RNA-bound reverse transcriptase in Escherichia coli and in vitro synthesis of a complementary DNA

1974 ◽  
Vol 12 (2) ◽  
pp. 163-180 ◽  
Author(s):  
Mirko Beljanski ◽  
Monique Beljanski
Keyword(s):  
Escherichia Coli ◽  
Reverse Transcriptase ◽  
In Vitro Synthesis ◽  
Complementary Dna

scholarly journals In Vitro Synthesis of Multicopy Single-Stranded DNA, Using Separate Primer and Template RNAs, by Escherichia coli Reverse Transcriptase

1998 ◽  
Vol 180 (11) ◽  
pp. 2999-3002 ◽  
Author(s):  
Tadashi Shimamoto ◽  
Hideki Kawanishi ◽  
Tomofusa Tsuchiya ◽  
Sumiko Inouye ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
Reverse Transcriptase ◽  
Bimolecular Reaction ◽  
Single Stranded Dna ◽  
Free System ◽  
In Vitro Synthesis ◽  
A Minor ◽  
Rna Transcript

ABSTRACT A minor population of wild strains of Escherichia colicontains a retron, a retroelement responsible for the synthesis of multicopy single-stranded DNA (msDNA). The retron is a genetic element consisting of the gene for reverse transcriptase (RT) and themsr-msd region under a single promoter. A single RNA transcript from the msr-msd region serves not only as a template but also as a primer for msDNA synthesis. Here, using a cell-free system with purified RT from retron Ec73, we examined whether the reaction can occur in a bimolecular reaction with use of separately expressed msr and msd transcripts. DNA sequencing of the cell-free product revealed that the sequence of the 5′-end region was identical to that of msDNA-Ec73, indicating that the cDNA synthesis was primed from the 2′-OH group of the specific internal G residue of the primer RNA, identical to the branching G residue in the RNA molecule of msDNA-Ec73. The present results raise an intriguing possibility for a role of bacterial retrons in vivo, the possibility that cellular mRNAs can be converted into cDNAs in retron-harboring cells if the mRNAs contain a sequence complementary to the sequence directly upstream of the branching G residue of the msr RNA transcript.

Cloning and expression of the glycerol-3-phosphate transport genes of Escherichia coli

1978 ◽  
Vol 56 (6) ◽  
pp. 611-617 ◽  
Author(s):  
Joel H. Weiner ◽  
Elke Lohmeier ◽  
Anthony Schryvers
Keyword(s):  
Escherichia Coli ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Cloning And Expression ◽  
In Vitro Synthesis ◽  
Whole Cells ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Plasmid Size ◽  
And Control

The two thousand Escherichia coli: Col E1 hybrid plasmid strains of the Clarke and Carbon colony bank (Clarke, L. &Carbon, J. (1976) Cell 9, 91–96) were screened by conjugation for those that correct the deficiency of a mutant unable to transport glycerol-3-phosphate. Six strains harbouring recombinant plasmids carrying the glpT region were identified and characterized with respect to plasmid size and transport properties. The initial rate of glycerol-3-phosphate transport in both whole cells and membrane vesicles prepared from such strains was elevated 3- to 10-fold over strains carrying random DNA inserts, whereas the Km of glycerol-3-phosphate transport was near 12 μM in both experimental and control strains. Four of the six glpT carrying plasmid strains demonstrated elevated levels of the anaerobic glycerol-3-phosphate dehydrogenase coded for by the neighbouring glpA gene.We have transferred the glpT hybrid plasmids into a minicell-producing strain of E. coli X1197 and have used the minicells for specific in vitro synthesis of plasmid-coded proteins. The glpT plasmids code for a 40 000 polypeptide which is localized in the periplasmic space. In addition, they code for a membrane-associated protein of 26 000 which may be the carrier polypeptide.

In vitro synthesis of β-galactosidase by membrane fractions of Escherichia coli

1970 ◽  
Vol 48 (8) ◽  
pp. 893-907 ◽  
Author(s):  
W. Chefurka ◽  
A. Yapo ◽  
B. Nisman
Keyword(s):  
Escherichia Coli ◽  
Actinomycin D ◽  
Heavy Fraction ◽  
Synthetic Activity ◽  
In Vitro Synthesis ◽  
Absolute Requirement ◽  
Discontinuous Sucrose Gradient ◽  
Reconstituted System ◽  
High Concentrations

The induction of β-galactosidase by a membranous fraction P1, prepared by digitonin lysis of spheroplasts of Escherichia coli, was studied in vitro. Electron micrographs of P1 show it to be a heterogeneous mixture of smooth vesicles, rough vesicles, rough vesicles attached to DNA, and ribosomes attached to DNA. P1 was subfractionated by differential centrifugation into an active heavy fraction, P4, and a relatively inactive light fraction, Pm. The P4 fraction consisted mainly of rough vesicles while the Pm fraction consisted mainly of smooth vesicles, but also of some rough vesicles. These vesicles of the Pm fraction were further separated by discontinuous sucrose gradient centrifugation.The induction of β-galactosidase by P1, P4, and Pm fractions was not related to mild contamination by unbroken viable spheroplasts. It was only partially sensitive to DNase and RNase. High concentrations of actinomycin D were required for complete inhibition of activity. This suggests that the transcription and translation components were shielded by the membranes. The synthetic activity of Pm was enhanced by fortification with DNA and/or S30. Only lac-containing DNA was active. The induction of β-galactosidase by this reconstituted system showed an absolute requirement for Pm membranes and for the inducer but only a partial requirement for nucleoside triphosphates. It was completely inhibited by puromycin and chloramphenicol.

scholarly journals In vitro synthesis of infectious RNA molecules from cloned hop stunt viroid complementary DNA.

1983 ◽  
Vol 59 (8) ◽  
pp. 251-254 ◽  
Author(s):  
Takeshi OHNO ◽  
Masayuki ISHIKAWA ◽  
Nobuhiko TAKAMATSU ◽  
Tetsuo MESHI ◽  
Yoshimi OKADA ◽  
...  
Keyword(s):  
In Vitro Synthesis ◽  
Complementary Dna ◽  
Rna Molecules ◽  
Stunt Viroid ◽  
Hop Stunt Viroid
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