A ~35 kDa polypeptide from insect cells binds to yeast ACS like elements in the presence of ATP

BACKGROUND: The S. cerevisiae origin recognition complex binds to the ARS consensus sequence in an ATP dependent fashion. Recently, the yeast Cdc6 has been reported to have DNA binding activity. Conservation of replication proteins among different species strongly supports their functional similarity. Here we report the results of an investigation into the DNA binding activity of human Cdc6 protein. Cdc6 was expressed and purified from baculovirus infected Sf9 (Spodoptera frugiperda) insect cells as GST fusion protein (GST-Cdc6) and its DNA binding activity was tested. RESULTS: Partially purified fractions containing GSTCdc6 or GST showed an ACS binding activity in an ATP dependent manner. However, further purification revealed the presence of a putative 35 kDa insect cell protein (p35) which was found responsible for the DNA binding activity. A close match to the 9/11 bases of the ARS consensus sequence was sufficient for p35 binding activity. A DNA fragment from the human c-myc origin region containing yeast ACS like elements also showed p35 binding activity. CONCLUSIONS: We have identified a Spodoptera frugiperda protein with ATP dependent DNA binding activity to ACS like elements. ACS like elements have been reported to be essential for ORC binding and replication initiation in yeast but their role in higher eukaryotes still remains elusive. Like the ARS consensus sequence elements of yeast, ACS like elements found in c-myc and lamin beta 2 origin regions may play similar roles in replication and indicate a conserved role for this DNA motif among eukaryotes

Functional characterization of Helicobacter pylori DnaB helicase

Helicobacter pylori causes gastric ulcer diseases and gastric adenocarcinoma in humans. Not much is known regarding DNA replication in H. pylori that is important for cell survival. Here we report the cloning, expression and characterization of H. pylori DnaB (HpDnaB) helicase both in vitro and in vivo. Among the DnaB homologs, only Escherichia coli DnaB has been studied extensively. HpDnaB showed strong 5 to 3′ helicase and ATPase activity. Interestingly, H. pylori does not have an obvious DnaC homolog which is essential for DnaB loading on the E. coli chromosomal DNA replication origin (oriC). However, HpDnaB can functionally complement the E. coli DnaB temperature‐sensitive mutant at the non‐permissive temperature, confirming that HpDnaB is a true replicative helicase. Escherichia coli DnaC co‐eluted in the same fraction with HpDnaB following gel filtration analysis suggesting that these proteins might physically interact with each other. It is possible that a functional DnaC homolog is present in H. pylori. The complete characterization of H. pylori DnaB helicase will also help the comparative analysis of DnaB helicases among bacteria

Hemoglobin receptor in Leishmania is a hexokinase located in the flagellar pocket

Hb endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket. To understand the nature of the Hb receptor (HbR), we have purified the 46-kDa protein to homogeneity from Leishmania promastigote membrane. Purified HbR specifically binds Hb. The gene for HbR was cloned, and sequence analysis of the full-length HbR gene indicates the presence of hexokinase (HK) signature sequences, ATP-binding domain, and PTS-II motif. Four lines of evidence indicate that HbR in Leishmania is a hexokinase: 1) the recombinant HbR binds Hb, and the Hb-binding domain resides in the N terminus of the protein; 2) recombinant proteins and cell lysate prepared from HbR-overexpressing Leishmania promastigotes show enhanced HK activity in comparison with untransfected cells; 3) immunolocalization studies using antibodies against the N-terminal fragment (Ld-HbR-ΔC) of Ld-HbR indicate that this protein is located in the flagellar pocket of Leishmania; and 4) binding and uptake of 125I-Hb by Leishmania is significantly inhibited by anti-Ld-HbR-ΔC antibody and Ld-HbR-ΔC, respectively. Taken together, these results indicate that HK present in the flagellar pocket of Leishmania is involved in Hb endocytosis

Transcriptional regulation of mouse PXR gene: an interplay of transregulatory factors.

Pregnane X Receptor (PXR) is an important ligand-activated nuclear receptor functioning as a 'master regulator' of expression of phase I, phase II drug metabolizing enzymes, and members of the drug transporters. PXR is primarily expressed in hepatic tissues and to lesser extent in other non-hepatic tissues both in human and in mice. Although its expression profile is well studied but little is known about the regulatory mechanisms that govern PXR gene expression in these cells. In the present study, we have cloned and characterized over 5 kb (-4963 to +54) region lying upstream of mouse PXR transcription start site. Promoter-reporter assays revealed that the proximal promoter region of up to 1 kb is sufficient to support the expression of PXR in the mouse liver cell lines. It was evident that the 500 bp proximal promoter region contains active binding sites for Ets, Tcf, Ikarose and nuclear factor families of transcription factors. Electrophoretic mobility shift assays demonstrated that the minimal region of 134 bp PXR promoter was able to bind Ets-1 and β-catenin proteins. This result was further confirmed by chromatin immunoprecipitation analysis. In summary, the present study identified a promoter region of mouse PXR gene and the transregulatory factors responsible for PXR promoter activity. The results presented herein are expected to provide important cues to gain further insight into the regulatory mechanisms of PXR function

Genome-wide expression profile of steroid response in Saccharomyces cerevisiae

The response of the yeast Saccharomyces cerevisiae to human steroid hormone progesterone was studied by genomic expression profiling. The transcription profile data revealed that steroid response was a global phenomenon wherein a host of genes were affected. For example, 163 genes were upregulated and 40 genes were downregulated, by at least more than twofold. The major categories of upregulated genes included protein destination (15%), metabolism (14%), transport facilitation (12%), cell growth, cell division, and DNA synthesis (8%), and transcription (7%), while metabolism (22%), transcription (11%), intracellular transport (10%), cell growth, cell division, and DNA synthesis (10%), energy (8%), cell rescue, defense, and cell death (6%), and protein synthesis (6%) encoding genes were downregulated. Notwithstanding the fact that yeast cells do not possess commonly occurring steroid response cascade similar to higher eukaryotes, our results demonstrate that a short-term exposure to progesterone results in differential regulation of predominantly stress responsive genes

Binding of β-catenin/LEF transcription factors to −243/−219 mouse PXR promoter region. A

<p>) Radiolabeled −243/−219 oligonucleotide was incubated with 15 μg of Hepa 1–6 nuclear lysate (lane 2). For competition experiments, unlabelled −243/−219 mouse PXR (lanes 3 and 4), unlabelled −297/−163 mouse PXR (lanes 5 and 6) and unlabelled non-self oligonucleotides (lanes 7 and 8) were added to the reactions in 50- and 200-fold molar excess. <b>B</b>) Antibodies against β-catenin (lane 2), LEF-1(lane 3) or pre-immune IgG (lane 1) were added to the DNA-protein complexes and incubated for an additional 15 minutes at room temperature. Supershifted bands are shown with an open arrow. NE = nuclear extract.</p

Responses of Pathogenic and Nonpathogenic Yeast Species to Steroids Reveal the Functioning and Evolution of Multidrug Resistance Transcriptional Networks▿ †

Steroids are known to induce pleiotropic drug resistance states in hemiascomycetes, with tremendous potential consequences for human fungal infections. Our analysis of gene expression in Saccharomyces cerevisiae and Candida albicans cells subjected to three different concentrations of progesterone revealed that their pleiotropic drug resistance (PDR) networks were strikingly sensitive to steroids. In S. cerevisiae, 20 of the Pdr1p/Pdr3p target genes, including PDR3 itself, were rapidly induced by progesterone, which mimics the effects of PDR1 gain-of-function alleles. This unique property allowed us to decipher the respective roles of Pdr1p and Pdr3p in PDR induction and to define functional modules among their target genes. Although the expression profiles of the major PDR transporters encoding genes ScPDR5 and CaCDR1 were similar, the S. cerevisiae global PDR response to progesterone was only partly conserved in C. albicans. In particular, the role of Tac1p, the main C. albicans PDR regulator, in the progesterone response was apparently restricted to five genes. These results suggest that the C. albicans and S. cerevisiae PDR networks, although sharing a conserved core regarding the regulation of membrane properties, have different structures and properties. Additionally, our data indicate that other as yet undiscovered regulators may second Tac1p in the C. albicans drug response

EMSA of mouse PXR proximal promoter.

<p>An end-labeled 134 bp promoter fragment (−297/−163) was incubated with AML-12 whole cell lysate. Lane <b>1</b>: free probe; Lanes <b>2, 3</b> & <b>4</b>: binding reactions performed with increasing amount of 3 μg, 6 μg and 12 μg of AML-12 lysate respectively. DNA-protein complexes are shown with arrows.</p