medical Sciences Budding, Department of Cell Physiology andPhawmcoiogy, University of Leicester, University Road, Leuester, LEI 9HN. UK Extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal h a s e s (JNK) have been implicated in the regulation of many cellular processes, including cell proliferation and apoptosis, and can be regulated by G protein-coupled receptors, as well as growth factor receptors. Here, we have examined the mechanisrn(s) by which Mj-muxarinic receptor (MyAChR) stimulation of ERK and JNK activities can be modulated by B2-adrenoceptor (Bz-AR) activation.CHO-62m3 cells stably expressing the M3-AChR (at 1200 fmol mg-l protein) and the B2-AR (at 300 fmol mg-l) Protein were utilised to investigate cyclic AMP modulation of ERK and JNK activation via Gq-coupled M3-AChR. Stimulation of B2-ARs with isoprenaline (ISO) causes a large, rapid increase in cyclic AMP accumulation (pEC50 (M), 8.4 +/-0.1; Em, (5min), approx. 100 fold-over basal), which was sufficient to activate almost maximally cyclic AMP-dependent protein kinase (PKA). The AChR agonist methacholine (MCh) caused a concentration-and time-dependent activation of ERK (pEC50.5.9 +/-0.2, Em, (5 min), 15.3 +/-2.6 fold-over-basal) and JNK (pEC50,5,9 +/-0.1, Em, (40 rnin), 8.9 +/-1.3 fold-over basal).

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