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At least 1,099 records · Page 61Linked to original sources
PubMed1996

Regulated assembly of connexin33 and connexin43 into rat Sertoli cell gap junctions.

Immunofluorescence and immunogold electron microscopy were employed to examine the assembly of connexins (Cx) 33, 37, and 43 into testis cell gap junctions in mature and postnatal rats. Cx37 was localized by immunofluorescence to the endothelia of blood vessels in both mature and immature testes and was not further characterized. Only Cx43 assembled into Leydig cell gap junctions, but Cx43 also co-assembled with Cx33 in some Sertoli-Sertoli gap junction plaques within and near Sertoli occluding junctions and on adluminal surfaces Assembly of Sertoli gap junctions appeared to be regulated according to the stage of the seminiferous epithelium since Cx33 (and Cx43) immunoreactivity was strong in Sertoli cells from stages II-VII but weak in stages IX-XIV. During postnatal maturation, assembly of Cx33 into gap junctions was regulated independently of Cx43 assembly. Cx43 was present on Sertoli cells of all tubules from postnatal Day 5 through Day 28. In contrast, Cx33 was not apparent on Sertoli cell surfaces until Day 15 and gradually accumulated in all tubules through Day 28. Between postnatal Days 38 and 43, the immunoreactivities of Cx33 and Cx43 became weak in Sertoli cells containing step 9-14 elongated spermatids. Thus, connexin abundance and gap junction composition in Sertoli cells is regulated during testis maturation and the cycle of the seminiferous epithelium.

Animals↗
PubMed2006

Alpha-crystallin expression affects microtubule assembly and prevents their aggregation.

The molecular chaperones alphaA- and alphaB-crystallins are important for cell survival and genomic stability and associate with the tubulin cytoskeleton. The mitotic spindle is abnormally assembled in a number of alphaA-/- and alphaB-/- lens epithelial cells. However, no report to date has studied the effect of alpha-crystallin expression on tubulin/microtubule assembly in lens epithelial cells. In the current work we tested the hypothesis that the absence of alphaA- and alphaB-crystallins alters microtubule assembly. Microtubules were reconstituted from freshly dissected explants of wild-type, alphaA-/-, alphaB-/-, and alpha(A/B) -/- (DKO) mouse lens epithelia and examined by electron microscopic and biochemical analyses. The wild-type microtubules were 4 mum long and approximately 25 nm wide and had a characteristic protofilament structure, but alphaB-/- microtubules were 2.5-fold longer. Microtubule-associated proteins (MAPs) extracted from microtubules by washing with salt included transketolase, alpha-enolase, and betaB2-crystallin. In DKO lens epithelial microtubules but not in wild-type, alphaA-/- or alphaB-/- microtubules, extraction of the MAPs gave very long (14-20 microm) "polyfilament" assemblies that were tightly bundled. Addition of exogenous alpha-crystallin (alphaA+ alphaB) was ineffective in preventing polyfilament formation. However, normal microtubule structure could be restored by including MAPs derived from wild-type lens epithelial cells during microtubule reconstitution. Intriguingly, these data suggest that alpha-crystallin may interact with MAPs to inhibit aggregation of microtubules in lens epithelial cells. Sedimentation analysis and 90 degrees light scattering measurements showed that alpha-crystallin suppressed tubulin assembly in vitro. Alpha-crystallin did not have a strong effect on the GTPase activity of purified tubulin. SDS-PAGE analysis showed that alpha-crystallin prevented heat-induced aggregation of tubulin, suggesting that alpha-crystallin may affect microtubule assembly by maintaining the pool of unassembled tubulin.

Animals↗
PubMed1996

Occupational disease surveillance using disability insurance at an automotive stamping and assembly complex.

Sickness and accident-insurance (sick leave) claims at an automotive stamping and assembly complex were analyzed using Poisson regression over a 4-year period to identify occupational health problems. The incidence of lower-respiratory disability (excluding asthma) was higher in painting operations (rate ratio [RR] = 2.9, 95% confidence interval [CI] = 1.2 to 6.8), and final assembly and processing areas (RR = 2.7, 95% CI = 1.0 to 7.4) at the assembly plant, and in metal assembly (welding) areas (RR = 2.8, 95% CI = 1.4 to 5.7) at the stamping plant. Disability rates for upper-extremity musculoskeletal disorders were statistically significantly higher (RR = 3.1 to 3.8) in major assembly plant production areas, as were back disability rates (RR = 1.5). During the first 6 months of new work assignments in painting or final assembly, respiratory problem rates were four times higher than in other areas. Upper-extremity musculoskeletal rate ratios ranged from 4.4 to 5.7 for new assignments in body, hard trim, and chassis areas. Higher rates in new assignments appeared to result from assignment changes precipitated by developing health problems, or from routine assignments to new tasks, some of which conferred high risk and were tolerated for less than 6 months. Musculoskeletal disability was consistent with known ergonomic hazards and paralleled that reported on the Occupational Safety and Health Administration log. Work-related musculoskeletal and other problems can be readily identified from disability insurance claims without dependence on plant medical visits or workers' compensation records. Disability insurance appears to absorb considerable work-related medical and absence costs.

Accidents, Occupational↗
PubMed2002

Site-specific alteration of actin assembly visualized in living renal epithelial cells during ATP depletion.

Disruption of normal actin organization in renal tubular epithelial cells is an important element of renal injury induced by ischemia. Studies of fixed cells indicate that the cytoskeleton is disrupted by both ischemia and ATP depletion in a site-specific manner. However, few studies have examined these effects in living cells, and the relationship between the time course of ATP reduction and alteration of the cytoskeleton remains unclear. Here, time-lapse video images of cultured renal epithelial cells expressing an enhanced green fluorescent protein (EGFP)-actin fusion protein were obtained, and the kinetics of fluorescence actin distribution before and during ATP depletion is quantified and compared with measured ATP levels. This study found that assembly of lamellar actin is inhibited rapidly as cellular ATP levels are reduced, whereas disruption of actin in stress fibers is more gradual and persistent. Actin associated with focal adhesions is largely resistant to ATP depletion in these experiments, and, consistent with previous studies, particulate aggregates of actin were formed within the cytoplasm of ATP-depleted cells. Most surprisingly, time-lapse imaging of EGFP-actin distribution, quantitative fluorescence imaging of phalloidin-stained cells, and ultrastructural studies indicate that assembly of actin filaments occurs at sites of epithelial cell-cell attachment in ATP-depleted cells. This assembly is initiated early during ATP depletion and continues after ATP levels are maximally reduced. Assembly of actin at sites of cell-cell attachment may be an element of the pathology of injury induced by ischemia, or alternatively, could reflect the function of a protective mechanism. These studies directly demonstrate site-specific alteration of actin assembly in living epithelial cells during ATP depletion. The results also reveal that actin reorganization continues after ATP levels are maximally decreased and that epithelial cell-cell attachments are sites of actin assembly in ATP-depleted cells.

Actin Cytoskeleton↗
PubMed2005

Intestinal lipoprotein assembly.

PURPOSE OF REVIEW: The assembly of intestinal lipoproteins is critical for the transport of fat and fat-soluble vitamins. In this review we propose a nomenclature for these lipoproteins and have summarized recent data about their intracellular assembly and factors that modulate their secretion. RECENT FINDINGS: The assembly and secretion of intestinal lipoproteins increases with the augmented synthesis of apoB, apoAIV and lipids. Chylomicron assembly begins with the formation of primordial, phospholipid-rich particles in the membrane, and their conversion to large chylomicrons occurs in the lumen of the smooth endoplasmic reticulum. Chylomicrons are transported from the endoplasmic reticulum via specialized vesicles to the Golgi for secretion. The identification of genetic mutations in chylomicron retention disease indicates that Sar1b may play a critical role in this process. In addition to chylomicron assembly, intestinal cells have been shown to transport dietary cholesterol via apoB-independent pathways, such as efflux. SUMMARY: Understanding the mechanisms involved in the intracellular transport of chylomicrons and chylomicron-independent secretion pathways are expected to be the next frontiers in the field of intestinal lipoprotein assembly and secretion.

Biological Transport, Active↗
PubMed2004

Saccharomyces cerevisiae Bni4p directs the formation of the chitin ring and also participates in the correct assembly of the septum structure.

In Saccharomyces cerevisiae cytokinesis is efficiently achieved when a concerted series of events take place at the neck region, leading to septum formation. Here it is shown that Bni4p plays a crucial role in this process. Deltabni4 mutants contain normal amounts of chitin and show normal chitin synthase III (CSIII) activity, but are partially resistant to Calcofluor White (CFW), probably due to the striking pattern of chitin distribution. CFW vital staining shows that chitin is synthesized in daughter cells and that it is also asymmetrically deposited at the mother-side of the neck in large-budded cells. This specific pattern coincides with that of Chs4p and Chs3p proteins. Alternatively, staining of unbudded cultures confirmed that Bni4p directs early chitin ring assembly, but is no longer required for the chitin deposition that occurs late in the cell cycle at cytokinesis. Consequently, this work provides a strategy to genetically discriminate between the absence of chitin synthesis (Deltachs3 mutant) and failure in chitin ring assembly (Deltabni4 mutants). The characterization of double mutants affected in chitin synthesis and primary septum (PS) assembly (Deltamyo1 and Deltachs2) provides evidence for the cooperation of Bni4p in PS formation besides its role in chitin ring assembly. In addition, it is shown that the chitin ring, but not the late deposition of chitin, cooperates in the correct assembly of the actomyosin ring and the PS when the biological function of the septins is compromised. We conclude that Bni4p is not only required for the assembly of the chitin ring, but is also involved in septum architecture and the maintenance of neck integrity.

Cell Cycle Proteins↗
PubMed1987

Spliceosome assembly in yeast.

Precursors to mRNA become substrates for splicing by being assembled into a complex multisubunit structure, the spliceosome. To study the assembly of the yeast spliceosome, intermediate complexes were separated by electrophoresis on nondenaturing polyacrylamide gels. Four splicing-dependent complexes, A1, A2-1, A2-2, and B, were observed. The order of assembly of these complexes was determined to be B----A2-1----A1----A2-2. The assembly process can be blocked at complex A1 by addition of 5 mM EDTA or by carrying out the assembly process in heat-inactivated rna2 extracts. The snRNA composition of the complexes was determined by hybridization with probes for five yeast snRNAs. snR14 (U4) was only found in complex A2-1, snR6 (U6) and snR7 (U5) were in complexes A1, A2-1, and A2-2, whereas snR20 (U2) was in all four of the complexes. snR19 (U1) was not present in any of the complexes. Hybridization with these probes was also employed to detect snRNPs present in yeast splicing extracts. We found that snR6, snR7, and snR14 were present together in a large complex. This complex underwent an ATP-dependent dissociation to give snR7 and snR6-snR14 complexes. snR19 and snR20 are present in distinct RNPs but the mobility of these is not affected by ATP. A mechanism for spliceosome assembly is proposed.

Animals↗
PubMed2006

Assembly of an active translation initiation factor complex by a viral protein.

Recruitment of the 40S ribosome to the 5' end of a eukaryotic mRNA requires assembly of translation initiation factors eIF4E, the cap-binding protein, together with eIF4A and eIF4G into a complex termed eIF4F. While the translational repressor 4E-BP1 regulates binding of eIF4E to eIF4G, the forces required to construct an eIF4F complex remain unidentified. Here, we establish that the herpes simplex virus-1 (HSV-1) ICP6 polypeptide associates with eIF4G to promote eIF4F complex assembly. Strikingly, release of eIF4E from the 4E-BP1 repressor is insufficient to drive complex formation, suggesting that ICP6 is an eIF4F-assembly chaperone. This is the first example of a translation initiation factor-associated protein that promotes active complex assembly and defines a new, controllable step in the initiation of translation. Homology of the N-terminal, eIF4G-binding segment of ICP6 with cellular chaperones suggest that factors capable of interacting with eIF4G and promoting eIF4F complex assembly may play important roles in a variety of processes where translation complexes need to be remodeled or assembled on populations of newly synthesized or derepressed mRNAs, including development, differentiation, and the response to a broad spectrum of environmental cues.

Cell Line↗
PubMed1988

A chemical modification/interference study of yeast pre-mRNA spliceosome assembly and splicing.

A chemical modification/interference assay was used to determine the yeast pre-mRNA sequence requirements for in vitro spliceosome assembly and splicing. Modifications of any of the nucleotides within the 5' splice site and branch point (TACTAAC box) consensus sequences as well as less conserved intron and exon positions were found to inhibit assembly and/or splicing. The interference pattern of the 5' splice site and TACTAAC box lesions increased as spliceosome assembly proceeded (complex III----complex I----complex II) and as splicing proceeded, suggesting that these sequence elements play multiple roles in the assembly of yeast spliceosomes and in the removal of intervening sequences. Furthermore, modification (or mutation) of a TACTAAC-like sequence upstream of the branch point was found to inhibit the rate of spliceosome assembly, implying a possible role for degenerate branch point sequences in modulating the efficiency of spliceosome assembly.

Base Sequence↗
PubMed1993

Replication-coupled chromatin assembly is required for the repression of basal transcription in vivo.

The chromatin assembly process coupled to DNA synthesis in the Xenopus oocyte nucleus is significantly more repressive toward basal transcription than chromatin assembly on duplex DNA. We show that chromatin assembly concurrent with DNA synthesis over the promoter region itself is causal for repression. However, the trans-activator Gal4-VP16 both relieves repression and activates transcription regardless of the chromatin assembly pathway. This activation is independent of whether Gal4-VP16 addition occurs before or after chromatin assembly. We propose that replication-coupled chromatin assembly represents a general mechanism to direct the efficient repression of basal transcription. However transcription induction by a specific activator, Gal4-VP16, occurs independent of this chromatin-mediated repression.

Animals↗
PubMed2002

ARACHNE: a whole-genome shotgun assembler.

We describe a new computer system, called ARACHNE, for assembling genome sequence using paired-end whole-genome shotgun reads. ARACHNE has several key features, including an efficient and sensitive procedure for finding read overlaps, a procedure for scoring overlaps that achieves high accuracy by correcting errors before assembly, read merger based on forward-reverse links, and detection of repeat contigs by forward-reverse link inconsistency. To test ARACHNE, we created simulated reads providing approximately 10-fold coverage of the genomes of H. influenzae, S. cerevisiae, and D. melanogaster, as well as human chromosomes 21 and 22. The assemblies of these simulated reads yielded nearly complete coverage of the respective genomes, with a small number of contigs joined into a smaller number of supercontigs (or scaffolds). For example, analysis of the D. melanogaster genome yielded approximately 98% coverage with an N50 contig length of 324 kb and an N50 supercontig length of 5143 kb. The assembly accuracy was high, although not perfect: small errors occurred at a frequency of roughly 1 per 1 Mb (typically, deletion of approximately 1 kb in size), with a very small number of other misassemblies. The assembly was rapid: the Drosophila assembly required only 21 hours on a single 667 MHz processor and used 8.4 Gb of memory.

Algorithms↗
PubMed2004

Analysis of segmental duplications and genome assembly in the mouse.

Limited comparative studies suggest that the human genome is particularly enriched for recent segmental duplications. The extent of segmental duplications in other mammalian genomes is unknown and confounded by methodological differences in genome assembly. Here, we present a detailed analysis of recent duplication content within the mouse genome using a whole-genome assembly comparison method and a novel assembly independent method, designed to take advantage of the reduced allelic variation of the C57BL/6J strain. We conservatively estimate that approximately 57% of all highly identical segmental duplications (>or=90%) were misassembled or collapsed within the working draft WGS assembly. The WGS approach often leaves duplications fragmented and unassigned to a chromosome when compared with the clone-ordered-based approach. Our preliminary analysis suggests that 1.7%-2.0% of the mouse genome is part of recent large segmental duplications (about half of what is observed for the human genome). We have constructed a mouse segmental duplication database to aid in the characterization of these regions and their integration into the final mouse genome assembly. This work suggests significant biological differences in the architecture of recent segmental duplications between human and mouse. In addition, our unique method provides the means for improving whole-genome shotgun sequence assembly of mouse and future mammalian genomes.

Animals↗
PubMed2004

The Atlas genome assembly system.

Atlas is a suite of programs developed for assembly of genomes by a "combined approach" that uses DNA sequence reads from both BACs and whole-genome shotgun (WGS) libraries. The BAC clones afford advantages of localized assembly with reduced computational load, and provide a robust method for dealing with repeated sequences. Inclusion of WGS sequences facilitates use of different clone insert sizes and reduces data production costs. A core function of Atlas software is recruitment of WGS sequences into appropriate BACs based on sequence overlaps. Because construction of consensus sequences is from local assembly of these reads, only small (<0.1%) units of the genome are assembled at a time. Once assembled, each BAC is used to derive a genomic layout. This "sequence-based" growth of the genome map has greater precision than with non-sequence-based methods. Use of BACs allows correction of artifacts due to repeats at each stage of the process. This is aided by ancillary data such as BAC fingerprint, other genomic maps, and syntenic relations with other genomes. Atlas was used to assemble a draft DNA sequence of the rat genome; its major components including overlapper and split-scaffold are also being used in pure WGS projects.

Animals↗
PubMed2006

Physical map-assisted whole-genome shotgun sequence assemblies.

We describe a targeted approach to improve the contiguity of whole-genome shotgun sequence (WGS) assemblies at run-time, using information from Bacterial Artificial Chromosome (BAC)-based physical maps. Clone sizes and overlaps derived from clone fingerprints are used for the calculation of length constraints between any two BAC neighbors sharing 40% of their size. These constraints are used to promote the linkage and guide the arrangement of sequence contigs within a sequence scaffold at the layout phase of WGS assemblies. This process is facilitated by FASSI, a stand-alone application that calculates BAC end and BAC overlap length constraints from clone fingerprint map contigs created by the FPC package. FASSI is designed to work with the assembly tool PCAP, but its output can be formatted to work with other WGS assembly algorithms able to use length constraints for individual clones. The FASSI method is simple to implement, potentially cost-effective, and has resulted in the increase of scaffold contiguity for both the Drosophila melanogaster and Cryptococcus gattii genomes when compared to a control assembly without map-derived constraints. A 6.5-fold coverage draft DNA sequence of the Pan troglodytes (chimpanzee) genome was assembled using map-derived constraints and resulted in a 26.1% increase in scaffold contiguity.

Animals↗
PubMed2003

Whole-genome sequence assembly for mammalian genomes: Arachne 2.

We previously described the whole-genome assembly program Arachne, presenting assemblies of simulated data for small to mid-sized genomes. Here we describe algorithmic adaptations to the program, allowing for assembly of mammalian-size genomes, and also improving the assembly of smaller genomes. Three principal changes were simultaneously made and applied to the assembly of the mouse genome, during a six-month period of development: (1) Supercontigs (scaffolds) were iteratively broken and rejoined using several criteria, yielding a 64-fold increase in length (N50), and apparent elimination of all global misjoins; (2) gaps between contigs in supercontigs were filled (partially or completely) by insertion of reads, as suggested by pairing within the supercontig, increasing the N50 contig length by 50%; (3) memory usage was reduced fourfold. The outcome of this mouse assembly and its analysis are described in (Mouse Genome Sequencing Consortium 2002).

Animals↗
PubMed1997

Adenosine 5'-triphosphate is required for the assembly of 11S seed proglobulins in vitro.

Seed protein proglobulins were synthesized from cDNAs in reticulocyte lysates. Most proglobulins were recovered as trimers when translation rates were low, but mostly monomers were recovered at high translation rates. The prevalence of monomers was accompanied by elevated amounts of insoluble protein recovered at the bottom of sucrose density gradients. Apyrase treatment of translation mixtures after synthesis, but before significant assembly occurred, drastically reduced trimer assembly and increased the proportion of insoluble aggregate. These observations indicated that ATP is required for protein folding and/or trimer assembly. The appearance of insoluble aggregated protein when rates of synthesis were elevated or when ATP was absent suggested that protein misfolding had occurred. Trimer assembly was stimulated when wheat germ translation mixtures defective in supporting efficient trimer assembly were supplemented with fractions isolated from endoplasmic reticula of developing pea (Pisum sativum) seeds. Molecular chaperones are likely involved in folding and/or assembly of proglobulin trimers both in reticulocyte lysates and in seeds. Consistent with this hypothesis, trimer formation was reduced when carboxymethylated bovine albumin and alpha-casein, considered to mimic proteins with extended chain and molten globular conformations and thereby compete for Hsp70- and Hsp60-type molecular chaperones, respectively, were introduced into translation mixtures.

Adenosine Triphosphate↗
PubMed1990

Effect of structural modifications on the assembly of a glycinin subunit.

A Gy4 glycinin cDNA was modified and used to produce structurally altered 11S storage protein subunits. We evaluated these modified subunits for their ability to assemble into oligomers. Alterations made in the acidic polypeptide changed the subunit solubility characteristics but did not eliminate assembly. Modifications in the basic polypeptide usually eliminated assembly of subunits into trimers. A region exhibiting high natural variability located at the COOH terminus of the acidic polypeptide that we have designated the hypervariable region was also studied. Extensive deletions and insertions were tolerated in the hypervariable region without perturbing subunit assembly. Some of the insertions significantly increased the methionine content in the Gy4 glycinin subunit. Together, our results indicated that the structure of the basic polypeptide was more critical for assembly of trimers than that of the acidic polypeptide, an observation that implies that the basic polypeptides direct trimer formation. The assembly assays described here will be useful in efforts to improve seed quality. Using them, the effects of modifications to the storage protein subunits can be rapidly evaluated before introducing the mutated genes into plants.

Base Sequence↗
PubMed1997

Role of the sulfhydryl redox state and disulfide bonds in processing and assembly of 11S seed globulins.

Seed legumins contain two conserved disulfide bonds: an interchain bond (IE) connecting the acidic and basic chains and an intrachain bond (IA) internal to the acidic chain. Mutant subunits were constructed in which these disulfide bonds were disrupted. Oxidized glutathione stimulated the rate of assembly of trimers with unmodified prolegumin subunits. Stimulation was not detected during assembly of IE mutant subunits and was diminished for the IA mutant. Hexamer assembly with trimers of mature unmodified subunits required oxidizing conditions. Trimers composed of mature IE mutants did not form hexamers. Both mutant and non-mutant subunits accumulated in hexamers when the cDNAs were expressed in tobacco. Hexamer assembly in seeds probably involved trimers with a mixture of mutant and non-mutant subunits. Similarly, mixed trimers that were a mixture of mutant and non-mutant subunits assembled into hexamers in vitro. The results demonstrate the importance of disulfide bonds during the assembly of 11S globulins.

Biopolymers↗