The conversion of [2-14C]mevalonic acid into triterpenes and tetraterpenes by cell-free extracts of a Neurospora crassa albino mutant

1977 ◽  
Vol 55 (15) ◽  
pp. 2137-2141 ◽  
Author(s):  
G. S. Bobowski ◽  
W. G. Barker ◽  
R. E. Subden
Keyword(s):  
Neurospora Crassa ◽  
Column Chromatography ◽  
Preliminary Analysis ◽  
Mevalonic Acid ◽  
Free System ◽  
Absolute Requirement ◽  
Albino Mutant ◽  
A Cell ◽  
Ultraviolet Absorption Spectroscopy ◽  
Layer Chromatography

Evidence for the conversion of [2-14C]mevalonic acid into triterpene and some tetraterpene components of Neurospora crassa in a cell-free system is presented. Radioactivity in the non-saponifiable neutral lipid extracts of the incubated cell-free extract was partitioned into three fractions by column chromatography. Purification of the radioactive component of one of these fractions by column chromatography and analysis by ultraviolet-absorption spectroscopy, thin-layer chromatography, and gas chromatography showed the radioactivity to be present primarily in squalene. Preliminary analysis of the remaining two fractions indicated the incorporation of mevalonate into phytoene and sterol intermediates. The conversion of mevalonate into squalene exhibited an absolute requirement for ATP and Mn2+ and was enhanced in the presence of NADP or NADPH.

Enzymatic Preparation of 14C-Labeled Phytoene, Squalene, and Geranylgeranyl Pyrophosphate from [2-14C]Mevalonic Acid

1980 ◽  
Vol 35 (11-12) ◽  
pp. 927-930 ◽  
Author(s):  
Gerhard Sandmann ◽  
Willy Hilgenberg ◽  
Peter Böger
Keyword(s):  
Column Chromatography ◽  
Mevalonic Acid ◽  
Preparative Thin Layer Chromatography ◽  
Enzymatic Preparation ◽  
Phycomyces Blakesleeanus ◽  
Free System ◽  
Geranylgeranyl Pyrophosphate ◽  
A Cell ◽  
White Mutant ◽  
Layer Chromatography

Methods are described for an enzymatic preparation of 14C-labeled terpenoids. With a cell-free system of a white mutant of Phycomyces blakesleeanus (Mucoraceae) [14C]squalene and [14C- cis]phytoene can be synthesized from [2-14C]mevalonate. The application of norflurazon, a phenyl- pyridazinone herbicide, helps to increase the yield of squalene. Furthermore, the liquid endosperm of Echinocystis lobata (Cucurbitaceae) was used for the formation of either [14C(-)]kaurene from [14C]mevalonic acid or [14C-/ra/w]geranylgeranyl pyrophosphate in the presence of Amo 1618. The hydrocarbons formed were purified by alumina-column chromatography and preparative thin-layer chromatography (TLC). Geranylgeranyl pyrophosphate was separated by DE-column chromatography followed by TLC.

A cell-free system from Streptococcus faecium for studies on the biosynthesis of triterpenoid carotenoids

1982 ◽  
Vol 60 (6) ◽  
pp. 675-683 ◽  
Author(s):  
Richard F. Taylor ◽  
Brian H. Davies
Keyword(s):  
Mevalonic Acid ◽  
Potassium Fluoride ◽  
Magnesium Ion ◽  
Free System ◽  
Isolation And Purification ◽  
Cell Free System ◽  
Manganese Ion ◽  
Absolute Requirement ◽  
Streptococcus Faecium ◽  
A Cell

A cell-free enzyme extract from Streptococcus faecium UNH 564P has been prepared. The extract incorporates either [2-14C]mevalonic acid (MVA) or [1-14C]isopentenyl pyrophosphate (IPP) into squalene and the carotenoids of the bacterium. ATP and manganese ion were found to be absolute requirements for MVA incorporation by the extract. Only manganese ion was found to be an absolute requirement for IPP incorporation by the extract. Other cofactors including magnesium ion, glutathione, potassium fluoride, NADP, and FAD significantly increase the incorporation of both substrates into the S. faecium terpenoids. Isolation and purification of the radioactive terpenoids from the cell-free system confirmed that the carotenoids of S. faecium are triterpenoids.

scholarly journals Transport of Cytoplasmically Synthesized Proteins into the Mitochondria in a Cell Free System from Neurospora crassa

1977 ◽  
Vol 81 (3) ◽  
pp. 533-544 ◽  
Author(s):  
Matthew A. HARMEY ◽  
Gerhard HALLERMAYER ◽  
Harald KORB ◽  
Walter NEUPERT
Keyword(s):  
Neurospora Crassa ◽  
Free System ◽  
Cell Free System ◽  
A Cell

The conversion of mevalonic acid into gibberellin A12-aldehyde in a cell-free system from Cucurbita pepo

Planta ◽  
1972 ◽  
Vol 102 (3) ◽  
pp. 261-271 ◽  
Author(s):  
J. E. Graebe ◽  
D. H. Bowen ◽  
J. MacMillan
Keyword(s):  
Cucurbita Pepo ◽  
Mevalonic Acid ◽  
Free System ◽  
Cell Free System ◽  
A Cell

The biosynthesis of a C19-gibberellin from mevalonic acid in a cell-free system from a higher plant

Planta ◽  
1974 ◽  
Vol 120 (3) ◽  
pp. 307-309 ◽  
Author(s):  
J. E. Graebe ◽  
P. Hedden ◽  
P. Gaskin ◽  
J. MacMillan
Keyword(s):  
Mevalonic Acid ◽  
Higher Plant ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Ribosome stalling is responsible for arginine-specific translational attenuation in Neurospora crassa.

1997 ◽  
Vol 17 (9) ◽  
pp. 4904-4913 ◽  
Author(s):  
Z Wang ◽  
M S Sachs
Keyword(s):  
Neurospora Crassa ◽  
Translational Regulation ◽  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Initiation Codon ◽  
Free System ◽  
Reading Frame ◽  
Ribosome Stalling ◽  
A Cell ◽  
Specific Regulation

The Neurospora crassa arg-2 upstream open reading frame (uORF) plays a role in negative arginine-specific translational regulation. Primer extension inhibition analyses of arg-2 uORF-containing RNA translated in a cell-free system in which arginine-specific regulation was retained revealed "toeprints" corresponding to ribosomes positioned at the uORF initiation and termination codons and at the downstream initiation codon. At high arginine concentrations, the toeprint signal corresponding to ribosomes at the uORF termination codon rapidly increased; a new, broad toeprint that represents additional ribosomes stalled on the uORF appeared 21 to 30 nucleotides upstream of this site; and the toeprint signal corresponding to ribosomes at the downstream initiation codon decreased. These data suggest that arginine increases ribosomal stalling and thereby decreases translation from the downstream initiation codon.

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

2019 ◽  
pp. 27-33
Author(s):  
YuE Kravchenko ◽  
SV Ivanov ◽  
DS Kravchenko ◽  
EI Frolova ◽  
SP Chumakov
Keyword(s):  
Phage Display ◽  
Selection Procedure ◽  
Free System ◽  
Phage Displays ◽  
High Affinity ◽  
Binding Domains ◽  
A Cell ◽  
Vhh Antibodies ◽  
Efficiency Of Selection ◽  
Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

Sign in / Sign up

Export Citation Format

Share Document