Statistical evidence for a biochemical pathway of natural, sequence-targeted G/C to C/G transversion mutagenesis in Haemophilus influenzae Rd.

Markov chain analysis of the Haemophilus influenzae Rd genome reveals striking under-representation of three palindromic tetranucleotide strings (CCGG, GGCC and CATG), accompanied by over-representation of six tetranucleotide strings that are derived from the former by exchanging strand location of the two residues making up a G/C nucleotide pair at the terminal palindrome position. Constraints are outlined for a molecular model able to explain the phenomenon as the result of sequence-targeted, enzyme-driven G/C to C/G transversion mutagenesis. Possible participation in the process by components of known DNA mismatch repair or restriction/modification systems (in particular, cytosine methylation) is discussed. The effect widens the spectrum of enzyme-driven, specific mutagenesis beyond the formerly described C/G to T/A transition (VSP repair of Escherichia coli). Potential evolutionary benefits of enzymatic pathways of specific mutagenesis can be envisioned.

Substrate preferences of Vsr DNA mismatch endonuclease and their consequences for the evolution of the Escherichia coli K-12 genome.

The substrate spectrum of Vsr DNA mismatch endonuclease of Escherichia coli K-12 was investigated using fluorescence-labelled oligonucleotide substrates and a DNA sequencer for detection and quantification of substrates and reaction products. Fourteen substrates were found to be processed by the enzyme, which differ in one or two positions from the canonical pentanucleotide sequence CTA/TGG (T mismatched to G). Relative second-order rate constants of these substrates were determined in groups of four by multiple substrate kinetics and compared to the underresentation of the corresponding pentanucleotides in the E. coli K-12 genome. The high quality of correlation further establishes active mutagenesis by VSP repair as a significant driving force of the evolution of the E. coli K-12 genome and provides clues to its possible selective value.

Fast quantitative assay of sequence-specific endonuclease activity based on DNA sequencer technology.

A quantitative assay of the sequence-specific endonuclease activity of Vsr DNA mismatch endonuclease is described. The procedure rests on fluorescently labelled oligonucleotide substrates and an automated DNA sequencer to determine amounts of both educt and product of the reaction; thus each individual measurement is internally standardized. The assay achieves high sample throughput by parallel measurement of multiple samples. Because of its capacity to produce and process large sets of experimental data, the system is particularly well suited for the determination of reaction kinetics. The procedure lends itself to further simplification by implementing software additions for direct peak integration. Obviously, the principle of the assay can be extended to the study of other enzymes, such as restriction endonucleases or sequence-specific proteases.

Statistical evaluation and biological interpretation of non-random abundance in the E. coli K-12 genome of tetra- and pentanucleotide sequences related to VSP DNA mismatch repair.

The abundance of all tetra- and pentanucleotide sequences is calculated for a set of DNA sequence data comprising 767,393 nucleotides of the E. coli K-12 genome. Observed frequencies are compared to those expected from a Markov chain prediction algorithm. Systematic and extreme non-random representations are found for special sets of sequences. These are interpreted as arising from incorporation of a 2'-deoxyguanosine residue opposite thymidine during replication which, in special sequence contexts, leads to a T/G mismatch that is simultaneously substrate for two competing DNA mismatch repair systems: the mutHLS and the VSP pathway. Processing by the former leads to error correction, by the latter to mutation fixation. The significance of the latter process, as demonstrated here, makes it unlikely that VSP repair has evolved mainly as a mutation avoidance mechanism. It is proposed that in E. coli K-12, VSP repair, together with DNA cytosine methylation, constitutes a mutagenesis/recombination system capable of promoting gene-conversion-like unidirectional transfer of short stretches of DNA sequence.

Protein design on computers. Five new proteins: Shpilka, Grendel, Fingerclasp, Leather, and Aida.

What is the current state of the art in protein design? This question was approached in a recent two-week protein design workshop sponsored by EMBO and held at the EMBL in Heidelberg. The goals were to test available design tools and to explore new design strategies. Five novel proteins were designed: Shpilka, a sandwich of two four-stranded beta-sheets, a scaffold on which to explore variations in loop topology; Grendel, a four-helical membrane anchor, ready for fusion to water-soluble functional domains; Finger-clasp, a dimer of interdigitating beta-beta-alpha units, the simplest variant of the "handshake" structural class; Aida, an antibody binding surface intended to be specific for flavodoxin; Leather--a minimal NAD binding domain, extracted from a larger protein. Each design is available as a set of three-dimensional coordinates, the corresponding amino acid sequence and a set of analytical results. The designs are placed in the public domain for scrutiny, improvement, and possible experimental verification.

Versatile controlling system for cryopreparation techniques in electron microscopy.

A new setup for freeze-substitution and a versatile controlling system has been developed. Our goal was to build a simple system allowing precise control of the physical parameters of freeze-substitution experiments to learn more about their influences on the cellular ultrastructure and immunoreactivity of macromolecules. An improved apparatus for freeze-substitution, based on liquid nitrogen cooling, and a universal software for controlling the complex preparation protocols from cryofixation to final polymerization are described. This controlling system has the following advantages: it allows precise control and registration of temperature profiles, reconstruction of each individual step of previous experiments, and optimization of working conditions. The setup of the freeze-substitution apparatus is designed to run many different substitution media in parallel; freeze-substitution (cryostat), embedding (working platform), and polymerization are carried out at separate places; therefore, more experiments can be done simultaneously. The ergonomic working platform allows exchange of media at controlled temperature and easy handling; survey of the temperature in individual tubes is possible, and the system is protected from water condensation and uncontrolled warming by the deep freezer.

[Ultra-high dose thrombolytic therapy with streptokinase in peripheral venous thrombosis]. / Ultrahochdosierte Thrombolysetherapie mit Streptokinase bei peripherer Phlebothrombose.

56 patients with deep vein thrombosis (lower limb) and two patients with vena subclavia thrombosis were treated for six hours/day according to an ultrahigh dosage scheme (1,500,000 U/h streptokinase). An average number of three cycles was applied. Complete recanalization could be demonstrated in 23 patients (43%) with lower limb thrombosis, whereas a partial recanalization was demonstrable in 40% (22 patients). A complete thrombolysis could be achieved in one patient with vena subclavia thrombosis. Severe side effects were one intracerebral hemorrhage and one major lung embolism, causing death in both patients. Ultrahigh dosage thrombolysis with streptokinase is an effective therapeutic regimen and is considered to be an alternative to standard fibrinolytic procedures.

Quantitative analysis of cell motility and chemotaxis in Dictyostelium discoideum by using an image processing system and a novel chemotaxis chamber providing stationary chemical gradients.

An image processing system was programmed to automatically track and digitize the movement of amebae under phase-contrast microscopy. The amebae moved in a novel chemotaxis chamber designed to provide stable linear attractant gradients in a thin agarose gel. The gradients were established by pumping attractant and buffer solutions through semipermeable hollow fibers embedded in the agarose gel. Gradients were established within 30 min and shown to be stable for at least a further 90 min. By using this system it is possible to collect detailed data on the movement of large numbers of individual amebae in defined attractant gradients. We used the system to study motility and chemotaxis by a score of Dictyostelium discoideum wild-type and mutant strains, including "streamer" mutants which are generally regarded as being altered in chemotaxis. None of the mutants were altered in chemotaxis in the optimal cAMP gradient of 25 nM/mm, with a midpoint of 25 nM. The dependence of chemotaxis on cAMP concentration, gradient steepness, and temporal changes in the gradient were investigated. We also analyzed the relationship between turning behavior and the direction of travel during chemotaxis in stable gradients. The results suggest that during chemotaxis D. discoideum amebae spatially integrate information about local increases in cAMP concentration at various points on the cell surface.

Post-translational glycosylation of the contact site A protein of Dictyostelium discoideum is important for stability but not for its function in cell adhesion.

The functions of type 1 and 2 carbohydrates of the contact site A (csA) glycoprotein of Dictyostelium discoideum have been investigated using mutants lacking type 2 carbohydrate. In two mutant strains, HG220 and HG701, a 68-kd glycoprotein was synthesized as the final product of csA biosynthesis. This glycoprotein accumulated to a much lower extent on the surfaces of mutant cells than the mature 80-kd glycoprotein did in wild-type cells. There was also no accumulation of the 68-kd glycoprotein observed within the mutant cells nor was a precursor of lower molecular mass detected, in accordance with previous findings that indicated cotranslational linkage of type 1 carbohydrate by N-glycosylation. Pulse-chase labelling showed that a 50-kd glycopeptide was cleaved off from the mutant 68-kd glycoprotein and released into the medium, while the fully glycosylated 80-kd glycoprotein of the wild type was stable. These results assign a function to type 2 carbohydrate in protecting the cell-surface-exposed csA glycoprotein against proteolytic cleavage. HG220 cells were still capable of forming EDTA-stable contacts to a reduced extent, consistent with the low amounts of the 68-kd glycoprotein present on their surfaces. Thus type 1 rather than type 2 carbohydrate appears to be directly involved in intercellular adhesion that is mediated by the csA glycoprotein. Tunicamycin-treated wild-type and mutant cells produce a 53-kd protein that lacks both type 1 and 2 carbohydrates. While this protein is stable and not transported to the cell surface in the wild type, it is cleaved in the mutants and fragments of it are released into the extracellular medium. These results suggest that the primary defect in the two mutants studied is relief from a restriction in protein transport to the cell surface, and that the defect in type 2 glycosylation is secondary.

Two-step glycosylation of the contact site A protein of Dictyostelium discoideum and transport of an incompletely glycosylated form to the cell surface.

Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cells of the 53-kDa precursor with no detectable exposure of this protein on the cell surface. It is concluded that type 1 carbohydrate, which is cotranslationally added in N-glycosidic linkages, is neither required for transport of the protein to the Golgi apparatus nor for type 2 glycosylation or protection of the protein against proteolytic degradation. Incapability of tunicamycin-treated cells of forming EDTA-stable cell contacts suggests a role for type 1 carbohydrate in cell adhesion. Type 2 carbohydrate is added posttranslationally. It is required in the absence of type 1 glycosylation for transport of the protein to the cell surface.

Probing an adhesion mutant of Dictyostelium discoideum with cDNA clones and monoclonal antibodies indicates a specific defect in the contact site A glycoprotein.

Expression of developmentally regulated membrane proteins of aggregating cells of Dictyostelium discoideum is subject to several control mechanisms. One of them involves periodic cyclic-AMP pulses as signals for gene expression. To increase the probability of selecting mutants specifically defective in the contact site A (csA) glycoprotein, one of the characteristic proteins of aggregating cells, we have bypassed the requirement for both cyclic-AMP pulses and another control element by two runs of mutagenesis. A ;double bypass' mutant, HG592, was obtained which aggregated in nutrient medium where wild-type did not develop. Mutants defective in expression of the csA-glycoprotein were selected from HG592 by fluorescence-activated cell sorting and colony immunoblotting using a monoclonal antibody specific for that protein. One among 51 csA-negative mutants, HG693, specifically lacked the capability of forming EDTA-stable intercellular contacts. It acquired chemotactic responsiveness and developed into fruiting bodies. Expression of the transcripts for eight developmentally regulated proteins was determined in HG693. Seven of the RNA species were normally expressed; they were recognized by cDNA clones which had been produced from poly(A) RNA isolated from membrane-bound polysomes. The single RNA species which was not substantially expressed in HG693 was recognized by a cDNA clone that was obtained by screening a lambdagt11 library with an antibody specific for the csA-glycoprotein. When probing RNA from wild-type cells, this clone hybridized with a single developmentally regulated RNA species of 1.9 kb whose expression was strongly enhanced by cyclic-AMP pulses. Appearance of this RNA coincided with the expression of the csA-glycoprotein.

Mutants of Polysphondylium pallidum altered in cell aggregation and in the expression of a carbohydrate epitope on cell surface glycoproteins.

Mutants of the cellular slime mold Polysphondylium pallidum have been selected using a cell sorter and a fluorescentlabeled monoclonal antibody, mAb 293. This antibody blocks cell adhesion when applied as Fab, and recognizes a carbohydrate epitope containing L-fucose. This epitope is expressed on the cell surface and is present on >10 membrane glycoproteins of different apparent mol. wts. Twenty mutants were obtained which did not bind mAb 293 when tested at 2 h of starvation. After longer periods of starvation the epitope became detectable in the mutants. In all these mutants aggregation patterns were atypical. Generally streams of cells that were radially orientated around aggregation centers were missing or were much shorter than in wild-type. Genetic analysis demonstrated that aberrant aggregation was linked to the alteration in carbohydrate epitope expression. One mutant was unstable and gave rise to subclones in which almost no antibody binding was observed, even after 24 h of starvation, and only few aggregation centers with no streams or very short ones were formed. These results indicate that the capability of the cells to aggregate is correlated with the exposure on their surfaces of the carbohydrate epitope recognized by mAb 293, whose function in development remains to be established.

Monoclonal antibodies against Dictyostelium plasma membranes: their binding to simple sugars.

Monoclonal antibodies raised against purified membranes from Dictyostelium discoideum were classified according to three criteria: type of antigen as revealed in immunoblots, developmental regulation of the target antigens, and location of the antigens on the cell surface. Some antibodies reacted with myosin, two with glycolipids. One group of antibodies bound to the protein moiety of the contact site A glycoprotein, whereas another group reacted with carbohydrate epitopes that the contact site A glycoprotein shared with a few other membrane glycoproteins. Binding of the latter antibodies to their antigens was either specifically blocked by N-acetylglucosamine or by maltose as well as methyl-alpha-mannoside and N-acetylglucosamine. These anti-carbohydrate antibodies bound specifically to agarose beads derivatized with some sugars. These results and competition studies with several carbohydrates suggest that the epitope recognized by the antibodies contains as major components N-acetylglucosamine, maltose and alpha-mannose residues. One monoclonal antibody, which reacts with N-acetylglucosamine, was used for affinity purification of the contact site A glycoprotein from a crude membrane extract. N-acetylglucosamine was used as a mild eluent of the antigen from the antibody column. No detergents were added during the entire purification procedure.

Nerve growth factor-mediated induction of choline acetyltransferase in PC12 cells: evaluation of the site of action of nerve growth factor and the involvement of lysosomal degradation products of nerve growth factor.

In previous experiments it has been demonstrated that nerve growth factor (NGF), subsequent to its binding to specific membrane receptors, is internalized. Ultrastructurally, this internalized NGF is localized in membrane-confined compartments which ultimately fuse with lysosomes. The present experiments were designed, first, to evaluate whether a very small but functionally important portion of the internalized NGF might reach the free cytoplasm (and subsequently the nuclear chromatin) and might be responsible for the induction of choline acetyltransferase (ChAT) in PC12 cells. Second, we investigated whether a lysosomal proteolytic degradation product of NGF might act as a second messenger in the NGF-mediated ChAT induction. In one series of experiments, guinea pig erythrocyte ghosts, loaded with NGF (or NGF antibodies), fluorescein isothiocyanate-coupled bovine serum albumin, and/or horseradish peroxidase (HRP) were fused with PC12 pheochromocytoma cells. Electron microscopy showed that [125I]NGF and HRP reaction product were located throughout the cytoplasm and the nucleus but did not penetrate membrane compartments such as the endoplasmic reticulum, the Golgi complex, the perinuclear space, or mitochondria. Biochemically, NGF injected into the cytoplasm did not produce an induction of ChAT, whereas NGF acting via cell surface receptors resulted in a 2-fold increase in ChAT. Conversely, injection of NGF antibodies did not prevent the receptor-mediated ChAT induction. In a second series of experiments, the half-life of internalized NGF was increased from 40 min to 24 hr by the administration of leupeptin, a protease inhibitor which is accumulated in lysosomes. However, the NGF-mediated ChAT induction was not affected by this treatment. It is concluded that NGF itself does not act directly on cytoplasmic or nuclear target sites, nor is a proteolytic degradation product of NGF responsible for the NGF-mediated ChAT induction. Thus, NGF must act via a second messenger mechanism, the nature of which remains to be established.

Monoclonal anti-glycoprotein antibody that blocks cell adhesion in Polysphondylium pallidum.

Polyclonal and monoclonal antibodies were prepared against a glycoprotein (gp 64) of Polysphondylium pallidum previously shown to act as a target site of adhesion-blocking Fab prepared from antisera against whole membranes of aggregation-competent cells. The purified glycoprotein, with a nominal Mr of 64000, could be fractionated into two subspecies, gp 64I and gp 64II, with apparent Mr of 66000 and 60000, as determined in 7.5% sodium dodecyl sulfate/polyacrylamide gels. Rabbit antibodies against purified gp 64 reacted not only with the two subspecies but also with many other membrane proteins. Almost all the cross-reactivity could be abolished by absorption of the antibodies with extensively purified gp 64. All monoclonal antibodies obtained by screening with gp 64 showed similar cross-reactivity. One monoclonal antibody specifically precipitating gp 64 was selected by screening with antigen that had been pretreated with anhydrous hydrogen fluoride for removal of carbohydrates. Fab from polyclonal anti-(gp 64) sera as well as one monoclonal Fab completely blocked cell adhesion of aggregation-competent P. pallidum cells. A carbohydrate fraction prepared by treatment of gp 64 with proteases and hydrazine completely neutralized the adhesion-blocking Fab. The product of hydrazinolysis contained less than 3% of the original peptide as based on the glucosamine recovered, but the specific neutralizing activity of the carbohydrate was essentially the same as that of the glycoprotein. In conclusion, monoclonal as well as polyclonal adhesion-blocking Fab reacted with carbohydrates; gp 64 shared the relevant carbohydrate moieties with other membrane proteins.

Responses of Dictyostelium discoideum amoebae to local stimulation by light.

Single amoebae of Dictyostelium discoideum were locally stimulated with microbeams of white and monochromatic light. Low illuminance stimulation favored formation of pseudopodia at the irradiated parts of the cells, high illuminance stimulation locally suppressed the extension of pseudopodia. When the high illuminance light spot was placed on any portion of the cell other than the moving front, no response could be observed. The results are compatible with the assumption that, during their phototactic response, single amoebae detect the direction of light by a shadowing effect caused by pigments like cytochromes, and/or by light scattering of particles in the cytoplasm.

Monoclonal antibodies against contact sites A of Dictyostelium discoideum: detection of modifications of the glycoprotein in tunicamycin-treated cells.

Tunicamycin acts on cell aggregation in Dictyostelium discoideum by changing cell movement and by inhibiting the EDTA-stable type of intercellular adhesion. Tunicamycin-treated cells show unco-ordinated pseudopodial activity such that pseudopods are simultaneously extended from all parts of the cell surface, and the cells are unable to move in straight paths. Concurrent with the inhibition of formation of EDTA-stable contacts, N-glycosylation of a glycoprotein specific for aggregation-competent cells is inhibited. This glycoprotein, previously called contact site A, has an apparent mol. wt. of 80 kilodaltons (kd). In membranes of tunicamycin-treated cells, two components are detected that react with certain monoclonal antibodies against contact sites A: one component of 66 kd, the other of 53 kd apparent mol. wt. Another group of monoclonal antibodies reacts only with the 80-kd glycoprotein and the 66-kd component. These results are in accord with the assumption that the glycoprotein carries two carbohydrate chains, and that the antibodies differ in their requirement for glycosylation of the antigen. Despite the coincidence between blockage of EDTA-stable cell adhesion and inhibited glycosylation of contact sites A, direct involvement of the carbohydrate moieties of this glycoprotein in intercellular adhesion seems questionable. EDTA-stable cell adhesion has not been blocked by Fab fragments from antibodies that specifically react with the glycosylated protein.

Stage-specific antigens reacting with monoclonal antibodies against contact site A, a cell-surface glycoprotein of Dictyostelium discoideum.

Monoclonal antibodies against a glycoprotein presumably involved in adhesion of aggregating Dictyostelium discoideum cells have been used for labeling of the antigen at the cell surface. The antigen is distributed over the whole surface of the cells, apparently in form of small clusters. The antigen appears concomitantly with the acquisition of EDTA-stable adhesiveness typical of aggregation competent cells. In contrast, discoidin I, a lectin whose accumulation during development parallels EDTA-stable adhesiveness in another strain (NC-4), is present in nearly the same amounts of growth phase and aggregating cells of AX2-214, the strain used by use. Thus, no correlation exists in this strain between the expression of discoidin I and the development of cell adhesiveness. The 80 kilodalton glycoprotein typical of aggregation competent cells has been purified by affinity chromatography on a monoclonal antibody column. The purified antigen absorbs adhesion-blocking Fab from rabbits. Another antigen strongly reacting with the same monoclonal antibodies has an apparent molecular weight of 106 000 and is not detectable before slugs are formed.