Adsorption of bacteriophage phi 29 to Bacillus subtilis through the neck appendages of the viral particle.

Phage phi 29 particles produced under restrictive conditions by mutants in gene 12 have normal amounts of all of the structural proteins except the appendage protein, p12*, which is missing. These particles are not infective and do not adsorb to Bacillus subtilis cells. By in vitro complementation of 12- particles with extracts containing protein p12* or with purified protein p12*, the defective particles could bind the appendage protein and become infective and able to adsorb to bacteria. Therefore, the neck appendages of phage phi 29, formed by protein p12*, are involved in the interaction of the phage with the cell wall receptors. Protein p12*, purified in its native state, competed with wild-type phage for adsorption to bacteria. Also, protein p12* could displace adsorbed phage from bacteria. Since the displaced phage was infective, protein p12* does not seem to be modified after phage adsorption.     

Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.     

Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29.

We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with [3H]thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation. During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA. The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope. We found two major types of molecules. One consisted of unit-length duplex DNA with one single-stranded branch at a random position. The length of the single-stranded branches was similar to that of one of the double-stranded regions. The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end. Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end. These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus.     

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.     

In vitro methylation of yeast tRNAAsp by rat brain cortical tRNA-(adenine-1) methyltransferase.

Rat brain cortices from young animals contain large amounts of tRNA (adenine-1)methyltransferase(s). The enzyme(s) can methylate E. coli tRNA and to a lower degree yeast tRNA. Among yeast tRNA species which can be methylated we have selected tRNAAsp as a substrate for the brain enzyme. The digestions of in vitro methylated [Me-3H]-tRNAAsp with pancreatic and/or T1 ribonucleases followed by chromatographies on DEAE-cellulose, 7 M urea, suggested that the methylation of tRNAAsp occurred at a single position within the D-loop. Further digestion of the radioactive oligonucleotide recovered after DEAE-cellulose chromatography by phosphomonoesterase and snake venom phosphodiesterase enzymes followed by bidimensional thin layer chromatography enabled us to determine the location of the adenine residue which becomes methylated by the brain enzyme. This one resulted to be the adenine 14 in the D-loop of yeast tRNAAsp.     

Comparison of the A-T rich regions and the Bacillus subtilis RNA polymerase binding sites in phage phi 29 DNA.

By using a modification of the BAC spreading method for mounting the DNA for electron microscopy, partial denaturation maps of protein-free phi 29 DNA and of phi 29 DNA containing protein p3 were obtained. In phi 29 p3-DNA1 the protein does not seem to influence the melting of the ends of the molecules. The comparison of the partial denaturation map and the B. subtilis RNA polymerase binding sites indicates that five of the seven early promoters (A1, A2, A3, B2 and C2) are located in A-T rich DNA regions whereas the other two early promoters (B1 and C1) are located in less A-T rich sites.     

Inhibition of the respiration of dormant and swollenStreptomyces spores by chloramphenicol

Chloramphenicol produces a decrease in the respiratory quotient of dormant and swollenStreptomyces antibioticus spores of about 20–25% and 18–24%, respectively, in the absence of protein synthesis as measured by [³H]leucine incorporation and by chemical methods. Rifampin and streptomycin had no inhibitory effect on respiration, thus excluding the possibility of an effect of all protein synthesis inhibitors on respiration. The inhibition of respiration by chloramphenicol was not a consequence of an increase in mortality due to toxicity, nor was it influenced by the developmental stage of the spores. It seems that chloramphenicol affects the activity of some component(s) related to the electron transport chain ofS. antibioticus spores, situated before the cytochrome oxidase level.     

Binding of microtubule proteins to DNA: specificity of the interaction.

Tubulin is detected among the DNA-binding proteins when an extract from fibroblasts is chromatographed on DNA-cellulose. Further purification of the colchicine-binding activity shows that purified tubulin from fibroblasts does not bind to DNA. Depolymerized brain microtubule proteins show a high affinity for DNA. The fraction bound is composed of tubulin and microtubule-associated proteins. Experiments with fractionated microtubule proteins indicate that tubulin-free microtubule associated proteins bind to DNA, while tubulin free of microtubule-associated proteins does not. Microtubule-associated proteins bind better to eukaryotic than to phage DNA suggesting a specificity of the interaction.     

Subcellular distribution of DNA-binding proteins from cultured hamster fibroblasts

The distribution between nuclei and cytoplasm of DNA-binding proteins from growing NIL cells was studied. To obtain the subcellular fractions, cell monolayers or cells previously detached from the culture dish were treated with the non-ionic detergent Nonidet P-40. Proteins with affinity for DNA were isolated from nuclear or cytoplasmic fractions by chromatography on DNA-cellulose columns and were further analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results show that P8, one of the major components in the 0.15 M NaCl-eluted proteins, is found predominantly in the cytoplasmic fractions, whereas P6, the other main protein peak in this eluate, is more prominent in the nuclear fraction. Among the other proteins eluted at 0.15 M NaCl from the DNA-cellulose column, P5 and P5′ are detected in both nuclear and cytoplasmic fractions. All the other proteins in the 0.15 M NaCl eluate are present almost exclusively in the cytoplasmic fraction. On the other hand, most of the proteins with higher affinity for DNA, eluted from the column at 2 M NaCl, are present in the nuclear fraction, although they are also detected in the cytoplasm in amounts similar to those observed in the nuclei.     

Deoxyribinucleic acid-binding proteins in virus-transformed cell lines.

The synthesis of proteins with affinity for DNA has been studied in clones of a Syrian hamster cell line (NIL) and subclones of this line transformed by polyoma virus (NIL-Py) or hamster sarcoma virus (NIL-HSV). The results show that the synthesis of DNA-binding proteins in NIL and in its virus-transformed derivatives NIL-Py and NIL-HSV is very similar in exponentially growing cells, but in dense culture there is a very significant difference in the level of a protein (P8), which is much higher in the transformed lines than in untransformed NIL. The high levels of P8 in dense transformed cells have been observed in all the clones of transformed cells examined, indicating that this behavior of P8 is related to transformation and not simply due to a fortuitous clonal selection from the NIL. Experiments with synchronized cells indicate that the time of maximal P8 synthesis relative to cellular DNA synthesis in NIL-HSV precedes that observed in NIL cells. P8 has a molecular weight of 30,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and is present in large amounts in the transformed cells in dense culture, where it makes up 0.5 to 1% of the total soluble protein.