No abstract

In this study, we investigated the role of interleukin-6 (IL-6) in Giardia lamblia infections in mice. Elevated IL-6 expression was found in wild-type mice 15 days postinfection. Furthermore, IL-6-deficient mice controlled infections only slowly although normal immunoglobulin A production was observed. Thus, IL-6 is necessary for early control of acute G. lamblia infections

Genes encoding adenine biosynthetic enzymes in Saccharomyces cerevisiae, including ADE5,7, ADE8, ADE4, ADE1, and ADE2, are coordinately repressed at the transcriptional level when adenine is added to the growth medium (11,33,34,44). The expression of HIS4, encoding three activities in the histidine pathway, is similarly regulated by exogenous adenine (45). Bas1p and Bas2p are DNA binding proteins required for transcription of HIS4 (3) and ADE genes (11, 44) in medium lacking adenine. Bas1p binds to DNA through an N-terminal Myb-related domain (23) to sequences containing 5Ј-TGAC TC-3Ј as a common core element (3,11,45). Binding sites for Bas1p were identified in the HIS4 (3, 45), ADE2, and ADE5,7 promoters in vitro and shown to be required for transcription of the corresponding genes in adenine-free medium (11, 34). Bas2p is a homeodomain protein (9) that binds in vitro to an AϩT-rich sequence in the HIS4 promoter located adjacent to the Bas1p binding site that is required for efficient HIS4 transcription in vivo (3, 45). It was also shown that Bas2p binds to an ADE2 promoter fragment containing the critical Bas1p binding site; however, the exact location of the Bas2p binding site(s) at ADE2 was not determined (11).Based on the fact that mutations in BAS1 or BAS2 lead to low, unregulated expression of ADE genes (11, 44) and HIS4 (3, 45), it was proposed that adenine repression occurs by reducing the ability of Bas1p or Bas2p to stimulate transcription of these genes. In accordance with this idea, we recently identified a 74-bp fragment from ADE5,7 containing two Bas1p binding sites, an Abf1p binding site, and a Bas2p binding site that is sufficient to confer adenine-regulated transcription on the heterologous CYC1 promoter (Ade upstream activating sequence [UAS Ade ] function). Mutations in the Abf1p, Bas1p, or Bas2p sites that impaired UAS Ade function in vivo abolished or weakened binding of the corresponding proteins to the ADE5,7 sequences in vitro. It is noteworthy that none of ca. 60 substitution mutations in UAS Ade produced the constitutively derepressed phenotype expected from inactivation of a negative control site (34). These results are consistent with the idea that adenine repression occurs by reducing the expression or function of Bas1p, Bas2p, or Abf1p, rather than through binding of a repressor protein to the promoter. Because addition of adenine to the medium did not affect expression of BAS1-lacZ (45) or BAS2 (11), the ability of Bas1p or Bas2p to activate transcription, rather than their expression, may be down-regulated by adenine.Among the three positive regulators implicated at ADE5,7, we considered Bas1p to be the most likely target for negative regulation by adenine. Abf1p and Bas2p both function at other promoters that presumably are unresponsive to purine levels. Abf1p was first identified by its binding to autonomous replicating sequences (12) and subsequently for its role in the silencing of HML and HMR (8). In addition, Abf1p binds to and activates the promoters of many genes involve...

Modifying the genomes of many organisms is becoming as easy as manipulating DNA in test tubes, which is made possible by two recently developed techniques based on either the customizable DNA binding protein, TALEN, or the CRISPR/Cas9 system. Here, we describe a series of efficient applications derived from these two technologies, in combination with various homologous donor DNA plasmids, to manipulate the Drosophila genome: (1) to precisely generate genomic deletions; (2) to make genomic replacement of a DNA fragment at single nucleotide resolution; and (3) to generate precise insertions to tag target proteins for tracing their endogenous expressions. For more convenient genomic manipulations, we established an easy-to-screen platform by knocking in a white marker through homologous recombination. Further, we provided a strategy to remove the unwanted duplications generated during the “ends-in” recombination process. Our results also indicate that TALEN and CRISPR/Cas9 had comparable efficiency in mediating genomic modifications through HDR (homology-directed repair); either TALEN or the CRISPR/Cas9 system could efficiently mediate in vivo replacement of DNA fragments of up to 5 kb in Drosophila, providing an ideal genetic tool for functional annotations of the Drosophila genome.