A series of ribosomal subparticles derived from the 50S subunit has been studied and compared in EF-T- and EF-G-dependent reactions. Three different 50S cores were prepared by CsC1 isophycnic centrifugation and one by NH(4)Cl-ethanol extractionm the 50S CsCl core a had lost proteins L1, L7, L8, L10, L12, L16, L25, L33, and some L6 and L11. The 50S CsCl core b additionally lacked protein L6, and 50S CsCl core c also lacked protein L5, L15, L18, L27, L28, L30, and most of L9, L14, L19, and L21. The 50S NH(4)Cl-ethanol core had lost up to 90 percent of proteins L7, L12 and 30-60 percent of proteins L8, L10, and L29. The 50S CsCl core a had much reduced activity in EF-G and none in EF-T GTPase reactions while 50S CsCl cores b and c were inactive. Addition of proteins L7, L12 restored the activity for both the EF-T- and EF-G-dependent GTPase with all of the three 50S CsCl cores, increasing stepwise from core c to core a; The 50S NH(4)Cl-ethanol core was partially active in the EF-G GTPase over the 2-30 mM MG-2+ range tested, while EF-T only showed some activity inthe upper portion of this range...

ABSTRACJA 38 kilobase pair region of the Escherichia coli K12 chromosome containing the replication origin has been physically mapped with restriction endonucleases EcoRI and HindIII. Replication starts within or very near a 1.3 kilobase pair HindIII fragment in the middle of this region and proceeds outward in both directions with apparently equal speed. This pattern was observed in both dnaA and dnaC temperaturesensitive (ts) initiation mutants at the start of the synchronous round of replication which occurs after downshift from the nonpermissive to the permissive temperature. The origin of replication of the Escherichia coli chromosome is located in the vicinity of the iiv operon (1-3), most likely between the dnaA and bglA genes (4, 5). This would place it at approximately 82 min on the revised genetic map of E. coli (6). From this site, replication proceeds in both directions around the large, circular chromosome and terminates within the region diametrically opposite the origin (1, 2, 7, 8).The isolation of conditional mutants defective in initiation has led to the identification of several genes specifically involved in this process: dnaA and dnaC (see ref. 9 for review), dnaI (10), and dnaP (11). The dnaH initiation mutant described by Sakai et al. (12) apparently is a double mutant with the defect in initiation due to a dnaA lesion (13). The dnaA and dnaC loci were the first discovered and are the best characterized. Mutations and 10-ml aliquots taken at 1-or 2-min intervals for pulse labeling with 0.4 ml of [3H]thymidine (0.5 mCi/ml and 60 Ci/ mmol, Schwarz/Mann). Pulses were quenched by adding nonradioactive thymidine to 100 ,ug/ml and pouring the aliquot over ice in a centrifuge tube containing enough KCN to make its final concentration 10 mM.Total cellular DNA was purified as follows: Pelleted cells were resuspended in 0.4 ml of 20% sucrose/100 mM NaCl/10 mM Tris-HCI (pH 8.2), treated for 20 min on ice with 0.1 ml of a 4 mg/ml solution of lysozyme in 120 mM Tris-HCl (pH 8,2)/S0 mM EDTA, and lysed with Sarkosyl (0.4 ml of a 2.5% solution). The lysate was heated to 650 for 5 min, cooled, diluted with water to 7.7 ml, and digested for 4 hr at 370 with 0.1 ml of a 1 mg/ml solution of Pronase (Calbiochem). Solid KI was added to bring the volume of the lysate to 10 ml and a density gradient was formed by centrifugation in a Beckman 50 Ti rotor at 37,000 rpm for 40 hr at 200. The DNA, which banded in the center of the gradient, was collected and dialyzed against 20 mM Tris-HCl (pH 7.8)/0.2 mM EDTA.Restriction Endonuclease Digestion. Restriction endonucleases were purchased from Miles Laboratories and New England Biolabs. A standard reaction mixture was used for all digestions [20 mM Tris-HCI (pH 7.8)/10 mM MgCl2/10 mM NaCI/7 mM 2-mercaptoethanol/0.2 mM EDTA/0.1% gelatin]. Sufficient enzyme was added to give a limit digest within a 4-hr incubation period at 370. When the DNA was digested with more than one enzyme, all were added at the beginning of the digestion period.Gel Electrophoresis and Fluorograph...

Previous investigators have reported the presence of two dominant proteins, tectonin I (25 kDa) and tectonin II (39 kDa), in nuclei and nuclear matrix from plasmodia of Physarum polycephalum. We demonstrate, by a modification of the nuclear isolation protocol and by protease sensitivity, that the tectonins are not nuclear proteins but rather are located on the exterior surface of the plasma membrane.We report the sequences of cDNAs of tectonins I and II, which encode 217 and 353 amino acids, respectively. Tectonin I is homologous to the C-terminal two-thirds of tectonin II. Both proteins contain six tandem repeats that are each 33-37 amino acids in length and define a new consensus sequence. Homologous repeats are found in L-6, a bacterial lipopolysaccharide-binding lectin from horseshoe crab hemocytes. The repetitive sequences of the tectonins and L-6 are reminiscent of the WD repeats of the ␤-subunit of G proteins, suggesting that they form ␤-propeller domains. Tectonin II has an additional N-terminal domain that includes a 47-residue sequence highly similar to the galactoside-binding sequence of the B-chain of ricin. The tectonins may be lectins that function as part of a transmembrane signaling complex during phagocytosis.In its plasmodial form, the myxomycete Physarum polycephalum exists as a multinucleated syncytium that feeds on bacteria and organic detritus by phagocytosis. The many nuclei within a single plasmodium progress through the cell cycle synchronously, and at the end of the G 2 phase undergo closed mitosis. Because of these characteristics, several investigators have examined the P. polycephalum nuclear matrix (1-4) and reported that, as with mammalian nuclear matrix, the P. polycephalum matrix contained a number of proteins ranging from approximately 40 kDa to more than 100 kDa but that it differed from mammalian nuclear matrix by having two dominant proteins with reported molecular masses of 23-28 and 35-38 kDa as determined by SDS-PAGE.1 The same proteins have also been found associated with purified rDNA chromatin (5). We have termed these proteins tectonins I and II, respectively.In the present study we report the cloning and sequencing of the cDNAs for the tectonins, and we use trypsin digestion of cell fractions to assess their localization in the plasmodium. We find that the tectonins are not nuclear proteins but instead are located on the plasmodial surface, and we report a method for purifying P. polycephalum nuclei not contaminated with the tectonins.The tectonins share with lectin L-6 of horseshoe crab hemocytes (6) six repeats of a novel consensus sequence that may form a ␤-propeller structure. Additionally, tectonin II contains in its N-terminal region a sequence similar to the galactosebinding domain of the B-chain of the plant toxin ricin (7). The tectonins may share with lectin L-6 the ability to recognize the outer membrane lipopolysaccharide of Gram-negative bacteria for phagocytosis and utilize an additional affinity for galactose to expand the number of ligands that they re...

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Plasmodial transglutaminase of Physarum polycephalum was purified by anion exchange and hydrophobic chromatography. Gel filtration and SDS-polyacrylamide gel electrophoresis indicate that it is a monomer of 96 -101 kDa. It is Ca 2؉ -dependent, with half-maximal activity at 0.7 mM Ca 2؉ . Optimal activity occurs at pH 7.5 and at 50 mM KCl. Inactivation by N-ethylmaleimide indicates that it is a thiol enzyme. With N,N-dimethylcasein as substrate, the K m for monodansylcadaverine is 33.9 ؎ 1.8 M. Damage of plasmodia by brief treatment with 15% ethanol activates the transglutaminase, with rapid accumulation of cross-linked proteins unable to enter gels during SDS-polyacrylamide gel electrophoresis. Added monodansylcadaverine is conjugated principally to LAV1-2, a plasmodia-specific 40-kDa protein with four EF-hand sequences believed to bind Ca 2؉ . Actin is seen as an additional substrate only in plasmodial homogenates. Immunoblots show that upon ethanol treatment, a portion of LAV1-2 is modified quickly and shifts to 36 kDa; another portion is cross-linked to itself or other proteins. The modification of LAV1-2 may lead to localized release of Ca 2؉ and activation of transglutaminase for walling off damaged areas of plasmodia. No significant increase in amount of the transglutaminase occurs during starvation-induced differentiation of plasmodia to form spherules, but a 50% reduction in the amount of total protein leads to a doubling in the specific mass of the TGase. Neither the transglutaminase nor LAV1-2 is found in the ameboid form of the organism.