Merck-AAAS

POMONA COLLEGE MERCK/AAAS Undergraduate Science Research Program

Understanding Ligand-Biomacromolecule Interactions at the Biophysical and Molecular Levels

PROJECTS

PROJECT 1: Discovery and Characterization of Novel Extremeophilic Homing Endonucleases. (Seligman, Crane). These studies will help us understand the prevalence of homing endonucleases (HEs) in a range of deeply branching extremophilic bacteria and archaea, while at the same time engaging in the characterization of these endonucleases at the molecular level. The overall goal of this work is to understand the role that homing endonculeases play in the evolution of genomes.

HEs have been found in several extremophilic archaea, and we propose that an examination of a range of 16S and 23S rRNA genes from microbes from extreme environments will yield unique HEs.

Sequence-based methods enable the survey of microbial populations without culturing individual organisms. Sequences commonly examined in such studies include the 16S and 23S rRNA genes. Since HEs are commonly found in introns in these genes, putative HEs have been fortuitously detected in environmental surveys.¹ Such sequences were found in hyperthermophiles closely related to Pyrobaculum from a subsurface environment 1,500 m beneath the earth, and from a mat in a gold mine hot water stream. Whether these sequences encode functional enzymes is an open question. Based upon these results, environmental studies directed at detecting the sequence of HEs are likely to be successful.

The Seligman lab uses E. coli based genetic assays and in vitro analyses to examine HE structure and function, and to engineer HEs with novel DNA target specificities.² While most studies have focused on a chloroplast derived HE, the lab has recently demonstrating that HEs from other sources function in E. coli. Thus, the lab is uniquely positioned to carry out both genetic and biochemical analysis on HE sequences identified by environmental surveys.

The Crane group is interested in microbial diversity at geothermal sites that range from acidic to basic and from 50–95°C. We will begin our search for novel HEs by examining the 16S and 23S genes in sequences that we have previously detected and in new sequences we discover. We can initiate our studies of novel archaea-based HEs by using genes already identified in other environmental surveys.¹

The discovery of introns and inteins in a range of procaryotic sources suggests a role for these elements and the HEs whose coding region is contained within them throughout the three domains in life. This view of the ubiquitous nature of these elements, as well as their presence in deeply branching members of the archaea, suggests a role for HEs in the early evolution of genomes, where their function appears to be to confer mobility to the introns and inteins that host them.

PROJECT 2: Polyphenolic Damage and Repair in Yeast and Mammalian DNA. (Cavalcanti, Hoopes, Johal, Negritto & Selassie). Polyphenolic compounds such as flavanoids which are ubiquitous in fruits and vegetables are natural anti-oxidants but also reveal potentially harmful pro-oxidant properties. Our objectives in the present study are to investigate the level of DNA damage induced by these compounds in yeast and mammalian cells, to analyze changes in DNA expression via microarrays, to assess the extent of DNA-polyphenol interactions, and to delineate the role of nucleotide excision repair (NER) pathway and homologous recombination (HR) in DNA repair in yeast.

The propensity of polyphenolics to readily form radicals in the presence of metal ions suggests an ability to induce DNA damage whose nature, extent and subsequent repair has not been systematically delineated. The deleterious effects of DNA damage and polyphenolic-induced DNA damage on aging and cell growth/viability, respectively, will be determined by an examination of DNA-polyphenol binding profiles, significant repair processes and analysis of the whole genome expression profile through microarray analysis.

The cytotoxicities and apoptosis-inducing abilities of a series of flavanoids (Kaempferol, Genistein, Epicatechin, Quercetin and Morin) will be determined in human HL-60 cells. Quartz-crystal microbalance (QCM) equipped with energy dissipation monitoring will then be used to measure the binding characteristics of flavanoids with and without the presence of Cu²⁺, to calf thymus DNA applied to the surface of a sensitive quartz crystal resonator. Life spans of yeast with isogenic deletions of components of the NER pathway will be compared to wild type growth with and without a flavanoid at an inhibiting dose < its ID₅₀. The levels of HR in flavanoid-treated yeast will also be determined by using a deletion assayl.³ Microarray analysis will be utilized to compare age-related and flavanoid-mediated changes in gene expression levels and these results will be validated using RT-PCR on a small set of affected genes.

The Cavalcanti laboratory will be responsible for the microarray analysis.⁴ The Hoopes laboratory will determine the extent of NER in flavanoid-treated DNA as well as aged DNA while the Johal laboratory will apply calf thymus DNA to the quartz crystal and assess the interactions of flavanoids in the presence/absence of Cu²⁺. The Negritto laboratory will be responsible for determining the degree of HR in the flavanoid-treated yeast and will also validate the microarray results by using RT-PCR. The Selassie laboratory will determine the cytotoxic and apoptotic ID₅₀ values of the flavanoids.⁵

Yeast are eukaryotic cells with cell division regulation similar to mammalian cells. Thus, the roles of DNA repair in yeast aging and polyphenolic exposure are relevant to cancer, aging studies and the consumption of nutraceuticals.

PROJECT 3: Interaction between Rab GDP Dissociation Inhibitor (GDI) and a PUG-UBX Protein, Gint3. (Cheney, Johal). Our objective is to investigate the nature of the interaction between two proteins involved in vesicle targeting using an ultra-sensitive quartz crystal resonator.

The eukaryotic cell cytoplasm has a very active traffic of vesicles that carry materials to be secreted from the cell, as well as vesicles moving material into the cytoplasm from the extracellular environment. It is imperative that these vesicles fuse with the correct target membrane. Rab GTPases are part of the machinery for ensuring correct vesicle targeting.⁶ When a vesicle fuses with its target membrane, the vesicle membrane, with its embedded rabs, becomes part of this membrane. To ensure that rabs do not build up on target membranes, Rab GDP dissociation inhibitor (GDI) removes rabs from target membranes and recycles rabs back to the donor membrane. However, there are many rabs (30 in Drosophila, 60 in humans) and far fewer GDI genes (one in Drosophila, two in humans).

How does GDI release the correct rab to the correct donor membrane? The current hypothesis is that donor membranes contain a receptor complex that binds the rab-GDI complex and triggers the dissociation of rab from GDI. The Cheney lab has identified a candidate receptor, Gint3, using a yeast two-hybrid system.⁷ The goal of the proposed project is to determine whether GDI and Gint3 bind in vitro and whether binding of a rab-GDI complex to Gint3 results in dissociation of rab from GDI.

We propose to use quartz crystal microbalance (QCM) to investigate GDI-Gint3 and rab-GDI-Gint3 interactions. Drosophila Gint3 will be expressed in bacteria as a glutathione-S-transferase (GST) fusion protein, purified and applied to the QCM. GST-GDI will be bacterially expressed and purified. Rab-GDI complexes will be purified from Drosophila containing a GST-GDI transgene. We will measure the binding of GST-GDI to Gint3. Likewise, we will measure the binding of rab-GDI to Gint3 and determine whether rab dissociates from GDI in the presence of Gint3. Bacterially expressed GST-GDI and GST-Gint3 will be purified by students in the Cheney lab. GST-GDI containing rab will be isolated from transgenic flies in the Cheney lab. The Cheney lab has recently generated these flies. In the Johal lab, students will apply Gint3 to the QCM resonator crystal and then measure the interaction of GST-GDI with Gint3 and the interaction of GDI-rab with Gint3. In particular, temperature-programmed QCM measurements will provide protein-protein binding energies, allowing the nature of these interactions to be better understood.

QCM measurements will help determine whether GDI and Gint3 bind in vitro and whether binding of a rab-GDI complex to Gint3 results in dissociation of rab from GDI. These studies will help elucidate the role of these interactions in vesicle targeting and in the subsequent dissociation of rab from GDI.

PROJECT 4: Modeling Diffusion-Limited Components of Insect Gas Exchange. (Garza-López, Wright). Although diffusion has long been considered the primary driving force for gas exchange in insect tracheal systems, recent synchrotron X-ray analysis has shown that tracheal systems are highly dynamic in vivo. This begs the question of whether supplementary convection is involved. Similar questions pertain to the cryptonephric system in the recta of mealworms that functions in fecal dehydration and water vapor absorption (WVA). During WVA, water diffuses into the open rectal canal and proceeds down the vapor pressure gradient across the rectal wall and down an osmotic pressure gradient into the Malpighian tubules.⁸ It must then be moved thermodynamically ‘uphill’ into the blood, possibly by hydrostatic pressure. WVA shows saturation kinetics in high humidity, indicating one or more rate-limiting factors.

Diffusional and hydrostatic water movements will be modeled in these two systems, incorporating physiological and anatomical measurements. It is hypothesized that the anatomy of the insect tracheal system is optimized for diffusional exchange but depends on supplementary forced convection in insects above a few milligrams in mass. Diffusion is predicted to impose upper size limits for insect wings. For WVA, we hypothesize that diffusion of water in the air or water phase will be rate-limiting.

In order to model these diffusion-reaction processes in compartmentalized systems, the defining space (e.g. the tracheal branching pattern) can be represented as a lattice, and the dynamics then studied using the theory of stochastic processes. To study this problem, we assign the exit site to be a trap (or sink) and solve the stochastic master equation: for the specific geometry characterizing the system.⁹ Here is the probability that a diffusing particle has reached site i at time t on a (lattice) system of N sites. is an NxNxN matrix whose elements are the transition probabilities between neighboring sites on the fractal structure. It is straightforward to incorporate temperature effects into these equations.

Tracheal path lengths, diameters, and sequential branching patterns will be measured from digital images of various insects. Metabolic rates and tracheal ventilation patterns will be measured by continuous-flow respirometry. Diffusion path lengths in the cryptonephric complex of mealworms will be measured from dissections and from sections of fixed, embedded specimens. WVA in different temperatures will be quantified gravimetrically.

The proposed modeling can study effects of different partial pressure gradients, tubule morphology, and diffusion of gases in air or water. By incorporating precise anatomical and physiological data, such models provide powerful tools for addressing these long-sought questions. This work should be of broad interest to physiologists, biophysicists and physical chemists, as well as training students in emerging fields at the physiology-chemistry interface.

References:

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2. Rosen, LE; Morrison, H; Masri, S; et al., Nucleic Acids Res. 34, 4791-800 (2006).

3. Schiestl, RH; Gietz, RD; et al., Carcinogenesis 10, 1445 (1989).

4. Kitagawa, E; Momose, Y and Iwahashi, H. Environ. Sci. Technol. 37, 2788 (2003).

5. Selassie, CD; Kapur, S;Verma, RP and Rosario, M. J. Med. Chem. 48, 7234 (2005).

6. Pfeffer, S and Aivazian, D. Nat Rev Mol Cell Biol 5, 886 (2004).

7. Fields, S and Sternglanz, R. Trends Genet. 10, 286 (1994).

8. O’Donnell, MJ and Machin, J. J. Exp. Biol. 155, 375(1991).

9. Garza-López, RA; Lee, C; Lin, D.; et al.. Chem. Phys. Lett. 356, 83 (2002).