Quantitative regulation of class switch recombination by switch region transcription.

The isotype specificity of immunoglobulin (Ig) class switching is regulated by a cytokine which induces transcription of a specific switch (S) region, giving rise to so-called germline transcripts. Although previous studies have demonstrated that germline transcription of an S region is required for class switch recombination (CSR) of that particular S region, it has not been shown whether the level of S region transcription affects the efficiency of CSR. We addressed this question by using an artificial DNA construct containing a constitutively transcribed mu switch (Smu) region and an alpha switch (Salpha) region driven by a tetracycline-responsive promoter. The construct was introduced into a switch-inducible B lymphoma line and the quantitative correlation between Salpha region transcription and class switching efficiency was evaluated. The level of Salpha transcription was linearly correlated with CSR efficiency, reaching a plateau at saturation. On the other hand, we failed to obtain the evidence to support involvement of either RNA-DNA heteroduplex or trans germline transcripts in CSR. Taken together, it is likely that S region transcription and/or transcript processing in situ may be required for CSR. We propose that because of the unusual properties of S region DNA, transcription induces the DNA to transiently be single stranded, permitting secondary structure(s) to form. Such structures may be recognition targets of a putative class switch recombinase.

The absence of ribonuclease H1 or H2 alters the sensitivity of Saccharomyces cerevisiae to hydroxyurea, caffeine and ethyl methanesulphonate: implications for roles of RNases H in DNA replication and repair.

BACKGROUND: RNA of RNA-DNA hybrids can be degraded by ribonucleases H present in all organisms including the eukaryote Saccharomyces cerevisiae. Determination of the number and roles of the RNases H in eukaryotes is quite feasible in S. cerevisiae. RESULTS: Two S. cerevisiae RNases H, related to Escherichia coli RNase HI and HII, are not required for growth under normal conditions, yet, compared with wild-type cells, a double-deletion strain has an increased sensitivity to hydroxyurea (HU) and is hypersensitive to caffeine and ethyl methanesulphonate (EMS). In the absence of RNase H1, RNase H2 activity increases, and cells are sensitive to EMS but not HU and are more tolerant of caffeine; the latter requires RNase H2 activity. Cells missing only RNase H2 exhibit increased sensitive to HU and EMS but not caffeine CONCLUSIONS: Mutant phenotypes infer that some RNA-DNA hybrids are recognized by both RNases H1 and H2, while other hybrids appear to be recognized only by RNase H2. Undegraded RNA-DNA hybrids have an effect when DNA synthesis is impaired, DNA damage occurs or the cell cycle is perturbed by exposure to caffeine suggesting a role in DNA replication/repair that can be either beneficial or detrimental to cell viability.

Reconstitution and characterization of a divergent plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica, expressed in Escherichia coli.

Plastocyanin (PC) is a copper protein that serves as a mobile electron carrier between cytochrome f and Photosystem I in the light reactions of photosynthesis. Despite large variability in amino acid sequences and isoelectric points, PCs from cyanobacterial and chloroplast sources reveal considerable similarities with respect to their secondary and tertiary structures. In this paper, we have expressed in Escherichia coli a PC from the prokaryote Prochlorothrix hollandica, and efficiently reconstituted the protein with copper under conditions yielding the characteristics of a native holoPC, as judged by redox titration (Eo' = +376 mV), near and far UV circular dichroism, and electron paramagnetic resonance (EPR) spectroscopy. By comparison of amino acid sequences, P. hollandica PC is the most divergent homolog identified to date, and analysis of this reconstituted preparation may reveal new insights as to the structural requirements for electron transport between the PC copper center and neighboring reaction partners.

Expression of the petE gene encoding plastocyanin in the photosynthetic prokaryote, Prochlorothrix hollandica.

The expression of the petE gene encoding plastocyanin (PC) in the prokaryote Prochlorothrix hollandica is dependent on the presence of copper in the medium. PC protein and petE mRNA are detectable only under copper (Cu) replete conditions, suggesting that control of PC accumulation can occur at the level of transcription or transcript stability. Addition of Cu (0.3 microM) to log-phase Cu-deficient cultures yields accumulation of PC to detectable levels within 12 h; transfer of Cu-replete cells to Cu-deficient medium results in a slow decrease in the level of protein likely due to dilution by cell growth. By contrast, addition of high-affinity Cu-specific chelators to rapidly deplete Cu-grown cells of copper yields a rapid loss of PC with 2 h. These data suggest that Cu-free apoPC is turned over rapidly by proteolysis. Overall, these data demonstrate that regulation of PC levels as Cu levels change involve events occurring at the level of both RNA and protein turnover.

Nucleotide sequence of the petE gene encoding plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica.

We have determined the nucleotide sequence of the petE gene encoding plastocyanin from the chlorophyll a/b-containing photosynthetic prokaryote, Prochlorothrix hollandica. Comparison of the deduced amino acid sequence encoded by the gene with the N-terminal sequence of the purified protein revealed that plastocyanin is synthesized as a precursor bearing an N-terminal domain of 34 amino acids having some structural similarity to thylakoid lumenal transit peptides identified in other organisms. The mature protein has an apparent isoelectric point of 8.37 and a molecular mass of 10,236 Da.