In vitro transcription by wheat germ ribonucleic acid polymerase II: effects of heparin and role of template integrity

Biochemistry ◽  
1979 ◽  
Vol 18 (21) ◽  
pp. 4581-4588 ◽  
Author(s):  
William S. Dynan ◽  
Richard R. Burgess
Keyword(s):  
Ribonucleic Acid ◽  
Wheat Germ ◽  
In Vitro Transcription

scholarly journals Requirement of the C-terminal proline residue for stability of the Ca2+-activated photoprotein aequorin

1993 ◽  
Vol 293 (1) ◽  
pp. 181-185 ◽  
Author(s):  
N J Watkins ◽  
A K Campbell
Keyword(s):  
Light Emission ◽  
Specific Activity ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Proline Residue ◽  
Long Term Stability ◽  
Recombinant Aequorin

cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.

scholarly journals Identification of a talin binding site in the cytoskeletal protein vinculin.

1989 ◽  
Vol 109 (6) ◽  
pp. 2917-2927 ◽  
Author(s):  
P Jones ◽  
P Jackson ◽  
G J Price ◽  
B Patel ◽  
V Ohanion ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Cytoskeletal Protein ◽  
Translation System ◽  
Cos Cells ◽  
Cell Matrix ◽  
Do So

Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.

scholarly journals Role of PhoP∼P in Transcriptional Regulation of Genes Involved in Cell Wall Anionic Polymer Biosynthesis in Bacillus subtilis

1998 ◽  
Vol 180 (15) ◽  
pp. 4007-4010 ◽  
Author(s):  
Ying Qi ◽  
F. Marion Hulett
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Teichoic Acid ◽  
Acid Synthesis ◽  
In Vitro Transcription ◽  
Anionic Polymer ◽  
Transcription System ◽  
Teichuronic Acid

ABSTRACT tagA, tagD, and tuaA operons are responsible for the synthesis of cell wall anionic polymer, teichoic acid, and teichuronic acid, respectively, in Bacillus subtilis. Under phosphate starvation conditions, teichuronic acid is synthesized while teichoic acid synthesis is inhibited. Expression of these genes is controlled by PhoP-PhoR, a two-component system. It has been proposed that PhoP∼P plays a key role in the activation oftuaA and the repression of tagA andtagD. In this study, we demonstrated the role of PhoP∼P in the switch process from teichoic acid synthesis to teichuronic acid synthesis, by using an in vitro transcription system. The results indicate that PhoP∼P is sufficient to repress the transcription of the tagA and tagD promoters and also to activate the transcription of the tuaA promoter.

Dlk1 Up Regulated Might Be an Important Event in Human MDS.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4842-4842
Author(s):  
Q.F. Xiao ◽  
Zi X. Chen ◽  
Dan D. Liu ◽  
Jian N. Cen ◽  
Jun He ◽  
...  
Keyword(s):  
Experimental System ◽  
K562 Cells ◽  
In Vitro Transcription ◽  
Clinical Samples ◽  
Mrna Levels ◽  
Amplification Protocol ◽  
Cd34 Cells ◽  
Normal Controls

Abstract The diagnosis of myelodysplastic syndrome (MDS) is made largely on the dysplastic morphology of BM cells from aspiration or biopsies. Prognosis scored by IPSS is depending on the percentage of marrow myeloblasts and the clonal cytogenetic abnormalities. To expand the understanding of genetic defects in hematopoietic cells of MDS in hope of finding novel genes correlated to pathogenesis and provide possible diagnostic marker for MDS, we have applied microarray to analyze the clinical samples from MDS patients. Total RNAs of CD34+ cells from 8 patients ( 2 RAEBt,2 RAEB,2 RA,1 RAS,1 CAA ) and one healthy people were extracted followed by a double in vitro transcription to circumvent the limited number of CD34+ cells. Following a modified Affymetrix target amplification protocol. Biotinylated cRNA was synthesized from 50 ng total RNA by double-round amplification and hybridized to an Human Genome U133 Plus 2.0 Array (Affymetrix). From the expression profile of 18404 different genes, we revealed that DNTT,MLL3,IL1R2,MAPK1,IGLL1 were down regulated while EGR-1, Rap1GAP or MAF were up regulated compared with normal controls. Most notably, Dlk1 was up regulated in MDS, while down regulated in AML and normal. By real-time RT-PCR we confirmed that in BMNCs the median levels of Dlk1 transcript in patients with RA and RAS were 2.55 (range, 0.00–23.7), RAEB and RAEBt were 8.24(range, 2.01–18.44), AML were 1.88 (range, 0.12–5.13), and other patients were 0.37(range, 0.00–1.79), respectively. The abundance of Dlk1 mRNA in MNCs from most MDS patients was markedly greater than that in the MNCs from others (P <0.05 ). Dlk1 expression in RAEB and RAEBt is markedly higher than AML (P <0.05 ) Forced expression of Dlk1 in transfected K562 cells resulted in faster growth than control cells, affected apoptosis induced by As2O3. and reduced the G2 arrested cells induced by TPA. By using the same experimental system we found that forced expression of Dlk1 can increase the mRNA levels of HES1 and p21WAF1 transcript variant 1. To elucidate the mechanisms we analyzed the levels of phosphorylated-p38 and p38 in Dlk1 transfected K562 cells treated with TPA. Dlk1 inhibited p38 phosphorylation while expression of p38 kept no change. These results support further investigation on the role of Dlk1 in abnormal hematopoiesis in MDSheterogeneous cell component. Diagnosis is currently depending on the dysplastic morphology of.

Translation of satellite tobacco necrosis virus ribonucleic acid by an in vitro system from wheat germ

Biochemistry ◽  
1976 ◽  
Vol 15 (22) ◽  
pp. 4943-4950 ◽  
Author(s):  
David W. Leung ◽  
Carl W. Gilbert ◽  
Robert E. Smith ◽  
Nancy L. Sasavage ◽  
John M. Clark
Keyword(s):  
Ribonucleic Acid ◽  
Wheat Germ ◽  
Tobacco Necrosis Virus ◽  
Necrosis Virus ◽  
In Vitro System

tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage

1994 ◽  
Vol 14 (6) ◽  
pp. 3596-3603
Author(s):  
H M Dunstan ◽  
L S Young ◽  
K U Sprague
Keyword(s):  
Rna Polymerase ◽  
Dna Cleavage ◽  
Rna Polymerase Iii ◽  
Low Frequency ◽  
In Vitro Transcription ◽  
Class Iii ◽  
Transcriptional Inhibition ◽  
Transcription In Vitro

tRNA(IleIAU) provides an activity, originally called TFIIIR, necessary to reconstitute transcription by silkworm RNA polymerase III in vitro from partially purified components. Here we report studies on the role of tRNA(IleIAU) in in vitro transcription. We show that tRNA(IleIAU) does not act positively but, rather, is required to prevent the action of a transcriptional inhibitor. We also show that the presence of tRNA(IleIAU) in transcription reaction mixtures prevents low-frequency DNA cleavage by the TFIIIB fraction. Studies on the mechanism of transcriptional inhibition suggest that this DNA cleavage could cause transcriptional inhibition through trans-inactivation of transcription machinery. The ability to block DNA cleavage, like the ability to facilitate transcription, is highly specific to silkworm tRNA(IleIAU).

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