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PubMed2003

Nickel (Ni2+) enhancement of microtubule assembly in vitro is dependent on GTP function.

Microtubule (MT) assembly in vitro is accompanied by hydrolysis of tubulin-bound GTP at E-site. Ni2+, a human carcinogen, has been shown to markedly perturb the MT system in cultured cells and enhance MT assembly in vitro. To further probe the mechanisms of such multiple Ni2+ damaging actions on MT, we have focused on dissecting the role of the Ni2+/GTP interaction in influencing MT assembly in vitro as monitored by a turbidity assay at A350 at 27 degrees C using purified bovine brain MT proteins containing 162 microM each of Mg2+ and EGTA. MT assembly was initiated by addition of GTP and progressed in a GTP dose-dependent manner. The minimal and optimal exogenous [GTP] required for MT assembly were 15.6 and 500 microM, respectively. Replacement of GTP (25-87%) with increasing [NiCl2] while keeping the sum of [GTP] and [Ni2+] constant at 500 microM enabled MT assembly to proceed with shortened "lags" but reaching the same maximum plateau levels or elongation rates as with 500 microM GTP only. However, in reactions with Ni2+ replacing >94% of GTP, marked inhibition of MT assembly (lower plateaus) occurred. Electron microscopic (EM) examinations showed that MT formed with high Ni2+ substitutions for GTP appeared shorter, more numerous, and resistant to Ca2+ disruption than those assembled with 500 microM GTP only. Notably, in the presence of 500 microM Ni2+ with no GTP added, no typical MT were observed under EM, despite increases in turbidity of the reaction. In addition, the critical concentration of MT proteins required for assembly was also considerably decreased under conditions of Ni2+ replacements of GTP. These results point to an important role of GTP/Ni2+ interaction in modulating the Ni2+ enhancement of MT assembly in vitro.

Animals↗
PubMed1997

Estimation of the diffusion-limited rate of microtubule assembly.

Microtubule assembly is a complex process with individual microtubules alternating stochastically between extended periods of assembly and disassembly, a phenomenon known as dynamic instability. Since the discovery of dynamic instability, molecular models of assembly have generally assumed that tubulin incorporation into the microtubule lattice is primarily reaction-limited. Recently this assumption has been challenged and the importance of diffusion in microtubule assembly dynamics asserted on the basis of scaling arguments, with tubulin gradients predicted to extend over length scales exceeding a cell diameter, approximately 50 microns. To assess whether individual microtubules in vivo assemble at diffusion-limited rates and to predict the theoretical upper limit on the assembly rate, a steady-state mean-field model for the concentration of tubulin about a growing microtubule tip was developed. Using published parameter values for microtubule assembly in vivo (growth rate = 7 microns/min, diffusivity = 6 x 10(-12) m2/s, tubulin concentration = 10 microM), the model predicted that the tubulin concentration at the microtubule tip was approximately 89% of the concentration far from the tip, indicating that microtubule self-assembly is not diffusion-limited. Furthermore, the gradients extended less than approximately 50 nm (the equivalent of about two microtubule diameters) from the microtubule tip, a distance much less than a cell diameter. In addition, a general relation was developed to predict the diffusion-limited assembly rate from the diffusivity and bulk tubulin concentration. Using this relation, it was estimated that the maximum theoretical assembly rate is approximately 65 microns/min, above which tubulin can no longer diffuse rapidly enough to support faster growth.

Animals↗
PubMed1986

Magnesium requirements for guanosine 5'-O-(3-thiotriphosphate) induced assembly of microtubule protein and tubulin.

Two conflicting interpretations on the role of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in microtubule protein and tubulin assembly have been previously reported. One study finds that GTP gamma S promotes assembly while another study reports that GTP gamma S is a potent inhibitor of microtubule assembly. We have examined the potential role of Mg2+ to learn if the conflicting interpretations are due to a metal effect. Turbidity, electron microscopy, and nucleotide binding and hydrolysis were used to analyze the effect of the Mg2+ concentration on GTP gamma S-induced assembly of microtubule protein (tubulin + microtubule-associated proteins) in the presence of buffer +/- 30% glycerol and in buffer with GTP added before or after GTP gamma S. GTP gamma S substantially lowers the Mg2+ concentration required to induce cross-linked or clustered rings of tubulin. These cross-linked rings do not assemble well into microtubules, and GTP only partially restores microtubule assembly. However, taxol will promote GTP gamma S-induced cross-linked rings of microtubule protein to assemble into microtubules. The effect of GTP gamma S on microtubule protein assembly in the presence of Zn2+ with and without added Mg2+ suggests that GTP gamma S also effects the formation of Zn2+-induced sheet aggregates. Purified tubulin was used in assembly experiments with Mg2+, Zn2+, and taxol to better understand GTP gamma S interactions with tubulin. The optimal Mg2+ concentration for assembly of tubulin is lower with GTP gamma S than with GTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Biochemical dissection of the role of the one-kilodalton carboxyl-terminal moiety of tubulin in its assembly into microtubules.

The 4-kDa C-terminal domain of both tubulin subunits plays a major role in the regulation of microtubule assembly [Serrano et al. (1984) Biochemistry 23, 4675]. Controlled proteolysis of tubulin with subtilisin produces the selective cleavage of this 4-kDa moiety from alpha- and beta-tubulin with a concomitant enhancement of the assembly. Here we show that gradual removal of the last six to eight amino acid residues of the C-terminal region of alpha and beta subunits by an exopeptidase, carboxypeptidase Y, produces a modified protein (C-tubulin) without relieving the modulatory effect of the C-terminal domain and the usual need of MAPs for microtubule assembly. Actually, treatment with this proteolytic enzyme did not change tubulin assembly as promoted by either MAP-2, taxol, MgCl2, dimethyl sulfoxide, or glycerol. The critical concentration for the assembly of C-tubulin remained the same as that for the unmodified tubulin control. Microtubule-associated proteins MAP-2 and tau incorporated into C-tubulin polymers. Clearly, pure C-tubulin did not assemble in the absence of MAPs or without addition of assembly-promoting compounds. However, proteolysis with the exopeptidase induced changes in tubulin conformation as assessed by biophysical methods and double-limited proteolysis. The cleavage with subtilisin after carboxypeptidase digestion did not result in enhancement of the assembly to the levels observed after the treatment of native tubulin with subtilisin. Interestingly, Ca2+ ions affected neither C-tubulin assembly nor depolymerized microtubules assembled from C-tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2000

Assembly of heterodimeric luciferase after de novo synthesis of subunits in rabbit reticulocyte lysate involves hsc70 and hsp40 at a post-translational stage.

Heterodimeric luciferase from Vibrio harveyi had been established as a unique model enzyme for direct measurements of the effects of molecular chaperones and folding catalysts on protein folding and subunit assembly after de novo synthesis of subunits in rabbit reticulocyte lysate. It was observed that luciferase assembly can be separated in time from synthesis of the two subunits and that under these post-translational conditions assembly was inhibited by either ATP depletion or inhibition of peptidylprolyl cis/trans isomerases, that is, by addition of cyclosporin A or FK506. Furthermore, it was observed that the inhibitory effect of FK506 on luciferase assembly can be suppressed by addition of purified cyclophilin, thereby providing the first direct evidence for the involvement of peptidylprolyl cis/trans isomerases in protein biogenesis in the eukaryotic cytosol. Here the ATP requirement in luciferase assembly has been characterized. Depletion of either Hsp90 or CCT from reticulocyte lysate did not interfere with luciferase assembly. However, addition of purified Hsc70 stimulated luciferase assembly. While addition of purified Hsp40 did not have any effect on luciferase assembly, the stimulatory effect of Hsc70 was further increased by Hsp40. Thus, after synthesis of the two subunits in reticulocyte lysate assembly of heterodimeric luciferase involves Hsc70 and its co-chaperone Hsp40. Therefore, Hsc70 aids protein biogenesis in the eukaryotic cytosol not only at the levels of nascent polypeptide chains and precursor proteins that have to be kept competent for transport into cell organelles, but also at the level of subunits that have to be kept competent for assembly.

Adenosine Triphosphate↗
PubMed2006

An algorithm for assembly of ordered restriction maps from single DNA molecules.

The restriction mapping of a massive number of individual DNA molecules by optical mapping enables assembly of physical maps spanning mammalian and plant genomes; however, not through computational means permitting completely de novo assembly. Existing algorithms are not practical for genomes larger than lower eukaryotes due to their high time and space complexity. In many ways, sequence assembly parallels map assembly, so that the overlap-layout-consensus strategy, recently shown effective in assembling very large genomes in feasible time, sheds new light on solving map construction issues associated with single molecule substrates. Accordingly, we report an adaptation of this approach as the formal basis for de novo optical map assembly and demonstrate its computational feasibility for assembly of very large genomes. As such, we discuss assembly results for a series of genomes: human, plant, lower eukaryote and bacterial. Unlike sequence assembly, the optical map assembly problem is actually more complex because restriction maps from single molecules are constructed, manifesting errors stemming from: missing cuts, false cuts, and high variance of estimated fragment sizes; chimeric maps resulting from artifactually merged molecules; and true overlap scores that are "in the noise" or "slightly above the noise." We address these problems, fundamental to many single molecule measurements, by an effective error correction method using global overlap information to eliminate spurious overlaps and chimeric maps that are otherwise difficult to identify.

Algorithms↗
PubMed2001

Assembly of RecA-like recombinases: distinct roles for mediator proteins in mitosis and meiosis.

Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30 degrees C but not 20 degrees C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G(1), consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.

Adenosine Triphosphatases↗
PubMed1974

A three-dimensional molecular assembly model of a lipoprotein from the Escherichia coli outer membrane.

From the complete amino-acid sequence of a lipoprotein from the outer membrane of E. coli, a three-dimensional molecular assembly model was constructed. It is proposed that the model provides a tubular hydrophilic channel through the outer membrane, which serves as a passive diffusion pore. An alpha-helix is constructed from the sequence, and six of them are arranged to form a superhelix with a hydrophilic interior and hydrophobic outer surface. The superhelical assembly is stabilized by seven ionic interactions between adjacent alpha-helices. Since the height of the assembly is 76 A, it could be inserted into the outer membrane and span the full 75-A thick membrane. The assembly is stabilized in the outer membrane not only by hydrophobic interaction between the surface of the assembly and the lipid bilayer, but also by three hydrocarbon chains of fatty acids linked to the amino-terminal end of the lipoprotein, which are flipped back along the assembly and inserted into the lipid bilayer of the outer membrane. Any two alpha-helices in an assembly are linked to the peptidoglycan at their carboxyl-terminal ends so that the outer membrane is anchored on the peptidoglycan layer. Six or more alpha-helices can form an assembly of this type. However, assuming that an assembly consists of six helices, there are 1.25 x 10(5) per cell hydrophilic channels of a diameter of 12.5 A and 35% of the cell surface is occupied by the assemblies.

Amino Acid Sequence↗
PubMed1989

Actin filaments mediate Dictyostelium myosin assembly in vitro.

Because myosin thick filaments form in the actin-rich cortex of nonmuscle cells, we have examined the role of Dictyostelium actin filaments in the assembly of Dictyostelium myosin (type II). Fluorescence energy transfer and light-scattering assembly assays indicate that self-association of Dictyostelium myosin into bipolar thick filaments is kinetically regulated by actin filament networks. Regulation is nucleotide dependent but does not require ATP hydrolysis. Myosin assembly is accelerated approximately 5-fold by actin filaments when either 1 mM ATP or 1 mM adenosine 5'-[beta,gamma-imido]triphosphate (AMP-P[NH]P) is present. However, actin filaments together with 1 mM ADP abolish myosin assembly. Accelerated assembly appears to require transient binding of myosin molecules to actin filaments before incorporation into thick filaments. Fluorescence energy-transfer assays demonstrate that myosin associates with actin filaments at a rate that is equivalent to the accelerated myosin assembly rate, evidence that myosin to actin binding is a rate-limiting step in accelerated thick filament formation. Actin filament networks are also implicated in regulation of thick filament formation, since fragmentation of F-actin networks by severin causes immediate cessation of accelerated myosin assembly. Electron microscopic studies support a model of actin filament-mediated myosin assembly. In ADP, myosin monomers rapidly decorate F-actin, preventing extensive formation of thick filaments. In AMP-P[NH]P, myosin assembles along actin filaments, forming structures that resemble primitive stress fibers. Taken together, these data suggest a model in which site-directed assembly of thick filaments in Dictyostelium is mediated by the interaction of myosin monomers with cortical actin filament networks.

Actins↗
PubMed1997

Inactivation of FtsI inhibits constriction of the FtsZ cytokinetic ring and delays the assembly of FtsZ rings at potential division sites.

A universally conserved event in cell division is the formation of a cytokinetic ring at the future site of division. In the bacterium Escherichia coli, this ring is formed by the essential cell division protein FtsZ. We have used immunofluorescence microscopy to show that FtsZ assembles early in the division cycle, suggesting that constriction of the FtsZ ring is regulated and supporting the view that FtsZ serves as a bacterial cytoskeleton. Assembly of FtsZ rings was heterogeneously affected in an ftsI temperature-sensitive mutant grown at the nonpermissive temperature, some filaments displaying a striking defect in FtsZ assembly and others displaying little or no defect. By using low concentrations of the beta-lactams cephalexin and piperacillin to specifically inhibit FtsI (PBP3), an enzyme that synthesizes peptidoglycan at the division septum, we show that FtsZ ring constriction requires the transpeptidase activity of FtsI. Unconstricted FtsZ rings are stably trapped at the midpoint of the cell for several generations after inactivation of FtsI, whereas partially constricted FtsZ rings are less effectively trapped. In addition, FtsZ rings are able to assemble in newborn cells in the presence of cephalexin, suggesting that newborn cells contain a site at which FtsZ can assemble (the nascent division site) and that the transpeptidase activity of FtsI is not required for assembly of FtsZ at these sites. However, aside from this first round of FtsZ ring assembly, very few additional FtsZ rings assemble in the presence of cephalexin, even after several generations of growth. One interpretation of these results is that the transpeptidase activity of FtsI is required, directly or indirectly, for the assembly of nascent division sites and thereby for future assembly of FtsZ rings.

Bacterial Proteins↗
PubMed1996

Reconstitution of the steroid receptor.hsp90 heterocomplex assembly system of rabbit reticulocyte lysate.

Rabbit reticulocyte lysate contains a multiprotein system that assembles steroid receptors into a heterocomplex with hsp90. In the case of the glucocorticoid receptor (GR), the receptor must be bound to hsp90 to bind steroid, and assembly of the GR.hsp90 complex restores the hormone binding domain of the receptor to the steroid binding conformation. Using both direct assay of heterocomplex assembly by Western blotting and indirect assay of assembly by steroid binding, it has previously been determined that the assembly system is both ATP/Mg2+-dependent and K+-dependent and that hsp70 and an acidic 23-kDa protein (p23) are required to form a functional GR.hsp90 complex. It is also thought that a 60-kDa protein (p60) may be required for progesterone receptor.hsp90 heterocomplex assembly, but a complete heterocomplex assembly system has never been reconstituted from individual components. In this work, we separate the proteins of rabbit reticulocyte lysate into three fractions by DEAE chromatography and then reconstitute the GR.hsp90 heterocomplex assembly system in a manner that requires the presence of each fraction. Fraction A contains most of the hsp70 and all of the p60 in lysate, and elimination of p60 by immunoadsorption inactivates this fraction, with bioactivity being restored by the addition of bacterially expressed human p60. The activity of fraction A is replaced by a combination of highly purified rabbit hsp70 and lysate from p60-expressing bacteria. Fraction B contains hsp90, and its activity is replaced by purified rabbit hsp90. Fraction C contains p23, and its activity is replaced in the recombined system by highly purified bacterially expressed human p23. A minimal GR.hsp90 heterocomplex assembly system was reconstituted with purified rabbit hsp70 and hsp90 and bacterially expressed human p23 and p60. This reports the first reconstitution of this apparently ubiquitous protein folding/heterocomplex assembly system.

Animals↗
PubMed2002

GABA(A) receptor composition is determined by distinct assembly signals within alpha and beta subunits.

Key to understanding how receptor diversity is achieved and controlled is the identification of selective assembly signals capable of distinguishing between other subunit partners. We have identified that the beta1-3 subunits exhibit distinct assembly capabilities with the gamma2L subunit. Similarly, analysis of an assembly box in alpha1-(57-68) has revealed an absolute requirement for this region in the assembly of alphabeta receptors. Furthermore, a selective requirement for a single amino acid (Arg-66), previously shown to be essential for the formation of the low affinity GABA binding site, is observed. This residue is critical for the assembly of alpha1beta2 but not alpha1beta1 or alpha1beta3 receptors. We have confirmed the ability of the previously identified GKER signal in beta3 to direct the assembly of betagamma receptors. The GKER signal is also involved in driving assembly with the alpha1 subunit, conferring the ability to assemble with alpha1(R66A) on the beta2 subunit. Although this signal is sufficient to permit the formation of beta2gamma2 receptors, it is not necessary for beta3gamma2 receptor formation, suggesting the existence of alternative assembly signals. These findings support the belief that GABA(A) receptor assembly occurs via defined pathways to limit the receptor diversity.

Amino Acid Sequence↗
PubMed1986

Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein.

We have developed an assay to monitor in vitro the posttranslational assembly of the chloroplast protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Most of the newly synthesized 55-kD catalytic ("large") subunits of this enzyme occur in a 29S complex together with 60- and 61-kD "binding" proteins. When the 29S complex is incubated with ATP and MgCl2 it dissociates into subunits, and the formerly bound large subunits now sediment at 7S (still faster than expected for a monomer). Upon incubation at 24 degrees C, these large subunits assemble into RuBisCO. The minority of newly made large subunits which are not bound to the 29S complex also sediment at 7S. When endogenous ATP was removed by addition of hexokinase and glucose, the dissociation of the 29S complex was inhibited. Nevertheless, the 7S large subunits assembled into RuBisCO, and did so to a greater extent than in controls retaining endogenous ATP. Thus the 7S large subunits are also assembly competent, at least when ATP is removed. Apparently, in chloroplast extracts, ATP can have a dual effect on the assembly of RuBisCO: on the one hand, even at low concentrations it can inhibit incorporation of 7S large subunits RuBisCO; on the other hand, at higher concentrations it can lead to substantial buildup of the 7S large subunit pool by causing dissociation of the 29S complex, and stimulate overall assembly. At both high and zero concentrations of ATP, however, antibody to the binding protein inhibited the assembly of endogenous large subunits into RuBisCO. Thus it appears that all assembly-competent large subunits are associated with the binding protein, either in the 7S complex or in the 29S complex. The involvement of the binding protein in RuBisCO assembly may represent the first example of non-autonomous protein assembly in higher plants and may pose problems for the genetic engineering of RuBisCO from these organisms.

Adenosine Triphosphate↗
PubMed1991

Analysis of early events in acetylcholine receptor assembly.

Mammalian cell lines expressing nicotinic acetylcholine receptor (AChR) subunit cDNAs from Torpedo californica were used to study early events in AChR assembly. To test the hypothesis that individual subunits form homooligomeric intermediates before assembling into alpha 2 beta gamma delta pentamers, we analyzed the sedimentation on sucrose density gradients of each subunit expressed separately in cell lines. We have shown previously that the acute temperature sensitivity of Torpedo AChR subunit assembly is due, in part, to misfolding of the polypeptide chains (Paulson, H.L., and T. Claudio. 1990. J. Cell Biol. 110:1705-1717). We use this phenomenon to further analyze putative assembly-competent intermediates. In nonionic detergent at an assembly-permissive temperature, the majority of alpha, beta, gamma, and delta subunits sediment neither as 3-4S monomers nor as 9S complexes, but rather as 6S species whether synthesized in fibroblasts, myoblasts, or differentiated myosyncytia. Several results indicate that the 6S species are complexes comprised predominantly of incorrectly folded subunit polypeptides. The complexes represent homoaggregates which form rapidly within the cell, are stable to mild SDS treatment and, in the case of alpha, contain some disulfide-linked subunits. The coprecipitation of alpha subunit with BiP or GRP78, a resident protein of the ER, further indicates that at least some of these internally sequestered subunits also associated with an endogenous protein implicated in protein folding. The majority of subunits expressed in these cell lines appear to be aggregates of subunits which are not assembly intermediates and are not assembly-competent. The portion which migrates as monomer, in contrast, appears to be the fraction which is assembly competent. This fraction increases at temperatures more permissive for assembly, further indicating the importance of the monomer as the precursor to assembly of alpha 2 beta gamma delta pentamers.

Animals↗
PubMed1994

The alpha and beta subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme.

Synthesis and assembly of most oligomeric plasma membrane proteins occurs in the ER. However, the role the ER plays in oligomerization is unknown. We have previously demonstrated that unassociated alpha and beta subunits of the Na,K-ATPase are targeted to the plasma membrane when individually expressed in baculovirus-infected Sf-9 cells. This unique property allows us to determine if assembly of these two polypeptides is restricted to the ER, or if it can also occur at the plasma membrane. To investigate the assembly of the Na,K-ATPase we have taken advantage of the ability of baculovirus-infected cells to fuse. Lowering the extracellular pH of the infected cells triggers an endogenously expressed viral protein to initiate plasma membrane fusion. When individual Sf-9 cells expressing either the Na,K-ATPase alpha or beta subunits are plated together and subjected to a mild acidic shock, they form large syncytia. In the newly continuous plasma membrane the separate alpha and beta polypeptides associate and assemble into functional Na,K-ATPase molecules. However, a hybrid ATPase molecule consisting of a Na,K-ATPase alpha subunit and a H,K-ATPase beta subunit, which efficiently assembles in the ER of coinfected cells, does not assemble at the plasma membrane of fused cells. When cells expressing the Na,K-ATPase alpha subunit are fused to cells coexpressing the Na,K-ATPase beta subunit and the H,K-ATPase beta subunit, the Na,K-ATPase alpha subunit selectively assembles with the Na,K-ATPase beta subunit. However, when cells are coinfected and expressing all three polypeptides, the Na,K-ATPase alpha subunit assembles with both beta subunits in the ER, in what appears to be a random fashion. These experiments demonstrate that assembly between some polypeptides is restricted to the ER, and suggests that the ability of the Na,K-ATPase alpha and beta subunits to leave the ER and assemble at the plasma membrane may represent a novel mechanism of regulation of activity.

Animals↗
PubMed1983

In vitro, rapid assembly of gap junctions is induced by cytoskeleton disruptors.

We report here rapid assembly of gap junctions in prostate epithelial cells in vitro. Assembly of gap junctions can be induced by incubation at 0 degrees C followed by incubation at 37 degrees C. Colchicine (10(-5) M, 10(-3) M) and cytochalasin B (25 micrograms/ml), 100 micrograms/ml) at room temperature or at 37 degrees C also induce assembly of gap junctions. Assembly of the junctions proceeds even in the presence of a metabolic inhibitor (dinitrophenol) or of an inhibitor of protein synthesis (cycloheximide). We conclude that assembly of gap junctions can proceed from a pool of pre-existing precursors. The experimental conditions that result in gap-junction assembly involve perturbation of the cytoskeleton. Therefore, we propose that the assembly of gap junctions requires convergent migration of precursor molecules whose positional control in the membrane is released by perturbation of the cytoskeleton. Aggregates of particles and rugosities, whose distribution size and shape is similar to that of gap junctions, may represent intermediate assembly stages. This would indicate that the final stages in the assembly take place only after convergence of the precursor molecules to the junctional site and involve profound conformational changes required for establishment of fully assembled connexons.

2,4-Dinitrophenol↗
PubMed2001

Fragment assembly with double-barreled data.

For the last twenty years fragment assembly was dominated by the "overlap - layout - consensus" algorithms that are used in all currently available assembly tools. However, the limits of these algorithms are being tested in the era of genomic sequencing and it is not clear whether they are the best choice for large-scale assemblies. Although the "overlap - layout - consensus" approach proved to be useful in assembling clones, it faces difficulties in genomic assemblies: the existing algorithms make assembly errors even in bacterial genomes. We abandoned the "overlap - layout - consensus" approach in favour of a new Eulerian Superpath approach that outperforms the existing algorithms for genomic fragment assembly (Pevzner et al. 2001 InProceedings of the Fifth Annual International Conference on Computational Molecular Biology (RECOMB-01), 256-26). In this paper we describe our new EULER-DB algorithm that, similarly to the Celera assembler takes advantage of clone-end sequencing by using the double-barreled data. However, in contrast to the Celera assembler, EULER-DB does not mask repeats but uses them instead as a powerful tool for contig ordering. We also describe a new approach for the Copy Number Problem: "How many times a given repeat is present in the genome?". For long nearly-perfect repeats this question is notoriously difficult and some copies of such repeats may be "lost" in genomic assemblies. We describe our EULER-CN algorithm for the Copy Number Problem that proved to be successful in difficult sequencing projects.

Algorithms↗
PubMed2025

Autocycler: long-read consensus assembly for bacterial genomes.

MOTIVATION: Long-read sequencing enables complete bacterial genome assemblies, but individual assemblers are imperfect and often produce sequence-level and structural errors. Consensus assembly using Trycycler can improve accuracy, but its lack of automation limits scalability. There is a need for an automated method to generate high-quality consensus bacterial genome assemblies from long-read data. RESULTS: We present Autocycler, a command-line tool for generating accurate bacterial genome assemblies by combining multiple alternative long-read assemblies of the same genome. Without requiring user input, Autocycler builds a compacted De Bruijn graph from the input assemblies, clusters and filters contigs, trims overlaps, and resolves consensus sequences by selecting the most common variant at each locus. It also supports manual curation when desired, allowing users to refine assemblies in challenging or important cases. In our evaluation using Oxford Nanopore Technologies reads from five bacterial isolates, Autocycler outperformed individual assemblers, automated pipelines, and other consensus tools, producing assemblies with lower error rates and improved structural accuracy. AVAILABILITY AND IMPLEMENTATION: Autocycler is implemented in Rust, open-source, and freely available at github.com/rrwick/Autocycler. It runs on Linux and macOS and is extensively documented.

Genome, Bacterial↗