Induction of heavy-metal binding phytochelatins by inoculation of cell cultures in standard media

A large increase in phytochelatin (PC) synthesis occurred when cell cultures of different plant species were transferred from spent medium to fresh standard media. Phytochelatin accumulation correlated with the initial concentration of zinc ions in the nutrient solution. After reaching stationary growth phase, phytochelatins had almost disappeared from the cells which indicates a high turnover of these molecules under normal conditions. No significant formation of the heavy-metal complexing phytochelatins was observed if the microelement ions zinc and copper were omitted from the nutrient solutions for plant cell cultures. Both the induction and degradation phenomena of these peptides indicate that phytochelatins are involved in metal ion homeostasis in plants.     

Prion protein PrPc interacts with molecular chaperones of the Hsp60 family.

Prions mediate the pathogenesis of certain neurodegenerative diseases, including bovine spongiform encephalopathy in cattle and Creutzfeldt-Jakob disease in humans. The prion particle consists mainly, if not entirely, of PrPSc, a posttranslationally modified isoform of the cellular host-encoded prion protein (PrPc). It has been suggested that additional cellular factors might be involved in the physiological function of PrPc and in the propagation of PrPSc. Here we employ a Saccharomyces cerevisiae two-hybrid screen to search for proteins which interact specifically with the Syrian golden hamster prion protein. Screening of a HeLa cDNA library identified heat shock protein 60 (Hsp60), a cellular chaperone as a major interactor for PrPc. The specificity of the interaction was confirmed in vitro for the recombinant proteins PrPc23-231 and rPrP27-30 fused to glutathione S-transferase with recombinant human Hsp60 as well as the bacterial GroEL. The interaction site for recombinant Hsp60 and GroEL proteins was mapped between amino acids 180 and 210 of the prion protein by screening with a set of recombinant PrPc fragments. The binding of Hsp60 and GroEL occurs within a region which contains parts of the putative alpha-helical domains H3 and H4 of the prion protein.     

Characterization of the single-strand-specific BPV-1 origin binding protein, SPSF I, as the HeLa Pur alpha factor.

SPSF I and II are two cellular proteins which bind specifically to single-stranded DNA. SPSF I and II binding sites are found in the minimal origin of replication of BPV-1 DNA and near the P2 promoter of the cellular c-myc gene. DNA-binding properties of the two proteins to single-stranded oligonucleotides of different lengths and sequences were quantified by determination of DNA-binding constants. The binding constant of SPSF proteins to the lower strand of the BPV-1 origin was determined to be 1.5 x 10(-10) M-1. Peptide sequences derived from purified SPSF I and II revealed the identity of at least one of the SPSF proteins with the so-called HeLa Pur alpha factor. The HeLa Pur alpha factor was identified previously by virtue of its capacity to bind to purine-rich strands of the PUR element found in initiation zones of DNA replication [Bergemann, A.D., Ma,Z.-W. and Johnson, E.M. (1992) Mol. Cell. Biol. 12, 5673-5682]. Expression of the Pur cDNA confirmed the identity of the Pur alpha protein with the 42 kDa SPSF I protein. Analysis of several Pur alpha cDNA clones revealed the existence of an extended 3'-untranslated region in all Pur mRNAs.     

Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems.

This article describes a procedure which permits for the first time the isolation of the prion protein PrPc from the Syrian golden hamster in heterologous systems. Using a glutathione S-transferase (GST) fusion approach, milligram amounts of stable, soluble, and homogeneous GST::PrPc protein were obtained in Escherichia coli and with baculovirus-infected insect cells. Authentic PrPc was released from the immobilized fusion protein by direct cleavage with thrombin. GST::PrPc expressed in these two expression systems and also authentic PrPc released by thrombin cleavage were recognized by a polyclonal antibody directed against amino acid 95 to 110 of the golden hamster PrPc protein. GST::PrPc was not detected by a monoclonal antibody recognizing the region encompassing amino acids 138 to 152 of the human prion protein. The fusion protein was sensitive to proteinase K digestion, demonstrating that the cellular rather than the proteinase K-resistant scrapie isoform was produced.     

RNA aptamers specifically interact with the prion protein PrP.

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.     

Nuclear factor I (NF I) binds to an NF I-type site but not to the CCAAT site in the human alpha-globin gene promoter.

Methylation interference and missing contact analyses demonstrate that nuclear factor I (NF I) recognizes an NF I-like site (5'-GGG(N)6GCCAG-3') within the alpha-globin promoter rather than the adjacent CCAAT box. Consistent with this, mutations within the CCAAT box do not alter significantly the affinity and specificity of the interaction whereas elimination of the 5'-GGG-3' half-site of the recognition sequence reduces the DNA binding strength of NF I by 2 orders of magnitude down to the range of unspecific interaction. On the other hand, the mutated alpha-globin promoter sequence that is no longer bound by NF I, although it retains an intact CCAAT box, interacts specifically with a protein component from nuclear extracts of HeLa cells. From these results we conclude that NF I is not the factor that interacts with the CCAAT box and that the second half of the canonical 5'-TGG(N)6GCCAA-3' NF I binding site cannot be regarded as identical with the CCAAT promoter element, as suggested previously.     

Interaction of the yeast Pso5/Rad16 and Sgs1 proteins: influences on DNA repair and aging.

The interaction trap method was used to isolate putative binding partners of Rad16/Pso5, a protein responsible for repair of silent DNA. One of the interactors found was Sgs1, a DNA helicase influencing the life span of Saccharomyces cerevisiae, with homology to the human BLM, WRN and RECQL4 proteins. Using the same fusion proteins from the two-hybrid screening, we show evidence that both proteins also interact in vitro. We tested isogenic strains, containing mutant alleles of the two genes in single and double mutant combination, for phenotypic similarity. Life span in sgs1Delta single and sgs1Delta rad16Delta double mutants is about 40% of that of WT, and the rad16/pso5Delta single mutant also had its life span reduced to 75%. Sensitivity to different mutagens, whose lesions are poorly repaired in rad16/pso5Delta mutants, was tested in sgs1Delta mutants. The sgs1Delta conferred sensitivity to MMS, H2O2 and was moderately sensitive to UV(254nm) (UVC) and 4-NQO. An epistatic interaction between rad16 and sgs1 mutations after UVC, 4-NQO and H2O2 was observed. Moreover, we found that in a top3 background, functional Sgs1p and Rad16p apparently channel MMS, 4-NQO and H2O2 induced lesions into aberrant DNA repair. Our results demonstrate that Sgs1 is not only involved in genome stability, somatic recombination and aging, but is also implicated, together with Rad16/Pso5, in the repair of specific DNA damage.     

Two cellular single-strand-specific DNA-binding proteins interact with two regions of the bovine papillomavirus type 1 genome, including the origin of DNA replication.

We have identified and purified to near homogeneity two specific single-stranded DNA-binding factors (SPSF I and II) with molecular masses of 42 and 39 kDa, respectively, from calf thymus. Gel retention analysis and competition experiments demonstrate that the ubiquitous proteins SPSF I and II specifically interact with single-stranded DNA derived from the minimal in vitro origin of replication of bovine papillomavirus type 1 and a region of the viral genome proposed to be involved in plasmid maintenance. Bovine papillomavirus type 1 proteins do not interfere with DNA binding of SPSF I and II. The exact location of the binding domains of SPSF I and II on the DNA has been determined by methylation interference and T4 DNA polymerase footprinting. A potential cellular binding site for SPSF I and II is the major promoter (P2) of the human c-myc gene.