Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection

Aptamers are short nucleic acid or peptide sequences capable of binding to a target molecule with high specificity and affinity. Also known as “artificial antibodies,” aptamers provide many advantages over antibodies. One of the major hurdles to aptamer isolation is the initial time and effort needed for selection. The systematic evolution of ligands by exponential enrichment (SELEX) is the traditional procedure for generating aptamers, but this process is lengthy and requires a large quantity of target and starting aptamer library. A relatively new procedure for generating aptamers using capillary electrophoresis (CE), known as CE-SELEX, is faster and more efficient than SELEX but requires laser-induced fluorescence (LIF) to detect the aptamer-target complexes. Here, we implemented an alternative system without LIF using real-time- (RT-) PCR to indirectly measure aptamer-target complexes. In three rounds of selection, as opposed to ten or more rounds common in SELEX protocols, a specific aptamer for bovine serum albumin (BSA) was obtained. The specificity of the aptamer to BSA was confirmed by electrophoretic mobility shift assays (EMSAs), an unlabeled competitor assay, and by a supershift assay. The system used here provides a cost effective and a highly efficient means of generating aptamers.

NPM-ALK Converts JUNB from a Tumor Suppressor to an Oncogene.

High expression of the tumor necrosis factor receptor CD30 and the AP-1 transcription factor JunB are the hallmark of anaplastic large cell lymphoma (ALCL). In contrast to the prototypic AP-1 factor c-Jun, JunB exerts an antioncogenic function in most cell types. Its functional role in ALCL remains uncertain. In about 50% of nodal ALCL the balanced chromosomal rearrangement t(2;5)(p23;q35), generating the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), can be detected. Expression of this fusion kinase induces malignancy in mice and also leads to IL-3 independent outgrowth of the murine hematopoietic cell line Ba/F3. Using NPM-ALK transduced Ba/F3 cells, we show that NPM-ALK induces JunB expression and activation as verified by quantitative RT-PCR, immunoblot and electro-mobility supershift assay. Interestingly, NPM-ALK transduced Ba/F3 cells also express CD30, which is undetectable in the corresponding wild type cells. Since NPM-ALK induces JunB and CD30 and also leads to growth factor independent proliferation in Ba/F3 cells, these cells mimic conditions present in ALCL. Knock down of JUNB in NPM-ALK expressing cells using RNA interference leads to downregulation of CD30. Moreover, this partial loss of JunB induces upregulation of p16INK4a and downregulation of CCND1, which directly affect the cell cycle at the G1/S transition. These observations indicate that JunB is an essential factor for CD30 regulation and for neoplastic transformation. To test if JunB by itself is sufficient to induce CD30 expression and IL-3 independence, we stably transduced Ba/F3 cells with JUNB. In Ba/F3 wild type (WT) cells, JunB expression leads to reverse effects compared to that observed in NPM-ALK transduced Ba/F3. Ba/F3 WT cells do not become IL-3 independent. In addition, compared to vector control, JUNB-transduced Ba/F3 cells show a decrease in proliferation. Furthermore, an induction of p16INK4a and a decrease of CCND1 expression are observed. Moreover, aberrant JunB expression does not trigger CD30 expression in this system. Taken together, we show that both NPM-ALK and JunB are essential to induce CD30 expression. Furthermore the opposing effects of JunB on p16INK4a and CCND1 in the presence or absence of NPM-ALK indicate that NPM-ALK converts JUNB from a tumor suppressor to an oncogene.

SMAD 8 binding to mice Msx1 basal promoter is required for transcriptional activation

The Msx1 gene in mice has been proven to be induced by BMP (bone morphogenetic protein) proteins, and three binding sites for SMAD, an intracellular BMP signalling transducer, have already been identified in its promoter. Gel shift analyses were performed and they demonstrated that the consensus found very near the transcription start site, a region designed BP (basal promoter), is functional for binding nuclear proteins from 10.5, 11.5 and 13.5 dpc (days post-coitum) embryos. Notably, this binding occurs only when the SMAD-binding consensus sequence is maintained, suggesting that it is required for the formation of a protein complex over BP. Binding of purified SMAD 1 and SMAD 4 as well as supershift assay with SMAD 1/SMAD 5/SMAD 8 antibody proved that a SMAD protein is present in this complex. Transfection assays in cell cultures with fragments from BP driving the expression of luciferase confirmed that only in the presence of the SMAD consensus site is Msx1 expression activated. A proteomic analysis of the complex components after immunoprecipitation identified several proteins necessary to activate transcription including SMAD 8. Our results suggest that BMP2/BMP4 signalling through SMAD 8 is required for transcriptional activation of the mouse Msx1 gene.

Oxalate-inducible AMBP gene and its regulatory mechanism in renal tubular epithelial cells

The AMBP [A1M (α1-microglobulin)/bikunin precursor] gene encodes two plasma glycoproteins: A1M, an immunosuppressive lipocalin, and bikunin, a member of plasma serine proteinase inhibitor family with prototypical Kunitz-type domain. Although previously believed to be constitutively expressed exclusively in liver, the present study demonstrates the induction of this gene by oxalate in porcine proximal tubular LLC-PK1 cells and rat kidney. In liver, the precursor protein is cleaved in the Golgi network by a furin-like enzyme to release constituent proteins, which undergo glycosylation before their export from the cell. In the renal tubular cells, A1M and bikunin co-precipitate, indicating lack of cleavage of the precursor protein. As the expression of the AMBP gene is regulated by A1M-specific cis elements and transcription factors, A1M protein was studied as a representative of AMBP gene expression in renal cells. Oxalate treatment (500 μM) resulted in a time- and dose-dependent induction of A1M protein in LLC-PK1 cells. Of the four transcription factors, HNF-4 (hepatocyte nuclear factor-4) has been reported previously to be a major regulator of AMBP gene expression in liver. Electrophoretic mobility-shift assay, supershift assay, immunoreactivity assay and transfection-based studies showed the presence of an HNF-4 or an HNF-4-like protein in the kidney, which can affect the expression of the AMBP gene. In situ hybridization and immunocytochemical studies showed that the expression of this gene in kidney was mainly restricted to cells lining the renal tubular system.

Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing. .

Co-operative regulation of the transcription of human dihydrodiol dehydrogenase (DD)4/aldo–keto reductase (AKR)1C4 gene by hepatocyte nuclear factor (HNF)-4α/γ and HNF-1α

Human dihydrodiol dehydrogenase (DD) 4/aldo-keto reductase (AKR) 1C4 is a major isoform of hepatic DD that oxidizes trans-dihydrodiols of polycyclic aromatic hydrocarbons to reactive and redox-active o-quinones and that reduces several ketone-containing drugs. To investigate the mechanism of transcriptional regulation of the human DD4 gene, the 5′-flanking region of the gene was fused to the luciferase gene. The results of luciferase assays using HepG2 cells and of 1,10-phenanthroline-copper footprinting indicated that two positive regulatory regions were located in regions from -701 to -684 and from -682 to -666. The former region contained a putative hepatocyte nuclear factor (HNF)-4 binding motif, and the latter region contained an HNF-1 consensus binding sequence. DNA fragments of the HNF-4 or HNF-1 motif gave a shifted band in a gel-shift assay with nuclear extracts from HepG2 cells. The formation of the DNA-protein complex was inhibited by the HNF-4 or HNF-1 motif of the α1-antitrypsin gene. A supershift assay using antibodies to human HNF-4α, HNF-4γ and HNF-1α showed that HNF-4α and HNF-4γ bound to the HNF-4 motif, and that HNF-1α interacted with the HNF-1 motif. Introduction of mutations into the HNF-4 or HNF-1 motif lowered the luciferase activity to 10 or 8% respectively of that seen with the intact human DD4 gene. These results indicate that HNF-4α, HNF-4γ and HNF-1α regulate co-operatively the transcription of the human DD4 gene in HepG2 cells.

DNA-binding activity in the excretory–secretory products ofTrichinella pseudospiralis(Nematoda: Trichinelloidea)

A novel DNA-binding peptide ofMr∼30 kDa was documented for the first time in the excretory–secretory (E–S) products of the infective-stage larvae ofTrichinella pseudospiralis.Larvae recovered from muscles of infected mice were maintained for 48 h in DMEM medium. E–S products of worms extracted from the medium were analysed for DNA-binding activity by the electrophoretic mobility shift assay (EMSA). Multiple DNA-protein complexes were detected. A comparison of theMrof proteins in the complexes indicated that they could bind to the target DNA as a dimer, tetramer or multiples of tetramers. Site selection and competition analysis showed that the binding has a low specificity. A (G/C-rich)-gap-(G/T-rich)-DNA sequence pattern was extracted from a pool of degenerate PCR fragments binding to the E–S products. Results of immunoprecipitation and electrophoretic mobility supershift assay confirmed the authenticity of the DNA-binding protein as an E–S product.