Phosphoenolpyruvate carboxylase mediated carbon flow in a cyanobacterium

1988 ◽  
Vol 66 (2) ◽  
pp. 93-99 ◽  
Author(s):  
George W. Owttrim ◽  
Brian Colman
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Protein Production ◽  
Carbon Fixation ◽  
Fixed Carbon ◽  
Free System ◽  
Pyruvate Orthophosphate Dikinase ◽  
Orthophosphate Dikinase ◽  
Phosphoglyceric Acid ◽  
A Cell

The source of the substrate phosphoenolpyruvate (PEP) for phosphoenolpyruvate carboxylase (PEP-case) activity in the cyanobacterium Coccochloris peniocystis has been investigated, as well as possible sinks for this carbon. PEP was not produced by pyruvate orthophosphate dikinase, as this activity was not detectable in cell-free lysates. PEP is supplied from photosynthetically or glycolytically produced 3-phosphoglyceric acid (3-PGA), as carbon was observed to flow from 3-PGA to C4 acids in a cell-free system. This indicates PEP-case activity is dependent on photosynthetically fixed carbon and thus two separate carbon fixation reactions occur in the cell in the light. Estimates of the in vivo concentrations of various metabolites indicates that neither substrate nor inhibitor concentrations limit enzyme activity in vivo. Thus PEP-case activity in vivo appears to be limited by the supply of PEP and is, therefore, high in the light and low in the dark. The nitrogen storage product cyanophycin was identified as one sink for carbon fixed by PEP-case. As a culture aged, cyanophycin production increased, while chlorophyll and protein production decreased.

scholarly journals Authentic Replication and Recombination of Tomato Bushy Stunt Virus RNA in a Cell-Free Extract from Yeast

2008 ◽  
Vol 82 (12) ◽  
pp. 5967-5980 ◽  
Author(s):  
Judit Pogany ◽  
Peter D. Nagy
Keyword(s):  
High Efficiency ◽  
Tomato Bushy Stunt Virus ◽  
Free System ◽  
Cell Free System ◽  
Virus Rna ◽  
A Cell ◽  
Viral Replicase ◽  
Template Specificity ◽  
Rna Complex

ABSTRACT To study the replication of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we have developed a cell-free system based on a Saccharomyces cerevisiae extract. The cell-free system was capable of performing a complete replication cycle on added plus-stranded TBSV replicon RNA (repRNA) that led to the production of ∼30-fold-more plus-stranded progeny RNAs than the minus-stranded replication intermediate. The cell-free system also replicated the full-length TBSV genomic RNA, which resulted in production of subgenomic RNAs as well. The cell-free system showed high template specificity, since a mutated repRNA, minus-stranded repRNA, or a heterologous viral RNA could not be used as templates by the tombusvirus replicase. Similar to the in vivo situation, replication of the TBSV replicon RNA took place in a membraneous fraction, in which the viral replicase-RNA complex was RNase and protease resistant but sensitive to detergents. In addition to faithfully replicating the TBSV replicon RNA, the cell-free system was also capable of generating TBSV RNA recombinants with high efficiency. Altogether, tombusvirus replicase in the cell-free system showed features remarkably similar to those of the in vivo replicase, including carrying out a complete cycle of replication, high template specificity, and the ability to recombine efficiently.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters

1981 ◽  
Vol 1 (7) ◽  
pp. 635-651
Author(s):  
D C Lee ◽  
R G Roeder
Keyword(s):  
Adenovirus Type ◽  
Free System ◽  
Ribonucleic Acids ◽  
Cell Free System ◽  
Cell Extracts ◽  
A Cell ◽  
Adenovirus Type 2

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA

1990 ◽  
Vol 10 (9) ◽  
pp. 4456-4465
Author(s):  
S M Carroll ◽  
P Narayan ◽  
F M Rottman
Keyword(s):  
Consensus Sequence ◽  
Specific Sequence ◽  
Free System ◽  
Consensus Sequences ◽  
Neplanocin A ◽  
A Cell ◽  
Steady State Levels ◽  
Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

scholarly journals Alloxan-induced luminol luminescence as a tool for investigating mechanisms of radical-mediated diabetogenicity

1981 ◽  
Vol 200 (3) ◽  
pp. 685-690 ◽  
Author(s):  
K Grankvist
Keyword(s):  
Superoxide Dismutase ◽  
Hydroxyl Radicals ◽  
Metal Ion ◽  
Reduced Glutathione ◽  
Chemical Reactivity ◽  
Free System ◽  
A Cell ◽  
Radical Generation ◽  
Metal Ion Chelators

Chemiluminescence of luminol in a cell-free system was used to investigate the mechanism of alloxan-dependent free-radical generation. In the presence of alloxan and reduced glutathione (GSH), luminescence was greatly stimulated by FeSO4. Replacing GSH by oxidized glutathione or NAD(P)(H), or replacing FeSO4 by CuSO4, ZNSO4 or FeCl3, did not yield chemiluminescence. The chemiluminescence of a mixture of alloxan. GSH, FeSO4 and luminol was inhibited by catalase, superoxide dismutase, scavengers of hydroxyl radicals (sodium benzoate, n-butanol, D-mannitol, dimethyl sulphoxide) or metal-ion chelators (EDTA, diethylenetriaminepenta-acetic acid, diethyldithiocarbamate. desferroxamine), D-glucose, L-glucose, D-mannose, D-fructose, 3-O-methyl-D-glucose, NAD+, NADH, NADP+ or NADPH, but not by urea or enzymically inactive superoxide dismutase. The results support the hypothesis that the diabetogenic action of alloxan is mediated by hydroxyl radicals generated in an iron-catalysed reaction. Protection against alloxan in vivo depends both on the chemical reactivity of protector with radicals or radical-generating systems and on the stereospecific requirement of some strategic site in the B-cell.

Yeast cell-free system that catalyses joint-molecule formation in a Rad51p- and Rad52p-dependent fashion

2000 ◽  
Vol 347 (2) ◽  
pp. 363-368 ◽  
Author(s):  
Vijayalakshmi NAGARAJ ◽  
David NORRIS
Keyword(s):  
Homologous Recombination ◽  
Specific Activity ◽  
Yeast Cells ◽  
Active Components ◽  
Free System ◽  
Cell Free System ◽  
Molecule Formation ◽  
A Cell

One of the central reactions of homologous recombination is the invasion of a single strand of DNA into a homologous duplex to form a joint molecule. Here we describe the isolation of a cell-free system from meiotic yeast cells that catalyses joint-molecule formation in vitro. The active components in the system required ATP and homologous DNA and operated in both 0.5 and 13 mM MgCl2. When the cell-free system was prepared from rad51/rad51 and rad52/rad52 mutants and joint-molecule formation was assayed at 0.5 mM MgCl2, the specific activity decreased to 6% and 13.8% respectively of the wild-type level. However, when the same mutant extracts were premixed, joint-molecule formation increased 4-8-fold, i.e. the mutant extracts exhibited complementation in vitro. These results demonstrated that Rad51p and Rad52p were required for optimal joint-molecule formation at 0.5 mM MgCl2. Intriguingly, however, Rad51p and Rad52p seemed to be more dispensable at higher concentrations of MgCl2 (13 mM). Further purification of the responsible activity has proven problematical, but it did flow through a sizing column as a single peak (molecular mass 1.2 MDa) that was co-eluted with Rad51p and RFA, the eukaryotic single-stranded DNA-binding protein. All of these characteristics are consistent with the known properties of the reaction in vivo and suggest that the new cell-free system will be suitable for purifying enzymes involved in homologous recombination.

scholarly journals Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

2003 ◽  
Vol 163 (4) ◽  
pp. 813-824 ◽  
Author(s):  
Kazuhito Tomizawa ◽  
Satoshi Sunada ◽  
Yun-Fei Lu ◽  
Yoshiya Oda ◽  
Masahiro Kinuta ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Critical Role ◽  
Ring Formation ◽  
Free System ◽  
Rich Domain ◽  
A Cell ◽  
Dynamin I ◽  
Deficient Mice

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation–induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with β-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

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