Microbial-invertebrate interactions and potential for biotechnology

Conclusion As the interactions between marine invertebrates and their bacterial commensals and symbionts are better understood, the application of biotechnology will enhance both environmental and economic benefit. In the immediate future, marine bacteria, either selected or genetically engineered, will play a significant role in enhancing the development of selected invertebrates in aquaculture and in the field. Luck may also favor discovery of mechanisms to suppress the development of biofouling species, perhaps by making it possible to coat submerged surfaces with bacterial films designed to repell larvae and/or interfere with larval morphogenesis. In any case, the future is appealing.     

Structural analysis of a maize gene coding for glutathione-S-transferase involved in herbicide detoxification

Summary We have used the cDNA clone encoding maize glutathione-S-transferase (GST I) to isolate a genomic DNA clone containing the complete GST I gene. Nucleotide sequence analysis of the cDNA and genomic clones has yielded a complete amino acid sequence for maize GST I and provided the exon-intron map of its gene. The mRNA homologous sequences in the maize GST I gene consist of a 107 bp 5 untranslated region, a 642 bp coding region and 340 bp of the 3 untranslated region. They are divided into three exons by two introns which interrupt the coding region. The 5 untranslated spacer contains an unusual sequence of pentamer AGAGG repeated seven times. The inbred maize line (Missouri 17) contains a single gene for GST I, whereas the hybrid line (3780A) contains two genes. Nucleotide sequence analysis of the primer extended cDNA products reveals that the 5 untranslated regions of the two genes in the hybrid 3780A are identical except for a 6 bp internal deletion (or insertion). The amino acid sequence of maize GST I shares no apparent sequence homology with the published sequences of animal GST's and represents the first published sequence of a plant GST. re]19850813 ac]19851126     

Ontogenetic development of corticotropin-releasing factor (CRF)-containing neural elements in the brain of the chicken during incubation and after hatching

Summary In chicken embryos of different ages and in young chickens after hatching, neural elements reacting with antibodies generated against synthetic ovine corticotropin-releasing factor (CRF) were studied by means of the peroxidase-anti-peroxidase (PAP) technique at the lightmicroscopic level. CRF-immunoreactivity was first observed in perikarya located in the periventricular part of the hypothalamus on the 14th day of the incubation period. CRF-containing neural elements were detected on the same day of incubation in the external zone of the median eminence, but not in all investigated animals. In extrahypothalamic sites, immunoreactive perikarya were demonstrable in the central gray of the mesencephalon on the 15th day of incubation. Furthermore, immunoreactive cells appeared in other brain regions such as nucleus accumbens and dorsomedial nucleus of the thalamus after hatching. The present observations provide information regarding the functional development of the hypothalamo-hypophyseal-adrenal axis in the chick embryo.     

Clastogen-induced chromosomal breakage as a marker for first trimester prenatal diagnosis of Fanconi anemia

Summary Using cultured trophoblast cells obtained by chorionic villus biopsy, we diagnosed Fanconi anemia (FA) in two pregnancies and excluded it in eight pregnancies at risk for the syndrome. Baseline chromosomal breakage and breakage induced by diepoxybutane (DEB) were analyzed. Increased breakage was used as a marker for the syndrome. Our results were unambiguous and provide a reliable method for prenatal detection of FA in the first trimester of pregnancy.     

The potential of biotechnology for developed and developing countries

Summary The extraordinary developments occurring in biotechnology throughout the world will lead to significant changes in world commerce, as well as in human health and welfare. A few dramatic advances have already been made in medical and agricultural biotechnology. Production of vaccines employing recombinant DNA methods is one example of the potential for genetic engineering applications in the treatment of hereditable and communicable diseases. Lesser understood, but with equally dramatic promise, is marine biotechnology, an example where the excellent possibilities for industry applications in both developing and developed countries can be elucidated. Recent successes in the cloning of growth hormone genes in salmon and other important genes and gene complexes in fin fish and shellfish promise major changes in production of commercially important fish and shellfish. Pharmacologically active compounds from marine animals and plants can now be harvested effectively, using biotechnology. Specialty chemicals and enzymes with unusual properties, unique to the marine environment, with potential for industrial applications, are being discovered. Thus, new products and new markets appear on the horizon. What has changed the picture significantly is the ready access which is now available to these new materials and compounds by the application of the methods of genetic engineering. No longer is it necessary to depend on sustained harvest, which may be subject to vagaries of weather or climate. By means of genetic engineering, genes coding for economically desirable traits can be cloned and those systems established in the laboratory in controlled culture and production. The enormous natural resources of developing countries cannot be productively harnessed by the developing countries themselves, since the industrial resources needed for the technology and the trained intellectual potential are not in abundant supply. Partnerships between developed and developing countries should be established so that the benefits of biotechnology can be achieved by both. An important role for applied microbiology, thus, is described and a dramatic impact at the global level can be predicted if effective biotechnology partnerships between the developing and developed countries of the world are created.

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Resumen El extraordinario desarrollo de la biotecnología en todo el mundo va a generar importantes cambios tanto en el comercio mundial como en la salud y bienestar de la humanidad. En las áreas de biotecnología médica y agrícola se han realizado importantes progresos. Un ejemplo de las posibilidades de la ingeniería genética en el tratamiento de enfermedades hereditarias y contagiosas es la produccion de vacunas utilizando técnicas de recombinación de ADN. La biotecnología marina es un área menos conocida pero no por ello con menos posibilidades de aplicaciones industriales tanto en países en desarrollo como en los desarrollados. Los éxitos obtenidos recientemente en la clonación de genes que codifican para las hormonas de crecimiento del salmón, así como de otros genes y conjuntos de genes de distintos peces y moluscos, suponen cambios importantes en la producción comercial de pescados y mariscos. Compuestos farmacológicamente activos procedentes de plantas y animales marinos pueden ahora cosechar se más eficientemente gracias a la biotecnología. Se estan descubiendo compuestos químicos y enzimas con propiedades especiales, unicas en el ambiente marino, cuya aplicación industrial tiene un futuro prometedor. Así pues, nuevos productos y nuevos mercados aparecen en el horizonte. La facilidad de acceso ahora disponible a estos nuevos materiales y compuestos, mediante la aplicación de los métodos de la ingeniería genética, ha cambiado significativamente el panorama. Ya no se ha de depender de cosechas sujetas a las variaciones del tiempo y del clima. Mediante la ingeniería genética los genes que codifican para características económicamente deseables pueden clonarse y los sistemas así obtenidos establecerse en laboratorio en condiciones de cultivo y producción controladas. La cantidad de recursos naturales que poseen los países en desarrollo no pueden ser utilizados productivamente por ellos mismos debido a la escasez de desarrollo tecnológico y potencial intelectual de los mismos. Deberián establecerse colaboraciones entre países en desarrollo y países desarrollados de manera que tanto unos como otros puedan beneficiarse de los progresos en biotecnología. Es este un papel importante que puede tener la microbiología aplicada y se puede predicir, a nivel global, un enorme impacto si se crean colaboraciones eficientes entre países en desarrollo y países desarrollados.

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Résumé Les extraordinaires développements de la biotechnologie dans le monde modifieront de façon significative le commerce mondial, aussi bien que la santé et le bien-être de l'homme. Des progrès considérables ont déjà été accomplis dans le domaine de la technologie médicale et agricole. La production de vaccins par les méthodes de l'ADN recombinant est un exemple du potentiel du génie génétique pour le traitement des maladies héréditaires et transmissibles. On connait moins le potentiel tout aussi considérable de la biotechnologie marine où on trouve d'excellents exemples d'applications industrielles réalisables aussi bien dans le pays développés que dans les pays en voie de développement. Les succès récents, obtenus par clonage des gènes d'hormones de croissance chez le saumon et par clonage d'autres gènes et groupes de gènes chez les poissons et les crustacés, laissent prévoir des changements majeurs dans le domaine de la production des poissons et crustacés d'intérêt commercial. Grâce à la biotechnologie, des composés pharmacologiquement actifs peuvent être obtenus à partir d'animaux et de végétaux marins. Des produits chimiques et des enzymes doués de propriétés nouvelles et spécifiques au milieu marin ont été découverts et sont suscéptibles d'applications industrielles. Ainsi, de nouveaux produits et de nouveaux marchés pointent à l'horizon. Ce qui a changé le tableau de façon décisive, c'est la facilité avec laquelle ces nouvelles substances peuvent être obtenues par les méthodes du génie génétique. Il n'est plus nécessaire d'avoir recours à des récoltes exposées aux aléas climatiques. Les gènes codant pour des caractères économiquement désirables peuvent être clonés au laboratoire et utilisés pour une production en cultures contrôlées. Faute de disposer des moyens technologiques et du potentiel scientifique nécessaires, les pays en voie de développement ne peuvent pas utiliser par eux-mêmes leurs énormes ressources naturelles. Des coopérations entre pays développés et moins développés doivent s'établir de façon à ce que la biotechnologie soit mutuellement profitable. La biotechnologie aura ainsi un rôle important et dont on peut attendre des effets considérables à l'échelle mondiale, à condition toutefois d'instituer une coopération effective entre les pays hautement industrialisés et le Tiers Monde.

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Invited paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki, 12–16.8.1985. Opening Session     

Effects of physical and chemical treatments on chloroplast manganese. NMR and ESR studies

Measurements were made of the water proton relaxation rate (T^?1₂ = R₂), electron spin resonance (ESR) six-line signal of ‘free’ Mn²⁺, and O₂-evolution activity in thylakoid membranes from pea leaves. The main results are: (1) Aging of thylakoids at 35°C causes a parallel decrease in O₂-evolution activity, in R₂ and in the content of bound Mn, suggesting that R₂ may be related to the loosely bound Mn involved in O₂ evolution. (2) Treatment of thylakoids with tetraphenylboron (TPB) at [TPB] > 2 mM produces a 2-fold increase in R₂, without release of Mn²⁺. The titration curve exhibits three sharp end points. The first end point occurs at a $\text{[}\text{TPB}\text{]}\text{[}\text{chlorophyll}\text{]}$ of 1.25, at which the O₂ evolution is completely inhibited. (3) Treatment of thylakoids with NH₂OH also increases R₂ by nearly 2-fold, either by the reduction of the higher oxidation states of Mn to Mn²⁺ and / or by exposing the Mn to solvent protons. Also, progressive release of bound Mn occurs at [NH₂OH] ≥ 1 mM as shown by an increase increase in the Mn²⁺ ESR signal and a decrease in R₂. (4) Addition of H₂O₂ (0.1–1.0%) to thylakoids causes an enhancement of R₂ similar to that by NH₂OH, but without the release of Mn²⁺. (5) Heat treatment of thylakoids at 40–50°C releases Mn²⁺ and increases R₂. Conversely, pH values of 7 to 4 release Mn²⁺ without changing R₂ while pH values of 7–9 increase R₂ without releasing Mn²⁺. Thus, both high and low pH values as well as the heat treatment cause structural changes enhancing the relaxivity of the bound Mn or of other paramagnetic species.     

Synthesis and maturation of cross-reactive glycoprotein in fibroblasts deficient in arylsulfatase a activity

The biosynthesis of arylsulfatase A was studied in cultured fibroblasts by pulse-chase labeling with [2-³H]mannose; the enzyme was isolated by immunoprecipitation and denaturing polyacrylamide gel electrophoresis. In normal fibroblasts, and in fibroblasts from a patient with multiple sulfatase deficiency, the enzyme was synthesized as a glycoprotein of apparent molecular weight of 59,000; half of it was processed over a period of 4 days to M_r= 57,000. The precursor chain of M_r= 59,000 was secreted in the presence of 10 mM NH₄Cl. An immunoprecipitable glycoprotein of normal size was synthesized by fibroblasts from two unrelated patients with metachromatic leukodystrophy, but this material disappeared within twenty hours. In fibroblasts from an individual with pseudodeficiency of arylsulfatase A, the immunoprecipitable precursor glycoprotein was smaller (M_r= 56,000). The synthesis of cross-reactive proteins with altered properties supports the concept of allelic mutations as the genetic basis of metachromatic leukodystrophy and of arylsulfatase A pseudodeficiency.     

Processing of the Encephalomyocarditis Virus Capsid Precursor Protein Studies in Rabbit Reticulocyte Lysates Incubated with N-Formyl-[35S]Methionine-tRNAfMet

Translation of encephalomyocarditis virus RNA in rabbit reticulocyte lysates in the presence of N-formyl-[(35)S]methionine-tRNA(f) (Met) revealed that a small polypeptide is cleaved from the N-terminus of the capsid protein precursor, preA, by virus-coded protease activity. Therefore, this N-terminal segment comprising the translation initiation site is not conserved in any of the mature capsid proteins.