Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid I. Tertiary Structure of Early Replicative and Recombining Deoxyribonucleic Acid

A replicative hybrid resulting from the infection of heavy (substituted with 5-bromodeoxyuridine) bacteria with light (not substituted with 5-bromodeoxyuridine) radioactive bacteriophage was isolated from a CsCl density gradient. Sedimentation studies indicate that 60% of the deoxyribonucleic acid (DNA) behaves as if it were in units more than four times as large as an intact reference molecule. Under the electron microscope, hybrid molecules appeared tangled, showed puffs and loops, occupied a small area, and often had a total length twice that of mature phage. This indicates that sucrose gradient sedimentation is not applicable as a method for estimating the relative molecular size of replicative forms of DNA. After denaturation, the separated strands of hybrid were of the same size as those of reference DNA. CsCl density gradient analysis revealed no terminal covalent addition of new material to the old parental strand. The possibility of a continuous growth of the DNA molecule, either on a single-stranded level or as a double helical structure, is disproved. When chloramphenicol (CM) was added at critical times after infection, DNA synthesis continued at a constant rate. The parental label soon assumed and retained a hybrid density, despite concomitant synthesis of DNA, throughout the rest of the period of incubation in CM. The hybrid moiety, however, actively participated in replication and exchanged its partner strand for a new one; this was demonstrated by changing the density label during incubation in CM. A new enzyme synthesized shortly after infection introduced single-stranded "nicks" into the parental DNA. Since nicking can be inhibited by chloramphenicol, the responsible enzyme is not of host origin. The time of the appearance of this enzyme coincided with the onset of molecular recombination. Another enzyme, which mediates the repair of the continuity of the polynucleotide chain after recombination, appeared after recombination. If selectively inhibited by chloramphenicol, recombinant molecules remained unrepaired, and, upon denaturation, the parental fragment was liberated in pure form.     

Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs

Our objective was to examine the ability of nucleate and anucleate fragments of artificially activated mouse eggs to transform sperm nucleus into male pronucleus. To this end, zona-free oocytes in metaphase II were activated by ethanol and bisected into halves (one with the spindle, the other anucleate) either within 10 to 20 min (series A) or 3 or 5 hr later (series B). In series A, the fragments were inseminated 3,5, and 8 h after activation, and in series B. 3 and 5 h after activation. Both nucleate and anucleate fragments lose the capability of transforming sperm nucleus into fully formed pronucleus sometime between 3 and 5 h after activation. In 8 h old parthenogenetic fragments, the majority of sperm nuclei remain unchanged or begin decondensation but never reach the stage of an early pronucleus. In over 1/3 of anucleate fragments of this age group, sperm nuclei develop defectively: chromatin decondenses inside the persisting nuclear envelope. In other experimental groups, the incidence of these abnormal sperm nuclei varies between 0 and 10%. In general, the anuclcate fragments retain the capability to transform sperm nuclei (fully or partially) longer than their nuclear counterparts. This difference may be accounted for by a different level of substances required for pronuclcar growth (extrachromosomal constituents of the germinal vesicle and nuclear lamins): high and constant in the cytoplasm of anucleate egg halves and low and progressively decreasing in the nucleate halves because of their putative uptake by the female pronucleus. However, the cytoplasmic factors responsible for the initial stages of transformation (nuclear envelope breakdown, chromatin decondensation) become eventually inactivated both in the presence and in the absence of a female pronucleus.     

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

Summary Time courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na⁺ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na⁺) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na⁺) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na⁺ on both membrane sides and with a clamped membrane potential,K _D values of 0.4 and 7.9 M were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence ofd-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na⁺(out>in). Low and high affinity transport could be fitted with identicalK _m values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparentK _m ofhigh affinity transport whereas the apparentK _m of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pighigh and low affinity Na⁺-d-glucose cotransporters are present which containlow and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport moleculc.     

Red-edge excitation fluorescence spectroscopy of proteins in reversed micelles

The dependence of fluorescence emission maxima ofl-tryptophan and single-tryptophan-containing proteins (ribonuclease T1, melittin, and parvalbumin) on excitation wavelength has been studied in reversed micelle systems of sodium bis(2-ethyl-1-oxyl) sulfosuccinate (AOT). No effect of fluorescence maximum shift for different excitation wavelengths is observed for ribonuclease T1, in which a single tryptophan residue is located in the nonrelaxating, nonpolar protein interior.l-Tryptophan and the rest of the studied proteins, which contain single tryptophan residues exposed to the solvent, exhibit the dipolar relaxational processes of partly immobilized water molecules in micelles. This effect depends on the molar H₂O/AOT ratio. Circular dichroism measurements prove that there have been no structural changes of the studied proteins in micellar systems. The results provide information about dynamic relaxational processes in proteins.     

Monte carlo simulations of protein folding. II. Application to protein A,ROP, and crambin

The hierarchy of lattice Monte Carlo models described in the accompanying paper (Kolinski, A., Skolnick, J. Monte Carlo simulations of protein folding. I. Lattice model and interaction scheme. Proteins 18:338–352, 1994) is applied to the simulation of protein folding and the prediction of 3-dimensional structure. Using sequence information alone, three proteins have been successfully folded: the B domain of staphylococcal protein A, a 120 residue, monomeric version of ROP dimer, and crambin. Starting from a random expanded conformation, the model proteins fold along relatively well-defined folding pathways. These involve a collection of early intermediates, which are followed by the final (and rate-determining) transition from compact intermediates closely resembling the molten globule state to the native-like state. The predicted structures are rather unique, with native-like packing of the side chains. The accuracy of the predicted native conformations is better than those obtained in previous folding simulations. The best (but by no means atypical) folds of protein A have a coordinate rms of 2.25 Å from the native Cα trace, and the best coordinate rms from crambin is 3.18 Å. For ROP monomer, the lowest coordinate rms from equivalent Cαs of ROP dimer is 3.65 Å. Thus, for two simple helical proteins and a small α/β protein, the ability to predict protein structure from sequence has been demonstrated. © 1994 John Wiley & Sons, Inc.     

Effect of the Tumor Promoter Phorbol 12-Myristate 13-Acetate (PMA) on Proliferation of Young and Senescent WI-38 Human Diploid Fibroblasts

The effects of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on the proliferation, protein kinase C activity (PKC), and c-fos gene expression were examined in cultures of young and senescent (90-95% lifespan completed) WI-38 human diploid fibroblasts. We observed that, following stimulation with medium containing 10% fetal bovine serum (FBS), the translocation of PKC from the cytosol to the particulate compartment was less efficient in senescent WI-38 cells than in young cells. However, when PMA was added to the medium, the intracellular distribution of PKC activity in old cells became nearly identical to that observed in young cells. The inducibility of c-fos mRNA by serum addition, which is a protein kinase C-dependent event [64], was significantly amplified in the presence of PMA. Moreover, the duration of peak c-fos expression, after stimulation by FBS and PMA, increased in senescent cells as compared to young cells. Our results reveal that the normal signal transduction pathway is altered in senescent, slowly proliferating human fibroblasts and that it can be partially restored in the presence of the tumor promoter PMA.     

Morphology and molecular phylogeny of the marine diatom genus Nagumoea (Bacillariophyceae) from Japan

The canal-bearing diatom genus Nagumoea, described based on only morphological evidence, was tentatively assigned to the order Bacillariales, although its phylogenetic position remained unclear. Because three isolates of Nagumoea (SK002, SK024 and SK053) were successfully established from Japanese coasts, we performed their morphological observations and molecular phylogenetic analyses to discuss the phylogeny and taxonomic position of this genus. Strains SK002 and SK024 were identified as Nagumoea africana, whereas SK053 conformed with Nagumoea serrata. There was high interspecific divergence between N. africana and N. serrata in the rbcL sequences (8.03–8.17%), indicating their distinctness. Furthermore, intraspecific variations were detected within N. africana (2.35%) in the rbcL, implying its cryptic diversity. The maximum likelihood and Bayesian phylogenetic trees inferred from the plastid rbcL, psbC and nuclear 18S rDNA genes recovered Nagumoea as monophyletic with strong statistical support and embedded within an unresolved, poorly supported lineage containing Achnanthes, Craspedostauros, Staurotropis and Undatella in the canal-bearing order Bacillariales (= the family Bacillariaceae). Although the constrained tree based on the monophyly of Nagumoea and the other canal-bearing clade (Surirellales and Rhopalodiales) was statistically rejected by the topology tests, the phylogenetic position of Nagumoea with other Bacillarialean members remains equivocal. The possession of two plastids positioned fore and aft, observed in the present study, and lack of keel, typical of the Bacillariales, indicate the possibility of Nagumoea being part of the ingroup of the Bacillariales or its closely related outgroup.