Showing papers in "Nucleic Acids Research in 1996"

A New Efficient Gene Disruption Cassette for Repeated Use in Budding Yeast

Abstract: The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.

Reliable Genotyping of Samples with Very Low DNA Quantities Using PCR

Abstract: Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii) that PCR-generated alleles or 'false alleles' will arise. A mathematical model has been developed to account for stochastic events when pipetting template DNA in a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results correlate with the mathematical model. The results of 150 typing experiments are consistent with the proposed model. Based on this model and the level of observed false alleles, an experimental procedure using the multiple tubes approach is proposed to obtain reliable genotypes with a confidence level of 99%. This multiple tubes procedure should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.

Complete Sequence Analysis of the Genome of the Bacterium Mycoplasma Pneumoniae

Abstract: The entire genome of the bacterium Mycoplasma pneumoniae M129 has been sequenced. It has a size of 816,394 base pairs with an average G+C content of 40.0 mol%. We predict 677 open reading frames (ORFs) and 39 genes coding for various RNA species. Of the predicted ORFs, 75.9% showed significant similarity to genes/proteins of other organisms while only 9.9% did not reveal any significant similarity to gene sequences in databases. This permitted us tentatively to assign a functional classification to a large number of ORFs and to deduce the biochemical and physiological properties of this bacterium. The reduction of the genome size of M. pneumoniae during its reductive evolution from ancestral bacteria can be explained by the loss of complete anabolic (e.g. no amino acid synthesis) and metabolic pathways. Therefore, M. pneumoniae depends in nature on an obligate parasitic lifestyle which requires the provision of exogenous essential metabolites. All the major classes of cellular processes and metabolic pathways are briefly described. For a number of activities/functions present in M. pneumoniae according to experimental evidence, the corresponding genes could not be identified by similarity search. For instance we failed to identify genes/proteins involved in motility, chemotaxis and management of oxidative stress.

TRANSFAC: a database on transcription factors and their DNA binding sites.

Abstract: TRANSFAC is a database about eukaryotic transcription regulating DNA sequence elements and the transcription factors binding to and acting through them. This report summarizes the present status of this database and accompanying retrieval tools.

Transfecting Mammalian Cells: Optimization of Critical Parameters Affecting Calcium-Phosphate Precipitate Formation

Abstract: DNA-calcium phosphate co-precipitates arise spontaneously in supersaturated solutions Highly effective precipitates for transfection purposes, however, can be generated only in a very narrow range of physico-chemical conditions that control the initiation and growth of precipitate complexes The concentrations of calcium and phosphate are the main factors influencing characteristics of the precipitate complex, but other parameters, such as temperature, DNA concentration and reaction time are important as well An example for this is the finding that almost all of the soluble DNA in the reaction mix can be bound into an insoluble complex with calcium phosphate in <1 min Extending the reaction time to 20 min results in aggregation and/or growth of particles and reduces the level of expression With improved protocols we gained better reproducibility and higher efficiencies both for transient and for stable transfections Up to 60% of cells stained positive for beta-gal and transient production of secreted proteins was improved 5- to 10-fold over results seen with transfections using standard procedures Similar improvements in efficiency (number of recombinant cell colonies) were observed with stable transfections, using co-transfected marker plasmids for selection Transient expression levels 2 days after DNA transfer and titers obtained from stable cell lines, emerging weeks later, showed strong correlation

Splice Site Prediction in Arabidopsis Thaliana Pre-mRNA by Combining Local and Global Sequence Information

Abstract: Artificial neural networks have been combined with a rule based system to predict intron splice sites in the dicot plant Arabidopsis thaliana. A two step prediction scheme, where a global prediction of the coding potential regulates a cutoff level for a local prediction of splice sites, is refined by rules based on splice site confidence values, prediction scores, coding context and distances between potential splice sites. In this approach, the prediction of splice sites mutually affect each other in a non-local manner. The combined approach drastically reduces the large amount of false positive splice sites normally haunting splice site prediction. An analysis of the errors made by the networks in the first step of the method revealed a previously unknown feature, a frequent T-tract prolongation containing cryptic acceptor sites in the 5' end of exons. The method presented here has been compared with three other approaches, GeneFinder, Gene-Mark and Grail. Overall the method presented here is an order of magnitude better. We show that the new method is able to find a donor site in the coding sequence for the jelly fish Green Fluorescent Protein, exactly at the position that was experimentally observed in A.thaliana transformants. Predictions for alternatively spliced genes are also presented, together with examples of genes from other dicots, monocots and algae. The method has been made available through electronic mail (NetPlantGene@cbs.dtu.dk), or the WWW at http://www.cbs.dtu.dk/NetPlantGene.html

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

Abstract: The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 · 10 ‐6 ) < Deep Vent (2.7 · 10 ‐6 ) < Vent (2.8 · 10 ‐6 ) < Taq (8.0 · 10 ‐6 ) << exo ‐ Pfu and UlTma (~5 · 10 ‐5 ). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2‐3 mM MgSO4 and 100‐300 μM each dNTP and at pH 8.5‐9.1. Under these conditions, the error rate of exo ‐ Pfu was ~40-fold higher (5 · 10 ‐5 ) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased ~2-fold, while the error rate of exo ‐ Pfu increased ~9-fold. An increase in error rate with pH has also been noted for the exonucleasedeficient DNA polymerases Taq and exo ‐ Klenow, suggesting that the parameters which influence replication error rates may be similar in pol I- and α-like polymerases. Finally, the fidelity of ‘long PCR’ DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but ~3‐4-fold higher than the error rate of Pfu DNA polymerase.

XRCC1 Polypeptide Interacts with DNA Polymerase β and Possibly Poly (ADP-Ribose) Polymerase, and DNA Ligase III Is a Novel Molecular ‘Nick-Sensor’ In Vitro

Abstract: The DNA repair proteins XRCC1 and DNA ligase III are physically associated in human cells and directly interact in vitro and in vivo. Here, we demonstrate that XRCC1 is additionally associated with DNA polymerase-beta in human cells and that these polypeptides also directly interact. We also present data suggesting that poly (ADP-ribose) polymerase can interact with XRCC1. Finally, we demonstrate that DNA ligase III shares with poly (ADP-ribose) polymerase the novel function of a molecular DNA nick-sensor, and that the DNA ligase can inhibit activity of the latter polypeptide in vitro. Taken together, these data suggest that the activity of the four polypeptides described above may be co-ordinated in human cells within a single multiprotein complex.

The Ribosomal Database Project (RDP)

Abstract: The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and World Wide Web (WWW)(http://rdpwww.life.uiuc.edu/). The electronic mail and WWW servers provide ribosomal probe checking, screening for possible chimeric rRNA sequences, automated alignment and approximate phylogenetic placement of user-submitted sequences on an existing phylogenetic tree.

SAGA: sequence alignment by genetic algorithm.

Abstract: We describe a new approach to multiple sequence alignment using genetic algorithms and an associated software package called SAGA. The method involves evolving a population of alignments in a quasi evolutionary manner and gradually improving the fitness of the population as measured by an objective function which measures multiple alignment quality. SAGA uses an automatic scheduling scheme to control the usage of 22 different operators for combining alignments or mutating them between generations. When used to optimise the well known sums of pairs objective function, SAGA performs better than some of the widely used alternative packages. This is seen with respect to the ability to achieve an optimal solution and with regard to the accuracy of alignment by comparison with reference alignments based on sequences of known tertiary structure. The general attraction of the approach is the ability to optimise any objective function that one can invent.

MITOMAP: A Human Mitochondrial Genome Database

Abstract: We have developed a comprehensive database (MITOMAP) for the human mitochondrial DNA (mtDNA), the first component of the human genome to be completely sequenced [Anderson et al. (1981) Nature 290, 457-465]. MITOMAP uses the mtDNA sequence as the unifying element for bringing together information on mitochondrial genome structure and function, pathogenic mutations and their clinical characteristics, population associated variation, and gene- gene interactions. As increasingly larger regions of the human genome are sequenced and characterized, the need for integrating such information will grow. Consequently, MITOMAP not only provides a valuable reference for the mitochondrial biologist, it may also provide a model for the development of information storage and retrieval systems for other components of the human genome.

Identification of a Saccharomyces Cerevisiae Ku80 Homologue: Roles in DNA Double Strand Break Rejoining and in Telomeric Maintenance

Abstract: Ku is a heterodimer of polypeptides of approximately 70 and 80 kDa (Ku70 and Ku80, respectively) that binds to DNA ends. Mammalian cells lacking Ku are defective in DNA double-strand break (DSB) repair and in site-specific V(D)J recombination. Here, we describe the identification and characterisation of YKU80, the gene for the Saccharomyces cerevisiae Ku80 homologue. Significantly, we find that YKU80 disruption enhances the radiosensitivity of rad52 mutant strains, suggesting that YKU80 functions in a DNA DSB repair pathway that does not rely on homologous recombination. Indeed, through using an in vivo plasmid rejoining assay, we find that YKU80 plays an essential role in illegitimate recombination events that result in the accurate repair of restriction enzyme generated DSBs. Interestingly, in the absence of YKU80function, residual repair operates through an error-prone pathway that results in recombination between short direct repeat elements. This resembles closely a predominant DSB repair pathway in vertebrates. Together, our data suggest that multiple, evolutionarily conserved mechanisms for DSB repair exist in eukaryotes. Furthermore, they imply that Ku binds to DSBs in vivo and promotes repair both by enhancing accurate DNA end joining and by suppressing alternative error-prone repair pathways. Finally, we report that yku80 mutant yeasts display dramatic telomeric shortening, suggesting that, in addition to recognising DNA damage, Ku also binds to naturally occurring chromosomal ends. These findings raise the possibility that Ku protects chromosomal termini from nucleolytic attack and functions as part of a telomeric length sensing system.

Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation.

Abstract: In 1994 we described a list of approximately 2500 point mutations in the p53 gene of human tumors and cell lines which we had compiled from the published literature and made available electronically through the file server at the EMBL Data Library. This database, updated twice a year, now contains records on 4496 published mutations (July 1995 release) and can be obtained from the EMBL Outstation-the European Bioinformatics Institute (EBI) through the network or on CD-ROM. This report describes the criteria for inclusion of data in this database, a description of the current format and a brief discussion of the current relevance of p53 mutation analysis to clinical and biological questions.

Optimized Codon Usage and Chromophore Mutations Provide Enhanced Sensitivity with the Green Fluorescent Protein

Abstract: The green fluorescent protein (GFP) from Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of cells and organisms. Despite many early successes using this reporter, wild type GFP is suboptimal for most applications due to low fluorescence intensity when excited by blue light (488 nm), a significant lag in the development of fluorescence after protein synthesis, complex photoisomerization of the GFP chromophore and poor expression in many higher eukaryotes. To improve upon these qualities, we have combined a mutant of GFP with a significantly larger extinction coefficient for excitation at 488 nm with a re-engineered GFP gene sequence containing codons preferentially found in highly expressed human proteins. The combination of improved fluorescence intensity and higher expression levels yield an enhanced GFP which provides greater sensitivity in most systems.

Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes.

Abstract: To improve the previous DNA/DNA nearest-neighbor parameters, thermodynamic parameters (deltaH degrees, deltaS degrees and deltaG degrees) of 50 DNA/DNA duplexes were measured. Enthalpy change of a helix initiation factor is also considered though the parameters reported recently did not contain the factor. A helix initiation factor for DNA/DNA duplex determined here was the same as that of RNA/RNA duplex (deltaG degrees(37) = 3.4 kcal/mol). The improved nearest-neighbor parameters reproduced not only these 50 experimental values used here but also 15 other experimental values obtained in different studies. Comparing deltaG degrees(37) values of DNA/DNA nearest-neighbor parameters obtained here with those of RNA/RNA and RNA/DNA, RNA/RNA duplex was generally the most stable of the three kinds of duplexes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA duplexes is sequence dependent.

A modified and improved method for bisulphite based cytosine methylation analysis.

Abstract: Sequencing of bisulphite modified genomic DNA is the most powerful method to determine methylation patterns in chromosomal DNA. In many experimental systems, the amount of material available for analysis is very small which makes it necessary to perform experiments at extreme levels of sensitivity and reproducibility. In this communication, we present an improved modification of the bisulphite based sequencing method. Our strategy is to perform the bisulphite treatment and subsequent PCR steps on material embedded into agarose beads. This prevents loss of DNA during the experimental procedure and ensures an optimal bisulphite reactivity by maintaining the DNA in the single stranded form. The modification improves previously published protocols in that it facilitates the handling of probes and reproducibly reaches a very high level of sensitivity.

Covalent Attachment of Synthetic DNA to Self-Assembled Monolayer Films

Abstract: The covalent attachment of thiol-modified DNA oligomers; to self-assembled monolayer silane films on fused silica and oxidized silicon substrates is described. A heterobifunctional crosslinking molecule bearing both thiol- and amino-reactive moieties was used to tether a DNA oligomer (modified at its terminus with a thiol group) to an aminosilane film formed on silica surfaces. A variety of aminosilanes, crosslinkers and treatment conditions have been tested to identify optimal conditions for DNA immobilization using this approach. The DNA films which result have been characterized using UV spectroscopy, water contact angle measurement, radiolabeling and hybridization methods.

The SWISS-PROT Protein Sequence Data Bank and Its New Supplement TREMBL

Abstract: SWISS-PROT is a curated protein sequence database which strives to provide a high level of annotation (such as the description of the function of a protein, its domain structure, post-translational modifications, variants, etc), a minimal level of redundancy and a high level of integration with other databases. Recent developments of the database include: an increase in the number and scope of model organisms; cross-references to seven additional databases; a variety of new documentation files; the creation of TREMBL, and unannotated supplement to SWISS-PROT. This supplement consists of entries in SWISS-PROT-like format derived from the translation of all coding sequences (CDS) in the EMBL nucleotide sequence database, except CDS already included in SWISS-PROT.

Inhibition of NF-κB DNA Binding by Nitric Oxide

Abstract: It has been suggested that the NF-kappaB transcription factor family may mediate expression of the gene encoding the cytokine-inducible form of nitric oxide synthase (iNOS). To establish if nitric oxide (NO) could in turn affect activity of NF-kappaB, the ability of NO-donor compounds to influence NF-kappaB DNA binding activity in vitro was investigated. NO-donor compounds sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) both inhibited the DNA binding activity of recombinant NF-kappaB p50 and p65 homodimers and of p50-p65 heterodimers. Inhibition of NF-kappaB p50 DNA binding by NO-donor compounds involved modification of the conserved redox-sensitive C62 residue, as a C62S p50 mutant was significantly more resistant to SNP-mediated inactivation. Non-reducing SDS-polyacrylamide gel electrophoresis demonstrated that SNP could inhibit p50 DNA binding by mechanisms other than the formation of intersubunit disulphide bonds involving p50 residue C62. Electrospray ionization mass spectrometry of a synthetic NF-kappaB p5O peptide containing the C62 residue suggested that NO gas can modify C62 by S-nitrosylation. This study indicates that NO-donors can directly inhibit the DNA binding activity of NF-kappaB family proteins, suggesting that cellular NO provides another control mechanism for modulating the expression of NF-kappaB-responsive genes.

APC Gene: Database of Germline and Somatic Mutations in Human Tumors and Cell Lines

Abstract: A database is described in which over 700 mutations in the human APC gene of tumors (colon cancer predominantly) are compiled from the literature. It includes both molecular informations about the mutations and also clinical data about the patients. A software have been designed in order to analyse all these informations in the database.

Identification of a (CUG)n Triplet Repeat RNA-Binding Protein and Its Expression in Myotonic Dystrophy

Abstract: Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease that is associated with a (CTG)n repeat expansion in the 3'-untranslated region of the myotonin protein kinase (Mt-PK) gene. This study reports the isolation and characterization of a (CUG)n triplet repeat pre-mRNA/mRNA binding protein that may play an important role in DM pathogenesis. Two HeLa cell proteins, CUG-BP1 and CUG-BP2, have been purified based upon their ability to bind specifically to (CUG)8 oligonucleotides in vitro. While CUG-BP1 is the major (CUG)8-binding activity in normal cells, nuclear CUG-BP2 binding activity increases in DM cells. Both CUG-BP1 and CUG-BP2 have been identified as isoforms of a novel heterogeneous nuclear ribonucleoprotein (hnRNP), hNab50. The CUG-BP/hNab50 protein is localized predominantly in the nucleus and is associated with polyadenylated RNAs in vivo. In vitro RNA-binding/photocrosslinking studies demonstrate that CUG-BP/hNab50 binds to RNAs containing the Mt-PK 3'-UTR. We propose that the (CUG)n repeat region in Mt-PK mRNA is a binding site for CUG-BP/hNab50 in vivo, and triplet repeat expansion leads to sequestration of this hnRNP on mutant Mt-PK transcripts.

Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers.

Abstract: Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.

PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR.

Abstract: PCR has become a powerful tool for genetic analysis and many applications for gene sequence quantitation are based on this technology ( 1–3). Standardized reaction conditions require accurate quantitation of input DNA as well as optimization of chemical and cycling parameters. In this study we discuss PicoGreen (Molecular Probes, Eugene, OR) fluorescence enhancement as a useful assay for template DNA quantitation and PCR product formation. Spectrophotometry is the principal method for evaluating quantity and quality of nucleic acids. In aqueous solution, DNA has maximal absorbance near 260 nm with an extinction coefficient of 50; protein absorbs light strongly near 280 nm. The concentration of a sample can be read directly (in μg/μl) by diluting it 1:20 in water or buffer; a practical lower limit of detection is 50–100 ng DNA in a 50–100 μl microcuvette. The A260/A280 ratio provides an estimate of DNA purity; values of 1.7–2.0 predict ‘clean DNA’. However, single-stranded DNA, RNA, PCR primers and dNTPs, or aromatic organic compounds such as phenol interfere by absorbing light in this range. Fixed tissue samples with substantial protein crosslinking and DNA preparations containing added enzymes or protein stabilizers are difficult to evaluate spectrophotometrically ( 4). Intercalating fluorochromes, such as ethidium bromide or Hoechst 33258, selectively bind to dsDNA. The sensitivity of Hoechst 33258 is ∼25 ng of DNA per assay, but preferential association with domains of high A–T content or reduced binding to DNA fragments <500 bp may result in skewed analysis ( 5). Accurate evaluation may require sophisticated or dedicated equipment since both dyes photobleach easily and fluorescence enhancement of DNA binding is low, leading to high background readings. These compounds are carcinogenic and pose handling and disposal problems. Electrophoretic array is the most common means of evaluating molecular distribution of both simple and complex DNA samples. When stained with ethidium bromide, transillumination with 254 nm UV light permits CCD camera visualization of a single agarose gel band containing ∼5 ng or a polydisperse sample containing 25–50 ng of dsDNA. SYBR-Green I  (Molecular Probes, Eugene, OR) is a proprietary fluorescent dsDNA-specific stain that has an emission peak at 520 nm following excitation at 254 or 497 nm. Image collection and analysis with 254 nm transillumination requires the use of an optical quality band-pass filter to eliminate infrared interference. SYBR-Green I is more sensitive than ethidium bromide with a limit detection of ∼50 pg per band or ∼250 pg per lane polydisperse dsDNA. Argon la er-activated gel scanning or capillary electrophoresis is more sensitive ( 6), but far more costly. Gel analysis allows evaluation of genomic DNA integrity, completeness of restriction endonuclease digestion and quantity of late cycle PCR products. However, this method is impractical for routine or high throughput DNA quantitation ( 7). PicoGreen is a fluorochrome that selectively binds dsDNA and has characteristics similar to that of SYBR-Green I. It has an excitation maximum at 480 nm (lesser peaks in the short-wave UV range) and an emission peak at 520 nm. When bound to dsDNA, fluorescence enhancement of PicoGreen is exceptionally high; little background occurs since the unbound dye has virtually no fluorescence. PicoGreen is very stable to photobleaching, allowing longer exposure times and assay flexibility. However, the molecular structure of the dye is proprietary and the mode of binding is not fully characterized, so potential handling hazard is unknown. We evaluated PicoGreen for quantitation of multiple DNA sample types. We examined the linearity of binding and the effective detection range for different species of ‘high molecular weight’ DNA standards (human placental, calf thymus and λ phage; with or without restriction digestion) and DNA isolated from a variety of tissue types preserved under different protocols. We also assayed ‘low molecular weight’ dsDNA (∼150 bp PCR products) in the presence or absence of reaction primers, dNTPs and Taq polymerase. Oligonucleotide primers were evaluated for interference with quantitation in some samples. Control DNAs were from commercial sources. EcoRI (New England Biolabs, Beverly, MA) digests were performed with 5 U per sample in a 10 μl reaction mix at 37 C for 2 h. We obtained sample DNA by organic extraction from flash-frozen or paraformaldehyde-fixed paraffin-embedded surgical remainder tissues. PCR mixtures contained 0.2 μM each primer, 50 μM each dNTP, 0.02 U/ μl AmpliTaq polymerase (Perkin-Elmer Corp., Wilton, CT) and TaqStart MAb (Clontech, Palo Alto, CA). Primers were removed from PCR reactions with Microcon 30 (Amicon, Beverly, MA). The A260/A280 of each sample was read against a TE blank in a Lambda-2 Spectrophotometer (Perkin Elmer Corp., Norwalk, CT), fitted with a 100 μl quartz microcuvette. DNA samples were diluted 1 μl into 100 μl of TE. A reading of 0.020, the lower confidence level of the instrument, represented 100 ng of DNA

Sequence Analysis and Characterization of Stutter Products at the Tetranucleotide Repeat Locus VWA

Abstract: The PCR amplification of tetranucleotide short tandem repeat (STR) loci typically produces a minor product band 4 bp shorter than the corresponding main allele band; this is referred to as the stutter band. Sequence analysis of the main and stutter bands for two sample alleles of the STR locus vWA reveals that the stutter band lacks one repeat unit relative to the main allele. Sequencing results also indicate that the number and location of the different 4 bp repeat units vary between samples containing a typical verses low proportion of stutter product. The results also suggest that the proportion of stutter product relative to the main allele increases as the number of uninterrupted core repeat units increases. The sequence analysis and results obtained using various DNA polymerases appear to support the slipped strand displacement model as a potential explanation for how these stutter products are generated.

DNA damage and DNA sequence retrieval from ancient tissues.

Abstract: Gas chromatography/mass spectrometry (GC/MS) was used to determine the amounts of eight oxidative base modifications in DNA extracted from 11 specimens of bones and soft tissues, ranging in age from 40 to >50 000 years. Among the compounds assayed hydantoin derivatives of pyrimidines were quantitatively dominant. From five of the specimens endogenous ancient DNA sequences could be amplified by PCR. The DNA from these specimens contained substantially lower amounts of hydantoins than the six specimens from which no DNA could be amplified. Other types of damage, e.g. oxidation products of purines, did not correlate with the inability to retrieve DNA sequences. Furthermore, all samples with low amounts of damage and from which DNA could be amplified stemmed from regions where low temperatures have prevailed throughout the burial period of the specimens.

RNA Aptamers That Bind l-Arginine with Sub-Micromolar Dissociation Constants and High Enantioselectivity

Abstract: A completely randomized RNA pool as well as a degenerate pool comprised of an RNA sequence which binds citrulline with a dissociation constant of 0 muM were used to select for tight binding arginine specific RNA aptamers. A modified in vitro selection scheme, based on affinity chromatography was applied to allow the enrichment of high affinity solution binders. The selection scheme included a negative selection with the non-cognate ligand citrulline, and a heat denaturation step prior to affinity elution with an excess of the cognate ligand arginine. After 20 cycles the majority of the pools bound specifically to the arginine matrix even after denaturation/renaturation in the presence of 20 mM of a non-cognate amino acid. When denatured and eluted in the presence of 20 mM arginine, the selected RNAs quantitatively washed off the column. These RNA aptamers were cloned and sequenced. Equilibrium dialysis performed with the most abundant clone among the selected sequence revealed Kd values of 330 nM for the RNA/arginine affinity, which is nearly a 200-fold improvement over the tightest binding arginine binding RNAs known to date. Arginine recognition by this RNA is highly enantioselectice: L- arginine is bound 12 000-fold better than D-arginine. Chemical modification analysis revealed that the secondary structure of the aptamer might contain a pseudoknot motif. Our tight binding arginine aptamers join a number of natural and in vitro selected RNAs which recognize arginine. The RNAs described here compare in their binding affinity with the tightest binding RNA aptamers for low molecular weight molecules isolated in other in vitro selection experiments.

The Helix-Hairpin-Helix DNA-Binding Motif: A Structural Basis for Non-Sequence-Specific Recognition of DNA

Abstract: One, two or four copies of the 'helix-hairpin-helix' (HhH) DNA-binding motif are predicted to occur in 14 homologous families of proteins. The predicted DNA-binding function of this motif is shown to be consistent with the crystallographic structure of rat polymerase beta, complexed with DNA template-primer [Pelletier, H., Sawaya, M.R., Kumar, A., Wilson, S.H. and Kraut, J. (1994) Science 264, 1891-1903] and with biochemical data. Five crystal structures of predicted HhH motifs are currently known: two from rat pol beta and one each in endonuclease III, AlkA and the 5' nuclease domain of Taq pol I. These motifs are more structurally similar to each other than to any other structure in current databases, including helix-turn-helix motifs. The clustering of the five HhH structures separately from other bi-helical structures in searches indicates that all members of the 14 families of proteins described herein possess similar HhH structures. By analogy with the rat pol beta structure, it is suggested that each of these HhH motifs bind DNA in a non-sequence-specific manner, via the formation of hydrogen bonds between protein backbone nitrogens and DNA phosphate groups. This type of interaction contrasts with the sequence-specific interactions of other motifs, including helix-turn-helix structures. Additional evidence is provided that alphaherpesvirus virion host shutoff proteins are members of the polymerase I 5'-nuclease and FEN1-like endonuclease gene family, and that a novel HhH-containing DNA-binding domain occurs in the kinesin-like molecule nod, and in other proteins such as cnjB, emb-5 and SPT6.

Chip PCR. I. Surface Passivation of Microfabricated Silicon-Glass Chips for PCR

Abstract: The microreaction volumes of PCR chips (a microfabricated silicon chip bonded to a piece of flat glass to form a PCR reaction chamber) create a relatively high surface to volume ratio that increases the significance of the surface chemistry in the polymerase chain reaction (PCR). We investigated several surface passivations in an attempt to identify 'PCR friendly' surfaces and used those surfaces to obtain amplifications comparable with those obtained in conventional PCR amplification systems using polyethylene tubes. Surface passivations by a silanization procedure followed by a coating of a selected protein or polynucleotide and the deposition of a nitride or oxide layer onto the silicon surface were investigated. Native silicon was found to be an inhibitor of PCR and amplification in an untreated PCR chip (i.e. native slicon) had a high failure rate. A silicon nitride (Si(3)N(4) reaction surface also resulted in consistent inhibition of PCR. Passivating the PCR chip using a silanizing agent followed by a polymer treatment resulted in good amplification. However, amplification yields were inconsistent and were not always comparable with PCR in a conventional tube. An oxidized silicon (SiO(2) surface gave consistent amplifications comparable with reactions performed in a conventional PCR tube.

Inducible site-directed recombination in mouse embryonic stem cells.

Abstract: The site-directed recombinase Cre can be employed to delete or express genes in cell lines or animals. Clearly, the ability to control remotely the activity of this enzyme would be highly desirable. To this end we have constructed expression vectors for fusion proteins consisting of the Cre recombinase and a mutated hormone-binding domain of the murine oestrogen receptor. The latter still binds the anti-oestrogen drug tamoxifen but no longer 17 beta-oestradiol. We show here that in embryonic stem cells expressing such fusion proteins, tamoxifen can efficiently induce Cre-mediated recombination, thereby activating a stably integrated LacZ reporter gene. In the presence of either 10 microM tamoxifen or 800 nM 4-hydroxy-tamoxifen, recombination of the LacZ gene is complete within 3-4 days. By placing a tamoxifen-binding domain on both ends of the Cre protein, the enzymatic activity of Cre can be even more tightly controlled. Transgenic mice expressing such an tamoxifen-inducible Cre enzyme may thus provide a new and useful genetic tool to mutate or delete genes at specific times during development or in adult animals.

A Quantitative Map of Nucleotide Substitution Rates in Bacterial rRNA

Abstract: A recently developed method for estimating the variability of nucleotide sites in a sequence alignment [Van de Peer, Y., Van der Auwera, G. and De Wachter, R. (1996) J. Mol. Evol. 42, 201-210] was applied to bacterial 16S, 5S and 23S rRNAs. In this method, the variability of each nucleotide site is defined as its evolutionary rate relative to the average evolutionary rate of all the nucleotide sites of the molecule. Spectra of evolutionary rates were calculated for each rRNA and show the fastest evolving sites substituting at rates more than 1000 times that of the slowest ones. Variability maps are presented for each rRNA, consisting of secondary structure models where the variability of each nucleotide site is indicated by means of a colored dot. The maps can be interpreted in terms of higher order structure, function and evolution of the molecules and facilitate the selection of areas suitable for the design of PCR primers and hybridization probes. Variability measurement is also important for the precise estimation of evolutionary distances and the inference of phylogenetic trees.