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PubMed2003

Permissive glycan support of photoreceptor outer segment assembly occurs via a non-metabolic mechanism.

PURPOSE: We have previously demonstrated that in RPE-deprived retinas, lactose, galactose, and structurally related glycans support the proper assembly of nascent photoreceptor outer segment membranes. Other glycans such as mannose and glucose have no effect on this process. While the ability to support outer segment assembly is highly specific, all of the permissive glycans we have tested are able to enter metabolic pathways within the retinal cells, thus the mechanism by which the glycans are functioning is still ambiguous. The present study was undertaken to determine if permissive glycan-mediated support of photoreceptor outer segment assembly occurred via a non-metabolic mechanism and if the phenomenon was reversible. METHODS: The RPE was removed from isolated Xenopus laevis embryonic eyes that were allowed to complete differentiation in (1) Niu-Twitty medium, (2) Niu-Twitty supplemented with 5x10-3 M lactose or mannose, (3) Niu-Twitty supplemented with lactose and 3H-galactose, (4) Niu-Twitty medium supplemented with isopropyl beta-D-thiogalactopyranoside (IPTG) at concentrations spanning five orders of magnitude, or (5) Niu-Twitty medium containing lactose or IPTG to which 3H-leucine was added for 2 days followed by an additional 2 days in Niu-Twitty alone. Control RPE-deprived and RPE-supported retinas were included for comparison. Under all experimental conditions, retinal photoreceptors were evaluated to determine the level of organized folding of outer segment membranes. RESULTS: Outer segment membranes of RPE-deprived retinas exposed to lactose were significantly more organized than both control RPE-deprived retinas and those exposed to mannose. In eyes exposed to the radiolabeled metabolizable glycan, the majority of the sugar was incorporated into Müller cells. IPTG, a non-metabolizable form of galactose, promoted the formation of organized outer segment assembly similar to lactose although at a 100 fold reduced concentration compared to metabolizable permissive glycans. Both IPTG and lactose supported outer segment assembly in a step-wise fashion with maximal support at 5x10(-5) M and 5x10(-3) M, respectively. Removal of the permissive glycans resulted in loss of support of outer segment assembly. CONCLUSIONS: The ability of both lactose and IPTG to support outer segment assembly in the absence of the RPE is dose-dependent and the effect of these sugars upon membrane folding is reversible. Moreover, this effect is supported by a non-metabolic mechanism and is therefore not accounted for by simple provision of an energy source.

Animals↗
PubMed2004

Spatial and temporal control of nuclear envelope assembly by Ran GTPase.

Using evidence derived primarily from studies using Xenopus egg extracts, a model for the role of Ran in multiple stages during NE assembly can be proposed (Figure 2). Ran is concentrated on chromatin prior to NE assembly and recruits RCC1 that generates Ran-GTP locally. Recruitment of RCC1 to chromatin may be a specialized mechanism to initiate NE assembly following fertilization of the egg, whereas in somatic cells, RCC1 may be present on chromatin throughout mitosis. Ran-GTP recruits vesicles to the surface of chromatin, and promotes vesicle fusion to form the double membrane of the NE. Ran-GTP may recruit membrane vesicles to chromatin through binding to integral membrane proteins through importin-beta. A transient complex would be formed between Ran-GTP, importin-beta and the target protein, which would be released locally to promote assembly of a precursor complex. GTP hydrolysis by Ran would release importin-beta, but may also play a role in vesicle fusion. Ran-GTP also promotes NPC assembly by releasing nucleoporins such as Nup107 from inhibitory complexes with importin-beta. In vertebrate cells undergoing mitosis, the majority of Ran molecules are excluded from the chromosomes and dispersed into the cytoplasm. Relocalization of Ran to chromatin at the end of mitosis may co-ordinate the initiation of NE assembly with disassembly of the mitotic spindle. The function of Ran in this transition is likely to be coupled to changes in the activity of cyclin-dependent protein kinases and other activities that control the progression of the cell cycle. Thus, changes in the localization of Ran and its regulators provide temporal and spatial control of NE assembly at the end of mitosis.

Animals↗
PubMed1991

Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly.

A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.

Amino Acid Sequence↗
PubMed1990

Kinetic analysis of the pre-equilibrium steps in the self-assembly of RecA protein from Escherichia coli.

Total intensity light scattering is employed to investigate the self-assembly kinetics of RecA protein. Reaction conditions are employed where the kinetics of self-assembly are slow enough to yield reliable scattered intensity measurements over the range of scattering angles from 40 to 130 degrees as a function of time. From these measurements the time-dependent behavior of the weight average molecular weight, Mr, and radius of gyration, RG, of the associating protein species as a function of [MgCl2], [NaCl], [RecA], and pH was determined. The temperature dependence of RecA self-assembly was also investigated and allowed an evaluation of the activation thermodynamic parameters of association. Results reveal RecA self-assembly is bi-phasic under all conditions examined. The first phase, referred to as "filamentation" is second-order in [RecA] and occurs via a quasi linear condensation scheme with an Arrhenius activation energy of 88.6 kcal/mol. Filamentation assembly involves the uptake of one proton, one MgCl2, the release of five to six NaCls, and is driven by the release of approximately 70 water molecules. The evaluated activation parameters of the first kinetic phase are consistent with the proposition that linear self-assembly of RecA protein into ordered filaments is entropically driven. The second kinetic phase, referred to as "bundling" is greater than second-order in both [RecA] and [MgCl2], is considerably slower that filamentation assembly, and is apparently initiated by 2nd order collisions of linear filaments.

Escherichia coli↗
PubMed1989

Processing and assembly of the integrin, glycoprotein IIb-IIIa, in HEL cells.

We examined the biosynthetic processing and assembly of the platelet glycoprotein (GP) IIb-IIIa complex in [35S]methionine-labeled HEL cells, a human cell line with features of megakaryocytes. Both GPIIb and GPIIIa were synthesized as single-chain precursors to which high mannose N-linked oligosaccharides were added in the endoplasmic reticulum (ER). A 5-fold excess of the major IIb precursor, preIIb, was synthesized relative to GPIIIa. Two smaller proteins immunologically related to GPIIb were synthesized in smaller amounts. Assembly of the GPIIb and GPIIIa precursors required 4-6 h for completion. All GPIIIa molecules were eventually assembled; the excess GPIIb precursors were degraded without reaching the cell surface. Following assembly, preIIb-IIIa complexes were rapidly transported to the Golgi apparatus where preIIb underwent modification of high mannose chains into complex oligosaccharides and proteolytic cleavage to yield disulfide-linked heavy and light chains. Pretreating cells with the ionophore monensin blocked cleavage of preIIb but not its carbohydrate modification or its assembly with GPIIIa. These studies suggest that 1) assembly of the precursors of GPIIb and GPIIIa in the ER is a slow process requiring conformational maturation of one or both subunits, and 2) only heterodimers assembled in the ER are transported to the Golgi apparatus for additional processing and, ultimately, expression on the cell surface.

Antibodies, Monoclonal↗
PubMed1989

Role of microtubule assembly in phenytoin teratogenic action in the sea urchin (Arbacia punctulata) embryo.

We evaluated the role of microtubule assembly in phenytoin (5-5-diphenylhydantoin) teratogenic activity in the sea urchin embryo. Zygotes were exposed to phenytoin or one of several phenytoin analogs within 15 min of fertilization and the frequency of the resultant malformations was assessed at the cleavage and late gastrula (prism) stages. Concomitant studies of drug uptake into zygotes and drug effects on both microtubule assembly in vitro and spindle morphology in situ were also performed. Phenytoin, 5-p-methylphenyl-5-phenylhydantoin, and 5-p-methoxyphenyl-5-phenylhydantoin were teratogenic (approaching 100% affected embryos) at both developmental stages were concentrated rapidly by the zygotes, and induced a shortened mitotic spindle in situ. In a separate in vitro system using porcine brain microtubular protein, these analogs were shown to inhibit microtubule assembly directly. The major human metabolite of phenytoin, 5-p-hydroxyphenyl-5-phenylhydantoin was teratogenic at the prism stage but induced only a 20% incidence of abnormal embryos at the first cleavage. This was attributed to the slow rate of uptake of this analog. This compound inhibited microtubule assembly in the in vitro assay and also shortened the mitotic spindle to an extent proportional to its observed weak effect on the first cleavage. Another analog, 5-p-hydroxyphenyl-5-p'-methylphenylhydantoin was not teratogenic at concentrations up to the limit of its solubility (285 microM). If this analog were as potent inside the cell as either phenyltoin or 5-p-hydroxyphenyl-5-phenylhydantoin, the intracellular concentrations achieved should have been sufficient to induce abnormal cleavage. Thus, the lack of teratogenic efficacy of this analog was correlated with its observed lack of effects on either microtubule assembly in vitro or spindle formation in situ. The anticonvulsant drug ethotoin was not teratogenic at concentrations up to 2.93 mM, apparently due to either poor uptake or inability to inhibit microtubule assembly or both. Overall, these studies are consistent with a hypothesis that phenytoin may induce abnormal development in this system by a direct inhibition of microtubule assembly.

Animals↗
PubMed1988

Fibronectin's amino-terminal matrix assembly site is located within the 29-kDa amino-terminal domain containing five type I repeats.

Fibronectin is organized into disulfide cross-linked, insoluble pericellular matrix fibrils by fibroblasts in vitro. Two sites, the Arg-Gly-Asp-Ser-containing cell attachment domain and a site located in the first 70 kDa of fibronectin, are required for matrix assembly. The first 70 kDa of fibronectin contain two structural motifs termed type I and type II homologies, which are repeated nine and two times, respectively. Previous work has implicated the amino-terminal region and the carboxyl terminus containing three type I repeats in matrix assembly, suggesting that type I repeats possess binding activity essential for fibronectin matrix assembly. To test this hypothesis, we developed a sensitive capture immunoassay to quantify insoluble matrix fibronectin and tested a panel of fibronectin fragments, containing all of the type I repeats found in the intact protein, for their ability to inhibit matrix assembly. Only fragments containing the first five type I repeats inhibited fibronectin matrix assembly, although sequences carboxyl-terminal to this domain enhanced this activity. Additional evidence for the specific recognition of the amino-terminal type I repeats by matrix assembling cells was found when the reversible, detergent-sensitive binding of a 125I-labeled fragment containing the first five type I repeats (29 kDa) to cell monolayers was studied. Only monolayers of cell lines that incorporate fibronectin into a fibrillar matrix specifically bound 125I-labeled 29 kDa. Binding of the radiolabeled amino-terminal fragment to matrix-forming cells was inhibited by unlabeled fragments containing the first five type I repeats but not by unlabeled fragments containing the remaining seven type I repeats. Matrix assembly is therefore not a generalized property of type I repeats. Rather, a critical site is located within the first 29 kDa of fibronectin.

Amino Acid Sequence↗
PubMed1987

Incorporation of six additional proteins to complete the assembly map of the 50 S subunit from Escherichia coli ribosomes.

In the course of 50 S ribosome assembly in vitro, the L-proteins associate consecutively with the rRNA in two main groups. The first assembly group is found on the reconstitution intermediate particle RI50(1), the second group being added to form the RI50(2) particle. The analysis presented here allows for the first time all the ribosomal proteins to be assigned to one or the other of these groups. The following 22 proteins are found in the RI50(1) particle: L1, (L2), L3, L4, (L5), L7/12, L9, L10, L11, (L13), L15, L17, (L18), L20, L21, L22, L23, L24, (L26), L29, (L33), (L34). The assembly map has been arranged accordingly. The assembly interdependences of six proteins (L28, L30, L31, L32, L33, and L34) were assessed and that of L14 revised by means of a large number of assembly mapping experiments, the final decisive steps of which are documented here. The interactions of these latter seven proteins and the incorporation of five additional assembly interdependences are integrated into the 50 S assembly map, which now contains all the components of the 50 S subunit, with the exception of protein L26; this protein is known to be the same as protein S20 and is predominantly associated with the 30 S subunit.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Biosynthesis and secretion of the rat core-specific lectin. Relationship of post-translational modification and assembly to attainment of carbohydrate binding activity.

A soluble lectin, the core-specific lectin (CSL), is synthesized and secreted by rat hepatocytes and the rat hepatoma cell line, H-4-II-E. This lectin binds mannose and N-acetylglucosamine residues in the "core" region of Asn-linked oligosaccharides. Secretion of the CSL was found to occur over an extended period of time, greater than 4 h being required for secretion of 50% of the lectin (Brownell, M. D., Colley, K. J., and Baenziger, J. U. (1984) J. Biol. Chem. 259, 3925-3932). We have determined that following synthesis in the endoplasmic reticulum, the CSL is rapidly transported to the Golgi where it is retained for an extended period of time prior to secretion. The lectin undergoes two post-translational modifications within the Golgi: an increase from Mr 24,000 to 25,000 and a progressive decrease in pI with an accompanying increase in Mr to a final value of 26,000. The lectin is also assembled into high molecular weight complexes of 150-260 X 10(3) and acquires the ability to bind carbohydrate in the Golgi. In hepatoma cells, the 24,000-25,000 modification is completed 20 min after initiation of synthesis. Assembly of the CSL subunits into high molecular weight complexes, acquisition of carbohydrate binding activity, and the 25,000-26,000 modification occur between 20 and 80 min after initiation of synthesis. These events have slower kinetics in primary hepatocytes and this allowed us to determine that the sequence of these biosynthetic events is: the 24,000-25,000 modification, complex assembly, the 25,000-26,000 modification, and acquisition of carbohydrate binding activity. The 24,000-25,000 modification occurs prior to complex assembly. Complex assembly may occur prior to, or concomitant with, the 25,000-26,000 modification. Assembly into the oligomeric form and the 25,000-26,000 modification correlate with the attainment of carbohydrate binding activity. The kinetics of CSL modification and assembly cannot account for its retention within the Golgi. Interaction with Golgi components either through carbohydrate binding or another interaction, may act to selectively retain the lectin within the Golgi.

Animals↗
PubMed1985

Differential association of the different brain microtubule proteins in different in vitro assembly conditions.

Microtubule protein was assembled in vitro for different time periods and at different protein concentrations near and far from the critical concentration needed for assembly. When the isolated polymers were characterized by electrophoresis, a higher association of high-molecular-weight microtubule-associated proteins, particularly of the larger microtubule-associated protein, was found in the polymers assembled close to the critical concentration, while tau factor polypeptides are predominantly present in those polymers assembled at higher protein concentration. Also, a higher proportion of high-molecular-weight microtubule associated proteins MAP1 and MAP2 was observed in microtubules assembled for short time periods, compared with those obtained after the monomer-polymer equilibrium was reached. When the assembled protein was further characterized by isoelectric focusing, no differences were found in the proportion of the different isotubulins present in the polymers assembled under different conditions tested.

Animals↗
PubMed1978

Stoichiometry and role of GTP hydrolysis in bovine neurotubule assembly.

A method is given for preparing tubulin with 1 mol of exchangeably bound [gamma-32P]GTP/mol of 6 S dimer. Bovine tubulin is shown to hydrolyze 1 mol of GTP/mol of 6 S dimer added to assembling microtubules at 37 degrees. Hydrolysis and assembly occur at the same rate and to the same extent. When microtubule-associated proteins (MAPs) are removed, both hydrolysis and assembly fail to occur. Readdition of the MAPs restores both activities. Tubulin with exchangeable GDP will co-assemble with GTP.tubulin even at equimolar levels. Exchangeability is demonstrated by pulse-chase experiments with GDP or GTP. GDP is also a potent inhibitor of assembly under these conditions, and the rate of assembly is reduced by 50% at 10 micron GDP. One mole of inorganic phosphate is released to the solvent per mole of exchangeable GTP hydrolyzed. An assembly mechanism is proposed in which exchangeable GTP is hydrolyzed without intermediate transphosphorylation of nonexchangeable GDP.

Animals↗
PubMed1983

Influence of trimethyl lead and inorganic lead on the in vitro assembly of microtubules from mammalian brain.

The influence of trimethyl lead (TriML) and inorganic lead (Pb) on the in vitro assembly of microtubules (MTs) from tubulin of porcine brain was studied using turbidity measurements and electron microscopy. At concentrations of 150 to 650 microM Pb, no significant effects on the in vitro assembly of MTs could be detected by both methods. On the other hand, TriML increasingly blocked MT assembly at 100 to 200 microM and completely inhibited assembly at 300 microM TriML and higher concentrations. Application of 400 microM TriML to preassembled MTs caused an immediate drop of the optical density. Electron microscopy showed that one minute after addition of TriML to assembled MTs only very few intact MTs could be detected whereas ten minutes later MTs were completely absent. The observed increase of optical density following the TriML-induced drop was found to be due to the formation of clusters of aggregates but not to assembly of MTs. The results demonstrate that inorganic lead and trimethyl lead have different effects on the in vitro assembly of MTs. It is assumed that the selective neurotoxic effects of organic lead might be due, at least in part, to impairments of neurotubular structures and functions.

Animals↗
PubMed1984

The effect of assembly and transit stressors on plasma fibrinogen concentration of beef calves.

Plasma fibrinogen concentration was measured in beef calves at various points within the system presently used to assemble, market and transport calves from one production point to another in order to determine the effect of the stresses encountered. A short haul of 160 km immediately after weaning did not significantly elevate fibrinogen concentration above the pretransit values. Yearling steers transported 400 km and confined in unfamiliar surroundings for 15 h did have an elevated (P less than 0.01) concentration of fibrinogen, but this increase was not significantly different from that of steers which were confined but not transported, thus confinement may be a significant portion of the stress associated with transit. The change in plasma fibrinogen concentration during assembly and transit was dependent upon the farm from which the calves originated. The magnitude of the change in fibrinogen concentration as a result of assembly and transit varied between the years studied. In one year pretransit assembly for ten days resulted in a higher fibrinogen concentration before and after transit than assembly for four days, but no difference was noted between the two groups in the second year. Bovine plasma fibrinogen concentration does increase in response to the stresses associated with assembly and transit. The stress of fasting and housing in unfamiliar surroundings also increase bovine plasma fibrinogen concentration and are present in the assembly and transit system. These two stresses may account for a majority of the stress associated with marketing and transit. The response of beef calves to the marketing and transit system varied between years.

Animals↗
PubMed1983

Assembly of unfertilized sea urchin egg tubulin at physiological temperatures.

Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by DEAE-column chromatography and cycles of temperature-dependent assembly and disassembly. Tubulin-containing column fractions self-assemble into intact microtubules in the absence of high molecular weight microtubule-associated proteins. Egg microtubules assembled during the third cycle of assembly following DEAE-chromatography are composed of 2 or 3 alpha tubulins and 2 beta tubulins as assayed by isoelectric focusing and two-dimensional electrophoresis. The critical protein concentrations necessary for assembly of egg tubulin at 37 and 25 degrees C are 0.15-0.24 and 0.24-0.28 mg/ml, respectively. At physiological temperatures, the critical protein concentrations are 0.81 mg/ml at 15 degrees C and 0.70-0.79 mg/ml at 18 degrees C. At 18 degrees C, bovine brain microtubule-associated proteins stoichiometrically stimulate the initial rate and final extent of egg tubulin assembly. These hybrid microtubules assemble at 18 degrees C at a critical protein concentration of 4-20 micrograms/ml.

Animals↗
PubMed1981

Collagen self-assembly in vitro. Differentiating specific telopeptide-dependent interactions using selective enzyme modification and the addition of free amino telopeptide.

The thermally induced in vitro self-assembly of collagen molecules to form active fibrils illustrates that collagen molecules themselves contain all of the structural information necessary for assembly. The molecule contains three structural domains, the NH2 and carboxyl-terminal extra helical regions (the telopeptides) and the major triple helical rod-like domain. Proteolytic removal of the short telopeptide domains drastically alters the in vitro self-assembly process. We have examined the specific contributions of each telopeptide to the initiation ("nucleation") and growth stages of self-assembly in collagens modified by selective proteinase treatment and by isolating a peptide containing the amino telopeptide and adding this to both normal and proteinase-modified collagen self-assembly systems. Pronase-modified collagen, devoid of both telopeptides, initiated self-assembly very poorly. Addition of small amounts of intact collagen accelerated the rate of nucleation of pronase-modified collagen. Addition of carboxypeptidase-modified collagen also accelerated the nucleation of pronase-modified collagen, suggesting that the remaining amino telopeptide was involved in nucleation. This was confirmed by isolating the cyanogen bromide fragment of the alpha 1(I) subunit containing the amino telopeptide and finding that it specifically accelerated the nucleation of intact pepsin- and pronase-modified to collagens. The amino telopeptide appears to bind to a specific region within the collagen triple helical domain. The isolated peptide requires thermal pretreatment to be active; hence, this interaction must involve a unique telopeptide conformation. This behavior is compatible with the recent model (Helseth, D. L., Jr., Lechner, J. H., and Veis, A. (1979) Biopolymers 18, 3005-3014) proposed for the conformation of the amino telopeptide and its interaction with a helical receptor site as a step in nucleation. Comparison of the behavior of leucine aminopeptidase- and carboxypeptidase-modified collagens suggests that the carboxyl telopeptide has its major role in the growth stages of self-assembly.

Animals↗
PubMed1993

The alpha 5 beta 1 integrin fibronectin receptor, but not the alpha 5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly.

The alpha 5 beta 1 integrin mediates cell adhesion and migration on fibronectin, a glycoprotein critical for normal vertebrate embryonic development. Indirect evidence reported to date suggests that this receptor also functions in the deposition of fibronectin matrices. We used a molecular genetic approach to critically evaluate this role of alpha 5 beta 1 integrins. Mutant Chinese hamster ovary (CHO) cells deficient in alpha 5 integrin expression could not assemble a fibronectin matrix. Reconstituting alpha 5 beta 1 integrin expression by transfecting them with a full-length cDNA encoding the human alpha 5 chain completely restored fibronectin matrix assembly. CHO cells expressing an alpha 5 chain lacking the cytoplasmic domain also assembled a fibronectin matrix. Removing the cytoplasmic domain of alpha 5 appears to increase its activity in fibronectin matrix assembly. In addition to alpha 5 beta 1 integrin binding to fibronectin's RGD-containing domain, cells must bind with high affinity to fibronectin's amino-terminal 29-kDa matrix assembly domain to form a fibronectin matrix. Studies with the alpha 5-deficient CHO cells show that the expression of alpha 5 beta 1 integrin is also necessary for cells to bind fragments containing this distinct site in fibronectin and that a fibronectin matrix increases binding of the 29-kDa fragment. Thus, alpha 5 beta 1 integrins not only mediate cell adhesion to fibronectin, but also play an essential role in the assembly of a fibronectin matrix. This role includes direct binding to fibronectin and modulating a distinct binding event involving the interaction of fibronectin's amino-terminal matrix assembly domain with the cell surface.

Amino Acid Sequence↗
PubMed1998

Automated clustering and assembly of large EST collections.

The availability of large EST (Expressed Sequence Tag) databases has led to a revolution in the way new genes are cloned. Difficulties arise, however, due to high error rates and redundancy of raw EST data. For these reasons, one of the first tasks performed by a scientist investigating any EST of interest is to gather contiguous ESTs and assemble them into a larger virtual cDNA. The REX (Recursive EST eXtender) algorithm described in this paper completely automates this process by finding ESTs that can be clustered on the basis of overlapping bases, and then assembling the contigs into a consensus sequence. By combining the clustering and assembly steps, REX can quickly generate assemblies from EST databases that are frequently updated without having to preprocess the data. A consensus assembly method is used to correct miscalled bases and remove indel errors. A unique feature of this method is that it addresses the issues of splice variants and unspliced cDNA data. Since REX is a fast greedy algorithm, it can address the problem of generating a database of assembled sequences from very large collections of EST data. A procedure is described for creating and maintaining an Assembled Consensus EST database (ACE) that is useful for characterizing the large body of data that exists in EST databases.

Algorithms↗
PubMed2002

High-resolution localization of clathrin assembly protein AP180 in the presynaptic terminals of mammalian neurons.

Synaptic vesicles (SVs) assemble at the presynaptic compartment through a clathrin-dependent mechanism that involves one or more assembly proteins (APs). The assembly protein AP180 is especially efficient at facilitating clathrin cage formation, but its precise ultrastructural localization in neurons is unknown. Using immunoelectron microscopy, we demonstrate the presynaptic localization of AP180 in axon terminals of rat cerebellar neurons. In contrast, the assembly protein AP2 was associated with both the presynaptic plasma membrane and the cytosolic side of the membrane at postsynaptic and extrasynaptic sites. Furthermore, ultrastructural analysis of primate retina showed that AP180 immunoreactivity was preferentially and highly enriched at ribbon synapses, where glutamate is released tonically at high levels and rapid vesicle turnover is essential. To maintain functional synaptic transmission, neurotransmitter-filled SVs must be readily available, and this requires proper reassembly of new vesicles. The expression of AP180, in addition to AP-2, in the clathrin-mediated endocytic pathway might add another level of control to SV reformation for efficient assembly of clathrin, effectively controlling the size of assembled vesicles and faithfully recovering SV-specific components.

Adaptor Protein Complex 2↗