Investigation of DNA complexes with histones F3 and F3+F2a2]

Characteristic viscosity, sedimentation constant and optical anisotropy were studied of the complexes formed between DNA and histone fractions F3 and F3+F2a2. The parameters mentioned continuously change with the increase of protein content within the complex. Analysis of experimental data shows that binding of a histone bads to a decrease of size and thermodynamic rigidity of the DNA molecule. On the basis of results obtained a model of F3 histone binding with DNA is suggested, amino acid sequence of this protein being taken into account. Comparison of behaviour of nucleohistones DNA+F3 and DNA+F1 studied previously testifies different way of binding of these histones to DNA.     

E2F7 and E2F8 keep the E2F family in balance

An article by Li and colleagues (in this issue of Developmental Cell) shows that the atypical E2Fs, E2F7 and E2F8, are critical for mouse development. One of the important functions of these family members stems from a negative feedback loop in which E2F7 and E2F8 limit the expression of E2F1 and prevent E2F1-dependent apoptosis.     

Deletion mutants of F, FΔ(8.5–17.6) and the mechanism of their formation

Heteroduplex analysis shows that a cryptic plasmid extracted from the male phage-sensitive strain, RP42, is a deletion mutant of F and lacks the segment with F-coordinates 8.5F to 17.6F. Consideration of the sequence deleted in F shows that the molecular recombination event between γδ and εζ DNA sequences actually occurs only between specific short segments at the ends.     

The gamma subunit of F1 and the PVP protein of F0 (F0I) are components of the gate of the mitochondrial F0F1 H(+)-ATP synthase

The gamma subunit of the F1 moiety of the bovine mitochondrial H(+)-ATP synthase is shown to function as a component of the gate. Addition of purified gamma subunit to F0-liposomes inhibits transmembrane proton conduction. This inhibition can be removed by the bifunctional thiol reagent diamide. Immunoblot analysis shows that the diamide effect is likely due to disulphide bridging of the gamma subunit with the PVP protein of the F0 sector.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: isolation of a new F-prime factor, F80, and its implication for the mechanism of F integration into the chromosome.

A new F-prime factor, F80, was isolated from an Escherichia coli strain harboring the F-prime factor F8 by selecting for transfer of the supE marker to a RecA- recipient. Genetic analysis shows that F80 carries a segment of the chromosomal DNA between lip and suc in addition to the tol-gal region normally in F8. Physical analysis by the electron microscope heteroduplex method suggests that the formation of F80 from F8 involves recombination between the alphabeta segment of F, which is present in F8, and the homologous sequence of F present in the E. coli chromosome at the site where F is supposed to integrate to form HfrP3. The implications of this result for the general mechanisms of F integration to form Hfr's are discussed.     

The kinetic and structural changes of the mitochondrial F1-ATPase with temperature

Mitochondrial F1-ATPase shows a break in the Arrhenius plot with an increase of the activation energy below 17 degrees C, this may imply that the F1-ATPase undergoes a conformational change at this temperature. Further, a structural change of the F1-ATPase is indicated by analysis of the intrinsic fluorescence at 307 nm between 33 and 11 degrees C and also by evaluation of the circular dichroism spectra of the enzyme at temperatures below and above the temperature corresponding to the discontinuity of the Arrhenius plot. It is therefore suggested that F1-ATPase exists in two temperature dependent conformational states to which different catalytic properties may be assigned.     

Structural and functional characterization of subunits of the F0 sector of the mitochondrial F0F1-ATP synthase.

Proteolytic digestion of F1-depleted submitochondrial particles (USMP), reconstitution with isolated subunits and titration with inhibitors show that the nuclear-encoded PVP protein, previously identified as an intrinsic component of bovine heart F0 (F01) (Zanotti, F. et al. (1988) FEBS Lett. 237, 9-14), is critically involved in maintaining the proper H+ translocating configuration of this sector and its correct binding to the F1 catalytic moiety. Trypsin digestion of USMP, under conditions leading to cleavage of the carboxyl region of the PVP protein and partial inhibition of transmembrane H+ translocation, results in general loss of sensitivity of this process to F0 inhibitors. This is restored by addition of the isolated PVP protein. Trypsin digestion of USMP causes also loss of oligomycin sensitivity of the catalytic activity of membrane reconstituted soluble F1, which can be restored by the combined addition of PVP and OSCP, or PVP and F6. Amino acid sequence analysis shows that, in USMP, modification by [14C] N,N'-dicyclohexylcarbodiimide of subunit c of F0 induces the formation of a dimer of this protein, which retains the 14C-labelled group. Chemical modification of cysteine-64 of subunit c results in inhibition of H+ conduction by F0. The results indicate that proton conduction in mitochondrial F0 depends on interaction of subunit c with the PVP protein.     

The photoreaction center of Rhodospirillum rubrum mutant strain F24.1

Rhodospirillum rubrum strain F24.1 is a spontaneous revertant of nonphototrophic mutant F24 derived from wild-type strain S1. Strain F24 shows no detectable photochemical activity and contains, at most, traces of the photoreaction center polypeptides. Strain F24.1 has a phototrophic growth rate close to that of the wild-type strain (Picorel, R., del Valle-Tascón, S. and Ramírez, J.M. (1977) Arch. Biophys. Biochem. 181, 665–670) but shows little photochemical activity. Light-induced absorbance changes in the near-infrared, photoinduced EPR signals and ferricyanide-elicited absorbance changes indicate that strain F24.1 has a photoreaction center content of 7–8% as compared to strain S1. Polyacrylamide gel electrophoresis of isolated F24.1 chromatophores shows the photoreaction center polypeptides to be present in amounts compatible with this value. Photoreaction center was prepared from strain F24.1 and showed no detectable difference with that of strain S1. It is concluded that strain F24.1 photosynthesis is due entirely to its residual 7–8% of typical photoreaction center.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. IX. Note on the deletion mutant of F, F delta(33-43)

Heteroduplex analysis shows that the plasmid extracted from cells of the F⁺ strain, W1655, is a deletion mutant of F, and lacks segments with F co-ordinates 32.6 to 42.9 kilobases. The genes on F for resistance to the female-specific phages T3, φII, and τ probably lie within this region. Consideration of the sequences deleted in several F-primes shows that the sequences from 0 to 42.9 kilobases on F are not necessary for autonomous replication nor for fertility.     

Membrane-Induced Structural Rearrangement and Identification of a Novel Membrane Anchor in Talin F2F3

Experimental challenges associated with characterization of the membrane-bound form of talin have prevented us from understanding the molecular mechanism of its membrane-dependent integrin activation. Here, utilizing what we believe to be a novel membrane mimetic model, we present a reproducible model of membrane-bound talin observed across multiple independent simulations. We characterize both local and global membrane-induced structural transitions that successfully reconcile discrepancies between biochemical and structural studies and provide insight into how talin might modulate integrin function. Membrane binding of talin, captured in unbiased simulations, proceeds through three distinct steps: initial electrostatic recruitment of the F2 subdomain to anionic lipids via several basic residues; insertion of an initially buried, conserved hydrophobic anchor into the membrane; and association of the F3 subdomain with the membrane surface through a large, interdomain conformational change. These latter two steps, to our knowledge, have not been observed or described previously. Electrostatic analysis shows talin F2F3 to be highly polarized, with a highly positive underside, which we attribute to the initial electrostatic recruitment, and a negative top face, which can help orient the protein optimally with respect to the membrane, thereby reducing the number of unproductive membrane collision events.     

Membrane-Induced Structural Rearrangement and Identification of a Novel Membrane Anchor in Talin F2F3

Experimental challenges associated with characterization of the membrane-bound form of talin have prevented us from understanding the molecular mechanism of its membrane-dependent integrin activation. Here, utilizing what we believe to be a novel membrane mimetic model, we present a reproducible model of membrane-bound talin observed across multiple independent simulations. We characterize both local and global membrane-induced structural transitions that successfully reconcile discrepancies between biochemical and structural studies and provide insight into how talin might modulate integrin function. Membrane binding of talin, captured in unbiased simulations, proceeds through three distinct steps: initial electrostatic recruitment of the F2 subdomain to anionic lipids via several basic residues; insertion of an initially buried, conserved hydrophobic anchor into the membrane; and association of the F3 subdomain with the membrane surface through a large, interdomain conformational change. These latter two steps, to our knowledge, have not been observed or described previously. Electrostatic analysis shows talin F2F3 to be highly polarized, with a highly positive underside, which we attribute to the initial electrostatic recruitment, and a negative top face, which can help orient the protein optimally with respect to the membrane, thereby reducing the number of unproductive membrane collision events.     

Wheat germ initiation factors 4F and (iso)4F interact differently with oligoribonucleotide analogues of rabbit alpha-globin mRNA

The binding of capped oligoribonucleotide analogues of the 5' terminus of rabbit alpha-globin mRNA to wheat germ protein synthesis initiation factors eIF-4F and eIF-(iso)4F was measured by direct fluorescence techniques. An analysis of the equilibrium association constants (Keq) indicates that both eIF-4F and eIF-(iso)4F recognize primarily the m7G cap structure but differ in the recognition of other structural features. eIF-4F is sensitive to the position and sequence of hairpin structures within the oligoribonucleotide, while eIF-(iso)4F shows a preference for linear sequences. These differences suggest that wheat germ eIF-4F and eIF-(iso)4F may have discriminatory activity for mRNA recognition.     

Human cyclin F.

Cyclins are important regulators of cell cycle transitions through their ability to bind and activate cyclin-dependent protein kinases. In mammals several classes of cyclins exist which are thought to co-ordinate the timing of different events necessary for cell cycle progression. Here we describe the identification of a novel human cyclin, cyclin F, isolated as a suppressor of the G1/S deficiency of a Saccharomyces cerevisiae cdc4 mutant. Cyclin F is the largest cyclin, with a molecular weight of 87 kDa, and migrates as a 100-110 kDa protein. It contains an extensive PEST-rich C-terminus and a cyclin box region that is most closely related to cyclins A and B. Cyclin F mRNA is ubiquitiously expressed in human tissues. It fluctuates dramatically through the cell cycle, peaking in G2 like cyclin A and decreasing prior to decline of cyclin B mRNA. Cyclin F protein accumulates in interphase and is destroyed at mitosis at a time distinct from cyclin B. Cyclin F shows regulated subcellular localization, being localized in the nucleus in most cells, with a significant percentage of cells displaying only perinuclear staining. Overexpression of cyclin F, or a mutant lacking the PEST region, in human cells resulted in a significant increase in the G2 population, implicating cyclin F in the regulation of cell cycle transitions. The ubiquitous expression and phylogentic conservation of cyclin F suggests that it is likely to coordinate essential cell cycle events distinct from those regulated by other cyclins.     

The relationship between metallothionein-1F (MT1F) gene and hepatocellular carcinoma

To investigate the expression of MT1F gene in hepatocellular carcinoma tissue and the growth suppression effect of exogenous introduction of MT1F gene on liver cell line HepG2 and to explore the potential application of MT1F gene in gene therapy of tumor. Eukaryotic expression vector of pCMV-MT1F plasmid was introduced into HepG2 line which expressed no MT1F protein originally with lipofectamine transfection method. The cell growth curve, soft agar colony formation rate and tumorigenicity in SCID mice were examined to demonstrate the growth suppression effect of exogenous MT1F gene on HepG2 cell line. The MT1F mRNA and MT1F protein were also detected in 60 pairs of surgical specimens of hepatocellular carcinoma by in situ hybridization and immunohistochemistry. The transfected HepG2 cell line grew more slowly than control HepG2 as shown by cell growth curves, the soft agar colony formation rate (3.8 percent vs. 7.4 percent, p <.01) and the average growth rate of tumor in SCID mice (30.9 +/- 6.9 vs. 70.3 +/- 5.6, p <.01). The expression level of MT1F mRNA and protein significantly increased in paracancerous tissue, normal tissue than in cancer tissues (75 percent, 70 percent vs. 16.7 percent by ISH and 66.7 percent, 60 percent vs. 10 percent by IHC, p <.01). Exogenous MT1F gene shows the strong effect of growth inhibition on HepG2 cell line. In the liver cancer tissue, MT1F shows down-regulated expression that supports the inhibited function of MT1F in cancer growth and suggests MT1F may have an important role in gene therapy of hepatocellular carcinoma.     

Further studies on F1-ATPase inhibition by local anesthetics

We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F1 extracted from these particles. All of these agents cause inhibition of ATPase in F1 as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F1. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F1 complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid.     

Gelatin binding to the 6F1(1)F2(2)F2 fragment of fibronectin is independent of module-module interactions

Fibronectin, a large modular protein, interacts with many other proteins in the extracellular matrix and on the cell surface. It has previously been shown that interactions between noncontiguous modules exist in the collagen binding region. It is shown here that the interaction between the sixth type I module ((6)F1) and the second type II module ((2)F2) can be disrupted by mutation of a residue in the intermodule interface of the (6)F1(1)F2(2)F2 fragment. The perturbation of the interface and the binding of collagen-derived peptides to individual modules were assessed by high-resolution nuclear magnetic resonance (NMR) spectroscopy. Cooperativity between the modules in binding ligand was assessed by analytical gelatin affinity chromatography of the mutant and wild-type proteins. Differential scanning calorimetry (DSC) was used to probe the influence of the interface on module stability. It is shown that while the (6)F1-(2)F2 interface confers significant thermal stability to the (2)F2 module, it has little effect on gelatin binding activity of the (6)F1(1)F2(2)F2 fragment.     

The relationship between histones F 2al and F 2a2 and the ancestral histone A peptide. Further evidence for the common origin of histones F 2al, F 2a2 and F 3.

The relationship between histones F 2al and F 2a2 becomes much more apparent if the alignment is not made between the total sequences but between the ancestral A peptide, reconstructed earlier for histone F 2al (IV) and F 2a2. 46.5% of the latter's sequence can thus be clearly connected with F 2al through this ancestral dodecapeptide. A parallel development of histones F 2al, F 2a2 and F 3 from the A peptide is proposed.     

The filter-feeders signal: response of ostracods to marine environmental changes in the Devonian F–F event

The percentage of filter-feeder species in benthic ostracod assemblages shows evident changes during the Late Devonian Frasnian–Famennian (F–F) transition with two sharp rises in the event stratal interval based on materials collected from the Yangdi section, South China. Neither anoxia nor oligotrophy, eustasy should have been the only primary cause of filter-feeders signal in the F–F event. The sea level fall and rise would create deteriorative environmental conditions such as lower oxygenation and less nutrients, which altogether reduced the diversity and abundance of the benthic ostracods in the F–F transition. The percentage of filter-feeders showed a decrease during the regression, and vice-versa. Therefore, the filter-feeders may be potentially an indicator of sea level changes in the F–F event.     

Genome sequence of the biocontrol strain Pseudomonas fluorescens F113

Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) that has biocontrol activity against fungal plant pathogens and is a model for rhizosphere colonization. Here, we present its complete genome sequence, which shows that besides a core genome very similar to those of other strains sequenced within this species, F113 possesses a wide array of genes encoding specialized functions for thriving in the rhizosphere and interacting with eukaryotic organisms.