Single Molecule Studies of Chromatin

In eukaryotic cells, DNA is packaged as chromatin, a highly ordered structure formed through the wrapping of the DNA around histone proteins, and further packed through interactions with a number of other proteins. In order for processes such as DNA replication, DNA repair, and transcription to occur, the structure of chromatin must be remodeled such that the necessary enzymes can access the DNA. A number of remodeling enzymes have been described, but our understanding of the remodeling process is hindered by a lack of knowledge of the fine structure of chromatin, and how this structure is modulated in the living cell. We have carried out single molecule experiments using atomic force microscopy (AFM) to study the packaging arrangements in chromatin from a variety of cell types. Comparison of the structures observed reveals differences which can be explained in terms of the cell type and its transcriptional activity. During the course of this project, sample preparation and AFM techniques were developed and optimized. Several opportunities for follow-up work are outlined which could provide further insight into the dynamic structural rearrangements of chromatin

Folding Elastic Thermal Surface - FETS

The FETS is a light and compact thermal surface (sun shade, IR thermal shield, cover, and/or deployable radiator) that is mounted on a set of offset tape-spring hinges. The thermal surface is constrained during launch and activated in space by a thermomechanical latch such as a wax actuator. An application-specific embodiment of this technology developed for the MATMOS (Mars Atmospheric Trace Molecule Occultation Spectrometer) project serves as a deployable cover and thermal shield for its passive cooler. The FETS fits compactly against the instrument within the constrained launch envelope, and then unfolds into a larger area once in space. In this application, the FETS protects the passive cooler from thermal damage and contamination during ground operations, launch, and during orbit insertion. Once unfolded or deployed, the FETS serves as a heat shield, intercepting parasitic heat loads by blocking the passive cooler s view of the warm spacecraft. The technology significantly enhances the capabilities of instruments requiring either active or passive cooling of optical detectors. This can be particularly important for instruments where performance is limited by the available radiator area. Examples would be IR optical instruments on CubeSATs or those launched as hosted payloads because radiator area is limited and views are often undesirable. As a deployable radiator, the panels making up the FETS are linked thermally by thermal straps and heat pipes; the structural support and deployment energy is provided using tape-spring hinges. The FETS is a novel combination of existing technologies. Prior art for deployable heat shields uses rotating hinges that typically must be lubricated to avoid cold welding or static friction. By using tape-spring hinges, the FETS avoids the need for lubricants by avoiding friction altogether. This also eliminates the potential for contamination of nearby cooled optics by outgassing lubricants. Furthermore, the tape-spring design of the FETS is also self-locking so the panels stay in a rigid and extended configuration after deployment. This unexpected benefit makes the tape-spring hinge design of the FETS a light, simple, reliable, compact, non-outgassing hinge, spring, and latch. While tape-spring hinges are not novel, they have never been used to deploy passive unfolding thermal surfaces (radiator panels, covers, sun shades, or IR thermal shields). Furthermore, because this technology is compact, it has minimal impact on the launch envelope and mass specifications. FETS enhances the performance of hosted payload instruments where the science data is limited by dark noise. Incorporating FETS into a thermal control system increases radiator area, which lowers the optical detector temperature. This results in higher SNR (signal-to-noise ratio) and improved science data

A Study of the Structure and Metabolic Processes of a Novel Membrane Cytochrome in an Extreme Microbial Community

The action of iron oxidizing microbes can generate acid mine drainage (AMD), characterized by acidic, toxic metal-tainted water that pollutes various water resources. The acidophilic biofilm community populating the Richmond mine, a pyrite (FeS{sub 2}) deposit in Northern California, is a key component of the oxidation of Fe(II) as well as subsequent pyrite dissolution. These natural biofilms contain many novel proteins that are being studied in order to understand how these microbes oxidize iron. The focus of this study is on the structure and characteristics of one novel, abundant outer membrane protein, cytochrome 572 (Cyt{sub 572}), which is perhaps important to the function of the entire community. To detect and study this cytochrome, monoclonal antibodies (mAb) were produced and screened for specificity to Cyt{sub 572}, both purified and membrane-bound. This was accomplished using enzyme linked immunosorbent assay (ELISA) and western blot analysis. Using western blotting, the presence of three high molecular weight bands at positions of dimer, trimer and tetramer corroborate chromatographic results that Cyt{sub 572} is a tetramer. Immunoprecipitation was used to detect a Cyt{sub 572} specific multiprotein complex, and these experiments are in progress. Apart from its novel amino acid sequence, Cyt{sub 572} binds to a heme group that exhibits unique spectral properties. To characterize the heme further, several biochemical methods were used to examine the purified cytochrome. Ethidium bromide was used in a novel way to detect proteins containing heme. The smallest heme-binding polypeptide fragment, about 23kDa, was identified by gel electrophoresis after proteolytic digestion of purified Cyt{sub 572}. The inability of these enzyme digests to completely degrade the protein reveals a secondary structure protective mechanism surrounding the heme group. This is perhaps associated with biofilm membrane proteins like Cyt{sub 572} that are in contact with an extremely acidic environment. Heme-protein interactions and higher order protein complexes have likely been selected for stability and the efficient transfer of electrons from Fe(II) to cells for energy, an essential function in the biofilm community

Influence of a Guanidine Riboswitch in Bacterial Cells

Bacteria live in environments containing complex ecologies of other microbes that communicate and survive through the action of a variety of small metabolic compounds. One common yet relatively unstudied metabolite is guanidine. Although it can be toxic to cells, recent studies have revealed that guanidine may function as a cellular metabolite through a specialized RNA sequence known as a riboswitch. Within our overall project on improving algal productivity, the focus of this study is to: (a) describe various guanidine riboswitch sequences in bacteria that interact with biofuel producing algae; and (b) determine if guanidine has a positive or negative influence on the growth of the bacteria containing these riboswitches. Over 2,000 species across four phyla of bacteria contain genes that help overcome guanidine toxicity. Recently it was discovered that guanidine, a small molecule with three nitrogen linked to a single carbon, regulates some of these genes by specific interactions with a segment of mRNA called a riboswitch. In this investigation, we used the largely uncharacterized cyanobacterium ESFC-1, and others across the four phyla, that contain the guanidine riboswitch, of which there are two subtypes. Both of these two subtypes regulate expression of proteins involved in the export and modification of guanidine inside the bacterial cell. Genome sequence analysis of our guanidine riboswitches indicate that our test bacteria differ in four key highly conserved residues for a guanidine-binding pocket in the model riboswitch. However, structures of the riboswitches may be similar, indicating their functions and guanidine-binding capabilities may be similar

A life in progress: motion and emotion in the autobiography of Robert M. La Follette

This article is a study of a La Follette’s Autobiography, the autobiography of the leading Wisconsin progressive Robert M. La Follette, which was published serially in 1911 and, in book form, in 1913. Rather than focusing, as have other historians, on which parts of La Follette’s account are accurate and can therefore be trusted, it explains instead why and how this major autobiography was conceived and written. The article shows that the autobiography was the product of a sustained, complex, and often fraught series of collaborations among La Follette’s family, friends, and political allies, and in the process illuminates the importance of affective ties as well as political ambition and commitment in bringing the project to fruition. In the world of progressive reform, it argues, personal and political experiences were inseparable

A Semi-Quantitative, Synteny-Based Method to Improve Functional Predictions for Hypothetical and Poorly Annotated Bacterial and Archaeal Genes

During microbial evolution, genome rearrangement increases with increasing sequence divergence. If the relationship between synteny and sequence divergence can be modeled, gene clusters in genomes of distantly related organisms exhibiting anomalous synteny can be identified and used to infer functional conservation. We applied the phylogenetic pairwise comparison method to establish and model a strong correlation between synteny and sequence divergence in all 634 available Archaeal and Bacterial genomes from the NCBI database and four newly assembled genomes of uncultivated Archaea from an acid mine drainage (AMD) community. In parallel, we established and modeled the trend between synteny and functional relatedness in the 118 genomes available in the STRING database. By combining these models, we developed a gene functional annotation method that weights evolutionary distance to estimate the probability of functional associations of syntenous proteins between genome pairs. The method was applied to the hypothetical proteins and poorly annotated genes in newly assembled acid mine drainage Archaeal genomes to add or improve gene annotations. This is the first method to assign possible functions to poorly annotated genes through quantification of the probability of gene functional relationships based on synteny at a significant evolutionary distance, and has the potential for broad application

Reconstitution of Human Excision Nuclease with Recombinant XPF-ERCC1 Complex

The human XPF-ERCC1 protein complex is one of several factors known to be required for general nucleotide excision repair. Genetic data indicate that both proteins of this complex are necessary for the repair of interstrand cross-links, perhaps via recombination. To determine whether XPF-ERCC1 completes a set of six proteins that are sufficient to carry out excision repair, the human XPF and ERCC1 cDNAs were coexpressed in Sf21 insect cells from a baculovirus vector. The purified complex contained the anticipated 5' junction-specific endonuclease activity that is stimulated through a direct interaction between XPF and replication protein A (RPA). The recombinant complex also complemented extracts of XP-F cells and Chinese hamster ovary mutants assigned to complementation groups 1, 4, and 11. Furthermore, reconstitution of the human excision nuclease was observed with a mixture of five repair factors (XPA, XPC, XPG, TFIIH, and RPA) and the recombinant XPF-ERCC1, thus verifying that no additional protein factors are needed for the specific dual incisions characteristic of human excision repair

Using quantitative proteomics of Arabidopsis roots and leaves to predict metabolic activity

Proteins isolated from developing roots and leaves of Arabidopsis thaliana were separated by high-resolution two-dimensional (2-D) electrophoresis. The resulting 2-D proteome maps are markedly different. Quantitative analysis of root and leaf protein spot pairs revealed that in most instances there was at least a 1.5-fold differential. Peptide mass fingerprint analysis of the 288 most abundant 2-D spots from each organ allowed 156 and 126 protein assignments for roots and leaves, respectively, 54 of which were common. Metabolismrelated proteins accounted for 20% of assignments in samples from both organs, whereas energy-related proteins comprised 25 and 18% of leaf and root samples, respectively. Proteins involved in disease resistance and defense encompass 13% of root proteins, but only 7% of leaf proteins. Comparison of protein abundance with transcript abundance, using previously reported microarray data, yielded a correlation coefficient of approximately 0.6, suggesting that it is inappropriate to make protein level or metabolic conclusions based solely upon data from transcript profiling. A comparative model of root and leaf metabolism was developed, based upon protein rather than transcript abundance. The model indicates elevated one-carbon and tricarboxylic acid metabolism in roots relative to leaves