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The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E
RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 106 infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses. 相似文献
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Apparent digestibility of crude protein, amino acids, lipid, carbohydrate and energy was measured for a range of feed ingredients fed to gilthead seabream, Sparus aurata L. — fish meal, poultry meal, meat meal, blood meal, squid meal, extracted soyabean and wheat flour. Chromic oxide was used as a non-absorbed reference substance and faeces were collected by stripping. Diets compounded from mixtures of these ingredients were then used to examine the possibility of predicting the digestibility of formulated diets.
Apparent digestibility of crude protein ranged from 79% to 90%, lipids from 83% to 95% and energy from 72% to 88% in the different ingredients. Apparent digestibility of carbohydrates was lower and ranged from 49% to 77%. Apparent digestibility of amino acids was higher than that of crude protein and differences were found among digestibilities of individual amino acids.
Tests conducted using five compound diets indicated that ingredient digestibility was additive for protein, amino acids, lipids and energy, whereas the digestibility of carbohydrates in the compound feeds was slightly lower than predicted.
Diets for Sparus aurata may thus be formulated on the basis of digestibility of individual ingredients. 相似文献
Apparent digestibility of crude protein ranged from 79% to 90%, lipids from 83% to 95% and energy from 72% to 88% in the different ingredients. Apparent digestibility of carbohydrates was lower and ranged from 49% to 77%. Apparent digestibility of amino acids was higher than that of crude protein and differences were found among digestibilities of individual amino acids.
Tests conducted using five compound diets indicated that ingredient digestibility was additive for protein, amino acids, lipids and energy, whereas the digestibility of carbohydrates in the compound feeds was slightly lower than predicted.
Diets for Sparus aurata may thus be formulated on the basis of digestibility of individual ingredients. 相似文献
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Book reviewed in this articles: Cereal Grain Protein Improvement. Shivanna, K. R., and B. M. Johri, The Angiosperm Pollen. Structure and Function. Gustafson, J. P., Gene Manipulation in Plant Improvement. Backer, A. W., Manual of Quantitative Genetics. Fishbeck, G., W. Plarre und W. Schuster (Hrag.) , Hoffmann , W., A. Plarre : Lehrbush der Züchtung landwirtschaftlicher Kulturflanzen, Bd. 2, Spezieller Teil, 2. Auflage. Dodds, J. H. (editor and author), Plant Genetic Engineering. Fiechter, A. (managing editor), Advanced in Biochemical Engineering/Biotechnology. Vol. 31. Plant Cell Culture. 相似文献
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A single receptor binds both insulin-like growth factor II and mannose-6-phosphate 总被引:23,自引:0,他引:23
R G MacDonald S R Pfeffer L Coussens M A Tepper C M Brocklebank J E Mole J K Anderson E Chen M P Czech A Ullrich 《Science (New York, N.Y.)》1988,239(4844):1134-1137
Amino acid sequences deduced from rat complementary DNA clones encoding the insulin-like growth factor II (IGF-II) receptor closely resemble those of the bovine cation-independent mannose-6-phosphate receptor (Man-6-P receptorCI), suggesting they are identical structures. It is also shown that IGF-II receptors are adsorbed by immobilized pentamannosyl-6-phosphate and are specifically eluted with Man-6-P. Furthermore, Man-6-P specifically increases by about two times the apparent affinity of the purified rat placental receptor for 125I-labeled IGF-II. These results indicate that the type II IGF receptor contains cooperative, high-affinity binding sites for both IGF-II and Man-6-P-containing proteins. 相似文献
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We examined collembolan food preference for fungal mycelium grown on copper-contaminated medium, and the relationship between copper content, food selectivity and collembolan fitness when fed contaminated mycelium.To clarify whether collembolan food selectivity is related to fitness parameters, Folsomia candida were fed mycelium of the dark-pigmented fungus Alternaria alternata grown on medium with different copper concentrations. Copper-contaminated food (fungus grown on 50, 125, 250 and 500 μg Cu g?1 medium, fresh wt.) was offered together with untreated food for 4 weeks. F. candida fed selectively on the provided mycelium and discriminated clearly between mycelium grown on high and low levels of contamination, distinctly preferring fungus grown on medium with a total copper concentration of 50 and 125 μg g?1. In contrast, fungus grown on highly contaminated medium (250 and 500 μg g?1) was avoided. Collembolan food preference generally matched fitness parameters. Reproduction was significantly affected by the total copper concentration of the fungal growth medium. When fed their preferred mycelium, collembolan reproduction was enhanced, whereas a diet of highly contaminated mycelium (250 or 500 μg g?1) resulted in a strong decrease in reproduction. Adult survival was affected only marginally. Even though heavy metal contamination is a potential stress factor for many soil microarthropods, F. candida is able to discriminate between high and low quality food sources, and even benefits from moderately elevated copper concentrations. 相似文献
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