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 reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

2006 ◽  
Vol 396 (3) ◽  
pp. 565-571 ◽  
Author(s):  
Takaomi Nomura ◽  
Kohji Nakano ◽  
Yasushi Maki ◽  
Takao Naganuma ◽  
Takashi Nakashima ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Synthetic Activity ◽  
23S Rrna ◽  
Elongation Factors ◽  
Hybrid Form ◽  
Pyrococcus Horikoshii ◽  
E Coli ◽  
Functional Features

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0·Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0·Ph-L12 complex and Ph-L11 could replace L10·L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

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.

DNA synthesis in the developing rat brain

1969 ◽  
Vol 47 (1) ◽  
pp. 47-50 ◽  
Author(s):  
Shan-Ching Sung
Keyword(s):  
Dna Synthesis ◽  
Rat Brain ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Rapid Rate ◽  
In Vitro Synthesis ◽  
Infant Rat ◽  
Developing Rat

The in vitro synthesis of DNA as measured by the incorporation of thymidine-2-14C into DNA has been studied for various regions of the infant rat brain. Both intact cerebellum and cell-free extracts of cerebellum from newborn and infant rat brain showed a very rapid rate of DNA synthesis which was highest around 6 days after birth and decreased rapidly thereafter up to 18 days. This DNA synthesis in developing rat brain was strongly inhibited by hydroxyurea but was much less sensitive than was RNA synthesis to inhibition by actinomycin D.

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
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