Single nucleotide in the MTF-1 binding site can determine metal-specific transcription activation.

Cells respond to changes in environment by shifting their gene expression profile to deal with the new conditions. The cellular response to changes in metal homeostasis is an important example of this. Transition metals such as iron, zinc, and copper are essential micronutrients but other metals such as cadmium are simply toxic. The cell must maintain metal concentrations in a window that supports efficient metabolic function but must also protect against the damaging effects of high concentrations of these metals. One way a cell regulates metal homeostasis is to control genes involved in metal mobilization and storage. Much of this regulation occurs at the level of transcription and the protein most responsible for this is the conserved metal responsive transcription factor 1 (MTF-1). Interestingly, the nature of the changes in the gene expression profile depends on the type of exposure. The cell somehow senses the kind of the metal challenge and responds appropriately. We have been using the Drosophila system to try to understand the mechanism of this metal discrimination. Using genome-wide mapping of MTF-1 binding under different metal stresses we find that, surprisingly, MTF-1 chooses different DNA binding sites depending on the specific nature of the metal insult. We also find that the type of binding site chosen is an important component of the capability to induce the metal-specific transcription activation.

Mapping assembly favored and remodeled nucleosome positions on polynucleosomal templates.

Positioning of nucleosomes regulates the access of DNA binding factors to their consensus sequences. Nucleosome positions are determined, at least in part by the effects of DNA sequence during nucleosome assembly. Nucleosomes can also be repositioned (moved in cis) by ATP-dependent nucleosome remodeling complexes. Most studies of repositioning have used short DNA fragments containing a single nucleosome. It is difficult to use this type of template to analyze the role of DNA sequence in repositioning, however, because the many remodeling complexes are strongly influenced by nearby DNA ends. Mononucleosomal templates also cannot provide information about how repositioning occurs in the context of chromatin, where the presence of flanking nucleosomes could limit repositioning options. This protocol describes a newly developed method that allows the mapping of nucleosome positions (with and without remodeling) on any chosen region of a plasmid polynucleosomal template in vitro. The approach uses MNase digestion to release nucleosome-protected DNA fragments, followed by restriction enzyme digestion to locally unique sites, and Southern blotting, to provide a comprehensive map of nucleosome positions within a probe region. It was developed as part of studies which showed that human remodeling enzymes tended to move nucleosomes away from high affinity nucleosome positioning sequences, and also that there were differences in repositioning specificity between different remodeling complexes.

Multiple distinct stimuli increase measured nucleosome occupancy around human promoters.

Nucleosomes can block access to transcription factors. Thus the precise localization of nucleosomes relative to transcription start sites and other factor binding sites is expected to be a critical component of transcriptional regulation. Recently developed microarray approaches have allowed the rapid mapping of nucleosome positions over hundreds of kilobases (kb) of human genomic DNA, although these approaches have not yet been widely used to measure chromatin changes associated with changes in transcription. Here, we use custom tiling microarrays to reveal changes in nucleosome positions and abundance that occur when hormone-bound glucocorticoid receptor (GR) binds to sites near target gene promoters in human osteosarcoma cells. The most striking change is an increase in measured nucleosome occupancy at sites spanning ∼1 kb upstream and downstream of transcription start sites, which occurs one hour after addition of hormone, but is lost at 4 hours. Unexpectedly, this increase was seen both on GR-regulated and GR-non-regulated genes. In addition, the human SWI/SNF chromatin remodeling factor (a GR co-activator) was found to be important for increased occupancy upon hormone treatment and also for low nucleosome occupancy without hormone. Most surprisingly, similar increases in nucleosome occupancy were also seen on both regulated and non-regulated promoters during differentiation of human myeloid leukemia cells and upon activation of human CD4+ T-cells. These results indicate that dramatic changes in chromatin structure over ∼2 kb of human promoters may occur genomewide and in response to a variety of stimuli, and suggest novel models for transcriptional regulation.

Process groups.

A Process Group is a group that studies its own behavior to enable its members to learn about group dynamics, individual dynamics, and interpersonal communications. (Swiller, Lang, & Halperin, 1993) Not only is social life identical with communication, but all communication (and hence all genuine social life) is educative. To be a recipient of a communication is to have an enlarged and changed experience. (Dewey, 1916).

Human SWI/SNF directs sequence-specific chromatin changes on promoter polynucleosomes.

Studies in humans and other species have revealed that a surprisingly large fraction of nucleosomes adopt specific positions on promoters, and that these positions appear to be determined by nucleosome positioning DNA sequences (NPSs). Recent studies by our lab, using minicircles containing only one nucleosome, indicated that the human SWI/SNF complex (hSWI/SNF) prefers to relocate nucleosomes away from NPSs. We now make use of novel mapping techniques to examine the hSWI/SNF sequence preference for nucleosome movement in the context of polynucleosomal chromatin, where adjacent nucleosomes can limit movement and where hSWI/SNF forms altered dinucleosomal structures. Using two NPS templates (5S rDNA and 601) and two hSWI/SNF target promoter templates (c-myc and UGT1A1), we observed hSWI/SNF-driven depletion of normal mononucleosomes from almost all positions that were strongly favored by assembly. In some cases, these mononucleosomes were moved to hSWI/SNF-preferred sequences. In the majority of other cases, one repositioned mononucleosome appeared to combine with an unmoved mononucleosome forming a specifically localized altered or normal dinucleosome. These effects result in dramatic, template-specific changes in nucleosomal distribution. Taken together, these studies indicate hSWI/SNF is likely to activate or repress transcription of its target genes by generating promoter sequence-specific changes in chromatin configuration.

Human SWI/SNF drives sequence-directed repositioning of nucleosomes on C-myc promoter DNA minicircles.

The human SWI/SNF (hSWI/SNF) ATP-dependent chromatin remodeling complex is a tumor suppressor and essential transcriptional coregulator. SWI/SNF complexes have been shown to alter nucleosome positions, and this activity is likely to be important for their functions. However, previous studies have largely been unable to determine the extent to which DNA sequence might control nucleosome repositioning by SWI/SNF complexes. Here, we employ a minicircle remodeling approach to provide the first evidence that hSWI/SNF moves nucleosomes in a sequence dependent manner, away from nucleosome positioning sequences favored during nucleosome assembly. This repositioning is unaffected by the presence of DNA nicks, and can occur on closed-circular DNAs in the absence of topoisomerases. We observed directed nucleosome movement on minicircles derived from the human SWI/SNF-regulated c-myc promoter, which may contribute to the previously observed "disruption" of two promoter nucleosomes during c-myc activation in vivo. Our results suggest a model wherein hSWI/SNF-directed nucleosome movement away from default positioning sequences results in sequence-specific regulatory effects.

Initial steps in Streptococcus pneumoniae interaction with and pathogenicity to the host.

Streptococcus pneumoniae (Pnc) is one of the leading pathogens in the world. Attachment to respiratory mucosal and lung surfaces is presumed to be involved in carriage, in disease and in the interaction with macrophages initiating innate immune responses. We hypothesized that bacterial adhesins mediate Pnc adhesion and host cell invasiveness. Initial studies have focused on the purification of cell wall and membrane proteins using fetuin affinity chromatography, SDS PAGE and western blot analysis probed with pooled healthy human sera. Using a Pnc clinical isolate, and a gpt mutant we have detected 10-lectin proteins isolated from the cell wall and adherent to the affinity column and 15 lectins isolated from membrane extracts. The fetuin-captured lectins agglutinated rabbit erythrocytes. 15 proteins in the cell wall and 18 proteins in the membrane that failed to bind to the fetuin column did not agglutinate rabbit erythrocytes. Further purification of the cell wall and membrane fetuin-separated fractions was achieved via anion exchange FPLC, was verified by SDS PAGE. These proteins maintained their agglutinating activity, and were subsequently tested for their ability to interfere with Pnc adhesion and invasion of epithelial cells in culture. Additional biochemical, immunological and molecular techniques are being used in attempt to identify relevant proteins.

Estudio de los aspectos de la contaminación de los suelos relacionados con la salud pública / Study of the aspects of soil pollution and public health

To give an idea of the extent and long history of soil pollution as a public health problem, reference is made to the parasitic condition of the sick rural population, the decrease in living space and the consequent pollution brought on by overpopulation and growth of urban areas and industry throughout the world; reference is also made to the attempts at solution of this situation begun in Biblical times when "the efficient hygienic disposal of excrement... to combat contamination of the soil" was called for. Soil pollution is produced by pathogenic microorganisms which may be clasified in three categories: those expelled by man (man-soil-man), those transmitted by animals (animal-soil-man), and those which naturally exist in the soild (soil-man). Precise details are given - including incidence, infectious agents, reservoirs and method of communication - for the twenty most important diseases brought on in man by these microorganisms

In addition to the pathogenic agents of biological origin, there are also chemical and radioactive agents which pollute the soil and expose man to new health hazards. In this connection, mention is made of the use of pesticides and herbicides, which has increased enormously in the last 25 years, and the accumulation of radioactive materials resulting from atomic explosions ...(AU)

Estudio de los aspectos de la contaminación de los suelos relacionados con la salud pública / Study of the aspects of soil pollution and public health

To give an idea of the extent and long history of soil pollution as a public health problem, reference is made to the parasitic condition of the sick rural population, the decrease in living space and the consequent pollution brought on by overpopulation and growth of urban areas and industry throughout the world; reference is also made to the attempts at solution of this situation begun in Biblical times when "the efficient hygienic disposal of excrement... to combat contamination of the soil" was called for. Soil pollution is produced by pathogenic microorganisms which may be clasified in three categories: those expelled by man (man-soil-man), those transmitted by animals (animal-soil-man), and those which naturally exist in the soild (soil-man). Precise details are given - including incidence, infectious agents, reservoirs and method of communication - for the twenty most important diseases brought on in man by these microorganisms

In addition to the pathogenic agents of biological origin, there are also chemical and radioactive agents which pollute the soil and expose man to new health hazards. In this connection, mention is made of the use of pesticides and herbicides, which has increased enormously in the last 25 years, and the accumulation of radioactive materials resulting from atomic explosions ...(AU)