RESUMEN

AIMS: To develop a rapid preparation method for real-time PCR analysis of cyanobacteria from cultures or field samples. METHODS AND RESULTS: Field samples and cultures containing Anabaena circinalis, Cylindrospermopsis raciborskii or Microcystis aeruginosa were subjected to three cell disruption treatments: (i) heating during thermocycling, (ii) microwave irradiation in the presence of detergent and (iii) probe sonication. Treated samples were directly added to the PCR reaction and analysed on two different real-time devices. A statistically significant difference was evident in the cycle thresholds for each of the treatments in all but one culture and one environmental sample, sonication and microwave treatments performing better than direct addition. The microwave treatment was also compared to the Qiagen DNA Mini kit and performance was equivalent when treated samples were analysed as above. CONCLUSIONS: Whilst microwave treatment was slightly less effective than probe sonication across all samples, it was more amenable to processing multiple samples and significantly better than heat treating the sample during thermocycling. SIGNIFICANCE AND IMPACT OF THE STUDY: The microwave method described here is a simple, rapid and effective preparation method for cyanobacterial DNA that can be easily deployed in the field, making the most of the speed and flexibility offered by fixed and portable real-time PCR devices.

Asunto(s)

Cianobacterias/aislamiento & purificación , ADN Bacteriano/genética , Reacción en Cadena de la Polimerasa/métodos , Técnicas Bacteriológicas , Cianobacterias/genética , Calor , Microondas , Radiación , Sensibilidad y Especificidad , Sonicación , Polimerasa Taq/genética

RESUMEN

BACKGROUND: Methods involving the analysis of nucleic acids have become widespread in the fields of traditional biology and ecology, however the storage and transport of samples collected in the field to the laboratory in such a manner to allow purification of intact nucleic acids can prove problematical. RESULTS: FTA databasing paper is widely used in human forensic analysis for the storage of biological samples and for purification of nucleic acids. The possible uses of FTA databasing paper in the purification of DNA from samples of wildlife origin were examined, with particular reference to problems expected due to the nature of samples of wildlife origin. The processing of blood and tissue samples, the possibility of excess DNA in blood samples due to nucleated erythrocytes, and the analysis of degraded samples were all examined, as was the question of long term storage of blood samples on FTA paper. Examples of the end use of the purified DNA are given for all protocols and the rationale behind the processing procedures is also explained to allow the end user to adjust the protocols as required. CONCLUSIONS: FTA paper is eminently suitable for collection of, and purification of nucleic acids from, biological samples from a wide range of wildlife species. This technology makes the collection and storage of such samples much simpler.

Asunto(s)

Animales Salvajes/sangre , Recolección de Muestras de Sangre/veterinaria , ADN/aislamiento & purificación , Papel/normas , Manejo de Especímenes/veterinaria , Animales , Animales Salvajes/genética , Aves , Recolección de Muestras de Sangre/instrumentación , Recolección de Muestras de Sangre/métodos , Recolección de Muestras de Sangre/normas , Braquiuros , Bovinos , Pollos , Columbidae , ADN/sangre , Decápodos , Galliformes , Lagartos , Moluscos , Mucosa Bucal/química , Reacción en Cadena de la Polimerasa/veterinaria , Ranidae , Mapeo Restrictivo/veterinaria , Saliva/química , Manejo de Especímenes/métodos , Manejo de Especímenes/normas , Atún

RESUMEN

Long-term aging of dry DNA is thought to be due to the attack of diverse cascades of reactive species with probably, no one single initiator of the cascades explaining all circumstances. Photosensitizer-initiated reactions from methylene blue and riboflavin were used to generate two model systems of reactive species around dry DNA in order to understand such systems and how to block them. Damage was assessed using plasmid DNA as a substrate with an in-situ microgel electrophoretic technique. Photodynamic methylene blue damage to DNA was very oxygen dependent but not that of riboflavin. This indicates that indirect type II pathways, probably via singlet oxygen were important for methylene blue but not for riboflavin. In both the absence and presence of oxygen, the DNA protection offered by dry caffeine and urate to both photodynamic agents indicated that most DNA attack was via electrophilic species. Overall, protection of dry archived DNA from spontaneously reactive species such as free radicals appears to be a real issue and, as expected, the predominant species in air appear to involve oxygen but not exclusively or necessarily so.

Asunto(s)

ADN/efectos de los fármacos , Azul de Metileno/farmacología , Fármacos Fotosensibilizantes/farmacología , Riboflavina/farmacología , Daño del ADN , ADN Bacteriano/efectos de los fármacos , Desecación , Escherichia coli/genética , Plásmidos/efectos de los fármacos

RESUMEN

The avian LINE CR1 generates multiply-superimposed insertions, resulting in apparently fortuitous inverted LINE repeat clusters (ILRCs). These loci display size micro-heterogeneity within populations, with few or no presence/absence polymorphisms, and yet only very closely related species share loci. Whilst the CR1 sequences that the ILRC loci are derived from are not species-specific, the loci themselves appear to be species-characteristic if not totally species specific. The mammalian LINE L1 is shown to act similarly to CR1 and also forms ILRCs. It is proposed that whilst the formation of these loci may be from a near-random process of super-insertion certain of them are in some way functional, explaining their conservation and rapid spread to population boundaries, whilst non-functional or inactive loci are quickly lost. ILRCs appear to decay from the element as formed by the accumulation of point mutations. ILRCs appear to an unusual example of non-polymorphic sequences being younger than polymorphic sequences with no obvious selective reason.

Asunto(s)

Genoma , Retroelementos/genética , Vertebrados/genética , Animales , Secuencia de Bases , Aves/genética , Gatos , Bovinos , Secuencia Conservada , ADN/química , ADN/genética , Humanos , Datos de Secuencia Molecular , Familia de Multigenes/genética , Mutación , Ratas , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie

RESUMEN

The instability of RNA in solutions during storage and travel is an impediment to its utilization in routine diagnostics. A robust and simple approach to the problem of RNA protection and processing is offered by storage of RNA desiccated with processing procedures that do not solublize the RNA until the beginning of reverse transcription. The feasibility of this general approach was tested with coxsackievirus B4 (CVB-4) from blood or culture fluid held on a storage and transport medium (FTA(R)) and analysed by reverse transcriptase PCR (RT-PCR) without removing the RNA from the FTA(R) until reverse transcription. Phase-trapping techniques based on water-miscible solvents such as ethanol or phenol were compared with simple buffers and concentrated lithium chloride solutions. RT-PCR detection of viral RNA reached a sensitivity of approximately 0.1 fg, which is comparable with other non-nested PCR techniques. Whole blood as a virus vehicle significantly interfered with CVB-4 detection, but to an acceptable degree. Desiccation-storage of the RNA of CVB-4 appears to be unaffected by weeks on the storage medium under ambient conditions. These characteristics indicate that this approach forms a credible developmental base for RNA-based pathogen diagnostics with particular application to the problem of transporting potentially infectious body fluids to a centralized laboratory for analysis.

Asunto(s)

ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Manejo de Especímenes/métodos , Recolección de Muestras de Sangre/instrumentación , Recolección de Muestras de Sangre/métodos , Cartilla de ADN , Enterovirus Humano B/genética , Enterovirus Humano B/aislamiento & purificación , Humanos , ARN Viral/química , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/metabolismo , Sensibilidad y Especificidad , Manejo de Especímenes/instrumentación

RESUMEN

Intense nuclear condensation with intense refractivity (pyknosis) is the ubiquitous terminus of all apoptosis and some necrosis of vertebrate cells, but its structural basis is unknown. Intense condensations were induced in a model system, the avian erythrocyte, and three different molecular processes distinguished from each other. Two of the hypercondensations, nucleolytic pyknosis, as in mammalian apoptosis, and anucleolytic pyknosis, as in necrosis, appear to be energetically spontaneous and appear to have a conformational basis with the third hypercondensation being a trans-nuclear membrane osmotic pressure compression effect. Nucleolytic pyknosis as per apoptosis was not intrinsic in this system and required exogenous nuclease. The pure anucleolytic pyknosis supported by this system was not induced by the apoptopic induction agents, staurosporine or antitopoisomerases (I and II), indicating a simple but unusual signaling pathway for anucleolytic pyknosis. Molecular weight determinations of the H5, H3, H4, H2a, and H2b, with final errors of +/-1 Da or less, seem to eliminate histone modifications as the basis of anucleolytic pyknosis. The molecular basis of pyknosis is proposed to be from internucleosomal rotational angle freedom that permits internucleosomal sharing of basic histone tails of adjacent nucleosomes and nucleofilaments. Much of the favorable conformational energy of pyknosis may be from the entropy increase of tail delocalization.

Asunto(s)

Eritrocitos/ultraestructura , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Pollos , Citoplasma/ultraestructura , ADN/análisis , Eritrocitos/efectos de los fármacos , Ósmosis

RESUMEN

Protein kinase C (PKC)-mediated phosphorylation of chromatin-associated proteins was studied in vitro. HL-60 and HeLa nuclear proteins were notably unresponsive to exogenously added brain PKC. In contrast, 3T3 fibroblasts and lymphocytes from primary cultures exhibited PKC-dependent phosphorylation of chromatin-associated proteins when chromatin was induced to expand. Unexpanded nuclei in all cell lines were unresponsive. Responsiveness was particularly obvious in the decondensed chromatin of primary lymphocytes, where a large number of proteins were phosphorylated in response to exogenous PKC. DNAase-I and micrococcal nuclease strongly modulated these phosphorylation patterns indicating that the substrates were DNA-associated. It was concluded that although substrate conformation, i.e. condensation state, was the primary determining factor in control of PKC-dependent nuclear protein phosphorylation, different cell lines greatly differ in their overall responsiveness to exogenous PKC.

Asunto(s)

Cromatina/metabolismo , Proteína Quinasa C/farmacología , Animales , Línea Celular , Cromatina/química , Células HeLa , Humanos , Ratones , Fosforilación , Conformación Proteica , Ratas , Especificidad por Sustrato , Células Tumorales Cultivadas

RESUMEN

Three avian highly repetitive tandem repeats were identified and examined. These repeats had similar unit lengths (about 42 bp long) but completely different sequences each containing particular protein binding sites. Each of these repeats was found within only one of the five closely related genera studied.

Asunto(s)

Aves/genética , ADN/genética , Proteínas Nucleares/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Composición de Base , Secuencia de Bases , Sitios de Unión , Pollos/genética , ADN/sangre , ADN/aislamiento & purificación , Patos/genética , Femenino , Gansos/genética , Masculino , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico

RESUMEN

Southern analysis of within-gel digested and restricted human cells has revealed very large Satellite III restriction fragments which show clear inter-individual length polymorphism. The Mb and sub-Mb length of these fragments indicate that they arise from regions of heterochromatin which contain homogeneous Satellite III sequences of peculiar resistance to common endonucleases. Based on sequence alone, such regions would be little digested by endonuclease digestion of chromatin in metaphase, regardless of its method of preparation. Polymorphic regions such as these might be expected to stain as part of the C-banding seen in endonuclease treated metaphase chromosomes, and may in part account for inter-individual C-band heteromorphisms.

Asunto(s)

ADN Satélite/genética , Células Cultivadas , Bandeo Cromosómico , Enzimas de Restricción del ADN , Heterocromatina/ultraestructura , Humanos , Masculino , Polimorfismo de Longitud del Fragmento de Restricción , Espermatozoides/ultraestructura

RESUMEN

HL-60 nuclear autophosphorylation was studied in vitro under circumstances in which the conformation of chromatin was manipulated with both polyamines and DNAse-I. A general re-arrangement of the phosphorylation patterns occurred as polyamines were removed and nuclei were observed to expand. DNAse-I treatment reduced these phosphorylation patterns to a much simpler configuration indicating that the responding substrates were DNA-associated. It was concluded that substrate conformation was the main determining factor in the control of nuclear protein phosphorylation. These results suggest a method of general utility for the identification of truly nuclear proteins by the characteristics of their phosphate acceptor activity.

Asunto(s)

Cromatina/química , Proteínas Nucleares/química , Línea Celular , Núcleo Celular/química , Desoxirribonucleasa I , Electroforesis en Gel de Poliacrilamida , Fosforilación , Espermidina , Espermina , Especificidad por Sustrato

RESUMEN

A 14 bp repeat isolated from the genome of a Mallard duck (Anas platyrhynchos) and a 13 bp repeat isolated from the genome of a Muscovy duck (Cairina moschata) were both tandem and genus-specific. There were 2.1 x 10(6) copies of the 14 bp repeat in the Mallard duck genome and 1.3 x 10(6) copies of the 13 bp repeat in the Muscovy duck genome. These two repeats were not obviously homologous but each contains different transcription protein binding sites. Experimentally, the two repeats were also observed to bind nuclear proteins specifically. A sequence-selective mechanism for the expansion of short tandem repeats from single ancestral sequences containing nuclear protein binding sites is presented.

Asunto(s)

ADN/química , Patos/genética , Proteínas Nucleares/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Autorradiografía , Secuencia de Bases , Sitios de Unión , ADN/metabolismo , Biblioteca Genómica , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Unión Proteica

RESUMEN

A putative old and ubiquitous interspersed DNA repeat family was identified from TaqI restriction, M13 cloning, and sequencing of the genomic DNA of a Mallard (Anas platyrhynchos), a Muscovy Duck (Cairina moschata), a Toulouse Goose (Anser anser), and a Black Swan (Cygnus atratus). A 425-bp consensus core sequence was obtained for the interspersed family. The 425-bp unit was about 2% of the avian genome and was found to be conserved in at least four genera of the order Anseriforme: Anas, Anser, Cairina, and Cygnus.

Asunto(s)

ADN/química , Patos/genética , Gansos/genética , Biblioteca Genómica , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Secuencia de Bases , Datos de Secuencia Molecular

RESUMEN

This paper examines the principal classes of repetitive DNA of the Toulouse goose (Anser anser) genome. There are four major classes and they are tandem repeats of less than 200 base pairs (bp). The longest repeat (class A) is 190 bp long and starts with a HinfI site. Class B is 43 bp long, commencing with a FokI site. Classes A and B show no extensive homology to DNA sequences held on a current data base (Genbank) but were confirmed to exist as major repeats in another strain of goose, the Emden goose (Anser anser) genome. Classes C and D are 5-bp repeats of 5' GAGAG 3' and 5' GGGAA 3', respectively. The macrosatellites C and D were compared with a current data base (Genbank) and were found to exist in a variety of other organisms as satellites.

Asunto(s)

ADN/genética , Gansos/genética , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Southern Blotting , Clonación Molecular , Biblioteca Genómica , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Mapeo Restrictivo

RESUMEN

Human Satellite III DNA is a major tandem repeat in the human genome and presents a TaqI-specific hypervariable restriction fragment length polymorphism when a Satellite III related sequence (228S) is used as a probe. In situ examination shows this sequence to be near specific for the region 9qh on chromosome 9 when it is used at low probe concentrations. However the region 9qh does not appear to be the only or even the primary source of the TaqI-deficient polymorphic sequences (TDPS). Rather, such sequences appear to be mostly present in chromosomes 20, 21, and 22, and these represent the largest regions of homogeneous Satellite III in the genome; they are also resistant to digestion with a range of other restriction endonucleases. The TDPS do not arise from either of the two currently recognized Satellite III-enriched genomic regions, namely autosomal 'K-domains', which form part of 15p in chromosome 15 or the heterochromatin of chromosome Y.

Asunto(s)

Secuencia de Bases , Cromosomas Humanos/ultraestructura , ADN Satélite/genética , Polimorfismo de Longitud del Fragmento de Restricción , Homología de Secuencia de Ácido Nucleico , Línea Celular , Mapeo Cromosómico , Sondas de ADN , Humanos

Asunto(s)

ADN Satélite , Filogenia , Primates/genética , Animales , Evolución Biológica , Southern Blotting , Sondas de ADN , Enzimas de Restricción del ADN , Gorilla gorilla/genética , Humanos , Masculino , Pan troglodytes/genética , Pongo pygmaeus/genética , Primates/clasificación

RESUMEN

Human DNA enriched in repetitive sequences specifically bound to a component(s) in purified preparations of rat brain protein kinase C (PKC). DNA which bound to protein was cloned in pUC-19 and one clone characterized as containing an approx. 140 bp insert. The band containing this insert (separated by acrylamide gel electrophoresis) was lost when the DNA was incubated with purified PKC preparations. Thus a protein in relatively pure PKC preparations is a sequence-selective DNA-binding protein. The results raise the possibility that PKC or a fragment of PKC binds selectively to specific DNA sequences.

Asunto(s)

Encéfalo/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , ADN/genética , Proteína Quinasa C/aislamiento & purificación , Animales , Clonación Molecular , ADN/metabolismo , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Humanos , Sueros Inmunes , Peso Molecular , Proteína Quinasa C/metabolismo , Ratas , Secuencias Repetitivas de Ácidos Nucleicos

RESUMEN

The various classes of human repetitive deoxyribonucleic acid (DNA) are described, with particular emphasis being given to their variation in the human genome. The significance of this information to forensic science is discussed.

Asunto(s)

ADN , Medicina Legal , Variación Genética , Secuencias Repetitivas de Ácidos Nucleicos , Humanos , Polimorfismo Genético , Cromosomas Sexuales

RESUMEN

DNA probes to multiple hypervariable human genomic loci are potentially highly informative as regards individual genome identification and parentage studies. However the resultant Southern blot patterns are generally highly complex and their interpretation difficult, with one important limitation being the extent of fragment separation and resolution. The separation of large DNA restriction fragments in agarose gels may be improved, in comparison to separations obtained by conventional electrophoresis, by the use of field inversion techniques. This is demonstrated by the analysis of the complex human Satellite III related DNA polymorphism. Detection of the Satellite III related restriction fragments is achieved either by using a [35S]-labelled probe (228S) or by using the same probe in a convenient non-isotopic form constructed by the photobiotin process. In addition, the probe 228S is useful for sexing the human genome, by the identification of a Y-specific restriction fragment.

Asunto(s)

ADN/análisis , Polimorfismo de Longitud del Fragmento de Restricción , Biotina , Southern Blotting , Sondas de ADN , Electroforesis en Gel de Agar/métodos , Humanos , Hibridación de Ácido Nucleico

RESUMEN

The disomal series generated by the digestion of chromatin with DNase I has been followed to its highest orders using Klenow end-labelling and field-inversion gel electrophoresis to maximise the resolution of large DNA fragments. The series is coherent to the 16N level and as such is incompatible with the most structurally acceptable coiling models. We propose that this is evidence for the general unsuitability of coiling models and is support for the existence of simple 'back-to-back' double-stranded structures within chromatin.

Asunto(s)

Cromatina/ultraestructura , Desoxirribonucleasa I , Animales , Pollos , ADN/sangre , ADN/ultraestructura , Eritrocitos/ultraestructura , Conformación de Ácido Nucleico

RESUMEN

Four separate features could be distinguished in Fe-DNAase-1 digestions of human lymphoblast nuclei: a di-nucleosomal (2N) repeat, a mono-nucleosomal (1N) repeat, a component of "random" DNA, and triple splitting of major peaks. The random component is major, is unlikely to be completely artifactual, and is what would be expected from the face to face layering model of Subirana et. al., (1). The 2N pattern appeared to be associated with compact, metaphase-type chromatin, whereas the 1N pattern was associated with more exposed chromatin. These two modes are explained in terms of orderly back-to-back folding of zig-zag nucleofilaments, and face-to-face folding respectively. Hybridization studies indicated that the centromeric classes of repetitive DNA had the same digestion spectra as the major interspersed classes of repetitive DNA, and DNA enriched in transcriptionally active sequences. It is suggested that current coil models are all inadequate explanations of higher order chromatin packing.

Asunto(s)

Cromatina/ultraestructura , Cromatina/metabolismo , Desoxirribonucleasa I , Humanos , Linfocitos/ultraestructura , Mitosis , Modelos Biológicos , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética