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.     

Shrub influences on seedling performance when restoring the slow‐growing conifer Pilgerodendron uviferum in southern bog forests

Forest restoration is most efficient if it can take advantage of facilitative interactions between established vegetation and planted trees. However, positive and negative interactions have been identified in a number of plant communities. After centuries of anthropogenic fires, forest recovery has been extremely slow in southern bog forests previously dominated by the slow‐growing and vulnerable conifer Pilgerodendron uviferum on Chiloé Island, Chile. Today, the landscape is dominated by secondary shrublands with scattered patches of Sphagnum moss and limited natural tree regeneration. We hypothesized that the retention of secondary shrubs facilitates the early performance of P. uviferum restoration plantings by providing better microsite conditions. To test this hypothesis, we compared the response of seedlings planted on sites prepared at two levels of intervention: after shrubs had been removed or where shrubs were retained. Shrub retention showed a nurse‐plant effect on P. uviferum seedlings 4 years after planting, which resulted in reduced physiological stress (measured as Fv/Fm) for seedlings, as well as reduced browsing. Consequently, the seedlings growing in areas with shrub retention had larger height increment and higher vitality than those in areas where shrubs had been removed. Thus, the more open micro‐site conditions created by shrub removal resulted in generally poorer seedling performance, although seedling mortality—which was low overall (approximately 2–4%)—showed no significant difference between the two levels of intervention. These findings have direct implications for the restoration of slow‐growing conifers that can tolerate extreme wet conditions in highly degraded forests.     

The effects of age,energy and protein intake on protein turnover and the expression of proteolysis-related genes in the broiler breeder hen

A study was conducted to determine the changes that occur to proteolysis and related genes due to age, protein, and energy intake in high-yield broiler breeder hens (Gallus gallus). Cobb 700 broiler breeders were randomly assigned to one of six diets in a 2 × 3 factorial fashion. Two levels of energy (390 and 450 kcal/day) and three levels of protein (22, 24, and 26 g CP/day) were utilized. Protein turnover was determined in the left pectoralis at 22, 26, 31 and 44 weeks. Relative mRNA expression of calpain 2 (CAPN2), proteasome C2 subunit (PSMA1), and F box protein 32 (FBXO32) were determined via RT-PCR at 20, 25, and 44 weeks. Contrasts indicate fractional synthesis rate (FSR) and FBXO32 increase to a maximum at 25–26 weeks and a decrease thereafter. A significant drop in PSMA1 and FBXO32 was observed between 25 and 44 weeks and matched the decrease observed in FBR. No differences were detected in the levels of fractional synthesis and degradation, or the expression of CAPN2, PSMA1, and FBXO32, due to protein or energy intake. In summary, protein turnover was upregulated during the transition into sexual maturity and decreased thereafter. The observed changes in degradation appeared to be mediated by the ubiquitin–proteasome pathway.