Increased histone acetylation has been correlated with increased transcription, and regions of heterochromatin are generally hypoacetylated. In investigating the cause-and-effect relationship between histone acetylation and gene activity, we have characterized two yeast histone deacetylase complexes. Histone deacetylase-A (HDA) is an Ϸ350-kDa complex that is highly sensitive to the deacetylase inhibitor trichostatin A. Histone deacetylase-B (HDB) is an Ϸ600-kDa complex that is much less sensitive to trichostatin A. The HDA1 protein (a subunit of the HDA activity) shares sequence similarity to RPD3, a factor required for optimal transcription of certain yeast genes. RPD3 is associated with the HDB activity. HDA1 also shares similarity to three new open reading frames in yeast, designated HOS1, HOS2, and HOS3. We find that both hda1 and rpd3 deletions increase acetylation levels in vivo at all sites examined in both core histones H3 and H4, with rpd3 deletions having a greater impact on histone H4 lysine positions 5 and 12. Surprisingly, both hda1 and rpd3 deletions increase repression at telomeric loci, which resemble heterochromatin with rpd3 having a greater effect. In addition, rpd3 deletions retard full induction of the PHO5 promoter fused to the reporter lacZ. These data demonstrate that histone acetylation state has a role in regulating both heterochromatic silencing and regulated gene expression.

In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group.

Using zero-length covalent protein-DNA crossinking, we have mapp the hstone-DNA cts In nucleosome core particles from which the C-and N-terminal domains of histone H2A were selectively tim by trypsin or clostripain. We found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axds, whereas its glbular domain contacts the end of DNA in the nceme core particle. The appearance of the histoe H2A contact at the dyad axis occurs only in the absence oflinker DNA and does not depend on the absence of linker hit . Our results show the ability of the histone H2A C-terminal domain to rearrange.This rearrangement might play a biological role in nuce disassembly and reassembly and the retention ofthe HZ2A-H2B dimer (or the whole octamer) during the passing ofpolymerases through the nucleosome.The nucleosome as a basic repeating subunit ofchromatin has been studied by various methods and many details of its structure are known (1). However, the questions of the arrangements and behaviors of the flexible histone terminal domains in nucleosomes are not resolved.Although histone tails (exposed and trypsin-sensitive terminal domains) do not affect the conformational saltdependent stability of core particles, they play a significant role in their thermal stability (2). Histone tails do not play a role in determining nucleosome positioning (3) and do not affect the helical periodicity of DNA in isolated nucleosomes (4). However, histone tails have been shown to participate in the folding of oligonucleosomes (5) and in the stabilization of higher-order chromatin structure (6). In addition, they contain sites for reversible post-translational modifications that can modulate chromatin structure (for review, see ref. 7). How histone terminal domains are involved in these interactions is still not clear.Current information about the structure of the histone octamer and the nucleosome is based on the arrangement of histone globular domains or whole histone molecules (8-10). There is little direct data concerning the localization of the flexible histone terminal domains in the nucleosome. Protein-DNA crosslinking experiments have revealed the binding ofthe histone H4 N-terminal domain to DNA at a distance of 1.5 helical turns from either side of the nucleosomal dyad axis (11). In the present study, using zero-length covalent histone-DNA crosslinking, we demonstrate that in isolated core particles the flexible trypsin-sensitive C-terminal domain of histone H2A is bound to the dyad axis, whereas the globular domain is bound to the end ofthe nucleosomal DNA. By taking into consideration the results of our previous histone-DNA crosslinking experiments (10,29,31) and the results of other investigators (26,30,32,34), we discuss the possible arrangement of the histone H2A C-terminal domain in chromatin and in intact nuclei and its rearrangement after the removal of linker DNA. MATERIALS AND METHODSPreparation of Hl-Depleted Chromatinad Core Particles. Soluble chromatin from chicken erythrocyte nuclei wa...

No abstract

We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double-and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.Traditional methods of bacterial identification are usually based on morphological and/or physiological features of a microorganism or on analysis of 16S rRNA gene sequences (59). These methods can require considerable amounts of time. Recently, PCR and other amplification technologies were introduced for bacterial identification (33). Immunological methods (16) and mass spectrometry (18) have also been adapted for this purpose but are expensive or cumbersome. DNA microchip technology (37) advantageously combines a rapid, highthroughput platform for nucleic acid hybridization with low cost and the potential for automation, although sample preparation procedures, including DNA and RNA isolation, fragmentation, and labeling, are still limiting steps (32,44). Another limitation of microarray technology is the lack of portable and inexpensive devices for the acquisition of hybridization patterns (5). We have addressed these shortcomings through the development of a rapid and simple system for sample preparation and microarray analysis. MATERIALS AND METHODSPreparation of silica syringe-operated columns. A silica suspension (50 l) was prepared as described previously (4) and loaded into a 25-mm-long sterile centrifuge device containing a polysulfone filter with a diameter of 6.5 mm and a pore size of 0.2 m (Whatman, Fairfield, N.J.). The column was sealed against the end of a 10-ml syringe without any glue, using the O-ring from a 1.5-ml screw-cap microcentrifuge tube introduced between the syringe and the top of the column, and washed once with 500 l of diethylpyro...

No abstract

No abstract

No abstract