Ethidium monoazide and propidium monoazide for elimination of unspecific DNA background in quantitative universal real-time PCR

Unspecific background DNA in quantitative universal real-time PCR utilizing a hydrolysis probe was completely suppressed by the addition of EMA or PMA to the PCR mix via cross-linking of the dyes to DNA during 650 W visible light exposure. The proposed procedure had no effect on the sensitivity of the real-time PCR reaction.     

Nucleotide pools of growing,synchronized and stressed cultures of Saccharomyces cerevisiae

High pressure liquid chromatography has been used to study the acid soluble nucleotide pool of Saccharomyces cerevisiae under different conditions of growth. ATP, ADP, AMP, NAD, GTP, UTP, UDP, CTP, CDP, and UDP-sugars plus UMP could be separated and were found in concentrations higher than 0.1 mumol per g yeast cell dry weight (= detection limit). During glucose-limited continuous culture the levels of individual nucleotides depended on the growth rate, which was most pronounced with pyrimidine (uridine, cytidine) nucleotides. The energy charge (E.C.) remained high (0.9) at all growth rates (0.07-0.3 h-1). During synchronized growth at a constant growth rate (0.11 h-1) almost all nucleotide levels and the E.C. remained at constant values with the only exception of UDP-sugars and UMP of which increased levels were found during the phase of budding. Under conditions of metabolic stress (addition of antimycin A, deoxyglucose plus iodoacetate) pronounced changes in the levels of purine (adenine and guanine) nucleotides and the E.C. were observed. All other nucleotides were less influenced by these conditions. Only under these conditions IMP accumulation was observed. The results strongly argue against the significance of purine nucleotide or E.C. measurements under viable conditions. In contrast, changes in the levels of pyrimidine nucleotides seem to be indicative of changes in the flux through the metabolic pathways where they act as coenzymes.     

Parasitic worms: strategies of host finding, recognition and invasion

Many parasitic worms enter their hosts by active invasion. Their transmission success is often based on a mass production of invasive stages. However, most stages show a highly specific host-finding behaviour. Information on host-finding mechanisms is available mainly for trematode miracidia and cercariae and for nematode hookworms. The larvae find and recognise their hosts, in some cases even with species specificity, via complex sequences of behavioural patterns with which they successively respond to various environmental and host cues. There is often a surprisingly high diversity of host-recognition strategies. Each parasite species finds and enters its host using a different series of cues. For example, different species of schistosomes enter the human skin using different recognition sequences. The various recognition strategies may reflect adaptations to distinct ecological conditions of transmission. Another question is how, after invasion, parasitic worms find their complex paths through their host's tissues to their often very specific microhabitats. Recent data show that the migrating parasite stages can follow local chemical gradients of skin and blood compounds, but their long-distance navigation within the host body still remains puzzling.

The high complexity, specificity and diversity of host-recognition strategies suggest that host finding and host recognition are important determinants in the evolution of parasite life cycles.     

The Bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 DNA replication, is a membrane-localized single-stranded DNA-binding protein.

The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.     

Mycobacterial disease—a challenge to biotechnology

Summary The two principal mycobacterial diseases — tuberculosis and leprosy — remain major causes of human suffering in many parts of the world. The struggle against these diseases has been hampered by the inadequacy of methods for their diagnosis, prevention and therapy. Improvements in diagnostic methods, especially for tuberculosis, are required as existing bacteriological techniques are time consuming and insensitive, but immunological tests suffer from a lack of specificity and a failure to distinguish active disease from past sensitization. Tests are required that will either detect the presence of mycobacterial antigens or the occurrence of immune reactions specific to active infection. Prophylaxis by BCG vaccine is never complete and in some regions appears virtually ineffective. In order to determine the reason for this variation, to produce better vaccines, and to use BCG more effectively, a deeper understanding of the immune reactions in mycobacterial disease is required. In particular, the bacterial determinants of virulence and protection and the mode of action of BCG require detailed study. The therapy of tuberculosis and, to a lesser extent, leprosy has undergone an extensive transformation since the introduction of rifampicin. Nevertheless the cost of modern short course regimens for tuberculosis limit their application. Even shorter regimens, made possible by more potent bactericidal drugs or by agents that stimulate the host's defences are thus required. Recent advances in biotechnology could therefore lead to significant advances in the control of these diseases but only if they are integrated into local, self-reliant public health initiatives.

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Enfermedades producidas por microbacterias. Un desafío a la biotecnologia

Resumen La dos principales enfermedades producidas por micobacterias — tuberculosis y lepra —persisten como las causas más importantes del sufrimiento humano en muchas partes del mundo. La lucha contra estas enfermedades se ha visto dificultada por lo inadecuado de los métodos para su diagnosis, prevención y terapia. Es necesaria una mejora de los métodos de diagnosis, especialmente en el caso de la tuberculosis, ya que las técnicas bacteriológicas existentes son poco sensibles y consumen mucho tiempo y los tests inmunológicos son inespecíficos y no permiten distinguir entre la enfermedad activa y la sensibilidad adquirida. Se necesitan tests capaces de detectar la presencia de antígenos microbacterianos o la existencia de reacciones de inmunización específicas de la infección activa. La profilaxis con la vacuna BCG no es nunca completa y en algunas regiones es virtualmente enefectiva. Con objeto de determinar las razones de esta variación, de producir mejores vacunas y de usar la BCG de forma más eficiente, se necesita entender con más profundidad las reacciones de inmunización en las enfermedades por micobacterias. En particular los determinantes bacterianos de la virulencia y de la protección y la forma de actuar de la BCG precisan de un estudio detallado. La terapia de la tuberculosis y en menor grado la de la lepra, han sufrido una extensa transformación desde la introducción de la rifampicina. Sin embargo, el costo de los modernos tratamientos a corto plazo de la tuberculosis limítan su aplicación. Se requieren tratamientos aún más cortos basados en el desarrollo de drogas bactericidas más potentes o de agentes que estimulen las defensas del enfermo. Los recientes avances en biotecnología podrían por tanto conducir a avances importantes en el control de estas enfermedades pero solamente si se integran en iniciativas médicas públicas consistentes.

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Les maladies à mycobactéries — Un enjeu pour les biotechnologies

Résumé Les deux principales maladies à mycobactéries — la tuberculose et la lèpre — sont toujours des causes majeures de souffrance humaine dans de nombreuses parties du monde. La lutte contre ces maladies est entravée par l'inadéquation des méthodes diagnostiques, préventives et thérapeutiques. Spéciallement en ce qui concerne la tuberculose, l'amélioration des moyens de diagnostic est rendue nécessaire par le fait que les techniques bactériologiques existantes sont longues et peu sensibles, et que les tests immunologiques, n'étant pas spécifiques, ne permettent pas de distinguer entre une maladie en évolution et une sensibilisation ancienne. On a besoin de tests permettant de déceler soit la présence d'antigènes mycobactériens, soit l'apparition de réactions immunitaires spécifiques au cours d'une infection évolutive. La prophylaxie par le BCG n'est jamais complète et parait être pratiquement inefficace dans certaines régions. Pour expliquer cette variation géographique, produire de meilleurs vaccins, et utiliser le BCG efficacement, il convient de mieux connaître les réactions immunitaires au cours des maladies à mycobactéries. En particulier, les déterminants bactériens de la virulence et de la protection, ainsi que le mode d'action du BCG sont à approfondir. La rifampicine a considérablement transformé la thérapeutique de la tuberculose et, à un moindre degré, de la lèpre. Néanmoins, le coût des nouveaux traitements intensifs de la tuberculose limite leur application. Il est nécessaire que des médicaments bactéricides plus puissants ou des agents stimulant les défenses de l'hôte puissent raccourcir encore davantage la durée des traitements. Le progrès biotechnologique peut donc améliorer de façon significative le contrôle des maladies considérées, à la seule condition que ce but soit volontairement intégré dans les initiatives locales en matière de santé publique.

     

Complete complementary DNA of rat tyrosine aminotransferase messenger RNA. Deduction of the primary structure of the enzyme

The primary structure of rat tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5), a liver-specific enzyme involved in gluconeogenesis, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 2362 nucleotides long (excluding the poly(A) tail) and codes for a polypeptide of 454 amino acids with a molecular weight of 50634. Unambiguous identification was obtained by comparison of this sequence with the amino acid sequences of several peptides obtained from the purified enzyme.     

Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from <Emphasis Type=Italic>Nicotiana benthamiana</Emphasis>for biotechnological application

Background  

Plant lipoxygenases (LOXs) have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in Nicotiana benthamiana was highly induced by agroinfiltration using a tobacco mosaic virus (TMV) based vector system.     

Tree killing by herbicide producing ants for the establishment of pureTococa occidentalis populations in the Peruvian Amazon

Populations ofTococa occidentalis (Melastomataceae) and the inhabiting ants (Myrmelachista sp.) were observed for more than eight months in the Peruvian Amazon (Sira mountains). They represent a complex coevolutionary system: the plants offer shelter (leaf domatia, hollow stems) and food (leaf glands), whereas the ants kill all surrounding plants, including large trees up to 10 m, by chemical weapons. Experiments with exposed plants revealed a highly specialized way to attack meristematic tissue and leaf nervature, which leads to a quick decay of the plant individuals. The clearing of the vegetation by the ants allows theTococa population to expand mostly by vegetative shoots to large monocultures (up to 30 m in diameter) free from any other plant species. Artificially introduced plant individuals, from differentT. occidentalis populations, are regarded as a foreign species by the ants.The succession of such aTococa-Myrmelachista system begins with one or a few founder plants on a light place in the midst of the vegetation.Myrmelachista soon inhabits their host plants which otherwise would not survive and begin to clear the place from all foreign plant species.Tococa expands quickly, forming circle shaped populations. Distantly situated canopy trees shade theTococa population after a number of years and cause their decay. The whole place appears contaminated for years and no other plant can establish itself. Some of the consequences of these open places are erosion and a severe influence on the regeneration of the forest.