Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA

<p>Abstract</p> <p>Background</p> <p>In a traditional electrophoresis mobility shift assay (EMSA) a ³²P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb) for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA (<10 kb) of known sequence.</p> <p>Results</p> <p>We have combined an EMSA-based selection step with subsequent denaturing PAGE for the localization of protein binding regions in long (up to10 kb) fragments of genomic DNA. Our strategy consists of the following steps: digestion of genomic DNA with a 4-cutter restriction enzyme (<it>Alu</it>I, <it>Bsu</it>RI, <it>Tru</it>I, etc), separation of low and high molecular weight fractions of resultant DNA fragments, ³²P-labeling with Klenow polymerase, traditional EMSA, gel elution and identification of the shifted bands (or smear) by denaturing PAGE. The identification of DNA fragments containing protein binding sites is carried out by running the gel-eluted fragments alongside with the full "spectrum" of initial restriction fragments of known size. Here the strategy is used for the identification of protein-binding regions in the 5' region of the rat p75 neurotrophin receptor (<it>p75NTR</it>) gene.</p> <p>Conclusion</p> <p>The developed strategy is based on a combination of traditional EMSA and denaturing PAGE for the identification of protein binding regions in long fragments of genomic DNA. The identification is straightforward and can be applied to shifted bands corresponding to stable DNA-protein complexes as well as unstable complexes, which undergo dissociation during electrophoresis.</p

Intronic L1 Retrotransposons and Nested Genes Cause Transcriptional Interference by Inducing Intron Retention, Exonization and Cryptic Polyadenylation

Transcriptional interference has been recently recognized as an unexpectedly complex and mostly negative regulation of genes. Despite a relatively few studies that emerged in recent years, it has been demonstrated that a readthrough transcription derived from one gene can influence the transcription of another overlapping or nested gene. However, the molecular effects resulting from this interaction are largely unknown.Using in silico chromosome walking, we searched for prematurely terminated transcripts bearing signatures of intron retention or exonization of intronic sequence at their 3' ends upstream to human L1 retrotransposons, protein-coding and noncoding nested genes. We demonstrate that transcriptional interference induced by intronic L1s (or other repeated DNAs) and nested genes could be characterized by intron retention, forced exonization and cryptic polyadenylation. These molecular effects were revealed from the analysis of endogenous transcripts derived from different cell lines and tissues and confirmed by the expression of three minigenes in cell culture. While intron retention and exonization were comparably observed in introns upstream to L1s, forced exonization was preferentially detected in nested genes. Transcriptional interference induced by L1 or nested genes was dependent on the presence or absence of cryptic splice sites, affected the inclusion or exclusion of the upstream exon and the use of cryptic polyadenylation signals.Our results suggest that transcriptional interference induced by intronic L1s and nested genes could influence the transcription of the large number of genes in normal as well as in tumor tissues. Therefore, this type of interference could have a major impact on the regulation of the host gene expression

A 9,372 bp BamHI fragment derived from 5' region of (positions from -9,645 to -274) cloned in pBS KS (sequence with lower case letters) was digested with RI and I

Sequence derived from UCSC Genome Browser on Rat Nov. 2004 Assembly (contigs AABR03076992.1 and AABR03076383.1). Protein binding was detected for fragments marked with green (BsuRI digest) and yellow highlight or underlined (AluI digest). Fragments marked with grey highlight were derived from pBS KS+. <p><b>Copyright information:</b></p><p>Taken from "Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA"</p><p>http://www.biomedcentral.com/1471-2164/9/272</p><p>BMC Genomics 2008;9():272-272.</p><p>Published online 4 Jun 2008</p><p>PMCID:PMC2435560.</p><p></p

A strategy for the detection of protein binding regions in genomic DNA fragments

<p><b>Copyright information:</b></p><p>Taken from "Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA"</p><p>http://www.biomedcentral.com/1471-2164/9/272</p><p>BMC Genomics 2008;9():272-272.</p><p>Published online 4 Jun 2008</p><p>PMCID:PMC2435560.</p><p></p

(A) Agarose gel electrophoresis of total digests (T) and gel-isolated fractions 30–300 bp (L) and 300–700 bp (H)

M, 1 kb ladder (Stratagene). (B) The expected digestion pattern generated by sequence analysis with DNAMAN software.<p><b>Copyright information:</b></p><p>Taken from "Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA"</p><p>http://www.biomedcentral.com/1471-2164/9/272</p><p>BMC Genomics 2008;9():272-272.</p><p>Published online 4 Jun 2008</p><p>PMCID:PMC2435560.</p><p></p

A poly (dI-dC) concentration range from 100 to 800 μg/ml was tested with 2-fold differences

At each concentration, the incubation of labeled restriction fragment with PC12 nuclear extract (NE) was performed using two different poly (dI-dC) preparations with the average sizes 250 and 500 bp. C, a 202 bp II-I fragment and C, a 384 bp I-I fragment derived from -1.8 kb and -1.6 kb regions of the rat promoter, respectively. Arrowheads point at the major specific DNA-protein complexes. M, P-labeled 100 bp ladder (Gibco-BRL).<p><b>Copyright information:</b></p><p>Taken from "Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA"</p><p>http://www.biomedcentral.com/1471-2164/9/272</p><p>BMC Genomics 2008;9():272-272.</p><p>Published online 4 Jun 2008</p><p>PMCID:PMC2435560.</p><p></p

A 275 bp fragment (F275), derived from L1 antisense promoter 20, was gel isolated by using QIAquickGel Extraction Kit (panel A) or gel-diffusion method described here (panel B), radiolabeled and incubated with ( ) or without (-) nuclear extract

The presence (+) or absence (-) of competitor, a 10-fold excess of the unlabeled fragment, is shown. Note that specific mobility shift, shown by arrowhead, is visible only in panel B.<p><b>Copyright information:</b></p><p>Taken from "Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA"</p><p>http://www.biomedcentral.com/1471-2164/9/272</p><p>BMC Genomics 2008;9():272-272.</p><p>Published online 4 Jun 2008</p><p>PMCID:PMC2435560.</p><p></p

(A) L and H molecular fractions of corresponding RI and I fragments were incubated with (lane ) and without (lane -) PC12 nuclear extract (NE) and analysed by EMSA

Direct phosphoimaging of the wet 1 mm-thick gel is shown. (B) Denaturing PAGE of the shifted fragments. Lanes - and + show the pool of fragments used in EMSA and shifted bands (smear), respectively. Connecting lines indicate the bands or smear analyzed on two different gels. M, P-labeled 100 bp ladder (Gibco-BRL).<p><b>Copyright information:</b></p><p>Taken from "Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA"</p><p>http://www.biomedcentral.com/1471-2164/9/272</p><p>BMC Genomics 2008;9():272-272.</p><p>Published online 4 Jun 2008</p><p>PMCID:PMC2435560.</p><p></p