Transcription of ColEl DNA by RNA polymerase in vitro starts at two sites in a region required for maintenance of the plasmid. Certain transcripts that start at one of the sites can be cleaved by RNase H and then act as primers for DNA replication. Transcription from the other site produces a RNA -108 nucleotides long (species I or RNA I). Transcripts analogous to the primer and RNA I of ColEl are produced when pl5A or small derivatives of two other ColE1-compatible plasmids, CloDF13 and RSF1030, are used as template. If purified RNA I is added to the transcription reaction containing RNase H, formation of primer is inhibited. Each RNA I can inhibit primer formation by the plasmid that specifies it but has no effect on primer formation by heterologous templates. Thus, the inhibition of primer formation by RNA I is incompatibility specific. Because RNA I does not inhibit initiation or propagation of transcription or the processing of preformed precursors, the step that is sensitive to inhibition is probably formation of the hybrid between the primer precursor and the template. This hybrid is the required substrate for RNase H. Experiments with recombinant plasmids show the region that determines the specificity of response to RNA I to be >300 base pairs upstream of the origin of DNA replication.

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The tescalcin gene is preferentially expressed during mouse testis differentiation. Here, we demonstrate that this gene encodes a 24 kDa Ca(2+)- and Mg(2+)-binding protein with one consensus EF-hand and three additional domains with EF-hand homology. Equilibrium dialysis with (45)Ca(2+) revealed that recombinant tescalcin binds approximately one Ca(2+) ion at physiological concentrations (pCa 4.5). The intrinsic tryptophan fluorescence of tescalcin was significantly reduced by Ca(2+), indicative of a conformational change. The apparent K(d) for Ca(2+) was 0.8 microM. A point mutation in the consensus EF-hand (D123A) abolished (45)Ca(2+) binding and prevented the fluorescence quenching, demonstrating that the consensus EF-hand alone mediates the Ca(2+)-induced conformational change. Tescalcin also binds Mg(2+) (K(d) 73 microM), resulting in a much smaller fluorescence decrease. In the presence of 1 mM Mg(2+), tescalcin's Ca(2+) affinity is shifted to 3.5 microM. These results illustrate that tescalcin should bind Mg(2+) constitutively in a quiescent cell, replacing it with Ca(2+) during stimulation. We also show that tescalcin is most abundant in adult mouse heart, brain, and stomach, as well as in HeLa and HL-60 cells. Immunofluorescence microscopy revealed that tescalcin is present in the cytoplasm and nucleus, with concentration in membrane ruffles and lamellipodia in the presence of serum, where it colocalizes with the small guanosine triphosphatase Rac-1. Tescalcin shares sequence and functional homology with calcineurin-B homologous protein (CHP), and we found that tescalcin, like CHP, can inhibit the phosphatase activity of calcineurin A. Hence, tescalcin is a novel calcineurin B-like protein that binds a single Ca(2+) ion.