[Educational program of type 2 diabetes].
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Body Mass Index↗
Biomedical subjects
S Schaper
Publications and source records attributed to S Schaper.
A yeast homolog of chromatin assembly factor 1 is involved in early ribosome assembly.
Cells have a recurrent need for the correct assembly of protein-nucleic acid complexes. We have studied a yeast homolog of the smallest subunit of chromatin assembly factor 1 (CAF1), encoded by YMR131c and termed "RRB1". Unlike other yeast homologs, Msi1p, and Hat2p, Rrb1p is essential for cell viability. Impairment of Rrb1p function results in decreased levels of free 60S ribosomal subunits and the appearance of half-mer polysomes, suggesting its involvement in ribosome biogenesis. Using tandem affinity purification (TAP ) combined with mass spectrometry, we show that Rrb1p is associated with ribosomal protein L3. A fraction of Rrb1p is also found in a protein-precursor rRNA complex containing at least ten other early-assembling ribosomal proteins. We propose that Rrb1p is required for proper assembly of preribosomal particles during early ribosome biogenesis, presumably by targeting L3 onto the 35S precursor rRNA. This action may resemble the mechanism by which CAF1 assembles histones H3/H4 onto newly replicated DNA.
Base Sequence↗
Bacterial replication initiator DnaA. Rules for DnaA binding and roles of DnaA in origin unwinding and helicase loading.
We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.
Bacterial Proteins↗
Identification of the chromosomal replication origin from Thermus thermophilus and its interaction with the replication initiator DnaA.
The chromosomal replication origin oriC and the gene encoding the replication initiator protein DnaA from Thermus thermophilus have been identified and cloned into an Escherichia coli vector system. The replication origin is composed of 13 characteristically arranged DnaA boxes, binding sites for the DnaA protein, and an AT-rich stretch, followed by the dnaN gene. The dnaA gene is located upstream of the origin and expresses a typical DnaA protein that follows the division into four domains, as with other members of the DnaA protein family. Here, we report the purification of Thermus-DnaA (Tth-DnaA) and characterize the interaction of the purified protein with the replication origin, with regard to the binding kinetics and stoichiometry of this interaction. Using gel retardation assays, surface plasmon resonance (SPR) and electron microscopy, we show that, unlike the E. coli DnaA, Tth-DnaA does not recognize a single DnaA box, instead a cluster of three tandemly repeated DnaA boxes is the minimal requirement for specific binding. The highest binding affinities are observed with full-length oriC or six clustered, tandemly repeated DnaA boxes. Furthermore, high-affinity DNA-binding of Tth-DnaA is dependent on the presence of ATP. The Thermus DnaA/oriC interaction will be compared with oriC complex formation generated by other DnaA proteins.
Adenosine Diphosphate↗
Evaluation of Costa Rican copepods (Crustacea: Eudecapoda) for larval Aedes aegypti control with special reference to Mesocyclops thermocyclopoides.
This study attempted to find organisms for the biological control of the mosquito Aedes aegypti in Costa Rica. Copepods of the genera Arctodiaptomus, Eucylops, Mesocyclops, Megacyclops, and Thermocyclops were collected in several parts of the country and cultured for laboratory evaluations. Mesocyclops thermocyclopoides was the most successful species in reducing the number of larval Ae. aegypti (7.3 larvae in 24 h at a density of 200 Aedes/liter). Arctodiaptomus dorsalis, Eucyclops cf. bondi, Eucyclops leptacanthus, Megacyclops sp., and Thermocyclops decipens were not effective predators. In cage simulation trials, M. thermocyclopoides showed 100% larval reduction after 4 wk and adult mosquitoes disappeared after 7 wk. The copepod was able to survive in Aechmea sp. bromeliads under laboratory conditions. In field trials under 3 different climatic conditions M. thermocyclopoides survived 2-5 months in bromeliad leaf axils and 3-6 months in used car tires. In tires, this species reduced the number of larval Ae. aegypti 79, 90, and 99% in tropical dry, moderate, and humid climates, respectively. An El Niño phenomenon affected the results by drought, which apparently also caused a decline in the population of the predatory mosquito Toxorhynchites haemorrhoidalis superbus. Considering these severe test conditions, M. thermocyclopoides might be a promising predator for mosquito control in Costa Rica.
Aedes↗
Crystallization and preliminary X-ray crystallographic studies of the 13-fold symmetric portal protein of bacteriophage SPP1.
Portal proteins are cyclical oligomers which play essential roles in bacteriophage pro-capsid formation, DNA packaging, and in connector formation. Bacteriophage SPP1 portal protein (gp6) is a turbine-like molecule with 13-fold symmetry [Dube et al. (1993) EMBO J. 12, 1303-1309]. The purified protein was crystallized with polyethylene glycol 400 as the precipitating agent using the vapor-diffusion method. Salt conditions were selected based on the properties of gp6 in different ionic environments. X-ray diffraction data up to a resolution of 7.85 A were measured from frozen crystals with orthorhombic space group C2221 and cell dimensions a = 180.5 (5), b = 223.5 (5), c = 417 (1) A. The asymmetric unit contains one tridecameric portal protein with 57.3 kDa subunits. The self-rotation searches confirm the 13-fold symmetry of the crystallized protein.
Capsid↗
Prediction of the structure of the replication initiator protein DnaA.
The secondary structure of DnaA protein and its interaction with DNA and ribonucleotides has been predicted using biochemical, biophysical techniques, and prediction methods based on multiple-sequence alignment and neural networks. The core of all proteins from the DnaA family consists of an "open twisted alpha/beta structure," containing five alpha-helices alternating with five beta-strands. In our proposed structural model the interior of the core is formed by a parallel beta-sheet, whereas the alpha-helices are arranged on the surface of the core. The ATP-binding motif is located within the core, in a loop region following the first beta-strand. The N-terminal domain (80 aa) is composed of two alpha-helices, the first of which contains a potential leucine zipper motif for mediating protein-protein interaction, followed by a beta-strand and an additional alpha-helix. The N-terminal domain and the alpha/beta core region of DnaA are connected by a variable loop (45-70 aa); major parts of the loop region can be deleted without loss of protein activity. The C-terminal DNA-binding domain (94 aa) is mostly alpha-helical and contains a potential helix-loop-helix motif. DnaA protein does not dimerize in solution; instead, the two longest C-terminal alpha-helices could interact with each other, forming an internal "coiled coil" and exposing highly basic residues of a small loop region on the surface, probably responsible for DNA backbone contacts.
Amino Acid Sequence↗
Interaction of the initiator protein DnaA of Escherichia coli with its DNA target.
Equilibrium and kinetic rate constants were determined for the binding of the initiator protein DnaA of Escherichia coli to its binding site, the non-palindromic 9-bp DnaA box, using gel retardation techniques. The dissociation constant for specific binding was between 1 and 50 nM for individual DnaA boxes on 21-bp double-stranded oligonucleotides. Only DnaA boxes of the sequence TT(A/T)TNCACA resulted in specific fragment retention. Both the 9-bp consensus sequence and flanking sequences determined the binding efficiency. One DnaA monomer was found to bind to a DnaA box and to induce a bend of about 40 degrees.
Bacterial Proteins↗
The complex for replication initiation of Escherichia coli.
We probed the complex between oriC and DnaA protein using two types of mutants in oriC. Base changes in the DnaA binding sites, DnaA boxes, had little effect on origin function. Mutations which change the distance between DnaA boxes R3 and R4, on the other hand, inactivated oriC unless the mutation deleted or inserted one complete helical turn. Origins with other 10 base pair insertions in the interval between DnaA boxes R2 and R3 were functional, but not insertions in the R1-R2 interval. FIS protein binds to a bipartite site in oriC between DnaA boxes R2 and R3. A model for the oriC/DnaA complex based on these results suggests an array of DnaA monomers with a 34 A spacing upon which oriC is arranged.
Bacterial Proteins↗
Isolation and some properties of a cathepsin E type proteinase from rat spleen.
Cathepsin E (EC 3.4.23.--) has been isolated from rat spleen. The procedure included autolysis at pH 4.2 which was probably the reason why we isolated a polypeptide of Mr 42 kDa instead of 90 kDa. The latter is reported in the literature to be the Mr of native cathepsin E. The enzyme dissociates under reducing conditions in two identical monomers. In our preparation a mechanism different from reduction must be active producing the 42 kDa polypeptide. This enzyme was hard to distinguish from cathepsin D (EC 3.4.23.5.) which shows similar properties such as size, substrate specificity, stability in 6 M urea, and dependence of the activity on pH. The clear distinction between the two enzymes was proven on the basis of immunochemical reactions. Antibodies to both cathepsins, D and E, did not show any crossreaction with the nonrelated antigen.
Animals↗
The azocasein-urea-pepstatin assay discriminates between lysosomal proteinases.
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Animals↗
Protein degradation in rat liver cells.
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Animals↗
Functional domains of DnaA proteins.
Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.
Amino Acid Sequence↗