Biochemistry Commons^™

Cbl And Cbl-B Dictate Csf-1r Endocytic Traffic And Signaling In Macrophages, Lu Huang

Electronic Theses and Dissertations

Macrophage colony stimulating factor receptor (CSF-1R or MCSFR) is a receptor tyrosine kinase essential for the growth and function of macrophages. Understanding the mechanisms that regulate CSF-1R activation and deactivation will provide insights to clinical treatment of macrophage related diseases including chronic inflammation and cancer. Previously, our laboratory showed that CSF-1R undergoes a novel membrane trafficking route that involves macropinocytosis to deactivate CSF-1R signaling. This thesis makes the discovery that the ubiquitin ligases Cbl and Cbl-b cooperate to regulate CSF-1R endocytosis and traffic to macropinosome in macrophages. Macrophages were derived from mice knocked out for Cbl, Cbl-b or the double …

Regulation Of Arf16-2 By Microrna160 During Soybean Root Nodule Development, Spencer Schreier

Electronic Theses and Dissertations

Soybean is an excellent candidate for sustainable agriculture due to its production of nutritious, versatile beans and the ability to form symbiotic organs called root nodules that perform nitrogen fixation. As demand for both yield and sustainable agriculture continue to increase, root nodules offer an attractive alternative to expensive and environmentally harmful nitrogen fertilizers. Understanding root nodule formation may open genetic engineering avenues for optimizing nitrogen fixation performance and transferring the nodule-formation ability to other plants. A major determinant of nodule numbers and quality in soybean is microRNA 160 (miR160), which dictates developmental stage-specific auxin sensitivity by targeting repressor auxin …

Design, Synthesis And Biological Screening Of Novel Cucsinspired Estrone Analogues Towards Treatment Of Hepatocellular Carcinoma, Mater Hussen Mahnashi

Electronic Theses and Dissertations

Cucurbitacins (CUCS) are natural products with highly oxygenated tetracyclic triterpenes produced mostly by Cucurbitaceae family plant. They are known for their therapeutic efficiency with different biological activities, such as anti-inflammatory, hepatoprotective and anti-cancer. Hepatocellular carcinoma (HCC) is the third leading cause of death worldwide. Previous reports have shown the ability of CUCS to inhibit the growth of hepatocellular carcinoma cell lines (HepG-2) significantly. Structural activity relationship studies suggested the potential of the 23, 24 enone side chain of CUCS to bind to the Epidermal Growth Factor Receptor (EGFR). Due to the limited quantities of CUCS upon isolation and the challenges …

Determining The Effects Of Methanol, Ethanol, Isopropanol, And Glycerol On Both Thermal Stability And Catalytic Activity Of Rv0045c, An Enzyme From M. Tuberculosis, Katelyn Baumer

Undergraduate Honors Thesis Collection

Tuberculosis (TB) is a highly infectious respiratory disease contracted through the inhalation of Mycobacterium tuberculosis. Serine hydrolases are abundant in M. tuberculosis and serve as a model for studying the inhibition of TB. Rv0045c is an example of such with little known regarding its biological function. Rv0045c was exposed to methanol, ethanol, isopropanol, or glycerol and the effects of varying concentration of these alcohols on the catalytic efficiency and thermal stability of the enzyme was determined. The thermal stability of Rv0045c was found to decrease with concentration of methanol, ethanol, or isopropanol. The opposite was true of the thermal stability …

Structural And Mechanistic Insights Into Hemoglobincatalyzed Hydrogen Sulfide Oxidation And The Fate Of Polysulfide Products, Victor Vitvitsky, Pramod K. Yadav, Sojin An, Javier Seravalli, Uhn-Soo Cho, Ruma V. Banerjee

Department of Biochemistry: Faculty Publications

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H₂S but, lacking organelles, cannot dispose of H₂S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H₂S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human …

Short Carboxylic Acid-Carboxylate Hydrogen Bonds Can Have Fully Localized Protons, Jiusheng Lin, Edwin Pozharski, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Short hydrogen bonds (H-bonds) have been proposed to play key functional roles in several proteins. The location of the proton in short H-bonds is of central importance, as proton delocalization is a defining feature of low barrier hydrogen bonds (LBHBs). Experimentally determining proton location in H-bonds is challenging. Here, bond length analysis of atomic (1.15–0.98 Å) resolution X-ray crystal structures of the human protein DJ-1 and its bacterial homolog YajL was used to determine the protonation states of H-bonded carboxylic acids. DJ-1 contains a buried, dimer-spanning 2.49 Å H-bond between Glu15 and Asp23 that satisfies standard donor-acceptor distance criteria for …

Dissecting The Histone-Binding Mechanism Of A Phd Finger Subtype, Daniel Boamah

Electronic Theses and Dissertations

Disordered tails of histones are critical information retrieval hub and thus, aberrations in the flow of information through these hubs are associated with a number of pathological consequences in human. Mechanism for retrieval of information from these hubs is achieved by protein-protein interaction, i.e. proteins dock onto histone tails to initiate chromatin signaling. Eukaryotes have a number of small peptide binding domains that have evolved to specifically interact with histone tails, and these domains called histone readers as they read the information encoded on histone tails. Plant homeodomain (hereafter PHD) finger, a binucleated zinc finger, family is one such histone …

Mechanistic And Inhibitory Analysis Of Clostridium Difficile Associated Diseases, Brianna Marie Jackman

Wayne State University Theses

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Using Fluorescence Lifetimes To Characterize Lipid Behavior In Nanodiscs, Cynthia Janku

Undergraduate Theses, Professional Papers, and Capstone Artifacts

Cellular uptake of molecules, including drugs, can be affected by the fluidity of the membrane. Nanoparticles have been hypothesized to alter membrane fluidity resulting in inflammation and its related clinical effects. Variations in phospholipids can alter membrane structure and its interaction with drugs or nanoparticles. To study membrane lipid differences and dynamics, we are using nanodiscs and liposomes as model systems. Nanodiscs are a lipid bilayer surrounded by a membrane scaffold protein, which is a derivative of Apolipoprotein A1, a protein involved in the removal of cholesterol from the body. There are important unresolved questions about how the belt protein …

Nitrosative Stress Sensing In Porphyromonas Gingivalis: Structure And Function Of The Heme Binding Transcriptional Regulator Hcpr, Benjamin R. Belvin

Theses and Dissertations

Porphyromonas gingivalis, a Gram negative anaerobe implicated in the progression of periodontal disease, is capable of surviving and causing infection despite high levels of reactive nitrogen species found in the oral cavity due to its efficient nitrosative stress response. HcpR is an important sensor-regulator that plays a vital step in the initiation of the nitrosative stress response in many Gram negative anaerobic bacteria. We employ a combination of X-ray crystallography, SAXS, resonance Raman spectroscopy, UV-Vis spectroscopy, and molecular biology techniques to better understand this key regulator. Knockout of the hcpR gene in W83 P. gingivalis results in the inability of …

Raman Spectroscopy And Chemometrics For Forensic Bloodstain Analysis : Species Differentiation, Donor Age Estimation, And Dating Of Bloodstains, Kyle C. Doty

Legacy Theses & Dissertations (2009 - 2024)

The field of forensic science is constantly growing, so the advancement of old and unreliable techniques is at the forefront of what will lead to future progress and improvement. Current methods for identification and analysis of bloodstains are underwhelming due to the insignificant amount of information provided in a destructive, unreliable, and unsafe manner. As is the purpose of this research, creating new methodologies that are rapid, nondestructive, robust, statistically reliable, and safe would significantly advance the way bloodstains are currently analyzed, while providing more useful and relevant information for investigations and criminal proceedings. Raman spectroscopy, along with advanced statistical …

Discovering Small Molecule Inhibitors Targeted To Ligand-Stimulated Rage-Diaph1 Signaling Transduction, Jinhong Pan

Legacy Theses & Dissertations (2009 - 2024)

The receptor of advanced glycation end product (RAGE) is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules, which plays an important role in immune responses. Full-length RAGE includes three extracellular immunoglobulin domains, a transmembrane domain and an intracellular domain. It is a pattern recognition receptor that can bind diverse ligands. NMR spectroscopy and x-ray crystallization studies of the extracellular domains of RAGE indicate that RAGE ligands bind by distinct charge- and hydrophobicity-dependent mechanisms. It is found that calgranulin binding to the C1C2 domain or AGEs binding to the V domain activates extracellular signaling, which triggers interactions of …

Novel Approaches To Mitigating Bacterial Biofilm Formation And Intercellular Communication, Stephen Kasper

Legacy Theses & Dissertations (2009 - 2024)

Long thought of as solitary single-cell organisms, it is now widely accepted that bacteria can act and cooperate as social organisms. Phenomena such as biofilm formation and quorum sensing (QS) are two intimately intertwined cooperative behaviors that significantly contribute to the pathogenesis of many bacteria. Biofilms are surface associated communities of bacteria encased in a secreted extracellular matrix, which provides several advantages over an individualized lifestyle, such as increased protection from antimicrobial agents as well as enhanced opportunity for the exchange of genetic material. Bacterial QS is a system of population-based communication through the production, sensing, and response to chemical …

Structural And Functional Characterization Of An Unusual Camp Responsive Transcription Factor, Cmr, In Tb Complex Mycobacteria, Sridevi Ranganathan

Legacy Theses & Dissertations (2009 - 2024)

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, is an intracellular pathogen that infects millions of people every year. Mtb can survive inside the host for extended periods of time by sensing and adapting to the host environmental stressors. Transcriptional gene regulation plays a critical role in this adaptation. This dissertation focuses on understanding the structural and functional aspects of one such transcriptional regulatory unit, Cmr (Rv1675c), in Mtb.

Study Of Biologically Important Macromolecules By Nuclear Magnetic Resonance, Christopher Michael Demott

Legacy Theses & Dissertations (2009 - 2024)

Intrinsically disordered proteins or unstructured segments within proteins play an important role in cellular physiology and pathology. A combination of peptide aptamers selected by using the yeast-two-hybrid scheme, and in-cell NMR identified high affinity binders to a transiently structured intrinsically disordered proteins (IDP). This method was validated using the prokaryotic ubiquitin-like protein, Pup, of the Mycobacterium proteasome. We discover two peptide aptamers that bind to opposite sites of a transient helix in Pup, an intrinsically disordered protein, that have vastly different effects on the survival of Mycobacterium bovis BCG.

Multifunctional Nanomaterials Utilizing Hybridization Chain Reaction For Molecular Diagnostics And Bioanalytical Applications, Md Muhit Rana

Legacy Theses & Dissertations (2009 - 2024)

DNA nanotechnology has shown great promise in molecular diagnostic, bioanalytical and biomedical applications. The great challenge of detecting target analytes, biomarkers and small molecules, in molecular diagnostics is low yield sensitivity. To address this challenge, different nanomaterials have been used for a long time and to date there is no such cost-effective bioanalytical technique which can detect these target biomarkers (DNA, RNA, circulating DNA/miRNA) or environmental heavy metal ions (Hg2+ and Ag+) in a cost-effective and efficient manner.

A Laser-Pulse Photolysis Study Of The Mechanism Of Potentiation Of The Glua2qflip Ampa Receptors By Cx546, Yu-Chuan Shen

Legacy Theses & Dissertations (2009 - 2024)

Insufficient activity of the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate ion channels is involved in neurological disorders and developmental problems, such as schizophrenia, one of the most serious psychiatric diseases. Use of regulatory agents to enhance AMPA receptor activity under the circumstance has been shown therapeutically beneficial. Specifically, small-molecule compounds generally termed as potentiators or positive modulators of AMPA receptors have long been pursued as drug candidates for a potential treatment of these neurological disorders and diseases. The goal of my MS thesis work is to investigate the mechanism of potentiation on AMPA receptor by a classic potentiator known as CX546. The …

The Role Of N-Myc Downstream Regulated Gene 1 In Breast Cancer Lipid Metabolism, Christopher James Sevinsky

Legacy Theses & Dissertations (2009 - 2024)

Altered carbohydrate and lipid metabolism are increasingly well characterized hallmarks of aggressive breast cancers. While aerobic glycolysis, or “the Warburg effect”, is a well-established metabolic adaptation exploited by tumor cells, the understanding of unique aspects of cancer lipid metabolism lags behind. This is especially true regarding the coordination of complex lipid synthesis and trafficking pathways, which remains poorly understood. N-Myc Downstream Regulated Gene1 (NDRG1) is overexpressed in many solid tumors, but its function is unclear. The importance of NDRG1 is best exemplified by the effect of null mutations on human physiology: inactivating mutations give rise to the severe autosomal recessive …

Mass Spectrometric Analysis And Machine Learning Enable Microorganism Classification Based On Rna Posttranscriptional Modifications, Colin Christopher Aldrich

Legacy Theses & Dissertations (2009 - 2024)

RNA post-transcriptional modifications (PTMs) are dynamic features that can be up- or down-regulated by the health and metabolic state of a cell. These covalent modifications are installed and removed on RNA nucleosides by enzymes controlled by the activation and deactivation of specific genes. The goal of this research was to demonstrate that RNA PTMs can serve as a unique feature for the classification/identification of microorganisms. We utilized a scheme based on electrospray ionization mass spectrometry (ESI-MS) to obtain global PTM profiles from total RNA extracted from various microorganisms in optimal growth conditions as well as Salmonella typhimurium (S. typhimurium) spiked …

A Kinetic Study Of Regulation Of Glua1 Homomeric And Heteromeric Ampa Receptors By 2,3-Benzodiazepines, Yin-Shuo (Andrew) Wu

Legacy Theses & Dissertations (2009 - 2024)

AMPA receptors are a subtype of the ionotropic glutamate receptor family. They are ligand-gated or glutamate-activated, transmembrane ion channels that mediate the majority of excitatory synaptic transmission in the central nervous system. AMPA receptors are essential for neuronal development and brain functions, including learning and memory. Over-activation of calcium permeable AMPA receptors has been linked to some neurological diseases such as stroke, Alzheimer’s disease and amyotrophic lateral sclerosis. Inhibitors of glutamate receptors are therefore drug candidates for the potential treatment of these neurological diseases. One of the most promising classes of AMPA receptor antagonists is 2,3-benzodiazepine derivatives, thought to be …

Further Development Of Raman Spectroscopy For Body Fluid Investigation : Forensic Identification, Limit Of Detection, And Donor Characterization, Claire K. Muro

Legacy Theses & Dissertations (2009 - 2024)

The challenges to forensic body fluid analysis have placed limitations on the type of information that investigators can acquire and how that information can be collected. In recent years, Raman spectroscopy has proven itself useful for characterizing body fluids. In 2008, a large-scale investigation was undertaken to explore the use of Raman spectroscopy as a means of identifying body fluids. This work resulted in multidimensional Raman spectroscopic signatures for the five main body fluids: semen, peripheral blood, saliva, vaginal fluid, and sweat. These studies were incredibly successful and created the foundation for years of continued research. Accordingly, the studies included …

Structural Characterization Of The Interactions Of Nicotinamide And Analogs With Human Sirt6 By Saturation Transfer Difference (Std) Nmr And Site Directed Mutagenesis, Beatriz Elena Bolivar-Vega

Legacy Theses & Dissertations (2009 - 2024)

The allosteric regulation of SIRT6 by nicotinamide (NAM), along with the growing evidence of this enzyme's key role in the immune response, prompted the mechanistic study of SIRT6 inhibition by pyrazinamide (PZA) and analogs. In our current study, PZA, an analog of NAM, was revealed to have a modest modulatory effect on SIRT6, an enzyme that regulates the NF-κB signaling pathway at the transcriptional level (a relevant pathway to inflammation). Similarly, the analogs of PZA, 5-Cl PZA, 5-MeO PZA, and POA exhibited a modulatory effect against SIRT6 in our in vitro studies, enabling identification of a potential new target for …

Benzylideneoxymorphone: A New Lead For Development Of Bifunctional Mu/Delta Opioid Receptor Ligands, Jason R. Healy, Padmavani Bezawada, Nicholas W. Griggs, Andrea L. Devereaux, Rae Reiko Matsumoto, John R. Traynor, Christopher W. Cunningham

Faculty Publications & Research of the TUC College of Pharmacy

Opioid analgesic tolerance remains a considerable drawback to chronic pain management. The finding that concomitant administration of delta opioid receptor (DOR) antagonists attenuates the development of tolerance to mu opioid receptor (MOR) agonists has led to interest in producing bifunctional MOR agonist/DOR antagonist ligands. Herein, we present 7-benzylideneoxymorphone (6, UMB 246) displaying MOR partial agonist/DOR antagonist activity, representing a new lead for designing bifunctional MOR/DOR ligands.

การคัดกรองและการประยุกต์เครื่องหมายไมโครแซทเทลไลท์เพื่อการวิเคราะห์ความหลากหลายทางพันธุกรรมของนกกระเรียนพันธุ์ไทย Grus Antigone Sharpii Blanford, 1895 ในสภาพกรงเลี้ยง, รังสินี สันคม

Chulalongkorn University Theses and Dissertations (Chula ETD)

Grus antigone sharpii จัดเป็นนกกระเรียนที่เคยสูญพันธุ์ไปจากธรรมชาติของประเทศไทย การเพาะเลี้ยงและขยายพันธุ์นกกระเรียนในกรงเลี้ยงเป็นขั้นตอนสำคัญที่สามารถเพิ่มจำนวนประชากรเพื่อนำไปปล่อยคืนสู่ธรรมชาติในอนาคตได้ โดยสิ่งสำคัญที่ต้องคำนึงถึงคือความหลากหลายทางพันธุกรรม โดยเฉพาะอย่างยิ่งในกลุ่มของพ่อแม่พันธุ์ตั้งต้น ในการศึกษาครั้งนี้มีวัตถุประสงค์เพื่อคัดกรองเครื่องหมายไมโครแซทเทลไลท์จำนวน 28 ตำแหน่ง ซึ่งเป็นไพรเมอร์ที่เคยใช้ศึกษาในนกกระเรียน G. americana และนกกระเรียน Anthropoides paradisea เพื่อใช้วิเคราะห์ความหลากหลายทางพันธุกรรมของนกกระเรียนพันธุ์ไทยจากสวนสัตว์เปิดเขาเขียว (KKOZ, n = 11) และสถานีวิจัยเพาะเลี้ยงนกน้ำบางพระ (BB, n = 17) จังหวัดชลบุรี ผลการศึกษาพบว่าไพรเมอร์ทั้ง 28 คู่สามารถเพิ่มปริมาณไมโครแซทเทลไลท์ดีเอ็นเอของนกกระเรียนพันธุ์ไทยได้ แต่มีเพียง 22 คู่ที่แสดงความเป็น polymorphism และมีตำแหน่งไมโครแซทเทลไลท์ที่ปรากฏค่า linkage disequilibrium 8 ตำแหน่ง การเบี่ยงเบนออกจากสมดุล Hardy-Weinberg พบที่โลคัส Gram8 และ Gpa38 ซึ่งเป็นผลมาจาก null allele และการเพิ่มขึ้นของอัลลีลแบบ heterozygous ตามลำดับ จากการวิเคราะห์ค่าความหลากหลายทางพันธุกรรมพบว่า ค่าเฉลี่ยของ expected และ observed heterozygosity มีค่าเท่ากับ 0.63 และ 0.69 ค่า inbreeding coefficient มีค่าเท่ากับ -0.100 ซึ่งแสดงว่านกกระเรียนมีความหลากหลายทางพันธุกรรมค่อนข้างสูง และเกิด inbreeding น้อย นอกจากนี้ยังพบว่านกกระเรียนจาก KKOZ และ BB มีความแตกต่างทางพันธุกรรมต่ำ (FST = 0.035) และไม่มีการแยกออกเป็นกลุ่มประชากรย่อย (K = 1) จากผลการศึกษาครั้งนี้บ่งบอกได้ว่านกกระเรียนพันธุ์ไทยจาก 2 พื้นที่การศึกษาน่าจะเหมาะสมที่จะใช้เป็นพ่อแม่พันธุ์เพื่อเพิ่มจำนวนประชากรในกรงเลี้ยงให้มากขึ้น และสามารถนำไปปล่อยคืนสู่พื้นที่ชุ่มน้ำตามธรรมชาติในอนาคตได้

Contribution Of Specific Amino Acids To Calcium-Dependent Dimerization Of Epithelial Cadherin, Xiaoyun Z. Howard

Electronic Theses and Dissertations

Cadherins are calcium-dependent molecules that play essential roles in embryonic development, tissue morphogenesis, and cancer progression. Epithelial (E-) cadherin is present at adherens junctions, structures that are important for maintaining tissue integrity. At adherens junctions, cell–cell adhesion complexes are formed as clusters of cadherin dimers through strand-swapped dimerization between two interacting protomers from adherent cells. Recently, a number of studies of dimer formation of E-cadherin supported a two-step adhesive binding model. First, an inter-molecular initial encounter complex (X-dimer) forms at the linker region of the first two extracellular domains, EC1 and EC2. Next, strand-swap dimerization occurs via exchange or ''swap'' …

Biochemical, Structural, And Drug Design Studies Of Norovirus And Zika Virus Proteases, Ben Kuiper

Wayne State University Dissertations

Noroviruses, which are the leading cause of acute gastroenteritis, cause an estimated 677 million infections and 213,000 deaths each year worldwide. Noroviruses are classified into seven genogroups (GI-GVII); GI, GII, and GIV have been shown to be infectious in humans. However, GII noroviruses cause the majority of outbreaks (89%). No pharmacologic treatment or vaccine currently exists to treat or prevent norovirus infections.

Recently, the development of a norovirus replicon system, a murine model of norovirus infection, and the development of a biochemical protease assay have allowed for the design and development of norovirus inhibitors. However, the replicon and biochemical assay …

The Effect Of Acetylation Of Cytochrome C On Its Functions In Prostate Cancer, Viktoriia Bazylianska

Wayne State University Theses

Prostate cancer is the second leading cause of cancer death among men in America. The progression of cancer goes along with the Warburg effect, a metabolic switch from depending primarily on mitochondrial respiration to glycolysis. In addition, cancer cells manage to evade apoptosis. Cell signaling, via posttranslational modifications (PTMs), is one of the most important means of regulation, and most commonly dysregulated in cancer. In prostate cancer, androgen signaling plays a crucial role in driving cell proliferation.

Mammalian Cytochrome c (Cytc) is a multifunctional protein involved in cellular life and death decision. It is an essential component of the electron …

Fam129b, A Novel Adherent Junction Protein, Forms A Complex With Keap1, Fatme Ali Hachem

Wayne State University Theses

FAM129B/Minerva, a protein implicated in melanoma cell invasion, has been shown to suppress TNFα induced apoptosis in cancer cells (Song, Evans & Evans (2010), JBC 286, 10201-09), thus contributing to the survival of metastatic cells. KEAP1, a substrate recognition molecule for the CUL3 E3 ubiquitin ligase, plays a central role in the activation of the TNFα pathway. We wish to test the hypothesis that the up-regulation of FAM129B in cancer cells suppresses apoptosis by sequestering KEAP1, thus preventing its participation in the apoptotic pathway. The first step is to demonstrate that FAM129B and KEAP1 form a complex. We have cloned …

Development Of Student Data Visualization Tool, Adaption Of Clostridium Difficile Toxin A Into Protein Delivery Vehicle, And Elucidation Of Tcdc Mechanism Of Toxin Control, Adam Boyden

Wayne State University Theses

Advancing student success in higher education is of paramount importance, and is in need for a tool that visualizes student data in a longitudinal manner. Student Circos plots achieve this by plotting student data in circular plots, depicting the timeline and grades for students selected by demographic or performance information. Cellular delivery of exogenous proteins is a bountiful area of research. However, most current systems have limited in vivo applications and most lack cellular specificity. By adapting Toxin A from Clostridium difficile, the goal was to create a cell specific protein delivery vehicle that would be robust in vivo. However, …

Determination Of The Direct Protein-Protein Interactions In The Drosophila Sin3a Complex, Ian Moore

Wayne State University Theses

The proteins that comprise the Drosophila SIN3 220 and SIN3 187 complexes are currently known. Limited information with regard to interacting complex member proteins has been described. Much of these data are results of high-throughput investigations, and there have been no studies done to reconstitute all direct interactions within the complex. The unique C-terminal region of the SIN3 220 isoform was used to test for interactions with other complex proteins using the bacterial expression system. Additionally, the region of Caf1-55 necessary for interaction with SIN3 in vivo was identified using a truncation mutant. The results of this work identified novel …