Expression domains of the Cf1a POU domain protein during Drosophila development

We have used antibodies directed against a unique portion of the Drosophila POU domain protein Cfla to localize its sites of expression in developing embryos. Cfla protein is first detected during germ band extension in the tracheal placodes and in the midline mesectoderm cells. Tracheal expression continues throughout embryonic development, especially in the main longitudinal tracheal trunks. Additional sites of high Cfla expression are in the anterior portion of the hindgut, the roof of the stomodeum, a subset of central nervous system cells, the oenocytes, and the ring gland. In addition, Cfla expression was localized in embryos mutant for several loci involved in determining fate along the midline of the CNS and the tracheal system. Cfla midline cell expression is dependent on proper single-minded gene function, and Cfla either regulates or acts in parallel to the genes pointed and rhomboid during midline CNS and tracheal development.     

Embryonic development in the primitive bilaterian Neochildia fusca: normal morphogenesis and isolation of POU genes Brn-1 and Brn-3

Neochildia fusca is a member of the taxon Acoela, a group of flatworms that, according to some recent molecular phylogenetic analyses, are distinct from other flatworms and constitute a basal branch with a sister taxon relationship to the rest of the Bilateria. In this paper, we analyze early neural development in this species and report the sequence and expression of two Pit-Oct-Unc (POU) genes, NeocBrn-1 and NeocBrn-3. Homologs of these highly conserved genes play a role in neural fate determination in vertebrates, Drosophila and Caenorhabditis elegans. Acoels, including Neochildia, have a unique invariant pattern of early cleavage called duet spiral cleavage. In subsequent cell divisions descendants of the first three micromere duets form an outer layer of epidermal and neural progenitors surrounding the meso/endoderm progenitors, which are themselves descended from the macromere duet 4A, B and the micromere duet 4a, b. Organ formation begins at mid-embryonic stages with the epidermal primordium adopting a ciliated epithelial shape. Sub-epidermally, a bilaterally symmetric brain primordium can be seen at the anterior pole. Laterally and posteriorly, myoblasts form a thin layer underneath the epidermis. In late embryos and juveniles of Neochildia, the brain is formed by a 3-4 cell-diameter-thick layer of neurons forming a cortex surrounding a neuropile that is relatively free of cell bodies. A highly regular "orthogonal" array of muscle fibers penetrates the brain. We have isolated and partially sequenced homologs of the vertebrate Brn-1 and Brn-3 genes, which we call NeocBrn-1 and NeocBrn-3, respectively. These sequences contain and span portions of the POU-specific domain and a homeodomain, and are sequence similar to their respective homologs in vertebrates and Drosophila. RT-PCR reveals that NeocBrn-1 and NeocBrn-3 are expressed from mid-embryonic to adult stages. Whole-mount in situ hybridization shows expression of both genes in distinct subsets of nerve cells in juvenile and adult worms. NeocBrn-1 also appears in a subset of intra-epidermal gland cells. These observations are an initial step towards reconstructing the neural development of a key group of bilaterians, the Acoela. These flatworms, by virtue of their distinct morphology, development and phylogenetically basal placement, are likely to provide key insights into the interpretation of the evolution of metazoan neural architecture.     

The amino terminal domain from Mrt4 protein can functionally replace the RNA binding domain of the ribosomal P0 protein

In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein.     

Conditional RNA interference achieved by Oct-1 POU/rtTA fusion protein activator and a modified TRE-mouse U6 promoter

RNA interference (RNAi) is a powerful technique and is widely used to down-regulate expression of specific genes in cultured cells and in vivo. In this paper, we report our development of a new tetracycline-inducible RNAi expression using a modified TRE-mouse U6 promoter in which the distal sequence element (DSE) was replaced by the tetracycline-responsive element (TRE). The modified TRE-mouse U6 promoter can be activated by a Tet-on version tetracycline-regulated artificial activator rTetOct which was constructed by fusing the rtTA DNA binding domain with the Oct-1 POU activation domain. This rTetOct/TRE-U6 system was successfully applied to conditionally and reversibly down-regulate the expression of endogenous p53 gene in MCF7 cells, and the expression of beta-defensin gene (mBin1b) either transiently expressed in COS7 cells or stably expressed in CHO cells.     

Mechanisms for flexibility in DNA sequence recognition and VP16-induced complex formation by the Oct-1 POU domain.

DNA binding by the Oct-1 protein is directed by its POU domain, a bipartite DNA-binding domain made up of a POU-specific (POUS) domain and a POU-homeo (POUH) domain, two helix-turn-helix-containing DNA-binding modules that cooperate in DNA recognition. Although the best-characterized DNA target for Oct-1 binding is the octamer sequence ATGCAAAT, Oct-1 also binds a number of different DNA sequence elements. For example, Oct-1 recognizes a form of the herpes simplex virus VP16-responsive TAATGARAT element, called the (OCTA-)TAATGARAT site, that lacks octamer site similarity. Our studies suggest two mechanisms by which Oct-1 achieves flexible DNA sequence recognition. First, an important arginine found in the Oct-1 POUS domain tolerates substitutions of its base contacts within the octamer site. Second, on the (OCTA-)TAATGARAT site, the POUS domain is located on the side of the POUH domain opposite from where it is located on an octamer site. This flexibility of the Oct-1 POU domain in DNA binding also has an impact on its participation in a multiprotein-DNA complex with VP16. We show that Oct-1 POUS domain residues that contact DNA have different effects on VP16-induced complex formation depending on whether the VP16-responsive element involved has overlapping octamer similarity or not.