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PubMed2001

Designing a polyvalent inhibitor of anthrax toxin.

Screening peptide libraries is a proven strategy for identifying inhibitors of protein-ligand interactions. Compounds identified in these screens often bind to their targets with low affinities. When the target protein is present at a high density on the surface of cells or other biological surfaces, it is sometimes possible to increase the biological activity of a weakly binding ligand by presenting multiple copies of it on the same molecule. We isolated a peptide from a phage display library that binds weakly to the heptameric cell-binding subunit of anthrax toxin and prevents the interaction between cell-binding and enzymatic moieties. A molecule consisting of multiple copies of this nonnatural peptide, covalently linked to a flexible backbone, prevented assembly of the toxin complex in vitro and blocked toxin action in an animal model. This result demonstrates that protein-protein interactions can be inhibited by a synthetic, polymeric, polyvalent inhibitor in vivo.

Acrylic Resins↗

PubMed2006

Buffering of crucial functions by paleologous duplicated genes may contribute cyclicality to angiosperm genome duplication.

Genome duplication followed by massive gene loss has permanently shaped the genomes of many higher eukaryotes, particularly angiosperms. It has long been believed that a primary advantage of genome duplication is the opportunity for the evolution of genes with new functions by modification of duplicated genes. If so, then patterns of genetic diversity among strains within taxa might reveal footprints of selection that are consistent with this advantage. Contrary to classical predictions that duplicated genes may be relatively free to acquire unique functionality, we find among both Arabidopsis ecotypes and Oryza subspecies that SNPs encode less radical amino acid changes in genes for which there exists a duplicated copy at a "paleologous" locus than in "singleton" genes. Preferential retention of duplicated genes encoding long complex proteins and their unexpectedly slow divergence (perhaps because of homogenization) suggest that a primary advantage of retaining duplicated paleologs may be the buffering of crucial functions. Functional buffering and functional divergence may represent extremes in the spectrum of duplicated gene fates. Functional buffering may be especially important during "genomic turmoil" immediately after genome duplication but continues to act approximately 60 million years later, and its gradual deterioration may contribute cyclicality to genome duplication in some lineages.

Arabidopsis↗

PubMed1994

Finding a needle in a haystack: detection of a small protein (the 12-kDa VP26) in a large complex (the 200-MDa capsid of herpes simplex virus).

Macromolecular complexes that consist of homopolymeric protein frameworks with additional proteins attached at strategic sites for a variety of structural and functional purposes are widespread in subcellular biology. One such complex is the capsid of herpes simplex virus type 1 whose basic framework consists of 960 copies of the viral protein, VP5 (149 kDa), arranged in an icosahedrally symmetric shell. This shell also contains major amounts of three other proteins, including VP26 (12 kDa), a small protein that is approximately equimolar with VP5 and accounts for approximately 6% of the capsid mass. With a view to inferring the role of VP26 in capsid assembly, we have localized it by quantitative difference imaging based on three-dimensional reconstructions calculated from cryo-electron micrographs. Purified capsids from which VP26 had been removed in vitro by treatment with guanidine hydrochloride were compared with preparations of the same depleted capsids to which purified VP26 had been rebound and with native (undepleted) capsids. The resulting three-dimensional density maps indicate that six VP26 subunits are distributed symmetrically around the outer tip of each hexon protrusion on VP26-containing capsids. Because VP26 may be readily dissociated from and reattached to the capsid, it does not appear to contribute significantly to structural stabilization. Rather, its exposed location suggests that VP26 may be involved in linking the capsid to the surrounding tegument and envelope at a later stage of viral assembly.

Capsid↗

PubMed1998

In vitro system for differentiating pluripotent neural crest cells into smooth muscle cells.

The change in vascular smooth muscle cells (SMC) from a differentiated to a dedifferentiated state is the critical phenotypic response that promotes occlusive arteriosclerotic disease. Despite its importance, research into molecular mechanisms regulating smooth muscle differentiation has been hindered by the lack of an in vitro cell differentiation system. We identified culture conditions that promote efficient differentiation of Monc-1 pluripotent neural crest cells into SMC. Exclusive Monc-1 to SMC differentiation was indicated by cellular morphology and time-dependent induction of the SMC markers smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM22alpha, and APEG-1. The activity of the SM22alpha promoter was low in Monc-1 cells. Differentiation of these cells into SMC caused a 20-30-fold increase in the activity of the wild-type SM22alpha promoter and that of a hybrid promoter containing three copies of the CArG element. By gel mobility shift analysis, we identified new DNA-protein complexes in nuclear extracts prepared from differentiated Monc-1 cells. One of the new complexes contained serum response factor. This Monc-1 to SMC model should facilitate the identification of nodal regulators of smooth muscle development and differentiation.

Animals↗

PubMed2025

Evolutionary Diversification and Functions of the Candidate Male Killing Gene wmk.

Symbiont-mediated male killing (MK) is a mechanism that selectively eliminates male offspring, often by disrupting sex-specific developmental processes. In Drosophila melanogaster, the WO-mediated killing gene wmk from Wolbachia prophage WO transgenically reproduces the MK phenotype, yet how the gene evolves and functions across diverse Wolbachia has not been systematically investigated. We analyzed 32 Wolbachia genomes available in the NCBI database to study wmk homologs across different arthropod hosts, reproductive parasitism functions, and Wolbachia supergroups. First, we report at least five distinct wmk phylogenetic clusters (Types I to V), often organized in multigenic dyads or triads. Second, among MK Wolbachia, there is a significantly higher number of wmk genes and diversity in Lepidoptera strains than in Drosophila strains, which exclusively harbor wmk Types I and III. Third, there are three patterns of wmk sequence and genomic organizational changes in Drosophila MK strains that associate with different evolutionary trajectories underpinning the MK phenotype. Fourth, single and combinatory transgenic expression of Types I and III in D. melanogaster uncovers male-biased lethality associated with Type I; however, dual expression of the Types together elicits a major reduction in offspring number. Fifth, wmk genes have low expression level across D. melanogaster developmental stages relative to the cifA and cifB genes, which could explain why cytoplasmic incompatibility is expressed in this system. These findings establish a complex and phylogenetically informed genetic basis of wmk-induced lethality, highlighting the role of gene copy number and expression, wmk Types, and host background in shaping the phenotype.

Animals↗

PubMed1997

A cluster of cuticle protein genes of Drosophila melanogaster at 65A: sequence, structure and evolution.

A 36-kb genomic DNA segment of the Drosophila melanogaster genome containing 12 clustered cuticle genes has been mapped and partially sequenced. The cluster maps at 65A 5-6 on the left arm of the third chromosome, in agreement with the previously determined location of a putative cluster encompassing the genes for the third instar larval cuticle proteins LCP5, LCP6 and LCP8. This cluster is the largest cuticle gene cluster discovered to date and shows a number of surprising features that explain in part the genetic complexity of the LCP5, LCP6 and LCP8 loci. The genes encoding LCP5 and LCP8 are multiple copy genes and the presence of extensive similarity in their coding regions gives the first evidence for gene conversion in cuticle genes. In addition, five genes in the cluster are intronless. Four of these five have arisen by retroposition. The other genes in the cluster have a single intron located at an unusual location for insect cuticle genes.

Animals↗

PubMed1980

The extraction by micrococcal nuclease of glucocorticoid receptors and mouse mammary tumor virus DNA sequences is dissociated.

Glucocorticoid receptors (RG) and mammary tumor virus (MM-TV) DNA sequences were extracted by micrococcal nuclease digestion from the nuclei of C3H mouse mammary tumor cells in order to specify their relative distribution in chromatin. RG was labelled and translocated into the nuclei by incubating cells with 3H Dexamethasone (3H Dex). The purified nuclei were then treated at 2 degrees C with micrococcal nuclease. Three chromatin fractions were successively obtained: an isotonic extract (ne3H1), ahypotonic extract (ne2) and the residual pellet (P). The Dex-RG complexes were measured by the hydroxyapatite technique. The MMTV DNA sequences were titrated by molecular hybridization with an excess of MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and the MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and MMTV DNA sequences were extracted in a concentration dependent manner while only 10-15% of nucleic acids became soluble in 10% perchloric acid. The extracted 3H Dex-RG complex was found to be partly bound to soluble chromatin and partly free. The free complex displayed similar sedimentation constants (4S, 7S) and DNA binding ability to the cytosol receptor. The 3H Dex-RG complexes were 2 to 8 fold more concentrated in ne1, which is known to be enriched in active chromatin, than in ne2. Conversely, the concentration of MMTV DNA sequences per microgram DNA was the same in the three nuclear fractions. These results suggest that the Dex-RG complexes are concentrated in an active fraction of chromatin. We propose that, among the 20-30 copies of MMTV genes per haploid genome, only a small proportion are transcribed or regulated.

Animals↗

PubMed1991

Stoichiometry of receptor-Gs-adenylate cyclase interactions.

Little is known about the relative stoichiometry of guanine nucleotide-binding (G) proteins relative to the effector systems to which they link. We addressed this question for the stimulatory G protein (Gs) linked to adenylate cyclase. Forskolin stimulates the catalytic subunit of adenylate cyclase (C), but it has a higher efficacy and potency when C also interacts with the G protein Gs. Accordingly, we measured high-affinity [3H]forskolin binding to intact cells to assay alpha s-C complexes. No high-affinity specific binding occurred with unstimulated cells. The beta-adrenergic agonist isoproterenol promoted the binding of [3H]forskolin to about 3000 sites per cell, suggesting that each receptor on average activates at least several Gs molecules. Activating Gs directly with cholera toxin maximally promoted [3H]forskolin binding to a similar number of sites, suggesting that this is the maximal number of alpha s-C complexes formed per cell. We conclude that each cell likely contains only a few thousand functional copies of C, and that the availability of C (rather than Gs, which exists in more than 100,000 copies per cell) is likely to be limiting for agonist stimulation of adenylate cyclase activity.

Adenylyl Cyclases↗

PubMed1997

Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants.

Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication.

CDC28 Protein Kinase, S cerevisiae↗

PubMed2000

Detecting gene copy number fluctuations in tumor cells by microarray analysis of genomic representations.

In this work, we explore the use of representations in conjunction with DNA microarray technology to measure gene copy number changes in cancer. We demonstrate that arrays of DNA probes derived from low-complexity representations can be used to detect amplifications, deletions, and polymorphic differences when hybridized to representations of genomic DNA. The method is both reproducible and verifiable, and is applicable even to microscopic amounts of primary tumors. We also present a mathematical model for array performance that is useful for designing and understanding DNA microarray hybridization protocols. The future applications and challenges of this approach are discussed.

Breast Neoplasms↗

PubMed1988

Structure, expression, and heterogeneity of the rice seed prolamines.

By screening two rice (Oryza sativa L.) seed cDNA libraries, recombinant cDNA clones encoding the rice prolamine seed storage protein were isolated. Based on cross-hybridization and restriction enzyme map analyses, these clones can be divided into two homology classes. All clones contain a single open reading frame encoding a putative rice prolamine precursor (molecular weight = 17,200) possessing a typical 14-amino acid signal peptide. The deduced primary structures of both types of prolamine polypeptides are devoid of repetitive sequences, a feature prevalent in other cereal prolamines. Clones of these two homology classes diverge mainly by insertions/deletions of short nucleotide stretches and point mutations. An isolated genomic clone about 15.5 kilobases in length displays a highly conserved 2.5-kilobase EcoRI fragment, repeated in tandem four times, each containing the prolamine coding sequence. Close homology is exhibited by the coding segments of the genomic and cDNA sequences, although the 5' ends of the untranslated regions are widely divergent. The sequence heterogeneity displayed by these genomic and cDNA clones and large gene copy number ( approximately 80-100 copies/haploid genome) indicate that the rice prolamines are encoded by a complex multigene family.

Journal Article↗

PubMed1992

Molecular rearrangement of lactose plasmid DNA associated with high-frequency transfer and cell aggregation in Lactococcus lactis 712.

High-frequency conjugation of the lactose plasmid pLP712 is associated with a constitutive cell aggregation phenotype and is facilitated by cointegration with a sex factor. Analysis of 23 independently derived enlarged lactose plasmids revealed that the sex factor DNA present in cointegrates varied in size. This suggested that more than simple cointegration with a sex factor plasmid was involved. Further analysis led to the discovery of a chromosomally located sex factor that could excise and be lost or exist as labile plasmid DNA. Cointegration with this sex factor was shown to be promoted by transposition of a copy of ISSI present on the lactose plasmid, and models are presented to account for the complex and variable structures of the resulting enlarged lactose plasmids.

Base Sequence↗

PubMed1981

Quantitation of transcribing native simian virus 40 minichromosomes extracted from CV1 cells late in infection.

Simian virus 40 transcriptional complexes could be extracted from CV1 cells late in infection and separated from the bulk of inactive viral chromatin. Sucrose gradient sedimentation, cesium sulfate equilibrium density gradient centrifugation, and electron microscopy indicated that the viral transcriptional complexes corresponded to at least 0.5 to 1% of the viral minichromosomes extracted, about 5,000 copies per cell.

Cells, Cultured↗

PubMed1995

Identification of human telomeric repeat motifs at the genome termini of human herpesvirus 7: structural analysis and heterogeneity.

Human herpesvirus 6 (HHV-6) and HHV-7 are closely related T-lymphotropic betaherpesviruses which share a common genomic organization and are composed of a single unique component (U) that is bounded by direct repeats (DRL and DRR). In HHV-6, a sequences have been identified at each end of the DR motifs, resulting in the arrangement aDRLa-U-aDRRa. In order to determine whether determine whether HHV-7 contains similar a sequences, we have sequenced the DRL-U and U-DRR junctions of HHV-7 strain JI, together with the DRR.DRL junction from the head-to-tail concatamer that is generated during productive virus infection. In addition, we have sequenced the genomic termini of an independent isolate of HHV-7. As in HHV-6, a (GGGTTA)n motif identical to the human telomeric repeat sequence (TRS) was identified adjacent to, but not at, the genome termini of HHV-7. The left genome terminus and the U-DRR junction contained a homolog of the consensus herpesvirus packaging signal, pac-1, followed by short tandem arrays of TRSs separated by single copies of a second 6-bp repeat. This organization is similar to the arrangement found at U-DRR in HHV-6 but differs from it in that the TRS arrays are considerably shorter in HHV-7. The right genome terminus and the DRL-U junction contained a homolog of the consensus herpesvirus packaging signal, pac-2, followed by longer tandem arrays of TRSs separated by single copies of either a 6-bp or a 14-bp repeat. This arrangement is considerably more complex than the simple tandem array of TRSs that is present at the corresponding genomic location in HHV-6 and corresponds to a site of both inter- and intrastrain heterogeneity in HHV-7. The presence of TRSs in lymphotropic herpesviruses from humans (HHV-6 and HHV-7), horse (equine herpesvirus 2), and birds (Marek's disease virus) is striking and suggests that these sequences may have functional or structural significance.

Base Sequence↗

PubMed2004

Recurrent chromosomal imbalances and structurally abnormal breakpoints within complex karyotypes of malignant peripheral nerve sheath tumour and malignant triton tumour: a cytogenetic and molecular cytogenetic study.

BACKGROUND: Cytogenetic studies of malignant peripheral nerve sheath tumours (MPNSTs) and malignant triton tumours (MTTs) are rare. AIMS: To undertake cytogenetic analysis of these tumours. METHODS: Conventional cytogenetic analysis of 21 MPNSTs and MTTs from 17 patients (nine with peripheral neurofibromatosis (NF1)) was carried out using standard culture and harvesting procedures. For a more precise identification of composite structural rearrangements and marker chromosomes, spectral karyotypic analysis (SKY) was applied to a subset of cases. In addition, EGFR gene copy number was assessed by fluorescence in situ hybridisation (FISH) analysis in a subset of cases. RESULTS: Cytogenetic analysis revealed predominantly complex karyotypes. SKY analysis was useful in further defining many structural anomalies. Structural aberrations most frequently involved chromosomal bands or regions 1p31-36, 4q28-35, 7p22, 11q22-23, 19q13, 20q13, and 22q11-13. Overall, loss of chromosomal material was much more common than gain. Loss of chromosomes or chromosomal regions 1p36 (48%), 3p21-pter (52%), 9p23-pter (57%), 10 (48%), 11q23-qter (48%), 16/16q24 (62%), 17(43%), and 22/22q (48%), and gains of 7/7q (29%) and 8/8q (29%) were most prominent. These gains and losses were distributed equally between MPNST and MTT, demonstrating that these entities are similar with respect to recurrent genomic imbalances. Similarly, none of the recurrent chromosomal breakpoints or imbalances was restricted to either NF1 associated or sporadic MPNSTs. FISH analysis was negative for amplification. CONCLUSIONS: These cytogenetic and molecular cytogenetic findings expand the knowledge of chromosomal alterations in MPNST and MTT, and point to possible recurring regions of interest.

Adolescent↗

PubMed2005

Applications of DNA microarrays in biology.

DNA microarrays have enabled biology researchers to conduct large-scale quantitative experiments. This capacity has produced qualitative changes in the breadth of hypotheses that can be explored. In what has become the dominant mode of use, changes in the transcription rate of nearly all the genes in a genome, taking place in a particular tissue or cell type, can be measured in disease states, during development, and in response to intentional experimental perturbations, such as gene disruptions and drug treatments. The response patterns have helped illuminate mechanisms of disease and identify disease subphenotypes, predict disease progression, assign function to previously unannotated genes, group genes into functional pathways, and predict activities of new compounds. Directed at the genome sequence itself, microarrays have been used to identify novel genes, binding sites of transcription factors, changes in DNA copy number, and variations from a baseline sequence, such as in emerging strains of pathogens or complex mutations in disease-causing human genes. They also serve as a general demultiplexing tool to sort spatially the sequence-tagged products of highly parallel reactions performed in solution. A brief review of microarray platform technology options, and of the process steps involved in complete experiment workflows, is included.

Animals↗

PubMed2005

Identification of molecular determinants that modulate trafficking of DeltaF508 CFTR, the mutant ABC transporter associated with cystic fibrosis.

Cystic fibrosis is a life-shortening inherited disorder associated primarily with a three-base in frame deletion that eliminates Phe508 in the ABC transporter, cystic fibrosis transmembrane conductance regulator (CFTR). Mutant CFTR, designated deltaF508 CFTR, is misprocessed and retained intracellularly. It is unclear what causes the trafficking impairment despite extensive investigative effort and the disease's prevalence. We hypothesize that the trafficking impairment is mediated by "receptors" of the cellular trafficking machinery that at three sequential "trafficking checkpoints" govern (1) exit from the endoplasmic reticulum (ER), (2) Golgi to the ER retrieval, and (3) targeting from post-Golgi compartments to lysosomes. We propose that, because of the Phe508 deletion and polypeptide misfolding: (1) a forward-directing signal recognized by the sec24 component of the COPII complex that mediates ER exit is eliminated; (2) a basic amino acid signal recognized by the COPI machinery involved in Golgi to ER retrieval becomes activated; and (3) a tyrosine-based sorting signal that targets to the lysosomes likewise becomes activated. We employed recently reported crystal structures of CFTR nucleotide binding domain 1 and sec24 in computational docking models to identify the most plausible CFTR-sec24 recognition domain. Site-directed mutagenesis and heterologous expression were also used to identify amino acid sequences that operate in Golgi to ER and post-Golgi to lysosome targeting. The importance of considering a multiple checkpoint model for trafficking is that rationale design of pharmaceutical interventions would require abrogation of all major checkpoints to deliver deltaF508 CFTR to the cell surface.

ATP-Binding Cassette Transporters↗

PubMed2005

Imaging alpha-hemolysin with molecular dynamics: ionic conductance, osmotic permeability, and the electrostatic potential map.

alpha-Hemolysin of Staphylococcus aureus is a self-assembling toxin that forms a water-filled transmembrane channel upon oligomerization in a lipid membrane. Apart from being one of the best-studied toxins of bacterial origin, alpha-hemolysin is the principal component in several biotechnological applications, including systems for controlled delivery of small solutes across lipid membranes, stochastic sensors for small solutes, and an alternative to conventional technology for DNA sequencing. Through large-scale molecular dynamics simulations, we studied the permeability of the alpha-hemolysin/lipid bilayer complex for water and ions. The studied system, composed of approximately 300,000 atoms, included one copy of the protein, a patch of a DPPC lipid bilayer, and a 1 M water solution of KCl. Monitoring the fluctuations of the pore structure revealed an asymmetric, on average, cross section of the alpha-hemolysin stem. Applying external electrostatic fields produced a transmembrane ionic current; repeating simulations at several voltage biases yielded a current/voltage curve of alpha-hemolysin and a set of electrostatic potential maps. The selectivity of alpha-hemolysin to Cl(-) was found to depend on the direction and the magnitude of the applied voltage bias. The results of our simulations are in excellent quantitative agreement with available experimental data. Analyzing trajectories of all water molecule, we computed the alpha-hemolysin's osmotic permeability for water as well as its electroosmotic effect, and characterized the permeability of its seven side channels. The side channels were found to connect seven His-144 residues surrounding the stem of the protein to the bulk solution; the protonation of these residues was observed to affect the ion conductance, suggesting the seven His-144 to comprise the pH sensor that gates conductance of the alpha-hemolysin channel.

Bacterial Toxins↗