 Non-enveloped T=3 icosahedral symmetry of identical sequence.  Single stranded positive sense RNA virus.  Coat protein (CP) coded by sub-genomic strand.

Slides:

Advertisements

Similar presentations

The Effect of the 5’UTR on in vivo Translation in TYMV Lisa Bauer Microbiology Mentor: Dr. Theo Dreher.

Advertisements

Expression of Cholera toxin B subunit in Banana callus culture Dawn Rivard.

Search for cellular attachment receptors used by a GII.4 Norovirus Dijon Gaurav Dutta Dwivedi Supervisors: Niklas Arnberg and Carin Årdahl.

Production of Turnip yellow mosaic virus nano-containers from Lactococcus lactis for zinc fortification Alma Laney Dr. Theo Dreher Lab Department of Microbiology.

Vaccinia Virus G1L Protein Expression and Purification HHMI Summer Undergraduate Research Program 2004 Laboratory of Dr. Dennis Hruby Oregon State University.

ABE Summer Workshop 2005 Southern & Western Blotting.

The ECM, Cell Adhesion, and Integrins 2/12/15 Lecture 6, ChE

BRIAN PHAN DR. JANE ISHMAEL DEPARTMENT OF PHARMACEUTICAL SCIENCES SUMMER 2010 Characterization of LC8 Interaction with NR1 subunit of NMDA Receptors.

PRESENTED BY: LAUREN SHIN MENTOR: DR. LUIZ BERMUDEZ MICROBIOLOGY DEPARTMENT Determining the Role of the luxR homolog in Mycobacterium avium subsp. paratuberculosis.

Student: Nicholas Pihl Mentor: Dr. Jeffrey Greenwood

The Growth Inhibitory Role and Cell Membrane Association of a Reptilian Insulin-Like Growth Factor Binding Protein Larissa Balogh Endocrine Laboratory.

The Interactions of Plant Hormones and rop Genes By Malem Gutema Mentor Dr. John Fowler Dr. John Fowler Summer 2004.

Production of Turnip yellow mosaic virus Capsids: The Future in Micronutrient Delivery Nicole Dzialowy Dr. Yannis Tzanetakis Dr. Theo Dreher Department.

NMR Studies of FXR-Prenylflavonoid Complexes HHMI Summer 2009 Ryan A. Moore Dr. Victor Hsu August 25, 2009.

LAB.9. SDS-PAGE, Sodium Dodecyl Sulfate, Polyacrylamide Gel Electrophoresis. describes a technique used to separate proteins according to their electrophoretic.

Turnip Yellow Mosaic Virus 3’UTR as a translational enhancer in Saccharomyces cerevisiae Lisa Bauer Microbiology Mentors: Daiki Matsuda Dr. Theo Dreher.

Chapter 3-Contd. Western blotting & SDS-PAGE

Unit 3: Viruses!.

Lab#6 Western Blotting BCH 462[practical].

Manufacture of Human Interleukin 13 Protein Using a Prokaryotic Expression System Ryan Rupp, York College of Pennsylvania, Department of Biological Sciences.

Table 5-1 Protein Purification Essential for characterizing individual proteins (determining their enzymatic activities, 3D structures, etc.) Two main.

Variations in the ELISA technique Used for testing the amount of antibody to an antigen in serum Lab 6. Preparation of rabbit IgG Enzyme linked Immunosorbent.

5. SEPARATION AND DETECTION OF PROTEINS II SDS-PAGE Jana Vobořilová, Anna Kotrbová-Kozak, Vlasta Fürstová, Tereza Kopská.

Virus Overview General characteristics of viruses

1 Genetics Faculty of Agriculture Instructor: Dr. Jihad Abdallah Topic 13:Recombinant DNA Technology.

Chapter 3 Proteins and Polypeptides George Plopper.

Western Blotting.

Chapter 20 Experimental Systems Dr. Capers.  In vivo ○ Involve whole animal  In vitro ○ Defined populations of immune cells are studied under controlled.

WHAT IS A WESTERN BLOT?.

Copyright © 2010 Pearson Education, Inc. General Characteristics of Viruses Differentiate a virus from a bacterium.  Obligatory intracellular parasites.

Results 1.Miller JR, Siripurkpong P, Hawes J, Majdalawieh A, Ro HS, McLeod RS. The trans-10, cis-12 isomer of conjugated linoleic acid decreases adiponectin.

Quantification and DNA Sequencing of IL-13Rα1 and IL-13Rα2 On Various Cancer Cell Lines Erin Dolac, Department of.

VIRUS STRUCTURE Basic rules of virus architecture, structure, and assembly are the same for all families Some structures are much more complex than others,

Antibody Detection. Part II Detection of Antigens Western Blotting “Dot-Blot Assay” Positive Control Primary antibody (IgY) Lysed E. Coli Cells Lysed.

Viruses are the smallest infectious diseases (ranging from nm) They are obligatory intracellular parasites without own metabolism (being parasites.

Proteomics The science of proteomics Applications of proteomics Proteomic methods a. protein purification b. protein sequencing c. mass spectrometry.

Antibodies Cells of the vertebrate acquired immune system produce antibodies with an exquisite specificity for molecules Biologists use antibodies to localize.

It's usually difficult to identify a protein of interest in a Commassie Blue-stained gel of cell extracts Coomassie Blue-stained gel MW stds. Cell extracts.

Western blotting. Antibodies in the Immune System Structure: 2 heavy chains + 2 light chains Disulfide bonds 2 antigen binding sites Isotypes: IgG, IgM,

Enzyme Linked Immunosorbent Assay. Purpose of ELISA  To detect antibodies in your blood or urine.  To see if you have been exposed to a disease.

Lecturer: David. * Reverse transcription PCR * Used to detect RNA levels * RNA is converted to cDNA by reverse transcriptase * Then it is amplified.

B c ratio (N/C) 5 15 d Fig. S1 Nuclear localization of NtE2F is assumed by its N terminal NLS. (a) Various constructs were transitory.

Phuong Pham Dr. Michael Freitag Summer 2012

Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription.

DNA Technology Ch. 20. The Human Genome The human genome has over 3 billion base pairs 97% does not code for proteins Called “Junk DNA” or “Noncoding.

Protein-protein interactions and western blotting MCB 130L Lecture 3.

The Antiviral Response in Plants: RNA Silencing Melissa Andreas Carrington Lab HHMI - Oregon State University Summer 2007.

Western blotting Pete Jones.

Figure S1 A MVNFVSAGLFRCLPVSCPEDLLVEELVDGLLSLEEELKDKEEEEAVLDGL LSLEEESRGRLRRGPPGEKAPPRGETHRDRQRRAEEKRKRKKEREKE EEKQTAEYLKRKEEEKARRRRRAEKKAADVARRKQEEQERRERKWRQ.

Lec1: General properties of viruses

Sesha Kiran Kollipara, Vikas Solanki and Bikash Mandal

Lab# 5 Western Blot BCH 462[practical].

Measurement of the p38 signaling pathway in the Pro253Arg mutation of FGFR2 in Apert syndrome Kaivalya Dandamudi.

The ECM, Cell Adhesion, and Integrins

General characteristics of viruses

Connective Tissue Growth Factor (CCN2) in Rat Pancreatic Stellate Cell Function: Integrin α5β1 as a Novel CCN2 Receptor Runping Gao, David R. Brigstock

Chapter 4 Recombinant DNA Technology

Chapter 20 – DNA Technology and Genomics

Transglutaminase-mediated oligomerization of the fibrin(ogen) αC domains promotes integrin-dependent cell adhesion and signaling by Alexey M. Belkin, Galina.

Introduction to Virus Structure and Diversity

Relationship between Genotype and Phenotype

Volume 56, Issue 2, Pages (August 1999)

Cell to Cell Communication

The plate cells are coated with rabbit antibodies to estradiol.

Quantification of Protein

Connective Tissue Growth Factor (CCN2) in Rat Pancreatic Stellate Cell Function: Integrin α5β1 as a Novel CCN2 Receptor Runping Gao, David R. Brigstock

Relationship between Genotype and Phenotype

Lab# 5 Western Blot BCH 462[practical].

V-2A-GFP/V-2A-coexpressing Mycobacterium tuberculosis antigens and GFP

Western immunoblot of Acanthamoeba whole-cell lysates reacted with normal human serum, demonstrating immunoreactivity against Acanthamoeba antigens. Western.

Presentation transcript:

 Non-enveloped T=3 icosahedral symmetry of identical sequence.  Single stranded positive sense RNA virus.  Coat protein (CP) coded by sub-genomic strand.  180 coat proteins (CP), 60 as 1 of 3 quasi-equivalent sub-units.  60 A sub-units: pentameric capsomeres.  60 B sub-units and 60 C sub-units: hexameric capsomeres.

 Arginine-glycine-aspartic acid (RGD)  A tripeptide motif used by Integrins as an attachment point.  RGD motifs are found within viral proteins and facilitate cellular adhesion.  Integrin (protein receptor)  Integrin is a ligand used by some cells and viruses for adhesion and cell signaling.  Stem cells up-regulate Integrin production.

 Utilize RGD motif as a binding site for Integrin attachment.  Multiple binding sites on exterior capsid.  Regulate stem cell adhesion.  Control stem cell growth and differentiation.  Mutant TYMV capsids are “vital for designing compatible biomaterials for tissue engineering purposes” (Gagandeep Kaur).

Arginine-Glycine-Aspartic acid (RGD) motifs can be engineered on the exterior of Turnip yellow mosaic virus (TYMV) coat proteins (CP) while maintaining the capsids: ability to assemble, structural stability, and allow binding of Integrin to the RGD motifs.

RGD 44 Monomer

RGD 44 Trimer

Mutants Sub- cloning Agro infiltrated Capsid Extraction Successful Assembly RGD 44Completed Yes RGD 55Completed RGD 61Completed RGD 102Completed RGD 103Completed RGD 161Completed RGD 162Completed RGD 184Incomplete

2x Low Speed Spins: 13G High Speed Spin: 50K RPM Low Speed Spin: 13G High Speed Spin: 50K RPM Resuspend Isolated Capsids 10X NaPO 4 Bentonite solution MgSO 4 RGD TYMV deveined plant tissue samples

Western blot Coomassie gel Ladder Wild-type RGD 44 RGD 55 RGD 103 RGD 162 Coomassie gel prepared using: Sodium dodecyl sulfate polyarcylamide gel electrophoresis (SDS-PAGE). Coomassie Brilliant Dye. Western blot prepared using: SDS-PAGE. Primary antibody: Rabbit anti-TYMV IgG. Secondary antibody: Goat anti-rabbit IgG Horseradish Peroxidase (HRP) conjugate. 27 kDa 15 kDa 27 kDa

Electron Microscopy Wild-type 28nm RGD 44 (60K) RGD 44 (125K) 50 nm

 RDG 44 expressed the mutant coat protein within the plant system, and self assembled forming capsids.  RDG 55, 103, or 162 coat proteins were not detected within the plant system. Future work will determine if the protein was or was not being expressed.  RDG 61, 62, 102, and 161 require further cloning techniques; transformation into Argobacterium, argoinfiltrated into the plant system, and capsid extraction to determine if coat protein can be expressed and self assemble within plant tissue.

HHMI (Howard Hughes Medical Institute) URISC (Undergraduate Research, Innovation, Scholarship and Creativity) Dr. Kevin Ahern The Dreher Lab Special thanks to Josh Powell

 Kaur, G. / Valarmathi, M.T. / Potts, J.D. / Wang, Q., Biomaterials, 29 (30), p ,Oct 2008