scholarly journals ViTroVo: in vitro assembly search for in vivo adaptive operator guidance

2021 ◽  
Author(s):  
Corrado Grappiolo ◽  
Raimon Pruim ◽  
Matthias Faeth ◽  
Paolo de Heer
Keyword(s):  
In Vitro Assembly

scholarly journals Modulations of SIR-nucleosome interactions of reconstructed yeast silent pre-heterochromatin by O-acetyl-ADP-ribose and magnesium

2017 ◽  
Vol 28 (3) ◽  
pp. 381-386 ◽  
Author(s):  
Shu-Yun Tung ◽  
Sue-Hong Wang ◽  
Sue-Ping Lee ◽  
Shu-Ping Tsai ◽  
Hsiao-Hsuian Shen ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Chromatin Condensation ◽  
Epigenetic Inheritance ◽  
Assembly System ◽  
Sir Proteins ◽  
Epigenetic Gene Silencing ◽  
In Vitro Assembly ◽  
The Individual

Yeast silent heterochromatin provides an excellent model with which to study epigenetic inheritance. Previously we developed an in vitro assembly system to demonstrate the formation of filament structures with requirements that mirror yeast epigenetic gene silencing in vivo. However, the properties of these filaments were not investigated in detail. Here we show that the assembly system requires Sir2, Sir3, Sir4, nucleosomes, and O-acetyl-ADP-ribose. We also demonstrate that all Sir proteins and nucleosomes are components of these filaments to prove that they are SIR-nucleosome filaments. Furthermore, we show that the individual localization patterns of Sir proteins on the SIR-nucleosome filament reflect those patterns on telomeres in vivo. In addition, we reveal that magnesium exists in the SIR-nucleosome filament, with a role similar to that for chromatin condensation. These results suggest that a small number of proteins and molecules are sufficient to mediate the formation of a minimal yeast silent pre-heterochromatin in vitro.

Inability to detect Chlamyodomonas microtubule assembly in vitro: possible implications to the in vivo regulation of microtubule assembly

1975 ◽  
Vol 17 (3) ◽  
pp. 669-681
Author(s):  
K.W. Farrell ◽  
R.G. Burns
Keyword(s):  
Column Chromatography ◽  
Divalent Cation ◽  
Microtubule Assembly ◽  
Mammalian Brain ◽  
Wide Range ◽  
In Vitro Assembly ◽  
Brain Extracts ◽  
Brain Tubulin

It has been demonstrated that the in vitro assembly of microtubules from Chlamydomonas preparations does not occur under a wide range of conditions, including those efficacious for mammalian brain tubulin. This incompetence of Chlamydomonas extracts to form microtubules is independent of the tubulin concentration, the presence of added nucleotides or an added seed, temperature, or the concentration of divalent cation. However, an amorphous aggregate was observed under certain conditions, who composition was mainly tubulin. The in vitro reassembly of microtubules in gerbil brain extracts is inhibited by Chlamydomonas preparations. Fractionation of the Chlamydomonas extracts by column chromatography suggests that the inhibitory component is Chlamydomonas tubulin itself. The mechanism of this inhibition is unknown, but reassembly experiments indicate that the 2 types of tubulins cannot copolymerize. We suggest that the Chlamydomonas tubulin, derived from a cytoplasmic pool, requires to be activated prior to its in vivo polymerization into microtubules.

Involvement of the consensus sequence motif at coil 2b in the assembly and stability of vimentin filaments

1992 ◽  
Vol 102 (1) ◽  
pp. 31-41 ◽  
Author(s):  
P.D. Kouklis ◽  
P. Traub ◽  
S.D. Georgatos
Keyword(s):  
Consensus Sequence ◽  
Sequence Motif ◽  
Sequence Motifs ◽  
3T3 Cells ◽  
Filament Assembly ◽  
In Vitro Assembly ◽  
Vimentin Filaments

Nearly all intermediate filament (IF) proteins share two sequence motifs located at the N- and the C-terminal ends of their helical rod domain (‘coil 1a’ and ‘coil 2b’, respectively). To examine the structural role of the coil 2b motif, we have performed in vitro assembly studies and in vivo microinjection experiments employing two site-specific reagents: (a) a 20-residue synthetic peptide (C-2) representing the conserved motif itself and (b) a monoclonal antibody (anti-IFA) that recognises an epitope within the conserved coil 2b sequence. We demonstrate here that vimentin protofilaments, when induced to assemble in the presence of C-2 or anti-IFA, show a lower propensity to polymerise and yield various abberant structures. The few filaments that are formed under these conditions appear much shorter than normal IFs and are unravelled or aggregated. Furthermore, when preformed vimentin filaments are exposed to C-2 or anti-IFA, most of the normal IFs are converted into shorter filamentous forms that possess an abberant morphology. None of these effects is seen when vimentin subunits are coincubated with control peptides. Microinjection of anti-IFA into the cytoplasm of interphasic 3T3 cells provokes collapse of vimentin IFs into a juxtanuclear mass and formation of numerous amorphous aggregates distributed throughout the cytoplasm. These two effects are not seen when the anti-IFA is microinjected into the cell nucleus. Our results provide experimental evidence supporting previous suggestions for a role for the conserved coil 2b sequence in filament assembly. We propose that this region is interacting with other sites along the vimentin molecule and that these interactions are essential for proper protofilament-protofilament alignment and filament stability.

Assembly of three major subclasses of mouse immunoglobulin G: a theoretical model for covalent assembly in vivo

1976 ◽  
Vol 54 (8) ◽  
pp. 688-698 ◽  
Author(s):  
J. R. Percy ◽  
M. E. Percy ◽  
R. Baumal
Keyword(s):  
Cell Lines ◽  
Immunoglobulin G ◽  
Mean Value ◽  
Individual Values ◽  
In Vitro Assembly ◽  
The Mean ◽  
The Individual ◽  
Mouse Myeloma

A mathematical model, based on second-order reaction kinetics, has been used to describe the covalent assembly of immunoglobulin G (IgG) in vitro from its heavy (H) and light (L) chains (Percy, M. E., Baumal, R., Dorrington, K. J. &Percy, J. (1976) Can. J. Biochem. 54, 675–687). In the present paper, the same model has now been applied to the steady-state assembly of IgG in vivo. This mathematical approach permits a quantitative comparison of the pathways of covalent assembly used by given immunoglobulins in vivo and in vitro. The assumptions in the model are: the species L, H, HL, HH, HHL and LHHL belong to a common pool; incompleted IgG intermediates may freely assemble to form HL, HH, HHL and LHHL; the reaction rate for covalent linkage between any two reacting species is proportional to the products of the number densities of the reactants and to a parameter P which takes the value PHH if the reaction joins two H chains, and PHL if it joins an H and L chain. In vivo values of PHH/PHL were determined for the 18 mouse myeloma tumours and cell lines studied by Baumal et al. (Baumal, R., Potter, M. &Scharff, M. (1971) J. Exp. Med. 134, 1316–1334). From these analyses, we have arrived at the following conclusions: (1) the three major IgG subclasses have distinctive values of PHH/PHL (mean value 53 for IgG1, 12 for IgG2a and 2.8 for IgG2b); (2) for IgGs of the same subclass, the values of PHH/PHL are similar; (3) the mean in vivo values of PHH/PHL are very close to those determined from in vitro assembly experiments. Finally, the individual values of PHH/PHL have been used to simulate pulse-chase experiments in the various tumours and cell lines. Considering the sources and magnitude of experimental error, the theoretical pathways of assembly agree with those determined qualitatively from the pulse-chase experiments.

scholarly journals Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: DNA-gp3 intermediate in in vivo and in vitro assembly.

1982 ◽  
Vol 41 (2) ◽  
pp. 508-517 ◽  
Author(s):  
M A Bjornsti ◽  
B E Reilly ◽  
D L Anderson
Keyword(s):  
Bacillus Subtilis ◽  
In Vitro Assembly

Fluorescent microtubules break up under illumination

1988 ◽  
Vol 46 ◽  
pp. 36-37
Author(s):  
G.P.A. Vigers ◽  
M. Coué ◽  
J.R. McIntosh
Keyword(s):  
Dynamic Behavior ◽  
Cultured Cells ◽  
Living Cells ◽  
Epifluorescence Microscopy ◽  
Fluorescent Molecule ◽  
In Vitro Assembly ◽  
Derivatives Of ◽  
Dic Microscopy

The dynamic behavior of microtubules (MTs) in living cells has recently been elucidated by the use of tubulin tagged with a fluorescent molecule and then injected into cultured cells. The dynamics of the labelled tubulin are then followed either by watching the incorporation of the fluorescence immediately after injection, or by monitoring fluorescence redistribution after photobleaching (FRAP).Until recently, the best fluorescent analogue of tubulin appeared to be dichlorotriazinyl-amino-fluorescein tubulin (DTAF-tubulin). We have now made fluorescein, rhodamine and X-rhodamine n-hydroxy-succinimidyl derivatives of tubulin. These analogues all have a higher fluorescence to protein (f-to-p) ratio and better in-vitro assembly characteristics than the DTAF-tubulin previously used. They have been injected into cultured PtKl cells, where they incorporate well into the microtubule cytoskeltons of the cells. However, while characterising the properties of these four analogues we discovered that they all suffer from a major problem: Microtubules formed from them, either in vitro or in vivo, are destroyed as the bound fluorophore becomes photobleached. We have characterised the photolability of the fluorescent microtubules both in vivo, using epifluorescence microscopy, and in vitro with DIC microscopy.

scholarly journals In Vivo and In Vitro Assembly of Sindbis Virus Nucleocapsid-Defective Mutants

2011 ◽  
Vol 100 (3) ◽  
pp. 403a
Author(s):  
Christian Berrios ◽  
Jonathan Snyder ◽  
Joyce Jose ◽  
Richard Kuhn
Keyword(s):  
Sindbis Virus ◽  
In Vitro Assembly

scholarly journals In Vitro Assembly of Human H/ACA Small Nucleolar RNPs Reveals Unique Features of U17 and Telomerase RNAs

2000 ◽  
Vol 20 (9) ◽  
pp. 3037-3048 ◽  
Author(s):  
François Dragon ◽  
Vanda Pogačić ◽  
Witold Filipowicz
Keyword(s):  
Full Length ◽  
Stem Loop ◽  
Rna Transcripts ◽  
Cell Extracts ◽  
In Vitro Assembly ◽  
Tail Structure ◽  
Human Telomerase ◽  
Nucleolar Rnas

ABSTRACT The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. They usually fold into a two-domain hairpin-hinge-hairpin-tail structure, with the conserved motifs H and ACA located in the hinge and tail, respectively. Synthetic RNA transcripts and extracts from HeLa cells were used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs) in vitro. Competition and UV cross-linking experiments showed that proteins of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Except for U17, RNPs could be reconstituted only with full-length H/ACA snoRNAs. For U17, the 3′-terminal stem-loop followed by box ACA (U17/3′st) was sufficient to form an RNP, and U17/3′st could compete other full-length H/ACA snoRNAs for assembly. The H/ACA-like domain that constitutes the 3′ moiety of human telomerase RNA (hTR), and its 3′-terminal stem-loop (hTR/3′st), also could form an RNP by binding H/ACA proteins. Hence, the 3′-terminal stem-loops of U17 and hTR have some specific features that distinguish them from other H/ACA RNAs. Antibodies that specifically recognize the human GAR1 (hGAR1) protein could immunoprecipitate H/ACA snoRNAs and hTR from HeLa cell extracts, which demonstrates that hGAR1 is a component of H/ACA snoRNPs and telomerase in vivo. Moreover, we show that in vitro-reconstituted RNPs contain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite for the assembly of the other H/ACA proteins.

scholarly journals Neurofilaments are obligate heteropolymers in vivo

1993 ◽  
Vol 122 (6) ◽  
pp. 1337-1350 ◽  
Author(s):  
MK Lee ◽  
Z Xu ◽  
PC Wong ◽  
DW Cleveland
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Dna Transfection ◽  
Tail Domain ◽  
In Vitro Assembly ◽  
Mature Neurons ◽  
Filament Network

Neurofilaments (NFs), composed of three distinct subunits NF-L, NF-M, and NF-H, are neuron-specific intermediate filaments present in most mature neurons. Using DNA transfection and mice expressing NF transgenes, we find that despite the ability of NF-L alone to assemble into short filaments in vitro NF-L cannot form filament arrays in vivo after expression either in cultured cells or in transgenic oligodendrocytes that otherwise do not contain a cytoplasmic intermediate filament (IF) array. Instead, NF-L aggregates into punctate or sheet like structures. Similar nonfilamentous structures are also formed when NF-M or NF-H is expressed alone. The competence of NF-L to assemble into filaments is fully restored by coexpression of NF-M or NF-H to a level approximately 10% of that of NF-L. Deletion of the head or tail domain of NF-M or substitution of the NF-H tail onto an NF-L subunit reveals that restoration of in vivo NF-L assembly competence requires an interaction provided by the NF-M or NF-H head domains. We conclude that, contrary to the expectation drawn from earlier in vitro assembly studies, NF-L is not sufficient to assemble an extended filament network in an in vivo context and that neurofilaments are obligate heteropolymers requiring NF-L and NF-M or NF-H.

Sign in / Sign up

Export Citation Format

Share Document