In vitro synthesis of 16S ribosomal RNA containing single base changes and assembly into a functional 30S ribosome

Biochemistry ◽  
1987 ◽  
Vol 26 (8) ◽  
pp. 2353-2364 ◽  
Author(s):  
W. Krzyzosiak ◽  
R. Denman ◽  
K. Nurse ◽  
W. Hellmann ◽  
M. Boublik ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
16S Ribosomal Rna ◽  
In Vitro Synthesis ◽  
Single Base

Oligo(A)-stimulated Tetrahymena rDNA synthesis in vitro

1981 ◽  
Vol 59 (6) ◽  
pp. 396-403 ◽  
Author(s):  
Peter R. Ganz ◽  
Gyorgy B. Kiss ◽  
Ronald E. Pearlman
Keyword(s):  
Rna Polymerase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Ribosomal Rna ◽  
Replication Proteins ◽  
General Applicability ◽  
In Vitro Synthesis ◽  
Restriction Fragments ◽  
Double Stranded Dna

The synthesis of Tetrahymena rDNA has been examined using purified DNA polymerase and partially purified preparations of homologous replication enzymes (fraction IV). DNA synthesis with purified DNA polymerase alone was less than that with fraction IV enzymes. This suggested that there were additional factors in fraction IV other than DNA polymerase which contributed to or enhanced rDNA synthesis in vitro. Neither hybridization of rDNA with Tetrahymena ribosomal RNA nor preincubation of rDNA with homologous or heterologous RNA polymerase served to stimulate in vitro synthesis by fraction IV enzymes. However, when rDNA was hybridized with oligoriboadenylate, DNA synthesis using fraction IV was stimulated approximately 4- to 4.5-fold over 150 min of incubation, relative to a similarly treated but unhybridized rDNA control. Using oligoriboadenylate-hybridized EcoR1 and HindIII restriction fragments of rDNA to localize the synthesis most of the in vitro synthesis occurred within a 2.4 × 106 Mr fragment encompassing the centre of the rDNA molecule. The approach of hybridizing a synthetic homooligoribonucleotide primer to double-stranded DNA should prove to be of general applicability in designing similar template–primers in other systems for the purpose of isolating replication proteins.

The in vitro Synthesis of Ribosomal RNA

1970 ◽  
Vol 35 (0) ◽  
pp. 415-418 ◽  
Author(s):  
A. Travers ◽  
R. Kamen ◽  
M. Cashel
Keyword(s):  
Ribosomal Rna ◽  
In Vitro Synthesis

scholarly journals Molecular Diagnosis of Periprosthetic Joint Infection by Quantitative RT-PCR of Bacterial 16S Ribosomal RNA

2013 ◽  
Vol 2013 ◽  
pp. 1-4 ◽  
Author(s):  
Mel S. Lee ◽  
Wen-Hsin Chang ◽  
Su-Chin Chen ◽  
Pang-Hsin Hsieh ◽  
Hsin-Nung Shih ◽  
...  
Keyword(s):  
16S Rrna ◽  
Molecular Diagnosis ◽  
Ribosomal Rna ◽  
Periprosthetic Joint Infection ◽  
Joint Infection ◽  
16S Ribosomal Rna ◽  
Purulent Discharge ◽  
Fistula Tract ◽  
Culture Negative

The diagnosis of periprosthetic joint infection is sometimes straightforward with purulent discharge from the fistula tract communicating to the joint prosthesis. However it is often difficult to differentiate septic from aseptic loosening of prosthesis because of the high culture-negative rates in conventional microbiologic culture. This study used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to amplify bacterial 16S ribosomal RNA in vitro and in 11 clinical samples. The in vitro analysis demonstrated that the RT-qPCR method was highly sensitive with the detection limit of bacterial 16S rRNA being 0.148 pg/μl. Clinical specimens were analyzed using the same protocol. The RT-qPCR was positive for bacterial detection in 8 culture-positive cases (including aerobic, anaerobic, and mycobacteria) and 2 culture-negative cases. It was negative in one case that the final diagnosis was confirmed without infection. The molecular diagnosis of bacterial infection using RT-qPCR to detect bacterial 16S rRNA around a prosthesis correlated well with the clinical findings. Based on the promising clinical results, we were attempting to differentiate bacterial species or drug-resistant strains by using species-specific primers and to detect the persistence of bacteria during the interim period before the second stage reimplantation in a larger scale of clinical subjects.

In vitro synthesis of Escherichia coli ribosomal RNA

1973 ◽  
Vol 75 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Linda S. Birnbaum ◽  
Sam Kaplan
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
In Vitro Synthesis

scholarly journals In Vitro Synthesis of Ribosomal RNA by Bacillus subtilis RNA Polymerase

1972 ◽  
Vol 69 (2) ◽  
pp. 407-411 ◽  
Author(s):  
C. Hussey ◽  
J. Pero ◽  
R. G. Shorenstein ◽  
R. Losick
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Ribosomal Rna ◽  
In Vitro Synthesis

The in vitro synthesis and characteristics of ribosomal RNA in imaginal discs of Drosophila melanogaster

1971 ◽  
Vol 110 (3) ◽  
pp. 245-262 ◽  
Author(s):  
William H. Petri ◽  
James W. Fristrom ◽  
Dan J. Stewart ◽  
E. W. Hanly
Keyword(s):  
Drosophila Melanogaster ◽  
Ribosomal Rna ◽  
Imaginal Discs ◽  
In Vitro Synthesis

Study of the function of Escherichia coli ribosomal RNA through site-directed mutagenesis

1990 ◽  
Vol 68 (1) ◽  
pp. 169-179 ◽  
Author(s):  
Daniel Leclerc ◽  
Léa Brakier-Gingras
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Large Body ◽  
Ribosomal Rna Genes ◽  
16S Ribosomal Rna ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Rna Genes

Various approaches have been developed to study how mutations in Escherichia coli ribosomal RNA affect the function of the ribosome. Most of them are in vivo approaches, where mutations are introduced in a specialized plasmid harboring the ribosomal RNA genes. The mutated plasmids are then expressed in an appropriate host, where they can confer resistance to antibiotics whose target is the ribosome. Conditions can be used where the host ribosomal RNA genes or the host ribosomes are selectively inactivated, and the effect of the mutations on ribosome assembly and function can be studied. Another approach, which has been developed mainly with 16S ribosomal RNA, can be used entirely in vitro. In this approach, a plasmid has been constructed which contains the 16S ribosomal RNA gene under control of a T7 promoter. Mutations can be introduced in the 16S ribosomal RNA sequence and the mutated 16S ribosomal RNAs are produced by in vitro transcription. It is then possible to investigate how the mutations affect the assembly of the 16S ribosomal RNA into 30S subunits and the activity of the reconstituted 30S subunits in cell-free protein synthesis assays. Although these approaches are recent, they have already provided a large body of interesting information, relating specific RNA sequences to interactions with ribosomal proteins, to ribosome function, and to its response to antibiotics.Key words: ribosomal RNA, ribosome, site-directed mutagenesis, antibiotic resistance.

On the role of small peptides in the regulation of RNA synthesis in Tetrahymena pyriformis

1980 ◽  
Vol 45 (1) ◽  
pp. 31-39
Author(s):  
H.A. Andersen ◽  
A.E. Lykkesfeldt ◽  
S.J. Nielsen
Keyword(s):  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Tetrahymena Pyriformis ◽  
Defined Medium ◽  
Specific Factor ◽  
Small Peptides ◽  
In Vitro Synthesis

Tetrahymena cells secrete a factor which inhibits RNA synthesis in vivo and in vitro. The factor is a relatively small peptide with a molecular weight between 300 and 1500 Daltons. Other, non-specific peptides in the broth medium or added to a chemically defined medium have a stimulatory effect on RNA synthesis in vivo and such peptides also stimulate the in vitro synthesis of RNA in a r-chromatin preparation. On the basis of these results we conclude that such extracellular small peptides compete with a specific factor which is part of the intracellular regulatory mechanism controlling the rate of RNA synthesis. The consequence of such competition is a high overproduction of ribosomal RNA in cells inoculated on peptide-rich broth media.

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