Translation is an important step in gene expression. The initiation of translation is phylogenetically diverse, since currently five different initiation mechanisms are known. For bacteria the three initiation factors IF1 – IF3 are described in contrast to archaea and eukaryotes, which contain a considerably higher number of initiation factor genes. As eukaryotes and archaea use a non-overlapping set of initiation mechanisms, orthologous proteins of both domains do not necessarily fulfill the same function. The genome of Haloferax volcanii contains 14 annotated genes that encode (subunits of) initiation factors. To gain a comprehensive overview of the importance of these genes, it was attempted to construct single gene deletion mutants of all genes. In 9 cases single deletion mutants were successfully constructed, showing that the respective genes are not essential. In contrast, the genes encoding initiation factors aIF1, aIF2γ, aIF5A, aIF5B, and aIF6 were found to be essential. Factors aIF1A and aIF2β are encoded by two orthologous genes in H. volcanii. Attempts to generate double mutants failed in both cases, indicating that also these factors are essential. A translatome analysis of one of the single aIF2β deletion mutants revealed that the translational efficiency of the second ortholog was enhanced tenfold and thus the two proteins can replace one another. The phenotypes of the single deletion mutants also revealed that the two aIF1As and aIF2βs have redundant but not identical functions. Remarkably, the gene encoding aIF2α, a subunit of aIF2 involved in initiator tRNA binding, could be deleted. However, the mutant had a severe growth defect under all tested conditions. Conditional depletion mutants were generated for the five essential genes. The phenotypes of deletion mutants and conditional depletion mutants were compared to that of the wild-type under various conditions, and growth characteristics are discussed.

It was long assumed that translation initiation in prokaryotes generally occurs via the so-called Shine Dalgarno (SD) mechanism. Recently, it became clear that translation initiation in prokaryotes is more heterogeneous. In the haloarchaeon Haloferax volcanii, the majority of transcripts is leaderless and most transcripts with a 5′-UTR lack a SD motif. Nevertheless, a bioinformatic analysis predicted that 20–30% of all genes are preceded by a SD motif in haloarchaea. To analyze the importance of the SD mechanism for translation initiation in haloarchaea experimentally the monocistronic sod gene was chosen, which contains a 5′-UTR with an extensive SD motif of seven nucleotides and a length of 19 nt, the average length of 5′UTRs in this organism. A translational fusion of part of the sod gene with the dhfr reporter gene was constructed. A mutant series was generated that matched the SD motif from zero to eight positions, respectively. Surprisingly, there was no correlation between the base pairing ability between transcripts and 16S rRNA and translational efficiency in vivo under several different growth conditions. Furthermore, complete replacement of the SD motif by three unrelated sequences did not reduce translational efficiency. The results indicate that H. volcanii does not make use of the SD mechanism for translation initiation in 5′-UTRs. A genome analysis revealed that while the number of SD motifs in 5′-UTRs is rare, their fraction within open reading frames is high. Possible biological functions for intragenic SD motifs are discussed, including re-initiation of translation at distal genes in operons.

Signal transducer and activator of transcription 5 (Stat5), a latent cytoplasmic transcription factor, becomes activated by phosphorylation upon cytokine, hormone, and growth factor interactions with their appropriate receptors and induces the transcription of target genes. It plays crucial roles in principal cell fate decisions and regulates cell differentiation, development, proliferation, apoptosis, and inflammation. It is active in the mammary gland, the liver, hematopoietic cells, and other organs and has pleiotropic functions, depending on its activation pathway and its site of action. We derived transgenic mice in which the expression of a LacZ reporter gene is directed by Stat5-specific response elements and visualized the activation of Stat5 in cells of mouse organs at different developmental stages. The reporter gene activity reflects the timing and the location of Stat5 activation and was documented in mammary epithelial cells during developmental stages of the gland, cells of the liver, kidney, spleen, thymus, and uterus and in granulocytes and macrophages of the transgenic lines.

Background:Signal transducer and activator of transcription 3 and 5 (Stat3 and Stat5) play important roles in cell differentiation, proliferation, apoptosis and inflammation. They are transiently activated by ligand-receptor interactions in normal cells but are often found to be constitutively active in cancer cells. Analysis of their activation pattern is therefore important for the description of developmental processes and the understanding of cellular transformation. Materials and methods: To visualize Stat3 and Stat5 transactivation activity in different cell types, we designed novel reporter constructs. These constructs comprise Stat3 or Stat5 specific promoter elements and reporter genes encoding bgalactosidase or fluorescent proteins. These constructs were integrated into lentiviral gene transfer vectors facilitating efficient transduction of most cell types. Results: The lentiviral reporter constructs were used to infect different cell types and their inducibility by activated Stat3 or Stat5 was measured. The Stat3-mCherry reporter was active in transduced tumor cells, which exhibit high levels of phosphorylated Stat3 and it was inducible in HepG2 liver cells by interleukin-6 treatment. The Stat5-LacZ reporter was active in cultured cells upon hormone induction of Stat5 and in primary mammary epithelial cells transplanted into cleared fat pads of mice during late pregnancy. Conclusion: These novel reporter constructs are valuable tools to investigate and to distinguish between Stat3 and Stat5 activity in primary cells and cancer cells. They will also be useful in the discovery of drugs targeting Stat3 or Stat5. They can also be employed to generate transgenic mice and track Stat activity during development.

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