Peter G. Schultz - US grants

Our specific aim is to develop strategies for introducing catalytic activity into the combining sites of immunoglobins. Because antibodies can be elicited against most biologically active macromolecules (proteins, nucleic acids and sugars) as well as small synthetic molecules, this approach may enable us to tailor- make semisynthetic molecules, this approach may enable us to tailor-make semisynthetic catalysts with enzyme-like specificities. We are purusing three strategies for introducing catalytic activity into antibody combining sites: (1) transition state stabilization by antibodies, (2) orientational catalysis by antibodies, and (3) site specific chemical modification of antibody binding sites with nucleophiles and cofactors. Antibodies will be elicited against tetrahedral phosphonates and planar bipyridyls as transition state analogues for hydrolysis of the corresponding esters and racemization of bridged-2,2'-bipyridyls, respectively. Antibodies will also be elicited against a cyclic seven-membered ring phosphonate as a transition state analogue for transesterification of an acyclic 6-hydroxy-ester. In addition, antibodies elicited to peptides and L-amino acids will be affinity labeled with thiols and pyridoxamine to afford catalysts for hydrolytic and transamination reactions, respectively. If successful, this work will be a first step in defining those elements necessary for the design and synthesis of catalytic antibodies with defined binding specificities.

This grant in the Organic Dynamics Program provides the 1988 Alan T. Waterman Award of the National Science Foundation to Professor Peter G. Schultz of the University of California at Berkeley. Professor Schultz's recent work has pioneered new approaches to the study of molecular recognition and catalysis, and the application of these studies to the design of selective catalysts. This work, which bridges chemistry and biology, includes the generation of catalytic antibodies ("abzymes"), the synthesis of hybrid enzymes, and the developmemt of methods for introducing unnatural amino acid analogs selectively into proteins.

Our long term goal is to develop and exploit new strategies for the design and synthesis of biological catalysts with tailored specificities. The specific aim of this proposal is to design, synthesize, and characterize sequence-specific hybrid ribonucleases and deoxyribonucleases with defined binding site sequences and binding site sizes. A new binding site consisting of an oligodeoxyribonucleotide of defined length and sequence has been site-selectively introduced outside the catalytic sites of the relatively nonspecific enzymes, bovine pancreatic ribonuclease A and staphylococcal nuclease. The resulting hybrid enzymes are able to sequence-specifically cleave single-stranded DNA and RNA. The design of this first generation of hybrid enzymes will be optimized and the catalytic properties and specificities characterized in detail. In addition, strategies will be explored for cleaving double- stranded DNA with these and other hybrid enzymes,including rec A mediated triple-strand formation, triple-helix formation, and derivatization of DNA binding proteins with staphylococcal nuclease. These hybrid sequence-specific phosphodiesterases will provide important tools for studies of both RNA and DNA structure and function as well as for mapping, isolating, and cloning nucleic acids. The strategies developed here may be applied to the design of other hybrid enzymes with tailored specificities for application in chemistry, biology, and medicine.

This research involves novel strategies to produce antibody polypeptides with optimal structures for immobilization to insoluble carriers. Each approach is designed to result in immobilized, monovalent antibody fragments with their combining sites facing out from the carrier surface, and attachment to the carrier will be achieved in a highly specific manner through a site on the polypeptide far removed from the combining site itself. Attachment in this fashion should allow complete retention of antibody binding activity and thus lead to improved techniques of affinity purification. In addition, immobilizing antibody fragments rather than the much large whole antibody should increase the number of antibody combining sites that can be immobilized on the carrier surface. In the case of Fv fragments, site-directed mutagenesis will be used to incorporate a single attachment site at a desired location on the polypeptide. The modified polypeptide will then be expressed in E. coli. Production of Fv fragments in E. coli may prove to be a superior alternative to present-day hybridoma technology, which is frequently the limiting factor in monoclonal antibody production and isolation. Moreover, both site- directed mutagenesis and random mutagenesis can be used to rapidly obtain antibody polypeptides with variant binding activities. Protein engineering may also be used to introduce pH or metal-regulated binding affinity into the combining site.

This collaborative project between workers at Berkeley and UCSF is designed to produce new polymeric peptides with useful properties. The investigators are all excellent scientists, although all well supported by other grants. In particular Dr. Craik has another NSF grant which duplicates his effort in this proposal. The P.I. has developed enzymatic methods for reacting amino acid esters in organic solvents with free amino acids to generate dipeptides. The approach has been extended to a tetra peptide, but the strategy seems unlikely to produce useful polymers, although it might be practical for short peptides. It should be noted that carboxypeptidase cannot be employed in such syntheses because it does not have and acyl enzyme intermediate. There are some interesting ideas in this proposal, but the practicality of synthesizing polypeptides by these approaches seems minimal.

A biosynthetic method has been developed that makes possible the site- specific incorporation of a large number of noncoded amino acids and analogues within proteins. In this approach, an amber suppressor tRNA chemically aminoacylated with the desired amino acid incorporates this amino acid site specifically into a protein in response to an amber codon introduced at the corresponding position in the protein's DNA sequence. Using this method, precisely tailored changes within a protein can be made to address specific structure-function questions, including changes in the size, acidity, nucleophility or hydrophobic properties of an amino acid . In addition, analogues with altogether new properties can be introduced including spin labels, photoactivable and redox active amino acids. We will apply this methodology to a series of specific problems associated with protein stability/folding and catalysis. These include the role of hydrogen bonding in stabilizing protein structure, low barrier hydrogen bonds in enzymic catalysis, the GTPase mechanism of small signal transduction proteins (Ras), mechanistic studies of the novel Claisen rearrangement catalyzed by the E. coli and B. substilis chorismate mutases, and the novel radical mechanism of ribonucleotide reductase. We have chosen these systems by virtue of their significance and the fact that noncoded amino acids may provide certain advantages in their study. Finally we also propose to continue to improve the methodology itself.

Our long term objective is to develop molecules capable of specifically recognizing and modifying defined sequences of DNA. These studies are expected to provide increased insight into the molecular basis for selective recognition of nucleic acids by proteins, oligonucleotides and small molecules. In addition, this work may result in new tools for manipulating and analyzing nucleic acid structure and function, as well as therapeutic agents based on selective gene inactivation. During the period of the previous application we addressed the problem of sequence specific DNA modification, specifically the design of molecules that hydrolyze nucleic acids at predefined sequences. A combination of chemical and biological mutagenesis was used to convert the relatively nonspecific phosphodiesterase, staphylococcal nuclease, into a molecule capable of sequence specifically hydrolyzing RNA, single-stranded DNA and duplex DNA. Adducts of staphylococcal nuclease with either oligonucleotides or DNA-binding proteins selectively bound and hydrolyzed RNA and DNA via Watson-Crick base pairing interactions, D-loop formation, triple-helix formation, or specific protein DNA complex formation. A major limitation to further progress in the development of these "engineered nucleases" is the availability of molecules capable of recognizing any predefined sequence of duplex DNA. Consequently, the focus of this continuation is (a) to increase our understanding of the molecular basis for the selective recognition of DNA by polypeptides and oligonucleotides and (b) to develop new strategies for generating oligonucleotides and proteins that selectively bind any predefined sequence of double-stranded DNA. This will be accomplished via a combination of genetic and chemical approaches. Large libraries (greater than 10-8) of proteins and oligonucleotides will be generated and screened (by in vitro affinity chromatography or in vivo selections) in order to identify "ligands" that bind specific target sequences. The resulting ligands will be characterized with respect to their affinity and specificity for DNA. The insight gained from this combinatorial approach to DNA recognition will be complemented by a detailed analysis of the interactions between DNA and sequence specific DNA binding proteins. Amino acids in the structurally characterized DNA binding proteins, 434 repressor and CAP, will be substituted with a series of unnatural amino acid variants with precisely defined electronic and steric properties. Characterization of these mutant repressors should allow us to more precisely define the structural features responsible for the selective recognition of DNA by proteins.

The SIR provided (I)-Se-Cys; 150mg The ribonucleotide reductase from Lactobacillus leichmannii catalyzed the reduction of NTPs to dNTPs using the cofactor adenosyllcobalamin. Understanding the mechanisms for NTP reduction is important not only because this enzyme is essential for the production of dNTPs and is therefore an important target for drug design, but also because the enzyme is proposed to reduce the NTPs via a novel, radical mechanism. Central to the proposed mechanisms is the formation of an active-site thiyl radical at position Cys 408. To provide evidence for this proposed mechanisms and to understand the role of the thiyl radical in catalysis, this cysteine is being replaced with a variety of amino acids using unnatural amino acid mutagenesis. This methodology allows an amino acid to be synthesized in the lab and then incorporated site-specifically in a protein. Given that the S-H bond strength is similar to that of the substrate C-H bond that is proposed to be homolyzed in the first step of the reduction, Cys 408 is being replaced by amino acids, such as selenocysteine, that are similar in structure to cysteine, but differ in X-H bond strength and kinetic reactivity. Once the amino acids are incorporated into ribonucleotide reductase, the mutant enzymes will be tested for their ability to catalyze the reduction of CTP to dCTP and the exchange of 3H from 3H-adenosyloblamin into 3H2) and may be examined using Electron Spin Resonance Spectroscopy. These replacements should provide insight into the thermodynamic and kinetic requirements for enzyme-catalyzed radical reactions.

Design and study of pyridine nucleotide ana- logues to probe the chemical and enzymatic hydrolysis of NAD.

DESCRIPTION (provided by applicant): This proposal is aimed at further developing and applying a general method that allows one to systematically add amino acids with novel physical, chemical and biological properties to the genetic codes of both prokaryotic and eukaryotic organisms. This methodology involves the generation of orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs that uniquely recognize a noncoding codon and do not cross react with any of the endogenous tRNAs and aminoacyl-tRNA synthetases in the host organism. The amino acid specificity of the aminoacyl-tRNA synthetase is then modified such that it aminoacylates its cognate tRNA with only the desired unnatural amino acid and no endogenous amino acid. Using this method, a large number of structurally diverse unnatural amino acids have been incorporated efficiently and with high fidelity into proteins in response to nonsense and frameshift codons. We now propose to extend these studies in several directions. The first is focused on developing robust, user-friendly methods to genetically encode a large number of these unnatural amino acids in mammalian cells and Streptomyces. If successful this effort will significantly expand the scope and impact of this technology. The second effort focuses on the synthesis and in vitro and in vivo characterization of chemically defined antibody conjugates for the treatment of cardiovascular disease and acute myeloid leukemia. These latter efforts should not only pave the way for new or significantly improved therapeutics for major unmet medical needs, they should also provide a generalizable, straightforward approach to create similar classes of antibody conjugates for metabolic, cardiovascular and inflammatory diseases.

Single molecule fluorescence detection methods can provide insights into the stochastic dynamics and structural distribution of proteins. Here we propose to develop both biochemical and spectroscopic tools to facilitate the application of single molecule methods to the study of protein structure and function. These include: (i) methods for selective derivatization of proteins with appropriate linkers and dyes for fluorescence studies and surface immobilization; and (ii) instrumentation and data analysis tools/methods for single molecule fluorescence detection. These methods will be applied to three well-defined biochemical systems: (i) the enzymatic hydrolysis of peptides and proteins by trysin; (ii) the folding of apomyoglobin; and (iii) studies of membrane protein association and diffusion with C5a receptor.

This project will test the general hypothesis that mitotic misregulation is the key determinant in chondrocyte aging. In the first phase, we propose to examine the transcript profiles of chondrocytes, and identify those genes whose expression changes with age, and osteoarthritis utilizing comprehensive DNA microarrays that cover more than 90% of the encoded genes in the human genome. A database will be established to manage this large amount of information and provide access to the public in a web-based interactive format. Some of the proposed capabilities will include hierarchical clustering, promoter analysis, and automated batch search to all public databases, including our comprehensive human and mouse transcriptome database. The second phase of this project will focus on functional analyses. Both computer-assisted sequence search and biochemical assays will be utilized to define the functional roles of genes whose expression changes with age and severity of osteoarthritic pathology. We expect a large proportion of genes identified in this study to be expressed-sequence tags (ESTs). We propose to develop high-throughput systems for cloning and protein expression, along with assays for functional characterization. Specific Aim 1: Examine the transcript profiles of chondrocytes in full thickness normal human articular cartilage from donors age 20-90, with at least 10 samples per decade. Specific Aim 2: Obtain transcript profiles of microdissected OA cartilage to distinguish early from advanced disease and fibrillated, calcified and fibrocartilage. Specific Aim 3: Establish a comprehensive database for articular cartilage aging and osteoarthritis development and provide access to the public in a web- based interactive format. Specific Aim 4: Clone differentially expressed ESTs. Specific Aim 5: Characterize gene functions in assays for chondrocyte proliferation and survival, differentiation, extracellular matrix remodeling, and inflammation. The proposed studies have the potential to identify genetic changes that determine joint aging and thus define new molecular markers for diagnosis and targets for prevention and therapeutic intervention.

[unreadable] DESCRIPTION (provided by applicant): This proposal brings together a number of efforts in the lab centered on nucleic acid structure, function and manipulation. All these projects are focused on generating nucleic acids and nucleic acid complexes with novel properties by a combination of rational design and molecular evolution. In particular we are attempting to design and characterize novel DNAs with altered base-pairing interactions based on metal complexation. Ultimately, our hope is to be able to enzymatically synthesize such pairs to use in applications for information storage and the in vitro evolution of nucleic acids with novel functions. At a higher level of structure, we will attempt to generate circular, folded RNAs that are capable as acting as viruses- are infective, can be replicated in vivo, and can modulate cellular function. Such molecules would find applications in cell-based functional genomics and possibly therapeutic applications. Finally, at the level of selective protein-nucleic acid complexes, we are attempting to generate a series of sequence specific recombinases that can be used for cloning experiments, in cellular genetic disruption/insertion experiments, or possibly transgenic animal experiments. In addition to the applications described above, these experiments will likely provide new insights into nucleic acid structure, function and recognition.

DESCRIPTION (provided by applicant): Research Area. This application addresses broad Challenge Area (14): Stem Cells and specific Challenge Topic, 14-AR-102: Discovery Technologies for Multipotent and Induced Pluripotent Stem Cells from Human Skin and Musculoskeletal Tissues. Adult stem cells represent a promising resource for the development of cell based therapies to treat a broad range of diseases. Their application is not hampered by ethical concerns and they can be used in a patient tailored manner. The accessibility of skin makes a multipotent adult stem cell population known as skin derived precursor (SKP) cells a particularly attractive cell type. Limiting the development of this technology is the lack of efficient methods to derive desired physiologically active therapeutic cell fates from SKPs. The work proposed in this project will delineate small molecule and protein factors that can direct the differentiation and reprogramming of SKP cells to a desired lineage. Long term objectives include the identification of factors that facilitate the efficient derivation of myelinating Schwann cells and physiologically active neurons from human SKP cells. Transplanted Schwann cells have been shown to promote functional recovery following spinal cord injury and are believed to hold promise in the development of treatments for demyelination diseases (e.g. multiple sclerosis). Neuron progenitor cells derived from SKPs could be used in neuron replacement therapies (e.g. for the treatment of Parkinson's disease) and factors that stimulate neuron induction in SKP cells could be used therapeutically to stimulate endogenous neurogenesis for the treatment of peripheral neuropathies. Unbiased high throughput screens of large libraries (>1 million) of drug-like small molecules and arrayed collections of ~4000 secreted proteins will be performed to identify factors that facilitate the derivation of desired cell fates. The physiological utility of promising leads will be validated using relevant in vivo mouse models and electrophysiology, where appropriate. To elucidate the biological mechanisms of factors identified in these screens, targets of small molecule hits will be determined using a combination of genomic and biochemical affinity based approaches. This work will facilitate the development of therapeutic applications for adult stem cells and will lead to the identification of biological mechanisms that mediate their differentiation. In addition, success in this project will serve to provide precedence and pave the way for additional efforts to identify factors that control stem cell fate. PUBLIC HEALTH RELEVANCE: The goal of this project is to identify molecules that facilitate the efficient derivation of therapeutic cell types from stem cells that reside in human skin. These molecules will advance the development of cell based therapies to treat a range of diseases related to the nervous system. Potential applications will be the development of cell transplantation strategies to treat spinal cord injury, multiple sclerosis, Parkinson's disease and various peripheral neuropathies.

DESCRIPTION (provided by applicant): Dystroglycan is a widely expressed transmembrane glycoprotein that acts as a high- affinity receptor for both extracellular matrix proteins containing laminin-G domains and certain arenaviruses. Secondary dystroglycanopathies encompass a collection of muscular dystrophies characterized by impaired post-translational processing of dystroglycan. Profound muscle weakness and wasting as well as potential central nervous system impariment are typical pathologies associated with secondary dystroglycanopathies. Causative mutations for these disorders are found in known or putative glycosyltransferases that participate in the O-glycosylation of alpha- dystroglycan, a modificaiton required for functionality. Despite extensive efforts to understand the genetics and pathology of these diseases, the genetic causes of over half of these cases remain a mystery. Furthermore, there are no treatments available for patients. This proposal outlines approaches to elucidate remaining genetic causes of dystroglycanopathies, discover and validate small molecule and peptide effectors of dystroglycan glycosylation and provide the muscular dystrophy field with improved mouse models to sustain rapid future progress. These goals will be successfully met through collaboration with the Schultz laboratory at The Scripps Research Institute. The objective of Specific Aim 1 is to identify novel dystroglycanophy genetic loci using both high-throughput in vitro complementation and knockdown screens. Elucidation of new candidate genes will offer new opportunities for improved genetic diagnosis, new viable therapeutic targets and a better understanding of dystroglycan post-translational processing. Specific Aim 2 is designed to identify novel small molecule and secreted peptide effectors of dystroglycan glycosylation in a cell culture based, high-throughput manner. This unbiased approach will provide new directions for the development of therapeutic interventions for muscular dystrophy. Specific Aim 3 is targeted at both validation of previously and newly identified therapeutic strategies and the development of conditional and knockdown mouse models of dystroglycanopathy. The new mouse models will better capture both the genetic and phenotypic complexity of dystroglycanophies than the currently available cohort of mouse models. These models will serve the muscular dystrophy research community in efforts to explain the cellular mechanism and to develop viable treatment strategies for each genetic cause of dystroglycanopathy. These studies will provide the muscular dystrophy research field with improved tools and progress towards suitable means of improving dystroglycan function. This research meets the challenge of the National Institute of Neurological Disorders and Stroke mission statement to support "research on the causes, prevention, diagnosis, and treatment of neurological disorders and stroke, and supports basic research in related scientific areas". PUBLIC HEALTH RELEVANCE: Muscular dystrophies are a diverse set of inherited diseases characterized by progressive skeletal muscle weakness and wasting. Dystroglycan, a cell surface protein, requires extensive modification to serve as a link between the intracellular and extracellular cellular support network in muscle such that, when disrupted, it results in several forms of muscular dystrophy. This proposal is designed to identify new gene mutations that can cause these types of muscular dystrophy, discover small molecules that can improve dystroglycan function, develop needed mouse models of the disease and to validate both newly identified and currently known treatment strategies.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Performing protein analysis from media and pull-down samples. We are attempting to identify proteins secreted from differentiated stem cells. In our previous research, we identify several small molecules which can induce differentiation of hESC. We also developed long term culture conditions using small molecules. Using proteomic mass spec approach, we want to identify secreted proteins from chemically differentiated stem cells.

DESCRIPTION (provided by applicant): We will attempt to use our unnatural amino acid mutagenesis methodology to genetically encode libraries of cyclic and linear peptides containing a diverse array of unnatural amino acids. These peptide libraries will be generated both intracellularly in bacteria and in a phage-displayed format to allow in vivo selections and display-based affinity panning, respectively. This strategy will enable direct genetic encoding of molecular structure, random or directed mutagenesis, selection, and biological amplification, while at the same time allowing chemical diversity beyond what the canonical 20 amino acid repertoire can offer. The integration of unnatural amino acids into genetically encoded peptide libraries should facilitate both evolutionary experiments and the generation of peptides with novel biological activities that may ultimately find therapeutic use. This will be achieved through the following specific aims: 1. The preparation and incorporation of novel unnatural amino acids with unique reactivities and with functional groups capable of targeting specific proteins involved in disease states (e.g., serine proteases, metalloproteases, etc.) 2. The use of intein-based intracellular cyclic peptide synthesis methodologies together with unnatural amino acid technology to generate novel cyclic peptide libraries and select specific enzyme inhibitors 3. The use of phage display systems to generate and screen libraries of peptides containing unnatural amino acids for inhibitors of specific protein targets.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Unnatural amino acids are incorporated through the suppression of the amber stop codon, as it is the less abundant stop codon in the genome of the E.coli. Thus it is likely to assume that the amber stop codon is supressed in different regions of the genome resulting in mutant proteins.

DESCRIPTION (provided by applicant): Antibody molecules are enormously important as therapeutic and diagnostic molecules. More recently, unique scaffolds like the VHH of camelids, or even non-antibody frameworks like knottins have become important in biomedicine. Antibody diversity and antigen binding in mammals is often restricted to the CDR loops of the immunoglobulin fold. In experiments challenging this paradigm, we have recently solved the crystal structures of two bovine antibodies containing ultralong CDR H3s (56 and 61 amino acids) and also deep sequenced the ultralong repertoire. Our data reveal that these CDR H3s form a very unusual architecture composed of a long ?-strand stalk which supports a disulfide rich knob that protrudes far from the immunoglobulin surface. Interestingly, the two different antibodies contain different patterns of disulfides, which result in different knob structures. Dee sequencing reveals extensive diversity in the ultralong CDR H3s where a multitude of different disulfides could potentially form within the knob. Thus, the bovine antibody system can produce an unprecedented repertoire of mega CDR H3s that may result in an impressive diversity of minifolds containing combinations of somatically generated disulfides. Thus, antibody diversity is located in a new minifold supported by the immunoglobulin domain. We will perform structural, functional, and engineering studies to investigate the properties of this new antibody class, as well as to lead the way to developing this unique structure into therapeutics.

? DESCRIPTION (provided by applicant): Spinal muscular atrophy is a genetic disease resulting from mutations in the SMN1 gene that results in impaired motor neuron function and can lead to death. Currently there are no approved drugs for this debilitating pediatric disease. Our goal is to generate an orally bioavailable drug for the treatment of SMA that upregulates SMN2 gene expression to compensate for the loss of the wild type SMN1 gene. Such drugs can likely be used in combination with the splicing modulators or neuroprotective agents (if approved) that are currently in clinical development to provide increased patient benefit. In preliminary work we have identified a lead compound with excellent in vitro activity and good pharmacokinetics that upregulates SMN levels in the SMN?7 neonatal mouse model without obvious adverse effects. New analogs will be designed, synthesized and assessed for in vitro activity, cytotoxicity and in vitro ADME and safety profiles. Analogues with improved cellular activity (full-length SMN levels), low cytotoxicity and favorable in vitro safety profiles will be selected for PK studies in adult and neonatal mice. These analogs will also be studied in rat PK models (oral administration, plasma and brain levels at different time points) to assess their pharmacokinetics across different species. Analogs that exhibit favorable brain PK profiles at tolerated doses (without obvious adverse effects in neonatal mice) will be progressed to pharmacodynamic (full-length SMN levels in brain tissue) and efficacy studies in SMN?7 neonatal mice. In parallel the biological target of select potent compounds will be identified using affinity probes and the mechanism by which SMN levels are upregulated will be investigated. Finally, once a preclinical candidate is selected (a) the dosing regimen will be optimized including efficacious dose range determination and dosing frequency; (b) a comprehensive set of in vitro physicochemical, ADME and safety profiling studies (detailed in Table 2, vide infra) will be carried out; and (c) th PCC will be assessed in rat and dog pharmacokinetic and toxicology studies. Upon the completion of the proposed studies, we will be positioned to initiate IND-enabling studies for the selected PCC, and move the program forward into clinical studies with an appropriate commercial partner.

Project Summary/Abstract We will attempt to recapitulate mitochondrial evolution based on the endosymbiotic theory. Eukaryotic organelles, like mitochondria and chloroplasts, are proposed to have evolved from bacterial endosymbionts during an early stage of evolution. Here we will begin to test this theory using two well established model organisms ? E. coli and S. cerevisiae. Specifically, we will generate bacterial endosymbionts in yeast cells. Once we establish stable endosymbionts we will systematically either knockout large parts of the bacterial genome or move specific genes to the host genome to obtain a minimal symbiont genome similar to the early stages of mitochondrial evolution. We will also attempt to further delete genes in the yeast mitochondria, explore those factors that affect the stability of this system and light utilizing endosymbionts using yeast and cyanobacteria. These studies have the potential to provide additional insights into the evolution of complex eukaryotic functions, such as energy generating mitochondria.