The Coat protein of RNA bacteriophages functions as a repressor of translation of the cistron coding for RNA synthetase (the phage-coded subunit of the RNA replicating enzyme) (1-8).Isolated RNA and coat protein can interact to form two kinds of complexes: Complex I in which a few molar equivalents of coat protein [one (9)-six (10) ] bind to one equivalent of RNA leading to formation of a complex that sediments at the same rate as free RNA, and Complex II in which about 180 coat-protein molecules bind to one molecule of RNA to form a complex that is phage-like, but not infectious (10). Complex I has been implicated in translational control of the RNA synthetase cistron since addition of coat protein to phage RNA reduces its messenger activity in an in vitro protein-synthesizing system and depresses the synthesis of the RNA synthetase (4-6, 9); it specifically inhibits the initiation step of that synthesis (8,(11)(12)(13). The mechanism by which the coat protein prevents translation of the RNA synthetase cistron is not known. We present evidence that the coat protein binds to the initiation site of the RNA synthetase cistron and directly prevents the ribosomes from translating that cistron. We show that when Complex I is formed in vitro and then incubated with Ti RNase, an RNA fragment is protected from nucleolytic degradation; this fragment (59 residues) contains the nucleotide sequence of the region of the R17 RNA preceding the cistron coding for the RNA synthetase and the first two codons of that cistron. The protection by the coat protein of this region from nuclease degradation is specific: when R17 RNA is mixed with the coat protein of Q,3 (an RNA phage serologically unrelated to R17) and the mixture is degraded by T1 RNase, no comparable fragment is found. MATERIAL AND METHODS[3H]Uridine-labeled R17 RNA (2.2 X 104 cpm/,g) and 2P-labeled R17 RNA (3 X 106 cpm/,ug) were prepared as in refs.14 and 15, respectively. Coat proteins of phages R17 and Q0were extracted by the acetic acid procedure (10); the ability of these protein preparations to repress in vitro synthesis of RNA 3033 synthetase was tested (4). Pancreatic RNase was obtained from Worthington and T1 and U2 RNases from Sankyo. Acid RNase and acid phosphatase B from spleen were a gift from Dr. G. Bernardi; they were prepared as described (16,17).Formation of Coat Protein-RNA Complex and Isolation of the Fragment Protected from Nuclease Degradation. The incubation mixture contained in a final volume of 0.6 ml: B2P-labeled R17 RNA (1.15 nmol; 3.2 X 109 cpm), R17 coat protein (4.5 nmol), 100 mM Tris HCl (pH 7.5), 10 mM Mg acetate, and 80 mM KCl (TMK buffer). After 10 min at 00, 0.18 ml of a T1 RNase solution (0.7 mg/ml in 10 mM TrisHCl pH 7.5-2 mM EDTA) were added. The mixture was incubated at 220 for 30 minm then chilled on ice and layered onto two sucrose density gradients (10-20% sucrose in TMK buffer) and centrifuged at 35,000 rpm for 18 hr at 40 in a SW41 Spinco rotor. The gradients were collected at 40 into plastic tubes (27 fractions). Aliq...

We have completed the pSC101 sequence. The coding capacities of the newly sequenced regions show the presence of two large open reading frames close to the oriT region. Their size and localization suggest that these polypeptide chains could be involved in the transfer process of pSC101.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to nervous system tumors. The schwannomin (also termed merlin) protein encoded by the NF2 gene shows a close relationship to the family of cytoskeleton-to-membrane proteins linkers ERM (ezrin-radixin-moesin proteins). Even though penetrance of the disease is >95% and no genetic heterogeneity has been described, point mutations in the NF2 gene have been observed in only 34-66% of the screened NF2 patients, depending on the series. In order to generate tools that would enable an exhaustive alteration screening for the NF2 gene, we have deduced its entire genomic sequence. This knowledge has provided the delineation of a mutation screening strategy which, when applied to a series of 19 NF2 patients, has revealed a high recurrence of large deletions in the gene and has raised the efficiency of mutation detection in NF2 patients to 84% of the cases in this series. The remaining three patients who express two functional NF2 alleles are all sporadic cases, an observation compatible with the presence of mosaicism for NF2 mutation.

This work describes the in vitro properties of fulllength CDC25 Mm (1262 amino acid residues), a GDP/GTP exchange factor (GEF) of H-ras p21. CDC25 Mm , isolated as a recombinant protein in Escherichia coli and purified by various chromatographic methods, could stimulate the H-ras p21⅐GDP dissociation rate; however, its specific activity was 25 times lower than that of the isolated catalytic domain comprising the last C-terminal 285 residues (C-CDC25 Mm285 ) and 5 times lower than the activity of the C-terminal half-molecule (631 residues). This reveals a negative regulation of the catalytic domain by other domains of the molecule. Accordingly, the GEF activity of CDC25 Mm was increased severalfold by the Ca 2؉ -dependent protease calpain that cleaves around a PEST-like region (residues 798 -853), producing C-terminal fragments of 43-56 kDa. In agreement with the presence of an IQ motif on CDC25 Mm (residues 202-229), calmodulin interacted functionally with the exchange factor. Depending on the calmodulin concentration an inhibition up to 50% of the CDC25 Mm -induced nucleotide exchange activity on H-ras p21 was observed, an effect requiring Ca 2؉ ions. Calmodulin also inhibited C-CDC25 Mm285 but with a ϳ100 times higher IC 50 than in the case of CDC25 Mm (ϳ10 M versus 0.1 M, respectively). Together, these results emphasize the role of the other domains of CDC25 Mm in controlling the activity of the catalytic domain and support the involvement of calmodulin and calpain in the in vivo regulation of the CDC25 Mm activity.The mouse CDC25 Mm 1 protein is a guanine nucleotide exchange factor (GEF) regenerating the active form of H-ras p21, the complex with GTP (1-3). Homologous products were found in rat (p140-rasGRF) (4) and human (H-GRF) (5, 6). These rasGEFs have been described to be specific for the central nervous system (4 -9). Some evidence has also been reported for the existence of full-length and truncated forms of these exchange factors in other tissues (10,11). Experiments in vivo suggest that the upstream connection of this GEF involves G-protein-coupled receptors (9, 12, 13) and not hormone-receptor-bound tyrosine kinases via the adaptor protein GRB2, as has been found for SOS, a ubiquitous rasGEF (14 -16). CDC25 Mm contains in the N-terminal moiety two domains of pleckstrin homology (PH1 and PH2), one of DBL homology (DH) and a coiled-coil region that follows the PH1 domain (cf. (21) and the DH is a domain sharing similarity with a GDP/GTP exchange factor of members of the Rho family (2,4,22,23). Farnsworth et al. (24) reported that in vivo the activity of the homologous p140-rasGRF from rat brain is enhanced by raising the calcium concentration, an effect associated with the binding of calmodulin, and that p140-rasGRF and calmodulin form a stable complex. A direct action of calmodulin was supported by the presence in the N-terminal region of CDC25 Mm of an IQ domain, a sequence frequently found in proteins interacting with calmodulin (25, 26). Very recent experiments in vivo have indicated that PH1, ...

BackgroundThe independent prognostic impact of diabetes mellitus (DM) and prediabetes mellitus (pre‐DM) on survival outcomes in patients with chronic heart failure has been investigated in observational registries and randomized, clinical trials, but the results have been often inconclusive or conflicting. We examined the independent prognostic impact of DM and pre‐DM on survival outcomes in the GISSI‐HF (Gruppo Italiano per lo Studio della Sopravvivenza nella Insufficienza Cardiaca‐Heart Failure) trial.Methods and ResultsWe assessed the risk of all‐cause death and the composite of all‐cause death or cardiovascular hospitalization over a median follow‐up period of 3.9 years among the 6935 chronic heart failure participants of the GISSI‐HF trial, who were stratified by presence of DM (n=2852), pre‐DM (n=2013), and non‐DM (n=2070) at baseline. Compared with non‐DM patients, those with DM had remarkably higher incidence rates of all‐cause death (34.5% versus 24.6%) and the composite end point (63.6% versus 54.7%). Conversely, both event rates were similar between non‐DM patients and those with pre‐DM. Cox regression analysis showed that DM, but not pre‐DM, was associated with an increased risk of all‐cause death (adjusted hazard ratio, 1.43; 95% CI, 1.28–1.60) and of the composite end point (adjusted hazard ratio, 1.23; 95% CI, 1.13–1.32), independently of established risk factors. In the DM subgroup, higher hemoglobin A1c was also independently associated with increased risk of both study outcomes (all‐cause death: adjusted hazard ratio, 1.21; 95% CI, 1.02–1.43; and composite end point: adjusted hazard ratio, 1.14; 95% CI, 1.01–1.29, respectively).ConclusionsPresence of DM was independently associated with poor long‐term survival outcomes in patients with chronic heart failure.Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT00336336.

The catalytic domain of the Saccharomyces cerevisiae SDC25 gene product, including the last 550 C-terminal residues (Sdc25p-C), was produced as an Escherichia culi recombinant protein fused with glutathione S-transferase. The highly purified (greater than 95 %) stable fusion protein, obtained by affinity chromatography, was very active in enhancing the dissociation rate or the GDP/GTP exchange of the GDP complex of Ras2p or human H-ras p21. This activity was further increased (three times) by glutathione S-transferase cleavage with thrombin. The stimulation of the guanine nucleotide release by Sdc2Sp-C was stronger for Ras2p . GDP than Ras2p . GTP, an effect that was less pronounced in the case of the p21 complexes. The association rate of the Ras2p. GDP (GTP) complex was also enhanced by Sdc2Sp-C. Monovalent and divalent salts inhibit the nucleotide-releasing activity of Sdc2Sp-C. Retention phenomena occurring on gel-filtration chromatography hindered the use of highly purified Sdc25p-C to study the formation of stable complexes with Ras2p. For this purpose, Sdc2Sp-C was produced as a non-glutathione-S-transferase fusion protein via pTTQ19. Upon partial purification, this product yielded a 54-kDa truncated form of Sdc2Sp-C (truncated Sdc2Sp-C) showing the same specific activity as the 64-kDa Sdc2Sp-C protein. On gel filtration, truncated Sdc25p-C and nucleotide-free Ras2p (or p21) formed a stable 1 : 1 stoichiometric complex that was dissociated by increasing concentrations of GDP. The properties of this complex were analyzed by using the mutant [S24N]Ras2p, the homologue of [S 17NIp21 known to induce a dominant negative phenotype, [R80D, N81D]Ras2p, a recessive negative mutant insensitive to the truncated form of Sdc2Sp-C in vitro. The complex with [S24N]Ras2p was greater than 100-fold less sensitive to the dissociating effect of GDP, whereas [R80D, N81DIRas2p was unable to form a stable complex with truncated Sdc25p-C. These results strongly suggest that the residues R80 and N81 are situated in or closely associated with the Ras2p specific site binding Sdc2Sp.

The interaction of Saccharomyces cerevisiae Ras2p with the catalytic domain of the GDP/GTP exchange factors (GEFs) mouse CDC25 Mm , yeast Cdc25p, and Sdc25p was analyzed by introducing the substitution R80D/N81D into Ras2p S24N, a mutant that is shown to interfere with the Ras2p wild type (wt)-GEF interaction by forming a stable complex. The triple mutant, like Ras2p R80D/N81D, did not interfere with the action of GEF on Ras2p wt (or H-Ras p21) and was unable to form a stable complex with GEF. The GEF stimulation of the nucleotide dissociation of the triple mutant was virtually abolished and strongly decreased with the double mutant. The affinity of Ras2p S24N/R80D/N81D for GDP and GTP was decreased 3 and 4 orders of magnitude, respectively, like that of Ras2p S24N, whereas the double mutant behaved as Ras2p wt. Like Ras2p S24N and unlike Ras2p R80D/N81D, the GTP-bound triple mutant did not activate adenylyl cyclase. Thus, the triple mutant and Ras2p S24N have opposite properties toward the binding to GEF but similarly modified behaviors toward GDP, GTP, and adenylyl cyclase. This work emphasizes the determinant role of the distal switch II region of Ras2p for the interaction with GEF and the different structural background of the interaction with adenylyl cyclase.Ras proteins are GTPases that regulate cell growth and differentiation by cycling between the active GTP-bound and the inactive GDP-bound states. The level of these two forms is determined by GTPase-activating proteins and guanine nucleotide exchange factors (GEFs) 1 (Boguski & McCormick, 1993). The yeast Saccharomyces cerevisiae Cdc25p (Parmeggiani et al., 1993) was the prototype for Ras GEFs, such as S. cerevisiae Sdc25p (Créchet et al., 1990b and mammalian tissuesspecific brain GEFs Jacquet et al., 1992;Schweighoffer et al., 1993). Due to difficulties in isolating the intact protein, only the catalytic domain of these GEFs has been used to investigate the mechanism of the exchange reaction in vitro so far (Créchet et al., 1990b;Lai et al., 1993;Shou et al., 1992;Schweighoffer et al., 1993;Haney & Broach, 1994;Jacquet et al., 1994Jacquet et al., , 1995Poullet et al., 1995). Several reports have dealt in these past years with the structures of Ras proteins involved in the interaction with GEF. Although no definite conclusions could be drawn concerning the location of the binding site of Ras proteins for GEF, site-directed mutagenesis of Ras proteins has given useful information on the role played by the various regions of the Ras molecule in the action of GEF. Specific substitutions in the phosphoryl binding I region, the switch I and II regions of human c-H-Ras p21 and Ras2p were identified that influence negatively the action of GEF; some of them have little effect on the affinity for GEF (Mistou et al., 1992), whereas others can induce dominant negative interferences on the interaction between wild type Ras and GEF, due to the formation of a stable complex between the mutant and GEF (Powers et al., 1989;Szeberényi et al., 1990;Stacey et al., 1991...

Human and Saccharomyces cerevisiae Ras proteins and their regulators GAP (GTPase activating protein)and GEF (guanine nucleotide exchange factor) display structural similarities and are functionally interchangeable in vivo and in vitro, indicating that the molecular mechanism regulating Ras proteins has been conserved during evolution. As the only exceptions, the two S.cerevisiae GAPs, Ira1p and Ira2p, are strictly specific for yeast Ras proteins and cannot stimulate the GTPase of mammalian Ras. This study searches for the reasons for the different sensitivity to Ira2p of human H‐ras p21 and yeast Ras2p. Construction of H‐ras/Ras2p chimaeras showed that Gly18 of Ras2p (Ala11 of H‐ras p21) is an important determinant for the specificity of Ira2p, revealing for the first time a function for this position. A second even more crucial determinant was found to be the 89–102 region of Ras2p (82–95 of H‐ras p21) including the distal part of strand beta4, loop L6 and the proximal part of helix alpha3. It was possible to construct Ras2p's resistant to Ira2p but still sensitive to human p120‐GAP and, conversely, a H‐ras p21 sensitive to Ira2p. This work helps clarify specific aspects of the conserved molecular mechanism of interaction between Ras proteins and their negative GAP regulators.