Evidences that human disturbance simplify the ant fauna associated a Stachytarpheta glabra Cham. (Verbenaceae) compromising the benefits of ant-plant mutualism.

Interaction among species, like ants and plants through extrafloral nectaries (EFNs), are important components of ecological communities' evolution. However, the effect of human disturbance on such specific interactions and its ecological consequences is poorly understood. This study evaluated the outcomes of mutualism between ants and the EFN-bearing plant Stachytarpheta glabra under anthropogenic disturbance. We compared the arthropod fauna composition between two groups of twenty plant individuals, one in an area disturbed by human activities and one in a preserved area. We also check the plant investment in herbivory defense and the consequential leaf damage by herbivore. Our results indicate that such disturbances cause simplification of the associated fauna and lack of proper ant mutualist. This led to four times more herbivory on plants of disturbed areas, despite the equal amount of EFN and ant visitors and low abundance of herbivores. The high pressure of herbivory may difficult the re-establishment of S. glabra, an important pioneer species in ferruginous fields, therefore it may affect resilience of this fragile ecological community.

Composition and functional characterization of yeast 66S ribosome assembly intermediates.

The pathway and complete collection of factors that orchestrate ribosome assembly are not clear. To address these problems, we affinity purified yeast preribosomal particles containing the nucleolar protein Nop7p and developed means to separate their components. Nop7p is associated primarily with 66S preribosomes containing either 27SB or 25.5S plus 7S pre-rRNAs. Copurifying proteins identified by mass spectrometry include ribosomal proteins, nonribosomal proteins previously implicated in 60S ribosome biogenesis, and proteins not known to be involved in ribosome production. Analysis of strains mutant for eight of these proteins not previously implicated in ribosome biogenesis showed that they do participate in this pathway. These results demonstrate that proteomic approaches in concert with genetic tools provide powerful means to purify and characterize ribosome assembly intermediates.

Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity.

Mass spectrometry is a powerful technique for the identification of proteins at nanogram quantities. However, some degree of sample preparation prior to mass spectrometry is required, and silver-stained protein gel samples are most problematic. Here we report our strategy to obtain peptide mass profiles from silver-stained protein gel samples from one- or two-dimensional gels by destaining prior to enzymatic digestion. This study demonstrates that by using the destaining method, the sensitivity and quality of mass spectra is increased for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis, permitting more proteins to be identified by peptide mass database analysis.

Neutron and x-ray scatter studies of the histone octamer and amino and carboxyl domain trimmed octamers.

The structure of the nucleosome has been under intense investigation using neutron crystallography, x-ray crystallography, and neutron solution scattering. However the dimension of the histone octamer inside the nucleosome is still a subject of controversy. The radius of gyration (Rg) of the octamer obtained from solution neutron scattering of core particles at 63% 2H2O, 37% 1H2O is 33 A, and x-ray crystallography study of isolated histone octamer gives a Rg of 32.5 A, while the reported values using x-ray crystallography of core particles from two individual studies are 29.7 and 30.4 A, respectively. We report here studies of isolated histone octamer and trypsin-limited digested octamer using both neutron solution scattering and small angle x-ray scattering. The Rg of the octamer obtained is 33 A, whereas that of the trimmed octamer is 29.8 A, similar to the structure obtained from the crystals of the core particles. The N-terminal domains of the core histones in the octamer have been shown by high resolution nuclear magnetic resonance (Schroth, G.P., Yau, P., Imai, B.S., Gatewood, J.M., and Bradbury, E.M. (1990) FEBS Lett. 268, 117-120) to be mobile and flexible; it is likely that these regions are disordered and "not seen" by x-ray crystallography.

A NMR study of mobility in the histone octamer.

The histone octamer from chicken erythrocytes was studied in 2 M NaCl using 500 mHz 1H NMR spectroscopy. We compared the spectrum of control octamers with that of octamers isolated from trypsinized nucleosome core particles. We observe that the sharp resonances found in the spectrum of the native octamer disappear completely after trypsinization. Therefore, within the time frame of the NMR experiment, all of the mobile amino acid residues in the histone octamer are found in the well defined trypsin sensitive domains. These results indicate that there is a very clear structural demarcation between the random coil N- and C-terminal tails and the globular domains of the histones.

Histone acetylation reduces nucleosome core particle linking number change.

Nucleosome core particles differing in their levels of histone acetylation have been formed on a closed circular DNA that contains a tandemly repeated 207 bp nucleosome positioning sequence. The effect of acetylation on the linking number per nucleosome particle has been determined. With increasing levels of acetylation, the negative linking number change per nucleosome decreases from -1.04 +/- 0.08 for control to -0.82 +/- 0.05 for highly acetylated nucleosomes. These results indicate that histone acetylation has the ability to release negative supercoils previously constrained by nucleosomes into a closed chromatin loop and in effect function as a eukaryotic gyrase.

Hyperacetylation of core histones does not cause unfolding of nucleosomes. Neutron scatter data accords with disc shape of the nucleosome.

Recent studies report that the frictional resistance of partially acetylated core particles increases when the number of acetyl groups/particle exceeds 10 (Bode, J., Gomez-Lira, M. M. & Schröter, H. (1983) Eur. J. Biochem. 130, 437-445). This was attributed to an opening of the core particle though other explanations, e.g. unwinding of the DNA ends were also suggested. Another possible explanation is that release of the core histone N-terminal domains by acetylation increased the frictional resistance of the particle. Neutron scatter studies have been performed on core particles acetylated to different levels up to 2.4 acetates/H4 molecule. Up to this level of acetylation the neutron scatter data show no evidence for unfolding of the core particle. The fundamental scatter functions for the envelope shape and internal structure are identical to those obtained previously for bulk core particles. The structure that gave the best fit to these fundamental scatter functions was a flat disc of diameter 11-11.5 nm and of thickness 5.5-6 nm with 1.7 +/- 0.2 turns of DNA coiled with a pitch of 3.0 nm around a core of the histone octamer. The data analysis emphasizes the changes in pair distance distribution functions at relatively low contrasts, particularly when the protein is contrast matched and DNA dominates the scatter. Under these conditions there is no evidence for the unwinding of long DNA ends in the hyperacetylated core particles. The distance distribution functions go to zero between 11.5 and 12 nm which gives the maximum chord length in a particle of dimension, 11 nm X 5.5 nm. The distance distribution function for the histone octamer contains 85% of the vectors within the 7.0-nm diameter of the histone core. 15% of the histone vectors lie between 7.0 and 12.0 nm, and these are attributed to the N-terminal domains of the core histones which extend out from the central histone core. Histone vectors extending beyond 7.0 nm are necessary to account for the measured radius of gyration of the histone core of 3.3 nm. A similar value of 3.2 nm is calculated for the recent ellipsoidal shape of 11.0 X 6.5 X 6.5 nm from the crystal structure of the octamer. However, the nucleosome model based on this structure is globular, roughly 11 nm in diameter, which does not accord with the flat disc shape core particle obtained from detailed neutron scatter data nor with the cross-section radii of gyration of the histone and DNA found previously for extended chromatin in solution.

Effect of HMG protein 17 on the thermal stability of control and acetylated HeLa oligonucleosomes.

Many studies have implicated histone acetylation and HMG proteins 14 and 17 in the structure of active chromatin. Studies of the binding of HMG 14 and 17 to chromatin core particles have shown that there are two binding sites for HMG 14 or 17 located within 20-25 bp of the DNA ends of the core particles [13-15]. Such binding sites may result from the free DNA ends in the core particle being available for the binding of HMG 14 and 17. We have studied the effects of the binding of HMG 17 on the thermal denaturation of DNA in mono, di and trinucleosomes. In each case the binding of 1 HMG 17 molecule per nucleosome reduces the DNA premelt region by 50%, while the binding of 2 HMG 17 molecules per nucleosome abolishes the premelt region. From this it is concluded that there are two HMG 17 binding sites per nucleosome which are located between the entry and exit points to the nucleosome and the strongly complexed central DNA region. Highly acetylated mono, di and trinucleosomes have been isolated from butyrate treated HeLa S3 cells. For this series of acetylated oligonucleosomes, it has been found that there are also two HMG 17 binding sites per acetylated nucleosome.

Thermal denaturation studies of acetylated nucleosomes and oligonucleosomes.

The thermal melting behaviors of control and acetylated mononucleosomes, dinucleosomes and trinucleosomes have been studied. Along each series of oligonucleosomes, the melting profiles change in a manner consistent with the increasing number of nucleosomes. For the control mononucleosome, the melting profile exhibits a premelting region at about 61-64 degrees C and a major cooperative transition at 75-77 degrees C. The melting profiles of the control dinucleosomes and trinucleosomes show a premelt at 61-62 degrees C (similar to that of the nucleosome core); an intermediate transition at 73-74 degrees C for the dinucleosome and at 76-77 degrees C for the trinucleosome and a major cooperative transition at 79-80 degrees C for the dinucleosome and at 81-82 degrees C for the trinucleosome. The major cooperative transition at the highest melting temperatures in the melting profiles of the mononucleosome, dinucleosome and trinucleosome comes from the melting of the central region of DNA in the nucleosome which complexed with the core histones; the premelt region is attributed to two DNA segments per nucleosome which flank this central DNA region and are free or weakly complexed with histones. The origin of the intermediate transition found for the dinucleosomes and trinucleosomes is not fully understood but probably results from the melting of DNA at the entry to and exit from the nucleosome and the linker DNA which are complexed with histones. A very similar pattern of behavior is observed for the acetylated oligonucleosomes. Direct comparison of the melting profiles of acetylated and control mononucleosomes, dinucleosomes and trinucleosomes show that the premelt region is unaffected by histone acetylation whereas the intermediate and major cooperative transitions for the acetylated oligonucleosomes are broader and occur consistently at lower temperatures than for the controls. These differences support proposals that the N-terminal regions of core histones interact within the nucleosome and on linker DNA.