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81.
Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination. 相似文献
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83.
A method for gene enrichment based on the avidin-biotin interaction. Application to the Drosophila ribosomal RNA genes. 总被引:4,自引:0,他引:4
A method of enriching, from the total DNA of an organism, for long DNA strands carrying a particular gene is described. The purified RNA corresponding to the gene is covalently attached to biotin via a cytochrome c bridge. This modified RNA is hybridized to the total DNA. Those DNA strands which hybridize are separated from all the other DNA, using the avidin-biotin interaction, by one of two methods. Avidin is covalently attached to submicroscopic polymer spheres; the complexes of avidin spheres with the DNA: RNA-biotin hybrids band in CsCl at a much lower buoyant density than does free DNA. Alternatively, the DNA:RNA-biotin hybrids are isolated by affinity chromatography on an avidin-solid support column. These methods have been used to prepare long single strands of Drosophila ribosomal DNA (rDNA) in high yield and 42 to 80% pure. 相似文献
84.
Silvia M.C. Dias João B. Fernandes José G.S. Maia Otto R. Gottlieb Hugo E. Gottlieb 《Phytochemistry》1982,21(7):1737-1740
The trunk wood of an Amazonian Aniba (Lauraceae) species contains, besides dillapiol and the benzodioxane-type neolignan eusiderin, four bicyclo(3.2.1)octanoid neolignans. These comprise representatives of the canellin-type: the known methoxycanellin-A and the novel compounds characterized as (1R, 3S, 4S, 5S, 6S, 7R)-1-allyl-4-hydroxy-3, 5-dimethoxy-7-methyl-6-(3′-methoxy-4′, 5′-methylenedioxyphenyl)-8-oxo-bicyclo(3.2.1)octane; (1R, 3S, 4S, 5S, 6S, 7R)-1-allyl-4-hydroxy-3, 5-dimethoxy-7-methyl-6-(3′, 4′, 5′-trimethoxyphenyl)-8-oxobicyclo(3.2.1)octane and (1R, 4R, 5R, 6S, 7R, 8S)-1-allyl-4, 8-dihydroxy-5-methoxy-7-methyl-6-(3′-methoxy-4′,5′-methylenedioxyphenyl)-3-oxobicyclo(3.2.1)octane. 相似文献
85.
Among more than 50 isolates ofBacillus thuringiensis Berliner (B.t.) tested, 7 incited 100% mortality when 2nd instar larvae ofSpodoptera littoralis Boisduval were fed on alfalfa leaves dipped in a spore-crystal suspension of 108 colony forming units/ml. Among those isolates,B.t. 24 demonstrated the highest activity. Larvae of instars 1 and 2 were the most susceptible toB.t. Susceptibility decreased with larval development. However, larvae of all instars were killed by isolateB.t. 24. Larvae that survived after feeding withB.t. 24 were retarded and fed less. Their weight relative to the controls was lower as the spore concentration on the leaves on which they fed was higher. Survival of the spores in the field dropped drastically to 2% after 4 days. Insecticidal activity of the sprayed suspension on those leaves, however, remained significant.B.t. 24 was also effective against larvae on cotton plants in the greenhouse and in a preliminary field experiment. Numbers of colony forming units recovered from leaves dipped in suspension of various spore concentrations showed significant correlation with the initial concentrations as did sprayed leaves. However, colony forming units recovered from sprayed leaves were 5–7.5 fold lower than from dipped leaves. Dipped cotton leaves showed 3.1×10?5 ml attached to 1 mm2 leaf surface whereas sprayed ones had 6×10?6 ml. Those data are important for the determination of spore concentrations in suspensions required for spraying. The isolateB.t. 24 was serotyped byH. de Barjac as H-6B. thuringiensis entomocidus. 相似文献
86.
Application of the avidin-biotin method of gene enrichment to the isolation of long double-stranded DNA containing specific gene sequences. 总被引:5,自引:5,他引:0
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A method of enriching for long double-stranded segments of eukaryotic DNA carrying particular genes is described. A purified RNA coded for by the gene is covalently attached to biotin via the protein, cytochrome c. This modified RNA is hybridized to total nuclear, double-stranded DNA under conditions that allow the formation of R-loops. Avidin, which has a high affinity for biotin, is covalently attached to polymer spheres. The complexes of avidin-spheres with DNA:RNA-biotin R-loop hybrids band in CsCl at a much lower bouyant density than does free DNA. This density is a function of the length of DNA coupled per avidin-sphere. This method was used to prepare very long double-strands of DNA highly enriched in the coding sequences for the large rRNAs of D. melanogaster and L. donovani and the histone mRNAs of S. purpuratus. 相似文献
87.
Collagen phenotypes were determined for rabbit articular chondrocytes in cartilage slices and first through fifth monolayer cultures. During the first 24 hr of slice culture, chondrocytes exhibited the following collagen phenotype: 96% type II, 3% X2Y and 1% type III. In primary monolayer culture, no other types of collagen were added to this differentiated chondrocyte phenotype; however, the synthesis per cell of each of the expressed collagens was stimulated. By the fifth day of primary culture, X2Y synthesis increased 10 fold, and by the eighth day, a further 4 fold. In contrast, the synthesis of collagen types II and III showed no change by the fifth day, but increased 7 fold by the eighth day. These results suggest independent regulation of X2Y in this situation. In a separate experiment, first through fifth cultures were studied. The synthesis per cell of type II collagen declined steadily and essentially ceased by the fifth culture, indicating the loss of differentiated function by these chondrocyte progeny. The loss of type II synthesis was not quantitatively replaced by the synthesis of type I trimer and type I collagen which was first detected in the third culture. While these qualitative changes in phenotype occurred, the stimulated rate of type III collagen synthesis did not change and that of X2Y declined only slightly. Thus the termination of type II synthesis did not significantly alter the synthesis of the other collagens produced by differentiated chondrocytes. The final “de-differentiated” phenotype was 41% type I, 25% X2Y, 20% type I trimer, 13% type III and 1% type II. 相似文献
88.
We measured a tubular brush-border enzyme (alanine aminopeptidase, AAP) and a lysosomal hydrolase (N-acetyl-beta-D-glucosaminidase, NAG) in morning urines from 15 healthy normal subjects to check if different storage times and temperatures could modify enzyme concentrations. Short-term (24 h) storage time at room temperature or 4 degrees C does not affect AAP and NAG activities. Both enzymes are well preserved at -70 degrees C. AAP dramatically falls after 1 month at -20 degrees C, lowering to about 8% of the initial value after only 4 days of storage. On the contrary, NAG is well preserved at these storage conditions. Centrifugation has revealed not critical for measurement of these two enzymes. 相似文献
89.
90.
A sensitive and precise automated assay of urinary lactate dehydrogenase (EC 1.1.1.27), alkaline phosphatase (EC 3.1.3.1) and gamma-glutamyltransferase (EC 2.3.2.2) is described. For this purpose, we used a BM/Hitachi System 704 model and reagents for automated analysis of serum enzymes from Boehringer Mannheim. However, the schedules of enzyme chemistry parameters recorded by the autoanalyzer and the spectrophotometric calibration are reprogrammed to meet requirements deriving from urine adoption and to optimize the enzyme assay in this unusual medium. 相似文献