Nucleic Acids Research

Motz, Michael; Sagner, Gregor; Pääbo, Svante; Kilger, Christian

doi: 10.1093/nar/gng112pmid: 14530454

Sequential DEXAS (direct exponential amplification and sequencing), a one step amplification and sequencing procedure that allows accurate, inexpensive and rapid DNA sequence determination directly from genomic DNA, is described. This method relies on the simultaneous use of two DNA polymerases that differ both in their ability to incorporate dideoxynucleotides and in the time at which they are activated during the reaction. One enzyme, which incorporates deoxynucleotides and performs amplification of the target DNA sequence, is supplied in an active state whereas the other enzyme, which incorporates dideoxynucleotides and performs the sequencing reaction, is supplied in an inactive state but becomes activated by a temperature step during the thermocycling. Thus, in the initial stage of the reaction, target amplification occurs, while in the second stage the sequencing reaction takes place. We show that Sequential DEXAS yields high quality sequencing results directly from genomic DNA as well as directly from human blood without any prior isolation or purification of DNA.

Zhang, Yuanli; Zhang, Dabing; Li, Wenquan; Chen, Jianqun; Peng, Yufa; Cao, Wei

doi: 10.1093/nar/gng123pmid: 14530456

A novel real‐time quantitative polymerase chain reaction (PCR) method using an attached universal template (UT) probe is described. The UT is an approximately 20 base attachment to the 5′ end of a PCR primer, and it can hybridize with a complementary TaqMan probe. One of the advantages of this method is that different target DNA sequences can be detected employing the same UT probe, which substantially reduces the cost of real‐time PCR set‐up. In addition, this method could be used for simultaneous detection using a 6‐carboxy‐fluorescein‐labeled UT probe for the target gene and a 5‐hexachloro‐fluorescein‐labeled UT probe for the reference gene in a multiplex reaction. Moreover, the requirement of target DNA length for UT–PCR analysis is relatively flexible, and it could be as short as 56 bp in this report, suggesting the possibility of detecting target DNA from partially degraded samples. The UT–PCR system with degenerate primers could also be designed to screen homologous genes. Taken together, our results suggest that the UT–PCR technique is efficient, reliable, inexpensive and less labor‐intensive for quantitative PCR analysis.

Kwan, Ann H. Y.; Czolij, Robert; Mackay, Joel P.; Crossley, Merlin

doi: 10.1093/nar/gng124pmid: 14530457

The rapid increase in the number of novel proteins identified in genome projects necessitates simple and rapid methods for assigning function. We describe a strategy for determining whether novel proteins possess typical sequence‐specific DNA‐binding activity. Many proteins bind recognition sequences of 5 bp or less. Given that there are 45 possible 5 bp sites, one might expect the length of sequence required to cover all possibilities would be 45 × 5 or 5120 nt. But by allowing overlaps, utilising both strands and using a computer algorithm to generate the minimum sequence, we find the length required is only 516 base pairs. We generated this sequence as six overlapping double‐stranded oligonucleotides, termed pentaprobe, and used it in gel retardation experiments to assess DNA binding by both known and putative DNA‐binding proteins from several protein families. We have confirmed binding by the zinc finger proteins BKLF, Eos and Pegasus, the Ets domain protein PU.1 and the treble clef N‐ and C‐terminal fingers of GATA‐1. We also showed that the N‐terminal zinc finger domain of FOG‐1 does not behave as a typical DNA‐binding domain. Our results suggest that pentaprobe, and related sequences such as hexaprobe, represent useful tools for probing protein function.

Géron‐Landre, Bénédicte; Roulon, Thibaut; Desbiolles, Pierre; Escudé, Christophe

doi: 10.1093/nar/gng125pmid: 14530458

Fluorescent labeling of a short sequence of double‐stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem–loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand‐induced triple helix formation, its extremities hybridize to each other, leaving a dangling single‐stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non‐repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 ± 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules.

Tichopad, Ales; Dilger, Michael; Schwarz, Gerhard; Pfaffl, Michael W.

doi: 10.1093/nar/gng122pmid: 14530455

We propose a computing method for the estimation of real‐time PCR amplification efficiency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real‐time PCR kinetics. PCR ground fluorescence phase, non‐exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for amplification efficiency estimation. The single reaction set‐up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective influence of researchers, and the estimation can therefore be fully instrumentalized.

Follows, George A.; Tagoh, Hiromi; Lefevre, Pascal; Morgan, Gareth J.; Bonifer, Constanze

doi: 10.1093/nar/gkg804pmid: 14530429

The c‐FMS gene encodes the macrophage colony‐stimulating factor receptor (M‐CSFR or CSF1‐R), which is a tyrosine kinase growth factor receptor essential for macrophage development. We have previously characterized the chromatin features of the mouse gene; however, very little is known about chromatin structure and function of the human c‐FMS locus. Here we present a side‐by‐side comparison of the chromatin structure, histone modification, transcription factor occupancy and cofactor recruitment of the human and the mouse c‐FMS loci. We show that, similar to the mouse gene, the human c‐FMS gene possesses a promoter and an intronic enhancer element (c‐fms intronic regulatory element or FIRE). Both elements are evolutionarily conserved and specifically active in macrophages. However, we demonstrate by in vivo footprinting that both murine and human c‐FMS cis‐regulatory elements are recognised by an overlapping, but non‐identical, set of transcription factors. Despite these differences, chromatin immunoprecipitation experiments show highly similar patterns of histone H3 modification and a similar distribution of chromatin modifying and remodelling activities at individual cis‐regulatory elements and across the c‐FMS locus. Our experiments support the hypothesis that the same regulatory principles operate at both genes via conserved cores of transcription factor binding sites.

Hertoghs, Kirsten M. L.; Ellis, Jonathan H.; Catchpole, Ian R.

doi: 10.1093/nar/gkg801pmid: 14530430

The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non‐sequence‐ specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double‐stranded plasmid DNA in a sequence‐specific manner are described for the first time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) ‘clamps’ for procedures such as particle‐mediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid‐mediated transfection into mammalian cells. LNA ODNs can bind to DNA in a sequence‐specific manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG‐based immune adjuvants to plasmid by ‘hybrid’ phosphorothioate–LNA ODNs induces tumour necrosis factor‐α production in the macrophage cell line RAW264.7. This observation exemplifies an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination.

Sertil, Odeniel; Kapoor, Rachna; Cohen, Brian D.; Abramova, Natalia; Lowry, Charles V.

doi: 10.1093/nar/gkg792pmid: 14530431

Two groups of anaerobic genes (genes induced in anaerobic cells and repressed in aerobic cells) are negatively regulated by heme, a metabolite present only in aerobic cells. Members of both groups, the hypoxic genes and the DAN/TIR/ERG genes, are jointly repressed under aerobic conditions by two factors. One is Rox1, an HMG protein, and the second, originally designated Rox7, is shown here to be Mot3, a global C2H2 zinc finger regulator. Repression of anaerobic genes results from co‐induction of Mot3 and Rox1 in aerobic cells. Repressor synthesis is triggered by heme, which de‐represses a mechanism controlling expression of both MOT3 and ROX1 in anaerobic cells; it includes Hap1, Tup1, Ssn6 and a fourth unidentified factor. The constitutive expression of various anaerobic genes in aerobic rox1Δ or mot3Δ cells directly implies that neither factor can repress by itself at endogenous levels and that stringent aerobic repression results from the concerted action of both. Mot3 and Rox1 are not essential components of a single complex, since each can repress independently in the absence of the other, when artificially induced at high levels. Moreover, the two repression mechanisms appear to be distinct: as shown here repression of ANB1 by Rox1 alone requires Tup1–Ssn6, whereas repression by Mot3 does not. Though artificially high levels of either factor can repress well, the absolute efficiency observed in normal cells when both are present—at much lower levels—demonstrates a novel inhibitory synergy. Evidently, expression levels for the two mutually dependent repressors are calibrated to permit a range of variation in basal aerobic expression at different promoters with differing operator site combinations.

Mendelsohn, Bryce A.; Li, Ai‐min; Vargas, Claudia A.; Riehman, Kristen; Watson, Alice; Fridovich‐Keil, Judith L.

doi: 10.1093/nar/gkg810pmid: 14530432

Scp160p is a multiple KH‐domain RNA‐binding protein in yeast known to associate with polyribosomes as an mRNP component, although its biological role remains unclear. As a genetic approach to examine Scp160p function, we applied an ethyl methanesulfonate (EMS) screen for loci synthetically lethal with scp160 loss, and identified a single candidate gene, EAP1, whose protein product functions in translation as an eIF4E‐binding protein, with additional uncharacterized spindle pole body functions. To reconfirm scp160/eap1 synthetic lethality, we constructed a strain null for both genes, supported by an SCP160 maintenance plasmid. We used this strain to establish a quantitative assay for both Scp160p and Eap1p functions in vivo, and applied this assay to demonstrate that Y109A EAP1, a previously described allele of EAP1 that cannot bind eIF4E, is markedly impaired with regard to its SCP160‐related activity. In addition, we explored the possibility of physical interaction between Eap1p and Scp160p, and discovered that Eap1p associates with Scp160p‐containing complexes in an RNA‐dependent manner. Finally, we probed the impact of EAP1 loss on Scp160p, and vice versa, and found that loss of each gene resulted in a significant change in either the complex associations or subcellular distribution of the other protein. These results clearly support the hypothesis that Scp160p plays a role in translation, demonstrate that the interaction of SCP160 and EAP1 is biologically significant, and provide important tools for future studies of the in vivo functions of both genes.

Li, Shuyi; Takeda, Yoshihiko; Wragg, Stéphanie; Barrett, John; Phillips, Andrew; Dynan, William S.

doi: 10.1093/nar/gkg775pmid: 14530433

The non‐homologous end joining pathway uses pre‐existing proteins to repair DNA double‐strand breaks induced by ionizing radiation. Here we describe manipulation of this pathway in living cells using a newly developed tool. We generated a single chain antibody variable fragment (scFv) that binds to the DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs), a key enzyme in the pathway. In contrast to existing pharmacologic inhibitors, the scFv binds a newly defined regulatory site outside the kinase catalytic domain. Although the scFv inhibits kinase activity only modestly, it completely blocks DNA end joining in a cell‐free system. Microinjection of the scFv sensitizes human cells to radiation, as measured by a reduction in efficiency of colony formation and induction of apoptosis at an otherwise sublethal dose of 1.5 Gy. The scFv blocks non‐homologous end joining in situ at a step subsequent to histone γ‐H2AX focus formation but preceding γ‐H2AX dephosphorylation. Blockage occurs in cells exposed to as little as 0.1 Gy, indicating that DNA‐PKcs is essential for double‐strand break repair even at low radiation doses. The ability to modify the radiation response in situ in living cells provides a link between biochemical, genetic and cytologic approaches to the study of double‐strand break repair intermediates.