DNA-based System Research Articles

Dye-sensitized biosystem sensing using macroporous semiconducting metal oxide films

Transparent semiconducting oxide (TiO2, SnO2 and ZnO) films having highly interconnected pores in the range of 30–200 nm were fabricated in one-pot using polystyrene-block-poly(ethylene oxide) (PS-b-PEO) diblock copolymer. Such porous structures were helpful for efficient transport of bulky guest species such as proteins. Biosensing protocols were constructed in the films by using bulky biomolecules, namely, adsorbed DNA, adsorbed antibody and adsorbed antigen–antibody (immunoassay). In the DNA-based system, the macroporous TiO2 film was the most useful among porous anatase films such as mesoporous and mesoporous/PS beads derived macroporous ones, leading to significant detection of 125 times higher photocurrent generated from a dye (Cy5) attached to DNA than those observed for the others. Macroporous SnO2 and ZnO films were also available for the construction of similar biomolecule immobilized electrodes. Extremely complicated immunoassay reactions involving bulky proteins (antigen, antibody, etc.) were also established using the macroporous TiO2 and SnO2 films. Especially photocurrent for electron transfer from Cy5 attached to the second antibody was successfully detected by using the SnO2 film for exceptionally selective sensing applications.

GENETIC VARIATION ANALYSIS OF THE NEWLY SYNTHESIZED ALLOTETRAPLOIDS CUCUMIS HYTIVUS USING RETROTRANSPOSON-BASED SSAP MARKERS

Ty1-copia-like retrotransposons have been found to comprise the most common class of transposable elements in plant and occur in high copy number in plant genomes. The dispersion, ubiquity and prevalence of Ty1-copia retrotransposons in plant genomes potentially provide an excellent system for developing a multiplex DNA-based marker system. Sequence-specific amplification polymorphism (SSAP) system is the most popular and displayed a higher level of polymorphism than that of AFLPs in cucumber. In this study, we studied the genetic variation of the early generations of the synthetic allotetraploids Cucumis hytivus using DNA-SSAP marker systems in Cucumis. The results showed that allopolyploid formation was associated with rapid and extensive genomic changes. Most genomic changes involved loss of parental restriction fragments and appearance of novel fragments. In addition, the variation of the allotetraploids early generations was a rapid process, the loss of , parental fragments and the gaining of novel fragments were mainly occurred in F 1 hybrid, the S 1 and S 2 generations, and then stabilized.

Preliminary clinical study using a multiplex real-time PCR test for the detection of bacterial and fungal DNA directly in blood

Early diagnosis of sepsis, rapid identification of the causative pathogen(s) and prompt initiation of appropriate antibiotic treatment have a combined impact on mortality due to sepsis. In this observational study, a new DNA-based system (LightCycler SeptiFast (LC-SF) test; Roche Diagnostics) allowing detection of 16 pathogens at the species level and four groups of pathogens at the genus level has been evaluated and compared with conventional blood cultures (BCs). One hundred BC and LC-SF results were obtained for 72 patients admitted to the intensive-care unit over a 6-month period for suspected sepsis. Microbiological data were compared with other biological parameters and with clinical data. The positivity rate of BCs for bacteraemia/fungaemia was 10%, whereas the LC-SF test allowed detection of DNA in 15% of cases. The LC-SF performance, based on its clinical relevance, was as follows: sensitivity, 78%; specificity, 99%; -positive predictive value, 93%; and negative predictive value, 95%. Management was changed for four of eight (50%) of the patients because organisms were detected by the LC-SF test but not by BC. LC-SF results were obtained in 7–15 h, in contrast to the 24–72 h required for BC. According to the LC-SF results, initial therapy was inadequate in eight patients, and antibiotic treatment was changed. Our results suggest that the LC-SF test may be a valuable complementary tool in the management of patients with clinically suspected sepsis.

A DNA-based method for detection ofPeridermium harknessii, the causal agent of western gall rust

Peridermium harknessii is not present in the southern hemisphere yet it poses a serious threat to Pinus radiata cultivation in exotic forest plantations there. If a suspected incursion were to occur, it would be necessary to rapidly confirm the presence of P. harknessii within non-sporulating galls. With this in mind, we have developed a DNA-based identification system, using the first intergenic spacer region (IGS-1), that is able to detect the presence of the pathogen within galled tissue. The PCR primers are highly specific and with the exception of the closest relative, Cronartium quercuum f. sp. banksianae, they did not cross react with any of the 11 species within the closely related genera Cronartium and Peridermium that were tested. Phylogenetic analysis of the IGS-1 region confirmed that C. quercuum f. sp. banksianae is the closest relative to P. harknessii. The PCR primers and protocol reported here should prove useful in the event of a suspected western gall rust disease outbreak in exotic P. radiata plantations.

Preface

Preface

Reversible Bridge-Mediated Excited-State Symmetry Breaking in Stilbene-Linked DNA Dumbbells

The excited-state behavior of synthetic DNA dumbbells possessing stilbenedicarboxamide (Sa) linkers separated by short A-tracts or alternating A-T base-pair sequences has been investigated by means of fluorescence and transient absorption spectroscopy. Electronic excitation of the Sa chromophores results in conversion of a locally excited state to a charge-separated state in which one Sa is reduced and the other is oxidized. This symmetry-breaking process occurs exclusively via a multistep mechanism-hole injection followed by hole transport and hole trapping-even at short distances. Rate constants for charge separation are strongly distance-dependent at short distances but become less so at longer distances. Disruption of the A-tract by inversion of a single A-T base pair results in a pronounced decrease in both the rate constant and efficiency of charge separation. Hole trapping by Sa is highly reversible, resulting in rapid charge recombination that occurs via the reverse of the charge separation process: hole detrapping, hole transport, and charge return to regenerate the locally excited Sa singlet state. These results differ in several significant respects from those previously reported for guanine or stilbenediether as hole traps. Neither charge separation nor charge recombination occur via a single-step superexchange mechanism, and hole trapping is slower and detrapping faster when Sa serves as the electron donor. Both the occurrence of symmetry breaking and reversible hole trapping by a shallow trap in a DNA-based system are without precedent.

Mapping of a Quantitative Trait Locus for Beef Marbling on Bovine Chromosome 9 in Purebred Japanese Black Cattle

The goal of this study is to detect quantitative trait loci (QTL) for carcass traits applicable for a DNA-based breeding system in a Japanese Black cattle population. A purebred paternal half-sib family from a commercial line composed of 65 steers was initially analyzed using 188 informative microsatellites giving a 16-cM average interval covering 29 autosomes. A significant QTL for marbling was detected in the centromeric portion of bovine chromosome (BTA) 9. After additional marker genotyping across a larger sample size composed of 169 individuals, this locus was refined to a 20-cM confidence interval between microsatellites BM1227 (24 cM) and DIK2741 (50 cM) at a 1% chromosome-wise threshold. The allele substitution effect between Q and q for a beef marbling standard score (1 to 12 range) on BTA9 was 1.0 (5.7% of total phenotypic variance in QTL contribution in this family). This result provides a primary platform for a marker-assisted selection system of the beef marbling trait within the Japanese Black (Wagyu) cattle population.

Detection of hepatitis delta virus recombinants in cultured cells co-transfected with cloned genotypes I and IIb DNA sequences

It was reported previously that hepatitis delta virus (HDV), the only animal virus in which replication is performed by cellular RNA polymerase(s), undergoes RNA recombination. However, the previous RNA transfection system was somewhat limited in terms of practical application. Cultured cells were transfected with plasmids expressing replication-competent genotypes I and IIb HDV genomic RNAs to develop a better system for studying the fundamental aspects of HDV RNA recombination and HDV-related RNA species were examined using restriction fragment length polymorphisms and sequence analysis of cloned RT-PCR products. This novel experimental system generated efficiently recombinants between the two parental HDV sequences, but not between replication-defective HDV constructs. The genome organization of the HDV recombinants produced in this system resembled that observed previously in cultured cells co-transfected with genome I and IIb RNAs. These data indicate that replication-dependent HDV RNA recombination can be catalyzed by host RNA polymerases in cultured cells co-transfected with two cloned HDV sequences. This new DNA-based system is simpler than the previous RNA-based method of study, and generates a higher recombination frequency, facilitating study of HDV RNA recombination.

470. Enhanced Transposition Activities of Mutant Sleeping Beauty Transposases

The development of efficient nonviral vectors that allow for long-term transgene expression may have significant impact on future gene therapy applications. Sleeping Beauty (SB) is a DNA-based transposon system derived from teleost fish sequences that has been shown to have activity within mammalian cells. In this two-part system, the transposase facilitates gene transfer by binding to specific sequences within the transposon (IR/DR elements) and catalyzing its integration into TA dinucleotide sequences within the target genome. The original SB transposase (SB10) was constructed based on consensus sequence alignments using several fish species. In attempt to further improve the transpositional activity of SB10, alignment analysis was conducted using transposase sequences across a wide range of species. Based upon this data, specific mutations were made within the SB10 protein and the functional activity of each mutant was evaluated.

DNA-based identification of the eastern subterranean termite, Reticulitermes flavipes (Isoptera: Rhinotermitidae).

A DNA-based system for the identification of Reticulitermes flavipes (Koller) was established based on the following criteria: adequate molecular variation, comprehensive specimen sampling, explicit morphological species identification, and cladistic analysis. Termite soldiers were identified by labral shape, and these specimens and associated pseudergates were used in subsequent molecular analyses. Mitochondrial DNA from the AT-rich region was sequenced for 173 R. flavipes, Reticulitermes hageni Banks and Reticulitermes virginicus (Banks) soldiers and pseudergates collected from several widely dispersed Texan localities. Cladistic analysis was performed after exploration of an optimal sequence alignment inclusive of DNA sequences from Texas and published sequences from Georgia and Canada. Among the Texan individuals, 29, 5, and 1 mitochondrial DNA haplotypes were identified respectively with R. flavipes, R. hageni, and R. virginicus. One haplotype from El Paso likely represented a different Reticulitermes species. Species were monophyletic; however, individual relationships were unresolved in a strict consensus of 700 most parsimonious trees. Texas haplotypes were isolated by distance, and a correlation between genetic and geographic distance was observed among the Texas, Georgia, and Canada haplotypes. These results suggest that the methods outlined in this study will allow for the identification of R. flavipes from localities between Texas and Canada.

Identification of north-western Atlantic Porphyra (Bangiaceae, Bangiales) based on sequence variation in nuclear SSU and plastid rbcL genes

Six species of Porphyra have commonly been recognized in the north-western Atlantic from Long Island Sound to the Canadian Maritimes: P. amplissima, P. leucosticra, P. linearis, P. miniata, P. purpurea, and P. umbilicalis. Distinguishing them with certainty has been problematic. A DNA-based system of molecular identification was developed using partial sequences of the nuclear small subunit ribosomal RNA gene (SSU) or the plastid ribulose-1,5-bisphosphate carboxylase–oxygenase large subunit gene (rbcL). Multiple samples of each taxon were surveyed for intraspecific variation. Intraspecific SSU divergences for Porphyra ‘leucosticta’, P. ‘miniata’, P. ‘umbilicalis’, and P. ‘purpurea’ ranged from 0% to 1%. There was more variation for P. ‘amplissima’ (0–2.1%) and P. ‘linearis’ (0–3.5%); however, each taxon was monophyletic. No intraspecific differences were observed for these taxa in rbcL (one to eight samples per taxon). These sequences were compared with P. yezoensis U51, introduced to Maine, and with P. ‘dioica’, a north-east Atlantic Porphyra easily confused with P. ‘purpurea’. To discriminate between P. ‘purpurea’, P. ‘umbilicalis’, and P. ‘leucosticta’, SSU variation was used to design primers for the Allele-Specific Polymerase Chain Reaction™. With molecular tools, we could classify over 80% of the monostromatic specimens surveyed, but the residue of unidentifiable specimens may indicate the existence of further monostromatic species in the north-west Atlantic. Porphyra ‘purpurea’ was found to occur further south than previously recorded. A morphologically cryptic Porphyra was discovered at Herring Cove, Nova Scotia, Canada.† Phylogenetic analyses using SSU or rbcL sequences showed ‘soft incongruence’ between gene trees, i.e. the topologies of the phylograms were similar but not identical, with only weak to moderate bootstrap support for the nodes that differed. Both trees strongly supported a clade including P. ‘purpurea’, P. ‘umbilicalis’, P. ‘linearis’, and P. ‘dioica’. Porphyra sp. Herring Cove was allied with the remaining Porphyra taxa in the SSU tree. The rbcL phylogeny was less well resolved, consisting of a polytomy of a P. ‘purpurea’–P. ‘umbilicalis’–P. ‘linearis’–P. ‘dioica’ clade, Porphyra sp. Herring Cove, a clade comprising P. ‘amplissima’ and P. ‘miniata’, and a P. ‘suborbiculata’–P. ‘leucosticta’–P. yezoensis clade.

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Argininosuccinate lyase (ASL) deficiency: mutation analysis in 27 patients and a completed structure of the human ASL gene.

Argininosuccinic aciduria is an urea cycle disorder caused by argininosuccinate lyase (ASL) deficiency and is inherited as an autosomal-recessive trait. To date, mutation analysis has been limited because of incomplete sequence information about the human ASL gene. As a consequence, only 12 different mutations in 12 patients have been reported, so far. This study aimed at the completion of the structure and the sequence of the human ASL gene, the development of a genomic DNA-based system for mutation analysis and, finally, the characterisation of the molecular genetic background of ASL deficiency in 27 unrelated patients. This report provides transcript variants, the complete sequence of the human ASL gene and a complete ASL homologue on chromosome 22. The homologue was formerly thought to be a pseudogene but was found, in this study, to be correlated with an immunoglobulin-lambda-like mRNA. On the basis of the novel sequence data, a polymerase reaction chain system for mutation-screening in all 16 coding exons of the ASL gene was established and applied to the analysis of the ASL-deficient patients. We found mutations in all of the 54 investigated alleles and identified 23 (19 novel) different mutations. Some mutational hot-spots were identified (mainly in exons 4, 5, and 7) as were several predominant mutations: IVS5+1G-->A (15 alleles), c.532G-->A (7), c.346C-->T (6), c.1153C-->T (4). This study introduces a system for mutation analysis in the ASL gene, thereby elucidating the genetic background of ASL deficiency, which was found to be associated with considerable allelic heterogeneity.

Coding properties of DNA languages

The computation language of a DNA-based system consists of all the words (DNA strands) that can appear in any computation step of the system. In this work we define properties of languages which ensure that the words of such languages will not form undesirable bonds when used in DNA computations. We give several characterizations of the desired properties and provide methods for obtaining languages with such properties. The decidability of these properties is addressed as well. As an application we consider splicing systems whose computation language is free of certain undesirable bonds and is generated by nearly optimal comma-free codes.

DNA typing for BoLA class I using sequence-specific primers (PCR-SSP).

The analysis of cattle MHC (BoLA) class I gene expression is an essential component of studies on immune responses and susceptibility to disease. International BoLA workshops have generated data and reagents that allow discrimination of class I molecules at the haplotype level, but progress has been limited by difficulties encountered in defining single alleles. Our aim in this study was to develop a DNA-based system for improved identification of expressed class I alleles, utilizing available cDNA sequences derived from cattle carrying a series of serologically defined class I specificities. This method has allowed more accurate typing of animals for expression of the class I genes present within a small number of haplotypes. The method has also reliably differentiated between allelic variants (identified by prior sequence analysis) and has split existing serological specificities. The data show that MHC class I genes in cattle are more polymorphic than demonstrated by serology and biochemical analysis.

Characterization of a set of variable number of tandem repeat markers conserved in Bovidae

Screening purpose-built libraries with minisatellite probes, we have isolated 36 bovine variable number of tandem repeat markers (VNTRs) characterized by a mean heterozygosity of 59.3 within the American Holstein breed. Matching probabilities and exclusion powers were estimated by Monte-Carlo simulation, showing that the top 5 to 10 markers could be used as a very efficient DNA-based system for individual identification and paternity diagnosis. The isolated VNTR systems should contribute significantly to the establishment of a bovine primary DNA marker map. Linkage analysis, use of somatic cell hybrids, and in situ hybridization demonstrate that these bovine VNTRs are scattered throughout the bovine genome, without evidence for proterminal confinement as in the human, and that at least some of them are organized as clusters. Moreover, Southern blot analysis and in situ hybridization demonstrate conservation of sequence and map location of minisatellites within Bovidae.

LIMITED HETEROGENEITY OF THE HLA CLASS II CONTRIBUTION TO SUSCEPTIBILITY TO RHEUMATOID ARTHRITIS IS SUGGESTED BY POSITIVE ASSOCIATIONS WITH HLA—DR4, DR1 AND DRwlO

Most patients with rheumatoid arthritis (RA) exhibit an HLA class II epitope found on most DR4 and DR1 molecules. We have investigated the possibility of other associations being present in the minority of patients lacking these antigens. One hundred and eighty patients with classical or definite RA and 100 controls were assigned HLA-DR, DQ and DP types by a DNA-based system using sequence-specific oligonucleotides to probe amplified class II alleles amplified by the polymerase chain reaction. The expected associations with DR4 (relative risk 10, P less than 0.0001) and DR1 (relative risk 2.3, P less than 0.001) were observed and only 13% of individuals lacked both of these alleles compared with 54% of age- and race-matched controls. A significant association with DRw10 (P = 0.02) was also observed in the DR4/DR1-negative RA group (3/23 patients compared with 0/54 DR4/DR1-negative controls). No novel associations with other DR, DQ, or DP alleles were evident in contrast to some previous studies. The third allelic hypervariable region of the DR beta chain of DRw10 contains a similar amino acid sequence to that found in many DR4 and DR1 molecules. These results extend this correlation and suggest that this susceptibility determinant may account for the HLA-linked susceptibility in 89% of our cases of RA.