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PubMed2025

Disassembly activates Retron-Septu for antiphage defense.

Retrons are antiphage defense systems that produce multicopy single-stranded DNA (msDNA) and hold promise for genome engineering. However, the mechanisms of defense remain unclear. The Retron-Septu system integrates retron and Septu antiphage defenses. Cryo-electron microscopy structures reveal asymmetric nucleoprotein complexes comprising a reverse transcriptase, msDNA (a hybrid of msdDNA and msrRNA), and two PtuAB copies. msdDNA and msrRNA are essential for assembling this complex, with msrRNA adopting a conserved lariat-like structure that regulates reverse transcription. Notably, the assembled Retron-Septu complex is inactive, with msdDNA occupying the PtuA DNA binding site. Activation occurs upon disassembly, releasing PtuAB, which degrades single-stranded DNA to restrict phage replication. This "arrest-and-release" mechanism underscores the dynamic regulatory roles of msDNA, advancing our understanding of antiphage defense strategies.

Cryoelectron Microscopy↗

PubMed2002

Requirement for two copies of RNA polymerase alpha subunit C-terminal domain for synergistic transcription activation at complex bacterial promoters.

Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at different promoters has been studied using RNA polymerase holoenzyme derivatives containing two full-length alpha subunits, or containing one full-length alpha subunit and one truncated alpha subunit lacking the alpha C-terminal domain (alpha CTD). At a promoter having a single DNA site for CRP, activation requires only one full-length alpha subunit. Likewise, at a promoter having a single DNA site for CRP and one adjacent UP-element subsite (high-affinity DNA site for alpha CTD), activation requires only one full-length alpha subunit. In contrast, at promoters having two DNA sites for CRP, or one DNA site for CRP and two UP-element subsites, activation requires two full-length alpha subunits. We conclude that a single copy of alpha CTD is sufficient to interact with one CRP molecule and one adjacent UP-element subsite, but two copies of alpha CTD are required to interact with two CRP molecules or with one CRP molecule and two UP-element subsites.

Base Sequence↗

PubMed2002

Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport.

Cytosolic coat proteins that bind reversibly to membranes have a central function in membrane transport within the secretory pathway. One well-studied example is COPI or coatomer, a heptameric protein complex that is recruited to membranes by the GTP-binding protein Arf1. Assembly into an electron-dense coat then helps in budding off membrane to be transported between the endoplasmic reticulum (ER) and Golgi apparatus. Here we propose and corroborate a simple model for coatomer and Arf1 activity based on results analysing the distribution and lifetime of fluorescently labelled coatomer and Arf1 on Golgi membranes of living cells. We find that activated Arf1 brings coatomer to membranes. However, once associated with membranes, Arf1 and coatomer have different residence times: coatomer remains on membranes after Arf1-GTP has been hydrolysed and dissociated. Rapid membrane binding and dissociation of coatomer and Arf1 occur stochastically, even without vesicle budding. We propose that this continuous activity of coatomer and Arf1 generates kinetically stable membrane domains that are connected to the formation of COPI-containing transport intermediates. This role for Arf1/coatomer might provide a model for investigating the behaviour of other coat protein systems within cells.

ADP-Ribosylation Factor 1↗

PubMed2001

Distinct roles for the cytoplasmic tail sequences of Emp24p and Erv25p in transport between the endoplasmic reticulum and Golgi complex.

Heteromeric complexes of p24 proteins cycle between early compartments of the secretory pathway and are required for efficient protein sorting. Here we investigated the role of cytoplasmically exposed tail sequences on two p24 proteins, Emp24p and Erv25p, in directing their movement and subcellular location in yeast. Studies on a series of deletion and chimeric Emp24p-Erv25p proteins indicated that the tail sequences impart distinct functional properties that were partially redundant but not entirely interchangeable. Export of an Emp24p-Erv25p complex from the endoplasmic reticulum (ER) did not depend on two other associated p24 proteins, Erp1 and Erp2p. To examine interactions between the Emp24p and Erv25p tail sequences with the COPI and COPII coat proteins, binding experiments with immobilized tail peptides and coat proteins were performed. The Emp24p and Erv25p tail sequences bound the Sec13p/Sec31p subunit of the COPII coat (K(d) approximately 100 microm), and binding depended on a pair of aromatic residues found in both tail sequences. COPI subunits also bound to these Emp24p and Erv25p peptides; however, the Erv25p tail sequence, which contains a dilysine motif, bound COPI more efficiently. These results suggest that both the Emp24p and Erv25p cytoplasmic sequences contain a di-aromatic motif that binds subunits of the COPII coat and promotes export from the ER. The Erv25p tail sequence binds COPI and is responsible for returning this complex to the ER.

Amino Acid Sequence↗

PubMed2005

Fluorescence in situ hybridization to localize transgenes in plant chromosomes.

Production of transgenic plants is now routine for many of our crop species. Methods for the detailed molecular analysis of transgenic plants are available, but often the exact location of the transgene within the crop genome is poorly understood. As a starting point to understanding more about the site of transgene insertion, transgenes can be physically located using fluorescence in situ hybridization (FISH). This technique allows transgenes to be located to specific chromosome regions following the hybridization of a fluorescent labelled probe to a chromosome spread. The technique is sensitive enough to detect single transgene copies and can reveal information about the complexity of a transgene insertion site as well as identifying plants homozygous for the transgene. A FISH method is described that has been used successfully to detect single-transgene copies in mitotic metaphase chromosome preparations of wheat and barley.

Chromosome Mapping↗

PubMedDate not supplied

Reverse engineering techniques applied to a human skull, for CAD 3D reconstruction and physical replication by rapid prototyping.

The production of a copy of an existing object of complex shape is one of the typical applications of the integration between two modern computer-based technologies, reverse engineering (RE) and rapid prototyping (RP). The method is extremely versatile and can be used in various applicative domains (e.g. replacement of anatomical parts with artificial prostheses, replication of skeletal remains). Two different acquisition techniques of images of a skull, by laser and by CT scan, were compared to ascertain which enabled more accurate reproduction of the original specimen. The skull was chosen due to it being the body part most often used in medico-legal investigations (for personal identification, skull-photo superimposition techniques, forensic art, etc). Comparison between the copy and the original yielded satisfactory results for both techniques. However, CT scanning demonstrated some advantages over the laser technique, as it provided a cleaner point cloud, enabling shorter pre-reproduction processing times, as well as data on the internal parts, which resulted in the reproduction of a more faithful copy.

Biomedical Engineering↗

PubMed2001

Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense-target RNA complex.

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. These RNAs are characterized by a long hairpin structure interrupted by several unpaired residues or bulged loops. In plasmid R1, the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by a four-way junction structure and a side-by-side helical alignment. This topology facilitates the formation of a stabilizer intermolecular helix between distal regions of both RNAs, essential for in vivo control. The bulged residues in CopA/CopT were shown to be required for high in vitro binding rate and in vivo activity. This study addresses the question of why removal of bulged nucleotides blocks stable complex formation. Structure mapping, modification interference, and molecular modeling of bulged-less mutant CopA-CopT complexes suggests that, subsequent to loop-loop contact, helix propagation is prevented. Instead, a fully base paired loop-loop interaction is formed, inducing a continuous stacking of three helices. Consequently, the stabilizer helix cannot be formed, and stable complex formation is blocked. In contrast to the four-way junction topology, the loop-loop interaction alone failed to prevent ribosome binding at its loading site and, thus, inhibition of RepA translation was alleviated.

Base Pairing↗

PubMed1991

Isolation of virus-like (VL30) elements from the Q10 and D regions of the major histocompatibility complex.

Previous studies from our laboratory have described two endogenous provirus-like sequences in a series of cosmids spanning the TL region of the major histocompatibility complex (MHC) of normal C57BL/10 mice. At least one of these viruses shares similarities with VL30 elements. To determine if additional VL30-like retroviral elements are integrated in the MHC, we constructed a cosmid library using DNA from a radiation leukemia virus (RadLV)-transformed cell line derived from C57BL/6 mice. The library was first screened using the H-2III (5') probe, which detects Class I genes of the H-2 complex. In the primary screening 163 H-2III positives were isolated. The H-2III-positive isolates were then hybridized with an AKR-derived virus probe, EcoB/S, which contains sequences from both the pol and the env genes of the virus. Nine virus-positive isolates were detected. Localization of these cosmid isolates containing viral sequences within the H-2 complex was done utilizing low-copy probes and confirmed using previously mapped cosmid isolates from other laboratories. We report here the isolation and characterization of VL30-like elements from the Qa and D regions of the MHC of several inbred mouse strains.

AKR murine leukemia virus↗

PubMed1988

Subunit stoichiometry of the chloroplast photosystem I complex.

A native photosystem I (PS I) complex and a PS I core complex depleted of antenna subunits has been isolated from the uniformly 14C-labeled aquatic higher plant, Lemna. These complexes have been analyzed for their subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results for both preparations indicate that one copy of each high molecular mass subunit is present per PS I complex and that a single copy of most low molecular mass subunits is also present. These results suggest that iron-sulfur center X, an early PS I electron acceptor proposed to bind to the high molecular mass subunits, contains a single [4Fe-4S] cluster which is bound to a dimeric structure of high molecular mass subunits, each providing 2 cysteine residues to coordinate this cluster.

Carbon Radioisotopes↗

PubMed1996

Molecular interplay of murein synthases and murein hydrolases in Escherichia coli.

Affinity chromatography using different lytic transglycosylases as a specific ligand revealed an interaction of both murein hydrolases and murein synthases. This interaction is taken as evidence for the assemblage into a multienzyme complex that could function as a murein replicase precisely copying the given three-dimensional structure of the murein sacculus. The sacculus of the mother cell would function as a template, which is identically replicated by copying the lengths of the existing glycan strands and the pattern of crosslinkages. A hypothetical enzyme complex specifically involved in cell division and a complex specifically involved in cell elongation are presented. It is postulated that PBPs 1a and/or 1b are present in both complexes, whereas the presence of PBP2 or PBP3 defines the specificity of the murein-synthesizing machinery as being involved in either cell elongation or septation. Moreover, the proposed "holoenzyme" suprastructure could explain why the specific inhibition of PBPs 1a/1b results in bacteriolysis and why inhibition of PBP2 and PBP3 causes the well-known morphological alterations, spherical growth, and filamentation, respectively.

Escherichia coli↗

PubMed1996

Solubilization of a complex of tryptic fragments of Na,K-ATPase containing occluded Rb ions and bound ouabain.

The nonionic detergent C12E10 (polyoxyethylene 10-laurylether) has been used to solubilize a complex of tryptic fragments of Na, K-ATPase containing occluded Rb ions and bound ouabain. The aim was to define which fragments are required to maintain Rb occlusion. The experiments utilize "19 kDa membranes" consisting of a 19 kDa and several smaller tryptic fragments (8-11.7 kDa) of the alpha subunit, which include trans-membrane segments M7/M10 and the pairs M1/M2, M3/M4, and M5/M6 [Capasso, J. M., et al (1992) J. Biol. Chem. 267, 1150-1158]. The beta subunit is partially split into a 16 kDa fragment and a glycosylated approximately 50 kDa fragment. Cation occlusion and ouabain binding are intact. After preincubation of "19 kDa membranes" with Rb (5 mM) and then ouabain (10 mM), 90-100% of occluded Rb was solubilized by C12E10 at 0 degrees C. All fragments of the alpha and beta subunits, and also the gamma subunit, were cosolubilized by C12E10, and were observed to sediment together on a sucrose density gradient as a complex containing occluded Rb ions. The soluble complex consists of a monomer containing one copy of each fragment, as indicated by size-exclusion HPLC, as well as estimates of specific Rb occlusion (20.0 +/- 1.2 nmol/mg of protein). In the absence of Rb ions and ouabain, the complex was unstable. Whereas the 19 kDa fragment (M7-M10) and beta subunit remained associated, the smaller fragments, containing M5/M6 and M3/M4 and M1/M2, and the subunit dissociated. Observations on the thermal inactivation of Rb occlusion, and effect of pH and ionic strength, suggest that the soluble complex is stabilized by multiple interactions, both within the lipid bilayer and in hydrophilic domains (e.g., salt bridges).

Amino Acid Sequence↗

PubMed1996

Yeast SUB1 is a suppressor of TFIIB mutations and has homology to the human co-activator PC4.

Activation of transcription in eukaryotes depends upon the interplay between transcriptional activators and general transcription factors. While direct contacts between activators and general factors have been demonstrated in vitro, an additional class of proteins, termed co-activators, is also required of transcriptional activation. Here we describe a yeast protein, SUB1, that was isolated as a suppressor of the cold-sensitive TFIIB R78H mutant. The N-terminal third of SUB1 is highly similar to the mammalian co-activator PC4. We show that increased expression of SUB1 suppresses two alleles of TFIIB (E62G, R78H) specifically and that the deletion of SUB1 is lethal in combination with these same two alleles. We show that SUB1 binds to TFIIB in vitro and that it specifically inhibits the formation of TBP-TFIIB-promoter complexes. Furthermore we show that increasing the copy number of SUB1 stimulates transcriptional activation in vivo. Based on our results and recent observations of others, we propose that SUB1 plays a role in the release of TFIIB from the transcription complex during transcription initiation.

Alleles↗

PubMed2003

MYCN gain and MYCN amplification in a stage 4S neuroblastoma.

Stage 4S neuroblastoma is a disease associated with spontaneous regression and good survival. We present a patient whose evolution has shown the variety and complexity of this disease in infants. Biologic factors, such as ploidy, MYCN copy number, loss of 1p36, and other chromosomal gains and losses were determined. A complex pattern of genetic abnormalities, such as near-diploidy, MYCN gain (2-4 copies per haploid genome) and imbalance/deletion of 1p36 was seen in the diagnostic sample. An extensive disseminated disease after a latent period of 26 months was associated with a special genetic evolution, such a tetraploidy, MYCN amplification (2:100-500 copies), 1p36 deletion, and gain of 17q. Our results provide evidence that either the primary tumor was heterogeneous in terms of gene amplification or that amplification was acquired later on as a transition from MYCN gain. We suggest that near-di-/tetraploid 4S tumors with MYCN gain and/or deletion 1p could be progressing 4S tumors.

Adrenal Gland Neoplasms↗

PubMed2000

rRNA operon copy number reflects ecological strategies of bacteria.

Although natural selection appears to favor the elimination of gene redundancy in prokaryotes, multiple copies of each rRNA-encoding gene are common on bacterial chromosomes. Despite this conspicuous deviation from single-copy genes, no phenotype has been consistently associated with rRNA gene copy number. We found that the number of rRNA genes correlates with the rate at which phylogenetically diverse bacteria respond to resource availability. Soil bacteria that formed colonies rapidly upon exposure to a nutritionally complex medium contained an average of 5.5 copies of the small subunit rRNA gene, whereas bacteria that responded slowly contained an average of 1.4 copies. In soil microcosms pulsed with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), indigenous populations of 2,4-D-degrading bacteria with multiple rRNA genes ( = 5.4) became dominant, whereas populations with fewer rRNA genes ( = 2.7) were favored in unamended controls. These findings demonstrate phenotypic effects associated with rRNA gene copy number that are indicative of ecological strategies influencing the structure of natural microbial communities.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1974

An intermediate complex in the dissociation of aspartate transcarbamylase.

The multisubunit enzyme aspartate transcarbamylase consists of six copies of two types of polypeptide chains, catalytic (C) and regulatory (R). A complex formed by the partial dissociation of this enzyme has been isolated. This species, which has the structure C(6)R(4), is a likely intermediate in the stepwise dissociation of aspartate transcarbamylase induced by mercurials. The formation of the complex is the result of the release of a single regulatory dimer (R(2)) from the parent molecule.The specific activity of the intermediate is essentially the same as that of aspartate transcarbamylase. By contrast, both homotropic and heterotropic interactions are reduced, but not abolished. These observations suggest that the allosteric transitions involved in the control mechanisms do not require the intact structure C(6)R(6).

Allosteric Regulation↗

PubMed1993

Isolation and characterization of membrane-associated form of penicillase plasmid (pI524) DNA in Staphylococcus aureus.

Our studies on the association of penicillinase plasmid (pI524) DNA with its host bacterial (Staphylococcus aureus) membrane revealed that the membrane-associated forms of this plasmid could be isolated from exponentially grown cells lysed on neutral sucrose gradient. Analysis of putative plasmid-membrane complexes isolated from the clear lysates on sucrose gradients indicated that approximately 23% of plasmid (pI524) DNA was stably associated with the bacterial cell membrane fractions. This suggested that one of the three or four copies of this plasmid per cell was complexed to the cellular membrane. Examination of the effect of various enzymes, e.g., ribonuclease and protease, as well as antibiotics (rifampicin and chloramphenicol), on complexing have shown the possible involvement of protein(s) rather than RNA in mediating the complexing of this plasmid to the cell membrane. The specificity of plasmid pI524 to its host cell membrane was observed in an experiment where R6k was included in binding assay.

Binding Sites↗

PubMed1982

Molecular analysis of argininosuccinate synthetase deficiency in human fibroblasts.

We have analyzed cultured skin fibroblasts derived from patients with argininosuccinate synthetase deficiency for alterations in gene structure, mRNA content, and protein structure. Genomic DNA was digested with the endonucleases EcoRI or HindIII, and the fragments were analyzed by Southern blotting and hybridization with a cDNA probe for argininosuccinate synthetase. The blot pattern is complex because there are at least 10 copies of argininosuccinate synthetase-like genes scattered over multiple human chromosomes. All nine patients studied showed patterns of DNA fragments that were indistinguishable from the normal control cell lines, and despite the possibility that the complexity could mask some changes, major deletions of the active gene(s) were not present. Blot hybridization of RNA indicated the presence of hybridizable mRNA of approximately normal size in seven of seven individuals examined with a suggestion of some heterogeneity. Analysis of enzyme antigen by protein transfer from NaDodSO4 containing polyacrylamide gels revealed considerable heterogeneity. This analysis revealed no cross-reacting material (CRM) in nine cell lines, CRM of normal molecular weight in one cell line, and CRM of reduced molecular weight in one cell line. These findings suggest that the genes for argininosuccinate synthetase in most citrullinemia patients are transcribed and produce stable mRNA. These mRNA either are not translated, or the translation product (enzyme) is rapidly degraded or is immunologically nonreactive. Defective gene expression in this disorder appears to involve abnormal mRNA, which may be altered by point mutations, frame shift mutations, deletions, insertions or particularly by abnormal RNA processing.

Argininosuccinate Synthase↗

PubMed1988

Topography and subunit stoichiometry of the coated vesicle proton pump.

We have previously shown that the coated vesicle (H+)-ATPase contains nine polypeptides of molecular weight 17,000-100,000 which form a single, macromolecular complex that can be immunoprecipitated using monoclonal antibodies which recognize the native enzyme (Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M. (1987) Biochemistry 26, 6632-6638). In the present paper, we have calculated from quantitative amino acid analysis that these polypeptides are present in the native complex in a stoichiometry of three copies each of the 73,000- and 58,000-dalton subunits, six copies of the 17,000-dalton subunit, and one copy each of the 100,000-, 40,000-, 38,000-, 34,000-, 33,000-, and 19,000-dalton subunits. To determine the disposition of the (H+)-ATPase subunits with respect to the membrane, we have carried out labeling studies using the membrane impermeant reagents Na125I/lactoperoxidase and 125I-sulfo-succinimidyl-3-(4-hydroxyphenyl)propionate and the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID). Polypeptides exposed to the cytoplasmic surface were identified by labeling with impermeant reagents in intact vesicles from which clathrin had been dissociated followed by immunoprecipitation of the native enzyme. Polypeptides exposed to the luminal surface were identified by increased labeling by these reagents following detergent solubilization under nondenaturing conditions. Labeling by [125I]TID was used to indicate which polypeptides are embedded in the lipid bilayer. Results of these experiments indicate that the principal polypeptides labeled from the cytoplasmic surface are those of molecular weight 73,000 and 58,000, although some cytoplasmic labeling of the 100,000, 40,000, 38,000 and 34,000/33,000 polypeptides was also observed. The polypeptides which show the greatest increase in labeling following detergent solubilization are those of molecular weight 100,000, 19,000, and 17,000, with some increase observed for the 40,000, 38,000, and 34,000/33,000 polypeptides. [125I]TID labeled the 17,000-dalton subunit most heavily, with significant labeling of the 100,000- and 40,000-dalton subunits also observed. In addition, we find that the 73,000-dalton polypeptide can be dissociated from the complex with 0.5 M KI in the absence of detergent, indicating a peripheral association of this subunit with the membrane. We have combined these results to construct a structural model of the coated vesicle (H+)-ATPase.

Amino Acids↗