Statistical analysis of vertebrate sequences reveals that long genes are scarce in GC-rich isochores

We compared the exon/intron organization of vertebrate genes belonging to different isochore classes, as predicted by their GC content at third codon position. Two main features have emerged from the analysis of sequences published in GenBank: (1) genes coding for long proteins (i.e., 500 aa) are almost two times more frequent in GC-poor than in GC-rich isochores; (2) intervening sequences (=sum of introns) are on average three times longer in GC-poor than in GC-rich isochores. These patterns are observed among human, mouse, rat, cow, and even chicken genes and are therefore likely to be common to all warm-blooded vertebrates. Analysis of Xenopus sequences suggests that the same patterns exist in cold-blooded vertebrates. It could be argued that such results do not reflect the reality because sequence databases are not representative of entire genomes. However, analysis of biases in GenBank revealed that the observed discrepancies between GC-rich and GC-poor isochores are not artifactual, and are probably largely underestimated. We investigated the distribution of microsatellites and interspersed repeats in introns of human and mouse genes from different isochores. This analysis confirmed previous studies showing that Ll repeats are almost absent from GC-rich isochores. Microsatellites and SINES (Alu, B1, B2) are found at roughly equal frequencies in introns from all isochore classes. Globally, the presence of repeated sequences does not account for the increased intron length in GC-poor isochores. The relationships between gene structure and global genome organization and evolution are discussed.     

On-line immunoanalysis of monoclonal antibodies during a continuous culture of hybridoma cells

The monoclonal-antibody production of an immobilized hybridoma cell line cultivated in a fluidized-bed reactor was monitored on-line for nearly 900 h. The monoclonal antibody concentration was determined by an immuno affinity-chromatography method (ABICAP). Antibodies directed against the product, e.g. IgG, were immobilized on a micro-porous gel and packed in small columns. After all IgG present in the sample was bound to the immobilized antibodies, unbound proteins were removed by rinsing the column. Elution of the bound antibodies followed and the antibodies were determined by fluorescence. The analytical procedure was automated with a robotic device to enable on-line measurements. The correlation between the on-line determined data and antibody concentrations measured by HPLC was linear. A sampling system was constructed, which was based on a pneumatically actuated in-line membrane valve integrated into the circulation loop of the reactor. Separation of the cells from the sample stream was achieved by a depth filter made of glass-fibre, situated outside the reactor. Rapid obstruction of the filter by cells or cell debris and contamination of the sample system was avoided by intermittent rinsing of the sample system with a chemical solution. The intermittent rinsing of the filter, which had a surface of 4.8 cm², resulted in an operational capacity of up to 40 samples (1.0 l total sample volume). Both the sampling system and the analytical device functioned without failure during this long-term culture. The culture temperature was varied between 34 and 40 °C. Raising the temperature from 34 up to 37 °C resulted in a simultaneous increase of growth and specific antibody production rate. Specific metabolic rates of glucose, lactate, glutamine and ammonium stayed constant in this temperature range. A further enhancement of temperature up to 40 °C had a negative effect on the growth rate, whereas the specific monoclonal antibody production rate showed a small increase. The other specific metabolic rates also increased in the temperature range between 38 to 40 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Theoretical Study of the TiO2 and MgO Surface Acidity and the Adsorption of Acids and Bases

We present ab-initio periodic Hartree–Fock calculations (crystal program) of small molecules on TiO₂ and MgO. The adsorption of the molecules may be molecular or dissociative. This depends on their acid and basic properties in the gas phase. For the molecular adsorption, the molecules are adsorbed as bases on Ti(+IV) sites, the adsorption energies correlate with the proton affinities. The dissociations on the surface correlate with the gas phase cleavages: thus, the dissociation of MeOH leads to a preferential basic cleavage (the fragment HO– is adsorbed on a Ti⁺⁴ ion and the fragment Me⁺ is adsorbed on a O²– ion of the oxide). The opposite result is obtained with MeSH. Another important factor is the adsorbate–adsorbate interaction: favorable cases are a sequence of H-bonds for the hydroxyl groups resulting from the water dissociation and the mode of adsorption for the ammonium ions. Lateral interactions also force the adsorbed CO₂ molecules to bend over the surface so that their mutual orientation resembles the geometry of the CO₂ dimer. With respect to water adsorption, MgO appears to be a basic oxide. As experimentally observed, NH₃ adsorbs preferentially on TiO₂ and CO₂ on MgO. However, this difference of reactivity should not be expressed in terms of acid vs. basic behaviour but in terms of hard and soft acidity. The MgO surface is a 'soft' acidic surface that reacts preferentially with the soft base, CO₂.     

Engineering of zinc finger and MHC motifs to locked-in tertiary folds

The assembly of helical and -sheet peptide blocks containing reactive chain ends results inhighly branched chain architectures (locked-in folds) mimicking native tertiary structures.This molecular kit strategy allows to bypass the protein folding problem in protein de novodesign and gives access to protein mimetics of high thermodynamic stability. The validity ofthis concept is exemplified for the design and synthesis of locked-in folds mimicking the zincfinger and MHC folding motifs.     

Post-translational modification of proteins and the discovery of new medicine

Post-translational modifications are fundamental to processes controlling behaviour, including cellular signaling, growth and transformation. As the molecular basis of protein modifications in normal and disease processes are becoming better defined, so new strategies for designing therapeutic entities to control complex disease processes are emerging.     

The oncofetal J28 epitope involves fucosylated O-linked oligosaccharide structures of the fetoacinar pancreatic protein

The fetoacinar pancreatic protein (FAP), characterized by themAb J28, is an oncofetal form of bile salt dependent lipase(BSDL), the expression of which is related to pancreatic differentiationand neoplastic processes. Because the J28 epitope, recognizedby imAb J28, is suggested to be dependent upon carbohydrates,we have attempted to gain information about the structure ofthis epitope. Indeed, treatment of FAP with sodium periodateabolished the reactivity of the protein to mAb J28, which demonstratesthe implication of oligosaccharides in the structure of theJ28 epitope. FAP offers both O-linked and N-linked carbohydratestructures, of which, as we have determined, one is involved.Peptides obtained after cyanogen bromide cleavage were desialylatedthen separated by affinity chromatography on an immobilizedpeanut agglutinin agarose column. The peptide retained on thiscolumn carried out the reactivity with the mAb J28. Althoughsome differences in amino acid analysis were observed, the N-terminalsequence of this peptide correlates with that of the C-terminalpart of the enzyme. Carbohydrate analysis of the peptide bearingthe J28 epitope revealed fucose, galactose, N-acetylgalactosamine,N-acetylglucosamine, and N-acetylneuraminic acid. The competitionobserved between mAb J28 and Ulex europaeus I lectin for bindingto the J28 epitope suggested that fucose residue a (1–2)linked to a galactose residue was implicated in the structureof the J28 epitope. Alternatively, the loss of the mAb J28 reactivityupon treatment of FAP either with bovine kidney or bovine epididymisfucosidase was observed indicating that fucose residues linkedat the     

ZmEsr, a novel endosperm-specific gene expressed in a restricted region around the maize embryo

A novel endosperm-specific gene named Esr (e mbryo s urrounding r egion) has been isolated by differential display between early developmental stages of maize endosperms and embryos. It is expressed in a restricted region of the endosperm, surrounding the entire embryo at early stages (4 to 7 days after pollination, DAP) and ever-decreasing parts of the suspensor at subsequent stages. The expression starts at 4 DAP and is maintained until at least 28 DAP. A minimum of three Esr genes are present in the maize genome and at least two of them map to the short arm of chromosome 1 at position 56. The Esr genes contain no introns and show no significant nucleotide or amino acid sequence homologies to sequences in the databases. The open reading frames encode basic proteins of 14 kDa with presumptive signal peptides at their N-termini followed by a hypervariable and a conserved region. The gene product may play a role in the nutrition of the developing embryo or in the establishment of a physical barrier between embryo and endosperm.     

Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.