Localization of the choline acetyltransferase (CHAT) gene to human chromosome 10

A cDNA clone encoding the complete sequence of porcine choline acetyltransferase (CHAT) isolated by S. Berrard et al. (1987, Proc. Natl. Acad. Sci. USA 84: 9280-9284) was hybridized to TaqI digests of a panel of 25 human-rodent somatic cell hybrids and to a complementary panel of 10 human-rodent hybrids in order to determine the chromosomal localization of human CHAT. To enhance the detection of the human signal, hybridization and washings were performed under low stringency conditions on membranes presaturated with sonicated DNA from parental rodent strains. All informative human fragments had the same distribution among the hybrids, mapping CHAT to a single human chromosome. CHAT was assigned to chromosome 10 because all other chromosomes were eliminated by exclusion based on the analysis of the signal segregation. This result indicates that mutation of the CHAT gene cannot be responsible for the primary defect in familial Alzheimer's disease.     

G-quadruplex-generating polymerase chain reaction for visual colorimetric detection of amplicons

We have developed a self-reporting polymerase chain reaction (PCR) system for visual colorimetric gene detection and distinction of single nucleotide polymorphisms (SNPs). Amplification is performed using target-specific primers modified with a 5′-end tail that is complementary to a G-quadruplex deoxyribozyme-forming sequence. At end-point, G-quadruplexes are forced to fold from PCR-generated duplex DNA and then are used to colorimetrically report the successful occurrence of PCR by assaying their peroxidase activity using a chromogenic substrate. Furthermore, primer design considerations for the G-quadruplex-generating PCR system have allowed us to visually distinguish SNPs associated with Mycobacterium tuberculosis drug resistance alleles.     

RecA-mediated strand invasion of DNA by oligonucleotides substituted with 2-aminoadenine and 2-thiothymine

Sequence-specific recognition of DNA is a critical step in gene targeting. Here we describe unique oligonucleotide (ON) hybrids that can stably pair to both strands of a linear DNA target in a RecA-dependent reaction with ATP or ATPγS. One strand of the hybrids is a 30-mer DNA ON that contains a 15-nt-long A/T-rich central core. The core sequence, which is substituted with 2-aminoadenine and 2-thiothymine, is weakly hybridized to complementary locked nucleic acid or 2′-OMe RNA ONs that are also substituted with the same base analogs. Robust targeting reactions took place in the presence of ATPγS and generated metastable double D-loop joints. Since the hybrids had pseudocomplementary character, the component ONs hybridized less strongly to each other than to complementary target DNA sequences composed of regular bases. This difference in pairing strength promoted the formation of joints capable of accommodating a single mismatch. If similar joints can form in vivo, virtually any A/T-rich site in genomic DNA could be selectively targeted. By designing the constructs so that the DNA ON is mismatched to its complementary sequence in DNA, joint formation might allow the ON to function as a template for targeted point mutation and gene correction.     

Dipstick-type biosensor for visual detection of DNA with oligonucleotide-decorated colored polystyrene microspheres as reporters

In recent years, there is a continuously growing interest in the development of biosensors for rapid, simple and inexpensive DNA tests suitable for the small laboratory or for on-site testing. Detection is accomplished through electrochemical, optical or gravimetric transduction. We report on the development of disposable dipstick-type DNA biosensors that employ oligonucleotide-decorated colored polystyrene microspheres as reporters and enable visual detection of DNA sequences without the use of instrumentation. The biosensors have been designed to detect DNA molecules that contain both, a biotin moiety and a segment that is complementary to the oligonucleotide attached on the surface of blue or red microspheres. Capture of the hybrids by immobilized streptavidin at the test zone results in the formation of a colored line. The biosensors were applied to: (a) detection of single-stranded DNA, (b) detection of PCR-amplified double-stranded DNA and (c) genotyping of single nucleotide polymorphisms (SNP). The results were compared with sensors based on gold nanoparticle reporters. It is also demonstrated that the microspheres offer the potential for multicolor detection of specific DNA sequences.     

A dynamic evolution of chromosome in subgenus Potamogeton revealed by physical mapping of rDNA loci detection

In this study, we analyzed 12 species in the subgenus Potamogeton (8 tetraploids and 2 diploids, 2 putative tetraploid hybrids) at the chromosomal level, including counting the chromosome number and physically mapping the rDNA. The extent of variation in the chromosome number and rDNA loci was determined in the Potamogeton species on both the inter- and intra-specific level. Moreover, one of the hybrid sets (P. perfoliatus ♂?×?P. wrightii ♀) was picked for performing artificial pollination for producing F1 generation adults and for sequential morphological character analysis and FISH detection. After comparing the parental species and natural hybrids from three different geographic locations, the intraspecific variations of rDNA loci were revealed in sampled plants; in addition, comparisons between artificial F1 and natural hybrids (P.?×?intortusifolius) showed a rapid change of 45S rDNA loci in response to the interspecific hybridization. We also compared our results concerning rDNA patterns with phylogenetic documents to derive complementary clues to karoytype evolution and interspecific relationships. Based on frequent hybridizations and active clonal reproduction with weak genetic selection, the rDNA chromosomal repatterning, such as the gain or loss of rDNA loci together with rDNA movements, might be one trend of chromosome evolution in this genus.     

Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence

Amplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chain-reaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA–DNA and DNA–RNA hybrids increased by up to 8°C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands.     

Attomole DNA detection assay via rolling circle amplification and single molecule detection

A bead-based assay was developed for highly sensitive single molecule DNA detection. Rolling circle amplification (RCA), an isothermal amplification technique that creates tandem repeated sequences, was used in combination with a fluorescent complementary DNA to create dense clusters of fluorescence. These clusters, each corresponding to a single target molecule, can be detected unambiguously due to their high signal/noise ratios. The limit of detection of this assay is approximately 1 amol. This simple single molecule assay allows high detection sensitivity without the use of complex equipment.     

Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster.

In situ hybridization of ¹²⁵I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ. This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead.     

Coevolution of visual signals and eye morphology in Polistes paper wasps

To be effective, signals must propagate through the environment and be detected by receivers. As a result, signal form evolves in response to both the constraints imposed by the transmission environment and receiver perceptual abilities. Little work has examined the extent to which signals may act as selective forces on receiver sensory systems to improve the efficacy of communication. If receivers benefit from accurate signal assessment, selection could favour sensory organs that improve discrimination of established signals. Here, we provide evidence that visual resolution coevolves with visual signals in Polistes wasps. Multiple Polistes species have variable facial patterns that function as social signals, whereas other species lack visual signals. Analysis of 19 Polistes species shows that maximum eye facet size is positively associated with both eye size and presence of visual signals. Relatively larger facets within the eye''s acute zone improve resolution of small images, such as wasp facial signals. Therefore, sensory systems may evolve to optimize signal assessment. Sensory adaptations to facilitate signal detection may represent an overlooked area of the evolution of animal communication.     

Extent of cross-fertilization in Orobanche cumana Wallr.

Sunflower broomrape (Orobanche cumana Wallr.) is considered a self-fertilizing species, but there is no indication as to whether it is strictly self-fertilized or that it presents some extent of cross-fertilization. The objective of this research was to measure the rate of cross-fertilization in O. cumana using an unpigmented recessive mutant as a visual marker. A pot and a field experiment in which single unpigmented plants were surrounded by a large number of pigmented plants were conducted. Occurrence of F₁ hybrids, readily distinguishable from unpigmented plants in the progenies of unpigmented plants provided a direct measurement of the cross-fertilization rate. Progenies of unpigmented plants contained 21.5 % of F₁ hybrids in the pot experiment and 28.8 % in the field experiment. The results revealed that O. cumana is a partially allogamous species, which has great relevance for understanding the genetic structure and dynamics of populations and, ultimately, race evolution in this parasitic plant.     

DNA biosensor based on hybridization refractory mutation system approach for single mismatch detection

We report on a simple approach to enhance solid-phase hybridization-based single base mismatch discrimination at high ionic strength based on the deliberate insertion of a natural DNA base mismatch in the surface-tethered probe. A large drop in hybridization signal of single base mismatched alleles using the designed probe as compared with the conventional probe, from 80% to less than 25% of the signal obtained with the fully complementary, non-mutation-containing sequence, when using colorimetric detection was further improved to 20% when using electrochemical detection, attributable to a difference of spacing of immobilized probes. Finally, the designed probe was used for the electrochemical detection of the DQA1*05:05 allele amplified from real human blood samples.     

A new approach to the isolation of RNA-DNA hybrids and its application to the quantitative determination of labeled tumor virus RNA

A procedure has been developed by which the hybrid formed between a labeled RNA and complementary DNA can be selectively separated from all other single and double-stranded nucleic acids. We describe the application of this procedure to the quantitative determination of labeled avian tumor virus RNA. Purified DNA complementary to avian myeloblastosis virus RNA is extended at its 3′ terminus with 40 to 60 dCMP residues, using terminal deoxynucleotidyl-transferase. The elongated DNA is annealed with the labeled nucleic acid preparation and the mixture is passed through a column of Sephadex to which poly(I) has been covalently bound. The poly(I) retains the specific RNA-DNA hybrids by virtue of their poly(C) extension. The column is washed with RNAase to degrade nonhybridized RNA, the RNA retained on the column is eluted with formamide and its radioactivity is determined. The background hybridization was reduced to 0.003 to 0.008% by addition of oligo(C)_5.20 to the hybridization mixture and by carrying out the adsorption to the poly(I)-Sephadex column in the presence of poly(U). The hybridization efficiency was about 50%. The content of radioactive Rous sarcoma virus-specific RNA was determined in infected and uninfected cells after labeling with [³H]uridine for two hours. The content of labeled virus-specific RNA in infected cells was 0.6 to 0.9% and 0.05% in uninfected cells. The value found for monkey cell RNA was 0.009%. This method can be used for the detection of hybrids between labeled RNA and complementary DNAs too short to allow quantitation by conventional methods. If the RNAase step is omitted the procedure can be used for the isolation of any RNA for which a complementary DNA is available, as well as for its precursor.     

Nanographite‐based fluorescent biosensor for detecting microRNA using duplex‐specific nuclease‐assisted recycling

The development of a nanographite (NG)‐based fluorescent biosensor for detecting microRNA (miRNA) is reported. Duplex‐specific nuclease (DSN)‐assisted signal amplification was key to its function. In the absence of a target, with the assistance of p‐stacking interactions, the NG adsorbed the double carboxyfluorescein (FAM)‐labelled probe (DFP) whose surface was perfectly complementary to miRNA, leading to quenching of FAM fluorescence. In the presence of a target, double‐stranded DNA/RNA hybrids were repelled by the NG and fluorescence was restored. Meanwhile, the considerable increase in signal strength and sensitivity suggests DSN‐mediated target recycling as an application. The detection limit of the proposed biosensor for miRNA was 10 pmol/L; there was a linear correlation when the miRNA concentration ranged from 50 pmol/L to 5 nmol/L. Additionally, the method could distinguish let‐7b from most let‐7 miRNA family members and was successfully used in a sample assay. This biosensor is a novel and highly sensitive tool for miRNA detection and has great potential for biochemical research, disease diagnosis, and therapy.     

Study of markers of human erythroid differentiation in hybrid cells.

Somatic cell hybrids exhibiting co-expression of the globin genes of two species were generated by fusion of mouse erythroleukemia cells with Chinese hamster or human marrow erythroid cells. In contrast, extinction of the mouse globin genes occurred in hybrids formed between the erythroleukemia cells and human fibroblasts. Direct detection of the human globin genes in human X mouse fibroblast hybrids was achieved by annealing of DNA from these cells to human globin complementary DNA. This method was developed to permit the chromosomal assignment of the human globin genes.     

Cloned riboprobe for detection of a mycoplasmalike organism

A [32P]-labeled single stranded-RNA probe (riboprobe) was constructed with plasmid vector pSP64 and used to detect and specifically identify an uncultured pathogenic mycoplasmalike organism in infected host. The riboprobe was more sensitive and reliable than complementary double stranded-DNA probe in detection of western X mycoplasmalike organism. When concentration of a double stranded-DNA probe was increased, nonspecific hybridization signal was observed with nucleic acid from healthy plants and from plants infected by other mycoplasmalike organisms. In contrast, sensitivity of detection with the complementary riboprobe was increased at elevated probe concentrations without nonspecific hybridization.     

High throughput analysis of grape genetic diversity as a tool for germplasm collection management

Using 20 SSR markers well scattered across the 19 grape chromosomes, we analyzed 4,370 accessions of the INRA grape repository at Vassal, mostly cultivars of Vitis vinifera subsp. sativa (3,727), but also accessions of V. vinifera subsp. sylvestris (80), interspecific hybrids (364), and rootstocks (199). The analysis revealed 2,836 SSR single profiles: 2,323 sativa cultivars, 72 wild individuals (sylvestris), 306 interspecific hybrids, and 135 rootstocks, corresponding to 2,739 different cultivars in all. A total of 524 alleles were detected, with a mean of 26.20 alleles per locus. For the 2,323 cultivars of V. vinifera, 338 alleles were detected with a mean of 16.9 alleles per locus. The mean genetic diversity (GDI) was 0.797 and the level of heterozygosity was 0.76, with broad variation from 0.20 to 1. Interspecific hybrids and rootstocks were more heterozygous and more diverse (GDI?=?0.839 and 0.865, respectively) than V. vinifera cultivars (GDI?=?0.769), Vitis vinifera subsp. sylvestris being the least divergent with GDI?=?0.708. Principal coordinates analysis distinguished the four groups. Slight clonal polymorphism was detected. The limit between clonal variation and cultivar polymorphism was set at four allelic differences out of 40. SSR markers were useful as a complementary tool to traditional ampelography for cultivar identification. Finally, a set of nine SSR markers was defined that was sufficient to distinguish 99.8% of the analyzed accessions. This set is suitable for routine characterization and will be valuable for germplasm management.     

Enhanced detection of staphylococcal genomes in positive blood cultures using a polymeric enzyme complex

This article describes a simple and inexpensive signal amplification method, termed polymeric enzyme detection (PED), which permits rapid and sensitive detection of conserved sequences in the tuf gene that identify Staphylococcus genus, conserved sequences in the femB gene that specifically detect Staphylococcus aureus species, and the methicillin resistance gene mecA directly from positive blood culture bottles. Microbe-specific capture probes were immobilized onto microtiter plates or silicon chips. Target sequences and biotin-labeled, target-specific probes were hybridized to complementary capture probes to create a biotin-labeled, surface-immobilized tripartite complex. In a two-step process, signal was amplified by incubating the surface-immobilized biotin with streptavidin followed by the addition of a 500-kDa dextran polymer conjugated with approximately 80 biotins. Signal was then developed by binding of a streptavidin-horseradish peroxidase conjugate followed by incubation with the substrate tetramethylbenzidine. Use of the PED method improved the lower limit of detection 10- to 100-fold in model DNA hybridization assays with limits of detection as low as 1 fmol/L target DNA. This level of sensitivity permits detection of genomic DNA from methicillin-resistant S. aureus positive blood cultures within 25 to 35 min using either a thin film biosensor chip or a microtiter plate-based assay.     

Taxonomic Identification of Mediterranean Pines and Their Hybrids Based on the High Resolution Melting (HRM) and trnL Approaches: From Cytoplasmic Inheritance to Timber Tracing

Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples.     

The role of attention in figure-ground segregation in areas V1 and V4 of the visual cortex

Our visual system segments images into objects and background. Figure-ground segregation relies on the detection of feature discontinuities that signal boundaries between the figures and the background and on a complementary region-filling process that groups together image regions with similar features. The neuronal mechanisms for these processes are not well understood and it is unknown how they depend on visual attention. We measured neuronal activity in V1 and V4 in a task where monkeys either made an eye movement to texture-defined figures or ignored them. V1 activity predicted the timing and the direction of the saccade if the figures were task relevant. We found that boundary detection is an early process that depends little on attention, whereas region filling occurs later and is facilitated by visual attention, which acts in an object-based manner. Our findings are explained by a model with local, bottom-up computations for boundary detection and feedback processing for region filling.     

Rapid identification and differentiation of Saccharomyces cerevisiae, Saccharomyces bayanus and their hybrids by multiplex PCR

AIMS: To develop a multiplex PCR assay for the specific identification and differentiation of Saccharomyces cerevisiae, S. bayanus and their hybrids. METHODS AND RESULTS: Two sets of primers with sequences complementary to the region YBR033w were used. A single amplicon of 1710 bp or 329 bp was obtained with species S. cerevisiae and S. bayanus, respectively, while the presence of both bands was observed in S. pastorianus because of its hybrid nature. Both amplification products were also obtained after amplification from DNA of several laboratory S. cerevisiae x S. bayanus hybrid strains. CONCLUSIONS: Multiplex PCR was optimized for the rapid and reliable identification of S. cerevisiae, S. bayanus and their hybrids. SIGNIFICANCE AND IMPACT OF THE STUDY: The procedure may be used for routine detection of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes, overcoming the problems of conventional techniques.