scholarly journals The Two-Step Purification of Ribosomal RNA and Plant Viral RNA by Polyacrylamide Slab Gel Electrophoresis

1978 ◽  
Vol 31 (1) ◽  
pp. 25 ◽  
Author(s):  
Robert H Symons
Keyword(s):  
Ribosomal Rna ◽  
Gel Electrophoresis ◽  
Ribosomal Rnas ◽  
Viral Rnas ◽  
Complementary Dna ◽  
Sequence Characterization ◽  
Ethanol Precipitation ◽  
Step Procedure ◽  
Rna Fragments ◽  
Slab Gels

The requirement for purified plant viral RNAs for sequence characterization by hybridization analysis using complementary DNA led to the development of a routine two-step procedure for their purification. The method was worked out using Escherichia coli ribosomal RNAs and then applied to four plant viral RNAs, all of which contain four major RNA components. The first of the two steps involves the electrophoresis of native RNA on 2�8 % polyacrylamide slab gels, the location of the RNA bands by brief staining and the recovery of the RNA from each gel band by electrophoretic elution. The eluted RNA, concentrated by ethanol precipitation, was then run on a second gel after a denaturation step to release nicked and aggregated RNA fragments. Each RNA band was again located by staining and recovered by electrophoretic elution and ethanol precipitation. The 16-S and 23-S ribosomal RNAs and the plant viral RNAs were obtained in overall yields of 40 and 8-16 % respectively. RNAs so purified have been successfully used for the preparation of complementary DNA by two different methods.

scholarly journals Detection of presumptive Bacillus cereus in the Irish dairy farm environment

2016 ◽  
Vol 55 (2) ◽  
pp. 145-151 ◽  
Author(s):  
A. O’Connell ◽  
E.M. Lawton ◽  
D. Leong ◽  
P. Cotter ◽  
D. Gleeson ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Ribosomal Rna ◽  
Gel Electrophoresis ◽  
Tap Water ◽  
Dairy Farm ◽  
Egg Yolk ◽  
Pulsed Field Gel Electrophoresis ◽  
Chromogenic Medium ◽  
Rrna Sequencing ◽  
Bulk Tank

AbstractThe objective of the study was to isolate potentialBacillus cereussensu lato (B.cereus s.l.)from a range of farm environments. Samples of tap water, milking equipment rinse water, milk sediment filter, grass, soil and bulk tank milk were collected from 63 farms. In addition, milk liners were swabbed at the start and the end of milking, and swabs were taken from cows’ teats prior to milking. The samples were plated on mannitol egg yolk polymyxin agar (MYP) and presumptiveB. cereus s.l. colonies were isolated and stored in nutrient broth with 20% glycerol and frozen at -80 °C. These isolates were then plated on chromogenic medium (BACARA) and colonies identified as presumptiveB. cereus s.l. on this medium were subjected to 16S ribosomal RNA (rRNA) sequencing. Of the 507 isolates presumed to beB. cereus s.l. on the basis of growth on MYP, only 177 showed growth typical ofB. cereus s.l. on BACARA agar. The use of 16S rRNA sequencing to identify isolates that grew on BACARA confirmed that the majority of isolates belonged toB. cereus s.l. A total of 81 of the 98 isolates sequenced were tentatively identified as presumptiveB. cereus s.l. Pulsed-field gel electrophoresis was carried out on milk and soil isolates from seven farms that were identified as having presumptiveB. cereus s.l. No pulsotype was shared by isolates from soil and milk on the same farm. PresumptiveB. cereus s.l. was widely distributed within the dairy farm environment.

scholarly journals Separation of 1–23-kb complementary DNA strands by urea–agarose gel electrophoresis

2009 ◽  
Vol 37 (17) ◽  
pp. e112-e112 ◽  
Author(s):  
Éva Hegedüs ◽  
Endre Kókai ◽  
Alexander Kotlyar ◽  
Viktor Dombrádi ◽  
Gábor Szabó
Keyword(s):  
Gel Electrophoresis ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Complementary Dna ◽  
Dna Strands

scholarly journals Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan.

1981 ◽  
Vol 1 (4) ◽  
pp. 347-357 ◽  
Author(s):  
M W Gray
Keyword(s):  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Ribosomal Rnas ◽  
High Potassium ◽  
Intact Cells ◽  
Crithidia Fasciculata ◽  
Low Magnesium ◽  
Chain Lengths

In a previous study from this laboratory, presumptive ribosomal ribonucleic acid (RNA) species were identified in the total cellular RNA directly extracted from intact cells of the trypanosomatid protozoan Crithidia fasciculata (M. W. Gray, Can. J. Biochem. 57:914-926, 1979). The results suggested that the C. fasciculata ribosome might be unusual in containing three novel, low-molecular-weight ribosomal RNA components, designated e, f, and g (apparent chain lengths 240, 195, and 135 nucleotides, respectively), in addition to analogs of eucaryotic 5S (species h) and 5.8S (species i) ribosomal RNAs. In the present study, all of the presumptive ribosomal RNAs were indeed found to be associated with purified C. fasciculata ribosomes, and their localization was investigated in subunits produced under different conditions of ribosome dissociation. When ribosomes were dissociated in a high-potassium (880 mM K+, 12.5 mM Mg2+) medium, species e to i were all found in the large ribosomal subunit, which also contained an additional, transfer RNA-sized component (species j). However, when subunits were prepared in a low-magnesium (60 mM K+, 0.1 mM Mg2+) medium, two of the novel species (e and g) did not remain with the large subunit, but were released, apparently as free RNAs. Control experiments have eliminated the possibility that the small RNAs are generated by quantitative and highly specific (albeit artifactual) ribonuclease cleavage of large ribosomal RNAs during isolation. In terms of RNA composition and dissociation properties, therefore, the ribosome of C. fasciculata is the most "atypical" eucaryotic ribosome yet described. These observations raise interesting questions about the function and evolutionary origin of C. fasciculata ribosomes and about the organization and expression of ribosomal RNA genes in this organism.

scholarly journals Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens

1988 ◽  
Vol 253 (1) ◽  
pp. 263-267 ◽  
Author(s):  
I Vancurová ◽  
J Volc ◽  
M Flieger ◽  
J Neuzil ◽  
J Novotná ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Size Exclusion ◽  
Streptomyces Aureofaciens ◽  
Hydrophobic Chromatography ◽  
Step Procedure

Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.

scholarly journals MITOCHONDRIAL RNA FROM CULTURED ANIMAL CELLS

1970 ◽  
Vol 46 (2) ◽  
pp. 245-251 ◽  
Author(s):  
Bland S. Montenecourt ◽  
Margaret E. Langsam ◽  
Donald T. Dubin
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Cell Lines ◽  
Ribosomal Rna ◽  
Gel Electrophoresis ◽  
Base Composition ◽  
Size Class ◽  
Mitochondrial Rna ◽  
Animal Cells ◽  
Molecular Weights

Discrete RNA fractions sedimenting slightly slower than 18s ribosomal RNA have been found in mitochondrial preparations from both hamster (BHK-21) and mouse (L-929) cells. This RNA could be separated into two components, present in approximately equimolar amounts, by prolonged zonal centrifugation or acrylamide gel electrophoresis. The hamster components had sedimentation constants averaging 16.8 and 13.4, and molecular weights (estimated by gel electrophoresis) averaging 0.74 and 0.42 x 106 daltons. Mixed labeling experiments showed that the mouse components sedimented and electrophoresed 3–6% more slowly than the corresponding hamster components. The RNA from both cell lines resembled mitochondrial ribosomal RNA from yeast and Neurospora in being GC poor, and in addition the larger and smaller components resembled each other in base composition. These results, taken with those of other recent studies, are compatible with the idea that our high molecular weight mitochondrial RNA is ribosomal; such RNA would then constitute a uniquely small size-class of ribosomal RNA.

A hidden break in the 28.0S rRNA from Diphyllobothrium dendriticum

1992 ◽  
Vol 66 (3) ◽  
pp. 193-197 ◽  
Author(s):  
K. A. Karlstedt ◽  
G. I. L. Paatero ◽  
J.-H. Mäkelä ◽  
B.-J. Wikgren
Keyword(s):  
Ribosomal Rna ◽  
Northern Blot ◽  
Gel Electrophoresis ◽  
Large Subunit ◽  
Small Subunit ◽  
Agarose Gel ◽  
Oligonucleotide Probes ◽  
Agarose Gel Electrophoresis ◽  
Diphyllobothrium Dendriticum ◽  
Large Subunit Ribosomal Rna

ABSTRACTNondenatured and denatured total RNA from the tapeworm Diphyllobothrium dendriticum (Cestoda) was analysed by agarose gel electrophoresis. It was found that the large subunit ribosomal RNA (lrRNA) is 28.0S and the small subunit ribosomal RNA (srRNA) is 19.5S. Following denaturation the 28.0S rRNA was disrupted into a 19.5S subfragment and a 20.7S subfragment due to the presence of a centrally located hidden break. By hybridization of Northern blot membranes with oligonucleotide probes specific for the 5′- and 3′-ends of the lrRNA respectively, we have shown that the 19.5S sub-fragment is from the 5′-end (the α-subfragment) and the 20.7S subfragment from the 3′-end (the β-subfragment) of the 28.0S rRNA of D. dendriticum.

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