CV:

Garshasb Rigi, Ph.D

Assistance professor of Molecular Genetics,

Email: garshasbbiotech@gmail.com

Education

Sep 2011- May 2014

National Institute of Genetic Engineering and Biotechnology

PhD student, Molecular Genetics

Tehran, Iran

Aug 2011– May 2012

Tarbiat Modares University · Department of Genetics (Faculty of Biological Sciences)

PhD student, Molecular Genetics

Iran · Tehrān

Oct 2007 – Sep 2009

Razi Vaccine and Serum Research Institute and Tehran PNU

MSc, Biotechnology and Molecular Genetics

Karaj, Iran

Aug 1996 – Feb 2000

Isfahan University of Technology

Bachelor of Science,

Eşfahān, Iran

Aug 1992 – Feb 1996

Excellent High school

Diploma, The Experimental Sciences

Borūjen, Chahār Maḩāll va Bakhtīārī, Iran

Teaching Experience

    Feb 2013–present

Microbiology

Behbahan  Khatam Alanbia University of Technology

 

Dec 2011 – present

Virology

Behbahan  Khatam Alanbia University of Technology

Dec 2011 – present

Biochemistry

Behbahan  Khatam Alanbia University of Technology

Dec 2011 – present

Molecular and Cellular Biology

Behbahan  Khatam Alanbia University of Technology

Dec 2011 – present

Genetics

Behbahan  Khatam Alanbia University of Technology

Dec 2009 -2011

Agricultural courses

 PNU of Boroujen

Research Experience

Jun 2014

Virtual screening for finding potential drug like chemicals which blocks extracellular domain of CD 47 as an anti-cancer target

Behbahan  Khatam AlanbiaUniversity of Technology, Department of Biology

Behbahan, Iran

Jun 2014

Virtual screening following molecular dynamics simulation for finding potential inhibitors of enterotoxin B from Staphylococcus aureus

Behbahan  Khatam AlanbiaUniversity of Technology, Department of Biology

Behbahan, Iran

Aug 2012 – May 2013

PhD Student

Tarbiat Modares University, Department of Genetics (Faculty of Biological Sciences)

Tehrān, Iran

Mar 2012 – present

Surface display of protein A on spore and vegetative cells of Bacillus subtilis, E. coli and chitosan later use as a biotechnological product

National Institute of Genetic Engineering and Biotechnology, Department of Industrial and Environmental Biotechnology

Tehrān, Iran

Feb 2011 – present

High level recombinant protein A production and detection of its effects on immune systeme

National Institute of Genetic Engineering and Biotechnology, Department of Molecular Genetics

Tehrān, Iran

Aug 2010 – present

Recombinant vaccine production project against Hyalomma anatolicum anatolicom

Razi Vaccine and Serum Research Institute, Department of Biotechnology

Karaj, Iran

Jul 2009

Recombinant influenza vaccine production project

Razi Vaccine and Serum Research Institute, Department of Biotechnology

Karaj, Iran

2011

The effect of Iranian medicine and lactobacillus supernatant on inhibition of Staphylococcus aureus sortase as a potential drug target (Seminar)

National Institute of Genetic Engineering and Biotechnology, Department of Molecular Genetics

Tehrān, Iran

2011

Protein A : Generation and Applications (Seminar)

National Institute of Genetic Engineering and Biotechnology, Department of Molecular Genetics

Tehrān, Iran

2011

Assisting in the research project of Sortase in vitro analysis and inhibition as a potential drug target Iranian medicine and lactobacillus supernatant

National Institute of Genetic Engineering and Biotechnology, Department of Molecular Genetics

Tehrān, Iran

2011

Assisting in the national research project of Invitro and invivo evaluation of antibacterial potential of Lactobacillus plantarum HKN 01 isolated from Iranian traditional dairy products

National Institute of Genetic Engineering and Biotechnology, Department of Molecular Genetics

Tehrān, Iran

2011

Assisting in the research project of Gene Isolation, Cloning, expression and characterization of a novel chitinase gene of Streptomyces sp.BJ03 (isolated from Caspian sea); An insight into its role in biological control

National Institute of Genetic Engineering and Biotechnology, Department of Molecular Genetics

Tehrān, Iran

2010

Assisting in administrating the project of investigating the effect of dearth on 12 varieties of Bermuda grass

Boroujen municipality, under supervision of Dr.Khorshid Razmjoo, lecturer of Isfahan University of Technology

2010

Administrating the study of investigating the effect of dearth on 10 varieties of Zoshia grass

Boroujen municipality, under supervision of Dr.Khorshid Razmjoo, lecturer of Isfahan University of Technology

2010

Administrating the study of investigating the effect of saltiness on 12 varieties of Bermuda grass

Boroujen municipality, under supervision of Dr.Khorshid Razmjoo, lecturer of Isfahan University of Technology

2010

Administrating the study of investigating the effect of saltiness on 10 varieties of Zoshia grass

Boroujen municipality, under supervision of Dr.Khorshid Razmjoo, lecturer of Isfahan University of Technology

2010

In vitro Tissue culture of Fritillaria imperialis (Seminar)

Tehran PNU, under supervision of Dr.Mohammad jafaraghayi, assistance professor of Seed and Plant Improvement Institute.

2010

Administrating the study of investigating the effect of saltiness on 12 varieties of Bermuda grass

Boroujen municipality, under supervision of Dr.Khorshid Razmjoo, lecturer of Isfahan University of Technology

2009

Acting as an assistant in the research project of the effect of aerating on the rice root in Hydroponic system

Under the supervision of Dr.Aghafakhr Mirlohi , the professor of Isfahan University of Technology

2009

Assisting in the national research project of improving the quality & quantity of Triticum turgidum l grain

Under the supervision of Dr. Ahmad arzani, the professor of Isfahan University of Technology.

2009

Cooperating in the research project of the outcomes of drought in Chaharmahal o Bakhtiari province in order to suggest the drought and salt resistant plant species

Shahrekord center of water – related studies

2009

Preparing the optimum utilizing plan of Siasard forest park

Boroujen municipality

2009

Preparing the optimum utilizing plan of Ahmadabad forest park

Boroujen municipality

2009

Preparing the complete plan of Boroujen area under the title of: “Green area the breathing lungs of the city “

Boroujen municipality

2009

Preparing the well organizing plan of Boroujen green area

Boroujen municipality

2009

Preparing the identity papers of Boroujen green area

Boroujen municipality

2009

Preparing the green house plan (designing, administration, care and economic justifications)

Boroujen municipality

2009

Preparing the women park plan of Boroujen

Boroujen municipality

2009

Preparation of the proposal of the isolation project of the green area water from drinkable water in Boroujen and management and supervising on preparing and administrating of the mentioned project after getting it approved

Boroujen municipality

2009

Suggesting the proposal for the wastes management, preparation, supervision and administrating after its approval

Boroujen municipality

 

Awards & Grants

Aug 2014

Winning the title of the Outstanding and Excellence student in Ph. D Research

Aug 2010

Award: Being respected and gloried in the festival of science and technology endeavours, organization of scientific endeavourer.

Aug 2010

Award: Gaining the title of the distinguished elite in the festival manifestation of scientific self – belief and innovation

Jul 2009

Award: Wining the title of the distinguished student in MA course

Jun 2009

Award: Membership in the national foundation of the elite.

Jun 2009

Scholarship: Full educational scholarship of ministry of sciences , researches & technology

Skills & Activities

Skills

Genetic Analysis, Gel Electrophoresis, LC-MS, MOLDI-TOF MS, Protein Analysis, Chromatographic Techniques, Immunoassay Development, Protein Characterization, Promoter Analysis, Flow Cytometer, Bacterial Genetics, Molecular Genomics, Immunogenetics, Classical Genetics, Genetic Analysis, Analytical HPLC, Bradford Assay, SDS-PAGE, CTAB DNA extraction, Cell Culture, Cloning, DNA Amplification, ELISA, PCR, Western Blot, Dialysis, DNA, Electrophoresis, Enzymes, Immunoassay, Mass Spectrometry, Protein Engineering, Gene Expression, Genomics, Bioinformatics and Computational Biology, Cell Culture Techniques, HPLC-MS, Spectrophotometer, Genomic PCR,

Genomic DNA Isolation, Statistical Design, High-Performance Liquid Chromatography (HPLC)

know lots of bioinformatic softwares and sites, Sites or online softwares such as:, Pymol, Mascot, EMBL, MATCH, PDB, PIR, Pfam, KEGG, GMO, SRS, Ensemble, BLOCKS, PRINTS, PubMed, Proteomics, Tigers, smart, Sage, GEO, Entrez, map Viewer, EPD, EPO, USPTO,  PROSITE,  NEB Cutter, swiss-prot, patch,  UNIPROT, REACTOME, INTERPRO, PRODOM, RefSeq, Phyre, CDD, SignalP, MegAlign, UCSC, CDART, Primerselect, Unigene, MapDraw, AGRICOLA, AGRIS, SurfIP, Patent Lens, PSORT, SWISS-2DPAGE database, FindMod, iProclass, Gene Indices, SNPdb, CAMBIA, TIGRFAMs, Gene Ontology (GO), NCBI, BLAST, mFold, Clustal w2, I-TASSER, TargetP, peptide property calculator, ExPaSy, msproduct, eTBLAST,

 SAFTWARES such as:, Vector.NTI Advance, CLONEWIN, MEGA, Bioedith,  SeqMan, SPSS, OLIGO, Chromas, Genetyx, Protean, GeneMan, EditSeq, SnapGene Viewer, Gene Runner,  Molegro Molecular Viewer, Design – Expert, SigmaPlot, MagTran, Xcalibor, Spdbv,

 

For other Skills, please refer to: https://www.researchgate.net/profile/Garshasb_Rigi3/ or

 http://www.  ir.linkedin.com/pub/garshasb-rigi-ph-d/48/365/ba2/ or

 http://garshasbbiotech.blogfa.com/

 

Languages

English
French
Persian

Scientific

Memberships

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Some Social, Cultural and political activities and executive positions and responsibilities in various situations & times

Bio safety Society of Iran

Genetics Society of Iran

Biotechnology Society of Iran

Iran Society for Development of Landscape & Environment Conservation

The Eastern Mediterranean Health Genomics and Biotechnology Network (EMGEN)

Bioinformatics society of Iran

The head of the scientific – researching committee of agronomy & plant breeding group in agriculture faculty of Isfahan University of technology

Scientific Committee of Biology at  National Institute of Genetic Engineering and Biotechnology

 

The head of green area portion in Boroujen municipality.

The head of the planning committee for the anniversary of municipalities’ foundation, in Boroujen municipality.

Manager of the optimal use of energy project in Boroujen municipality and successful management especially during the year of utilization pattern reform.

The promotion assistant in the agriculture org of Chaharmahal o Bakhtiari province.

Supervising engineer of the wheat project in Boroujen County.

The head of the glorification and celebrating the Fajr ceremonies in Boroujen and winning the excellence level in 3 successive years.

The head of education & instruction in Imam Hosseyn Basidj as a distinguished member.

Assisting and cooperating in Shohada Basidj site council namely in Salehin project.

Member of religious group of Ansarilmahdi in Imam Hosseyn mosque – Boroujen and acting as the head of Islamic researches project between all.

The manager of the Rana ziba cultivating engineering company.

The expert of Golshan cooperative productive company in the fields of Glass house produced plants & flowers.

Producing a huge change & transforming in Boroujen green space and receiving several letters of appreciation.

Playing a great role & having a great effect in sowing trees in Boroujen Township and receiving several letters of appreciation from different governors & Bosses of Boroujen natural resource administration.

Gaining success in fighting against & controlling the calamities plagues and epidemic diseases in Boroujen Township & receiving several letters of appreciation from the headmaster of agriculture office of Boroujen Township.

Serving as the councilor & adviser of the governor of Boroujen Township in the fields of employment of agriculture graduators.

Membership in the center for the Basidji (The Islamic revolution semi - military and cultural special forces) elite of Chaharmahal o Bakhtiari province.

Chief - editor of Payam Magazine, a political, economic and cultural magazine.

The brother successor of university student basij at National Institute of Genetic Engineering and Biotechnology.

Membership of  central committee of Nanotechnology at Behbahan Khatam Alania University of Technology

Referee

Preparative Biochemistry & Biotechnology (ISI Journal)

Iranian Journal of breast disease (Journal)

Organic and Medicinal Chemistry Letters (ISI Journal)

The first Iranian festival – fair of invention – innovation (Conference)

04th National Bioinformatic Congress of  Iran (Conference)

Scientific

Appreciations

Being respected and gloried in the festival of science and technology             endeavourers of the Islamic revolution guards, organization of scientific

Being appreciate at Department of Molecular Genetics, National Institute of        Genetic Engineering and Biotechnology

Being appreciate at National Institute of  Genetic Engineering and Biotechnology in honor of outstanding performance and dedication and “Excellence” in the research projects and for innovative and high-quality research

Scientific Recommendations

Gholamreza Bakhshi Khaniki

Professor,

Head of Agricultural Biotechnology department,

Tehran PNU

Aghafakhr Mirlohi

Professor,

Department of Agronomy and Pant Breeding,

Isfahan University of Technology

Khorshid Razmjoo

Associated Professor ,  

Department of Agronomy and Pant Breeding,

Isfahan University of Technology

Ahmad Arzani

Professor,

Department of Agronomy and Pant Breeding,

Isfahan University of Technology

Mohammad Reza Khajehpour

Associated Professor , 

Department of Agronomy and Pant Breeding,

Isfahan University of Technology

Hossain Samadi

Assistance Professor,

Director of Water Recourses Research Center,

Shahrekord University

Gholam Reza Ahmdian

Assistance Professor,

Department of Industrial and Environmental Biotechnology,

National Institute of Genetic Engineering and Biotechnology

Kambiz Akbari

Assistance Professor,

Director of Division of  Industrial and Environmental Biotechnology,

National Institute of Genetic Engineering and Biotechnology

Mohammad Ali Malboobi

Associated Professor , 

Department of Plant Biotechnology,

National Institute of Genetic Engineering and Biotechnology

Amir Mousavi

Associated Professor , 

Department of Plant Biotechnology,

National Institute of Genetic Engineering and Biotechnology

Ali Asghar Karkhaneh

Associated Professor , 

Department of Industrial and Environmental Biotechnology,

National Institute of Genetic Engineering and Biotechnology

Gholam Ali Kardar

Associated Professor , 

Immunology, Asthma & Allergy Research Institute (IAARI),

 Children's Medical Center,

 Tehran University of Medical Sciences,

Books

Real-Time PCR, under publishing

Common laboratory techniques of molecular and cellular biology and genetic engineering (Being Compilation)

Journal Publications

Garshasb Rigi, Samira Ghaed Mohammadi, Maryam Rezaei Arjomand, Gholamreza Ahmadian, Kambiz Akbari Noghabi: Optimization of extracellular truncated Staphylococcal Protein A (SpA) expression in Escherichia coli BL21 (DE3). Biotechnology and Applied Biochemistry 2014 Mar; 61(2):217-25;

Majid Vahed, Ebrahim Motalebi, Garshasb Rigi, Kambiz Akbari Noghabi, Mohammad Reza Soudi, Mehdi Sadeghi, Gholamreza Ahmadian: Improving chitinolytic activity of Bacillus pumilus SG2 by random mutagenesis. Journal of Microbiology and Biotechnology 2013 Nov 28; 23(11):1519-28;

Farideh Zakeri, Majid Sadeghizadeh, Mohammad Reza Kardan, Hossein Shahbani Zahiri, Gholamreza Ahmadian, Fatemeh Masoumi, Hakimeh Sharafi, Garshasb Rigi, Hojatollah Vali, Kambiz Akbari Noghabi: Differential proteome analysis of a selected bacterial strain isolated from a high background radiation area in response to radium stress.. Journal of proteomics 05/2012; 75(15):4820-32.

H Sharafi, L Alidost, H Abassi, G Rigi, H Shahbani Zahiri, G Ahmadian, H Vali, K Akbari Noghabi: Antibacterial activity of probiotic Lactobacillus plantarum HK01: Effect of divalent metal cations and food additives on production efficiency of antibacterial compounds. Preparative Biochemistry & Biotechnology 01/2012;

Garshasb Rigi, Gholam Ali Kardar, Kambiz Akbari Noghabi, Gholamreza Ahmadian: Detection of protein A effects on immune system. Iranian journal of Immunology; 05/2014; 11: 786-787

Garshasb Rigi, Morteza Esfahanian, Khosrow Aghayipur, Reza Behrouzi, Gholamreza Ahmadian: Cloning and Sequencing of HAO3 gene of Hyalomma anatolicum anatolicum in unsecretory shuttle vector pPICZB. Modern Genetics Journal; 05/2012

Samira Ghaedmohammadi, Garshasb Rigi, Reza Zadmard, Gholamreza Ahmadian: Display of Staphylococcus aureus protein A on the Bacillus subtilis spore surface: Development of novel immunoadsorptive particles. Under review;

Kambiz Morabbi Heravi, Maryam Rezaei Arjomand, Garshasb Rigi, Majid Vahed, Samira Ghaedmohammadi, Gholamreza Ahmadian: Expression of β-galactosidase (lacZ) using a modified version of Bacillus pumilus chitinase promoter. Under review;

Garshasb Rigi, Parisa Beyranvand, Samira Ghaedmohammadi, Gholamreza Ahmadian, Kambiz Akbari Noghabi: Comparison of the extracellular full-length and truncated recombinant protein A production in Escherichia coli BL21 (DE3). Under review;

Garshasb Rigi, Mohsen Farhadpour, Alireza Ghasempour, Kambiz Akbari Noghabi, Gholamreza Ahmadian: Purification of full–length and truncated forms of recombinant Staphylococcal protein A (SpA) using hydrophilic interaction liquid chromatography. Under review;

 

Patents

High level and high pure recombinant protein A production insight into biological and drug applications. US Patent (Filing)

Conference Proceedings

Garshasb Rigi, Gholam Ali Kardar, Kambiz Akbari Noghabi, Gholamreza Ahmadian: Detection of protein A effects on immune system. 12th International Congress of Immunology and Allergy of Iran, Tehran, Iran; 05/2014

Zeynab Azad, Elahe Kamalzadeh, Anis Nozari, Forough Bakhtiyari, Hamideh Heydari, Garshasb Rigi: Insilico study of Gag polyprotein: potential target for investigating on HIV-diagnosis and therapy. First International and 13th Iranian Genetics Congress, Tehran, Iran; 05/2014

Sahar Ghasemi, Farzaneh Yazdani, Mahnaz Vahidian, Nahid Boveyrati, Garshasb Rigi: Bioinformatic Analysis of Homo sapiens ArfGAP with FG repeats 1 (AGFG1). First International and 13th Iranian Genetics Congress, Tehran, Iran; 05/2014

Samira Ghaedmohammadi, Garshasb Rigi, Majid Vahed, Gholamreza Ahmadian: Cloning, expression and secretion of protein A from S.aureus in B.subtilis. 8th Iranian Biotechnology Congress, Tehran, Iran; 09/2013

Garshasb Rigi, Samira Ghaed Mohammadi, Maryam Rezaei Arjomand, Majid Vahed, Gholamreza Ahmadian, Kambiz Akbari Noghabi: Cloning and expression of truncated Staphylococcal Protein A (SpA) in E. coli. 8th Iranian Biotechnology Congress, Tehran, Iran; 06/2013

Reza Behroozi, Nesa Jafari, Kambiz Akbari noghabi, Gholamreza Ahmadian, Kosar Rahimzadeh, Garshasb Rigi, Esmaeil Frozan: Biological control of charcoal rot (Macrophomina phaseolina) using Streptomyces sp. BJ03 isolated from North of Iran. 12th Iranian Genetics Congress, Tehran, Iran; 05/2013

Samira Ghaedmohammadi, Garshasb Rigi, Majid Vahed, Gholamreza Ahmadian: Bacillus subtilis genome-wide screening to search sortase and its potential substrates. 1st Tabriz International Life Science Conference, Tabriz, Iran; 05/2013

Garshasb Rigi, Samira Ghaed Mohammadi, Maryam Rezaei Arjomand, Gholamreza Ahmadian, Kambiz Akbari Noghabi: Cloning and expression of Full - length form of Staphylococcal Protein A (SpA) in E. coli. 1st Tabriz International Life Science Conference, Tabriz, Iran; 05/2013

Mohammad Sadegh Koosha, Ali Mirjalili, Majid Tebianian, Garshasb Rigi, Gholamreza Ahmadian: Cloning and Sequencing of Mycobacterium tuberculosis ESAT-6 gene in pEGFP-N1 as an eukaryotic expressions vector. 12th Iranian Genetics Congress, Tehran,Iran; 05/2012

Fatemeh Rafieenia, Shamseddin Ahmadi, Jalal Rostamzadeh, Sabrieh Amini, Garshasb Rigi: The Role Of NMDA Receptors In Tolerance And Dependence To The Morphine In The Striatum And The Prefrontal Cortex Of Rat Brain. 12th Iranian Genetics Cogress, Tehran, Iran; 05/2011

Hadi. Zarei Mahmoodabady, Garshasb Rigi, M.Morshedy: Investigating the Effect of Industrial Tile and ceramic Wastewater on Groundwater Pollution (Case Study: Yazd Province). 2nd International Conference on Environmental Science and Technology, Singapore; 02/2011

Garshasb Rigi, H. Zarei, h Mahmoodabady, M. Chabok, Fa. Morady: Investigating the Potential of Recycling Urban Solid Wastes (Case Study: Ardakan City). The First International Conference on Sustainable Urbanization, Faculty of Construction and Land Use , Hong Kong Polytechnic University; 06/2010

Garshasb Rigi, H. Zarei Mahmoodabadi: Investigation of Wastewater Treatment Sludge, Yard Trimming Waste, and Food Waste Combination in Vermicompost Quality, a Case Study of Yazd, Iran. The First International Conference on Sustainable Urbanization, Faculty of Construction and Land Use, The Hong Kong Polytechnic University; 06/2010

Garshasb Rigi, Mashayekhpoor Saied, Aghaiepour Khosrow: Cloning and sequencig of the Influenza A (H9N2) M2e - HSP70 fusion protein in prokaryotic expression vector in order to recombinant vaccine production. 2nd International Veterinary Poultry Congress, Tehran, Iran; 02/2010

Garshasb Rigi, Aghayipor Khosrow, Mashayekhpour Saeid, Ebrahimi Seyed mahmoud: cloning & the sequencing of HAO3 gene ( Hyalomma Anatolicum Anatolicum) in secretory shuttle vector pPICZαA. The First National Congress on of Veterinary Laboratory Science, Tehran, Iran; 10/2009

Conference Attending

          4th congress of agronomy & plant breeding in Iran – Karaj

          6th congress of agronomy & plant breeding in Iran – Mazandaran University, Babolsar

          7th congress of agronomy & plant breeding in Iran – Karaj

          5th national conference of waste and residue management in Mashhad

Workshop and Course Participating

          The green area management course at the 3rd national conference on green space and metropolitan sights at Kish Island and receiving the certificate from the organization of the state municipalities

          The watering management and drought crisis management in green area course in Isfahan

          The waste management course in Isfahan and receiving certificate from the state organization of municipalities and Isfahan waste org

          The waste management course in Shiraz and receiving certificate from the state organization of municipalities and Shiraz waste org

          Growing and production of decorating flowers course of the Education Complex of Agriculture org of Chaharmahal o Bakhtiari province

           Good Laboratory Practice (GLP) workshop, National Institute of Genetic Engineering and Biotechnology

Workshop Teaching

Genetic Engineering workshop instructor at Iranian Academic Center for Education Culture and Research (ACECR), Tehran University of Medical Science (TUMS) Branch

Recombinant Protein Production workshop instructor at Iranian Academic Center for Education Culture and Research (ACECR), Tehran University of Medical Science (TUMS) Branch

Gene Cloning and Molecular techniques workshop instructor at Iranian Academic Center for Education Culture and Research (ACECR), Tehran University of Medical Science (TUMS) Branch

Primer design and PCR workshop instructor at Iranian Academic Center for Education Culture and Research (ACECR), Tehran University of Medical Science (TUMS) Branch

Gene Expression  workshop instructor at Iranian Academic Center for Education Culture and Research (ACECR), Tehran University of Medical Science (TUMS) Branch

 

Teacher Evaluation Scores by students

 

Other notable scores

TOEFL ibt Score: 85/120

MSRT Score: 62/100

M.Sc. Thesis score: 19.25/20

Degree of M.Sc.  Thesis: Excellent

Ph.D. comprehensive exam score: 19.38/20

Ph.D. Thesis score: 19.95/20

Degree of Ph. D Thesis: Excellent

 

 

+ نوشته شده توسط گرشاسب ریگی چشمه علی در سه شنبه 1393/04/03 و ساعت 19:10 |

Journal of Proteomics

Available online 22 May 2012


Differential proteome analysis of a selected bacterial strain isolated from a high background radiation area in response to radium stress

  • Farideh Zakeria, b, c, Majid Sadeghizadeha, Mohammad Reza Kardand, Hossein Shahbani Zahirib, Gholamreza Ahmadianb, Fatemeh Masoumib, Hakimeh Sharafib, Garshasb Rigib, Hojatollah Valie, Kambiz Akbari Noghabib, ,
  • a Department of Genetics, Faculty of Biological Science, Tarbiat Modares University, P.O. Box 14115-154, Tehran, Iranb Department of Molecular Genetics, National Institute of Genetic ngineering and Biotechnology (NIGEB), P.O. Box 14155-6343, Tehran, Iranc Agriculture, Medicine and Industry Research School, Nuclear Science and Technology Research Institute, Tehran, Irand Radiation Applications Research School, Nuclear Science and Technology Research Institute- Iranian nuclear regulatory authority, Tehran, Irane Facility for Electron Microscopy Research, McGill University, 3640 Street, Montreal, Quebec, Canada H3A 2B2
  • Received 10 January 2012. Accepted 13 May 2012. Available online 22 May 2012.

Abstract

The present study describes the response of a bacterial strain, isolated from a hot spring in an area with the highest levels of natural radiation, under radium (226Ra) stress. The bacterium has been characterized as a novel and efficient radium biosorbent and identified as a variant of Serratia marcescens by biochemical tests and molecular recognition. In order to gain insights into key cellular events that allow this strain to survive and undergo 226Ra adaptation and biosorption, the strain was tested under two experimental conditions of 1000 and 6000 Bq 226Ra stress. A proteomic approach involving two-dimensional polyacrylamide gel electrophoresis and mass spectrometry was used to identify the differentially expressed proteins under 226Ra stress. Functional assessment of identified proteins with significantly altered expression levels revealed several mechanisms thought to be involved in 226Ra adaptation and conferring resistant phenotype to the isolate, including general stress adaptation, anti-oxidative stress, protein and nucleic acid synthesis, energy metabolism, efflux and transport proteins. It suggests that this strain through evolution is particularly well adapted to the high background radiation environment and could represent an alternative source to remove 226Ra from such areas as well as industrial radionuclide polluted wastewaters.

Keywords

Serratia marcescens; 226Ra stress; Proteomics; Biosorption; High background radiation area

+ نوشته شده توسط گرشاسب ریگی چشمه علی در شنبه 1391/04/03 و ساعت 16:11 |

Biological control of charcoal rot (Macrophomina phaseolina) using Streptomyces sp. BJ03 isolated from North of Iran

Reza Behroozi1, Nesa Jafari1, Kambiz Akbari noghabi1, Gholamreza Ahmadian*1, Kosar Rahimzadeh1, garshaseb Rigi1 and Esmaeil Frozan1 1 National Institution of Genetic Engineering and Biotechnology (NIGEB) *Corresponding author: gholamrezaahmadian@yahoo.ca

Macrophomina phaseolina is a plant pathogenic fungus which have a broad range of host such as soybean, bean, chickpea, pine and sesame. This fungus is a soil born pathogen that infects its hosts in primary growth stages like seedling growth. In this study, antifungal activity against Macrophomina phaseolina, was examined and inhibition percentage values was calculated. According to activity and broad spectrum of isolates, three isolates showed a high activity against charcoal rot and one of them had the highest activity. Some morphologically appearance suggested that these species belong to genus Streptomyces and phylogenetic studies using 16SrDNA confirmed it. Morphological changes such as hyphal branching and abnormal shape were observed in fungi grown on ISP2 medium. Hence, Streptomyces sp. BJ03 highly produced extracellular chitinase on colloidal chitin agar plates, it seems that this antifungal activity of this strain is related to production of extracellular chitinase. Therefore, the selected strain could be used for biocontrol of phytopathogenic fungi.

Keywords: Macrophomina phaseolina , Streptomyces sp. BJ03, biological control

+ نوشته شده توسط گرشاسب ریگی چشمه علی در جمعه 1391/03/05 و ساعت 20:52 |

Cloning and Sequencing of HAO3 gene of Hyalomma anatolicum anatolicum in unsecretory shuttle vector pPICZB

Garshasb Rigi Cheshmehali1, Morteza Esfahanian2, Khosrow Aghayipur3, Reza Behrouzi4, Nezam Armand 5 ,Gholamreza Ahmadian6*

1- PhD student in Molecular Genetics in National Institute of Genetic Engineering and Biotechnology 2- M.Sc in Biotechnology in Razi Vaccine and Serum Research Institute3- Assistant professor of Department of Biotechnology, Razi Vaccine and Serum Research Institute 4- M.Sc in Biotechnology in National Institute of Genetic Engineering and Biotechnology 5- Lecturer  of  Department of Biology in Behbahan Khatamolanbia University of Technology 6- Assistant professor of Department of  Molecular Genetics in National Institute of Genetic Engineering and Biotechnology (gholamrezaahmadian@yahoo.ca*)

Abstract

Introduction :Hyalomma anatolicum anatolicum is parasite of farm and wild animals in tropical and sub tropical region of the world include south of Iran .Ticks of genus Hyalomma are vector of many disease also this parasite cause decrease of milk, meat and low quality leaser which produce by its host.

Objective: HAO3 gene is candid to invent new vaccine against this tick.

Material and Methods: In this study we use two ORF specific primers to amplify HAO3 in Termocycler then it has ligated with pPICZB expression vector and transformed in E.Coli DH5α then pPICZB has extracted from E.Coli .on next stage HAO3 has made apart from pPICZB by two restriction enzyme (EcoRI and XbaI).

Results: Sequencing shows that our gene is exactly like the HAO3 which is in gene bank and it is partly like BM86 and HA86.

 

Keywords: HAO3 gene, cloning, sequencing, vaccine

+ نوشته شده توسط گرشاسب ریگی چشمه علی در یکشنبه 1390/12/21 و ساعت 9:11 |

The Role Of NMDA Receptors In Tolerance And Dependence To The Morphine In The Striatum And The Prefrontal Cortex Of Rat Brain

 

Fatemeh Rafieenia1*, Shamseddin Ahmadi2, Jalal Rostamzadeh2, Sabrieh Amini3, Garshasb Rigi Cheshmehali4

 

1M.Sc in Cellular and Molecular Biology, University of Kurdistan, Sanandaj, Iran

Assistant professor of  Department of Biological Science & Biotechnology, Faculty of Science, University of Kurdistan, Sanandaj, Iran

Assistant professor  of  Department of Biological, Faculty of Science, Islamic Azad University, sanandaj branch, iran

4 PhD student in Molecular Genetics in National Institute of Genetic Engineering and Biotechnology, Tehran, iran

 

Introduction: N-Methyl-D-Aspartate (NMDA) receptors have important roles in learning, memory formation, tolerance and dependence to the morphine.

Aim: In the present study, after induction of morphine dependence in male wistar rats, changes in gene expression of NR1 subunit of NMDA receptors, the main subunit of these receptors, in the striatum and the prefrontal cortex of rat brain were examined.

Material and Methods: For induction of morphine dependence, a dose of 10 mg/kg of morphine was administrated intraperitoneally for seven consecutive days. Control group received saline replace to morphine similarly. Tolerance to the morphine was examined by a hot plate test of analgesia, 1 day after the final repeated administrations. A semi-quantative RT-PCR was used for evaluating gene expression in different periods after withdrawal.

Results: The results showed that the level of mRNA of NR1 in the striatum on day 1 after the final repeated administrations of morphine was significantly increased, while in the prefrontal cortex was significantly decreased. on the other interval times of 3, 7, 14 and 21 days after the final repeated administrations of morphine, changes in the gene expression in the two areas compared with control group were not significant, and almost returned to the basal level.

 

Keywords: Morphine, NMDAR, Striatum, Prefrontal,cortex

+ نوشته شده توسط گرشاسب ریگی چشمه علی در یکشنبه 1390/12/21 و ساعت 9:6 |

Cloning and Sequencing of Mycobacterium tuberculosis ESAT-6 gene in pEGFP-N1 as an eukaryotic expressions vector

 

Mohammad Sadegh Koosha1*, Ali Mirjalili2,Majid Tebianian2, Garshasb Rigi Cheshmehali3,Gholamreza Ahmadian4

M.Sc in Biotechnology Engineering in Razi Vaccine and Serum Research Institute1

Assistant professor of Department of Biotechnology, Razi Vaccine and Serum Research Institute2

PhD student in Molecular Genetics in National Institute of Genetic Engineering and Biotechnology3

Assistant  professor of  Department of  Molecular Genetics in National Institute of Genetic Engineering and Biotechnology4

Introduction: Tuberculosis which has killed many people and caused many financial damages since many years ago is one of the major health problems. In spite of serious efforts to prevent disease, it is one of the important causes of death throughout world with two million death annually. One of the most important immunizing proteins of Mycobacterium tuberculosis is  ESAT–6 which is a 9.8KDa polypeptide and is  recognized  by T cells and stimulates  strong cell immunity responses  and  produces high amount  of  IFN–γ.

Objects and methods: This research was done in order to clone and sequence the ESAT–6 protein from Mycobacterium  tuberculosis  into a eukaryotic expression vector named pEGFP– N1.Characterizing of this gene by PCR cloning, restriction enzyme analysis and sequencing the gene can be the basis to produce a type of Stable Cell Line to be used for evaluation of ESAT-6 immune responses.

Results: Comparison of the nucleotide sequence of this gene and sequence of amino acid product of that with registered sequence in gene bank shows that this gene with isolated type (Rv3875) has high identity. This recombinant plasmid will be express on the CT-26 cell line which is a tumor cell from the BaIb/C mouse.

Key words: ESAT –6, pEGFP-N1, Cloning , Mycobacterium  tuberculosis.

+ نوشته شده توسط گرشاسب ریگی چشمه علی در یکشنبه 1390/12/21 و ساعت 9:0 |

Cloning and sequencig of the Influenza A (H9N2) M2e - HSP70 fusion protein in prokaryotic expression vector in order to recombinant vaccine production

*Rigi cheshmehali Garshasb - Mashayekhpoor Saied-Aghaiepour Khosrow

            

Iran-Razi Vaccine and Serum Research Institute

*Corresponding author,s E. mail: garshasbbiotech@gmail.com

Abstract

      One of the concerns about influenza A vaccine based on M2e protein is their limited potency; hence, optimal approaches to enhance immunogenicity of M2e protein immunization remain to be established. It seems by linking this M2e-peptide to an appropriate carrier such as mycobacterium tuberculosis C-terminal 28-kDa domain of HSP70 (HSP70 359-610), we can render it very immunogenic.This study was designed to produce a novel influenza A virus recombinant fusion protein consisted of M2e , a potent immunogenic protein from influenza A virus, fused to C-terminal domain of mycobacterium tuberculosis HSP70, HSP70359-610 , as a carrier and adjuvant. We fused the genes of M2e and HSP70359-610 then inserted in pQE-60, prokaryotic expression vector PQE-60. Identification of the cloned gene was confirmed by PCR, restriction analysis and gene sequencing.Then cloned products were sent for sequencing to check the correct ORF and direct origin of the cloned gene. Comparison of the nocleotid sequence of this gene and sequence of amino acid product of that with registered sequences in genbank shows that this gene with total isolated types has much resemblance and after gene expression in prokaryotic system , it’s protein product can be a good candidate for recombinant vaccine against the Influenza.

 

Keywords: cloning-sequencing-Influenza A-M2e gene-HSP70fusion protein-PQE-60 vector-PCR.recombinant vaccine

+ نوشته شده توسط گرشاسب ریگی چشمه علی در سه شنبه 1390/09/08 و ساعت 9:58 |

cloning & the sequencing of HAO3 gene ( Hyalomma Anatolicum Anatolicum) in secretory shuttle vector pPICZαA , Rigi cheshmehali  Garshasb , Aghayipor Khosrow , Mashayekhpour Saeid , Ebrahimi Seyed mahmoud , Razi Vaccine and Serum Research Institute

Abstract

     Hyalomma anatolicum anatulicum (Acari: Ixodidae) parasitize farm and wild animals in tropical and sub tropical region of the world. Ticks of the genus Hyalomma are well – khown vectors of viruses and avid parasits of man. One of the most important disease transmited by Hyalomma ticks is Crimean – Congo Hemorragic Fever (CCHF). H.anatolicum is one of the main vector ticks. In order to develop a broad – spectrum protection agianst different Hyalomma variants, some recent studies have been aimed at the HAO3 gene of Hyalomma anatolicum anatulicum.In this study, open reading frame of Hyalomma HAO3 gene was amplified by PCR using specific primers with restriction sites for XbaI and EcoRI restriction enzymes, cloned to pPICZαA expression vector in E.Coli DH5α. recimbinant vector was confirmed by PCR and restriction enzymes. Then cloned products were sent for sequencing to check the correct ORF and direct origin of the cloned HAO3 gene. Comparison of the nucleotid sequence of this gene and sequence of amino acid product of that with registered sequences in genbank shows that this gene with total isolated types HAO3, BM86, HA98 has much resemblance and after gene expression in eukaryotic yeast (pichia pastoris), it’s protein product can be a good candidate for recombinant vaccine against the Hyalomma.

 

Keywords: cloning-sequencing- PCR -HAO3gene- pPICZαA vector

 

+ نوشته شده توسط گرشاسب ریگی چشمه علی در سه شنبه 1390/09/08 و ساعت 9:56 |
 

Review article

 Influenza vaccine production in plants

Rigi Cheshmehali Garshasb1 – Mousavi Seyyed Amir2

1- PhD student in Biology – Molecular Genetics in National Institute of Genetic Engineering and Biotechnology (NIGEB)- Iran

2-Director, Department of Plant Biotechnology & International Relations Coordinate of National Institute of Genetic Engineering and Biotechnology (NIGEB)- Iran

 

A b s t r a c t

              Plants have been identified as promising expression systems for commercial production of vaccine antigens. Transgenic plants, including edible plant parts are uggested as excellent alternatives for the production of vaccines and economic scale-up through cultivation. As an important case the spectre of an influenza pandemic has led to a revision of national and global pandemic preparedness plans and has stressed the need for more efficient influenza vaccines and manufacturing practices. This article reviews the current status of developments in the area of use of plants for the development of vaccine antigens against influenza.

 

Keywords: transgenic plants, vaccine antigens, influenza, pandemic.

 

 Introduction

       Infectious diseases account for more than 45% of total deaths in developing countries (9). Vaccination is the most effective means to prevent infectious diseases. More than 30 million children in the world are not immunized against treatable or preventable diseases (www.care.org/campaigns/childrenpowerty/facts.asp) because the currently used approaches to vaccine production are technologically complex and expensive. Specialized requirements of packaging, cold chain and mode of delivery add to the cost.

      Currently used mammalian cell line based vaccine manufacturing requires large investment and expertise. These factors limit their scale up and thus, global availability. Advances in molecular biology techniques during the 1980s, helped in the development of new strategies for the production of subunit vaccines. These comprised of proteins derived from pathogenic viruses, bacteria or parasites. Although mammals, their tissue sand cell lines are currently utilized for commercial production of vaccines, these systems are expensive and their scale up is not easy (9). Toxins, infectious agents and other noxious compounds get carried in animal cell based processes and are often difficult to remove. Such production systems are prone to microbial contamination which sometimes escapes detection even in purified vaccines. Expression of recombinant antigen proteins in E. coli is often not feasible because of lack of a variety of post translational modifications and folding requirements.

          Some of the mammalian-type post translational processing and modifications in protein happen in yeast and insect cell lines. However, immunologically significant differences in the pattern of post translational modifications limit their deployment in the expression of vaccine antigens. As a major alternative, plants are emerging as a promising system to express and manufacture a wide range of functionally active proteins of high value to health industry. Various plant biotechnological techniques, such as, modern breeding methods, clonal propagation , somatic hybridization, protoplast/cell suspension culture, hairy root culture and genetic transformation can play a vital role in establishing the use of plants as “surrogate production organisms”. One or more immunoprotective antigens of pathogens can be produced in plants by the expression of gene(s) encoding the protein(s). In recent years, plant-based novel production systems aimed at developing “edible” or “oral” vaccines have also been discussed(9). Compared to traditional vaccines, edible vaccines offer simplicity of use, lower cost, convenient storage, economic delivery and mucosal immune response. Successful development of vaccine antigens against human and animal pathogen(s) in plants requires selection of one or more immunoprotective antigens and designing of genes and promoters that would express the antigen(s) at a high level in target plant tissue. Genetic transformation methods are then utilized for introducing the gene in the target plant species (figure 1).

This review focuses on applications of plants for vaccine production against Influenza.

 Introducing the Influenza and importance of it’s plant- based vaccine

      Influenza is a highly contagious and acute respiratory disease with a high degree of morbidity and mortality. It is estimated that influenza is responsible for 36,000 deaths and more than 200,000 hospitalizations in the United States annually. Globally, influenza epidemics result in 3–5 million hospitalizations and 300,000–500,000 deaths each year (4). New epidemic strains of influenza A arise due to point mutations within two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (figure 2). These changes in HA and NA enable emerging virus strains to evade the host’s immune system and therefore necessitates the annual revision of vaccine to include the new viruses (3, 4). Influenza type A virus is classified into several subtypes which vary in two of their transmembrane proteins, hemagglutinin (HA)1 and neuraminidase (NA) and possess similar nucleoprotein and matrix protein (3,4). A constant antigenic reassortment in surface-located HA and NA, which are, respectively, responsible for binding of the virus to its cellular receptor and promoting release of the mature virus from infected cells (4), makes vaccine development a complicated process. There are 16 known HA subtypes and nine known NA subtypes of influenza A viruses and many different combinations are possible. Three subtypes, (H1N1, H1N2, and H3N2), commonly circulate among humans and all known subtypes of influenza A viruses can be found in birds (4). Influenza A subtype H5N1 virus, known as avian influenza or bird flu, is endemic mainly in birds and does not usually infect people. However, because of its high virulence, H5N1 may be transmitted from infected birds to humans and cause fatal disease.

 Figure 1 . Steps in the production of plant-derived vaccine antigens (9)

       The recent swine H1N1 influenza pandemic (pH1N1) revealed the limitations of the current influenza vaccine manufacturing technologies. Regardless of the origin, mechanism of emergence or precise genetic makeup of the ‘next’ pandemic strain, recent experience with pH1N1demonstrate clearly that the current, egg-based manufacturing system would not be able to respond quickly enough in the face of a highly pathogenic influenza virus adapted for rapid human-tohuman pread.

     A plant-based manufacturing technology that can produce vaccine doses within one month of

the sequencing of a pandemic strain should be describe.

 Figure 2 . Schematic representation of the structural characteristics of viral particles and plant-made VLPs (virus-like particles) .  A. Cross-section showing internal differences. Hemagglutinin (HA) (green) andneuraminidase (NA) (orange) are the two major viral proteins protruding outside of the viral envelope (pink) , HA being the main antigenic determinant of the virus.  B. Transmission electron microscopy images of influenza viruses and plant-made VLPs (3).

 Influenza vaccines produced in plants

       Evidence of influenza antigen production in plants was only recently disclosed, in 2004, through an international patent application (1). In this application, it is concluded that influenza HA accumulates in calluses of NT1 tobacco cells transformed to express the complete coding sequence of influenza H5 from strain A⁄ turkey ⁄ Wisconsin ⁄ 1968 (H5N9). When extracted without detergent, the apoplastic fluid of the transgenic cells exhibited hemagglutination activity, indicating that plant-produced H5 was secreted and active. Rabbits immunized with a crude preparation of extracellular fluid from transgenic NT-1 cells producing H5 from strain A⁄ turkey ⁄ Wisconsin ⁄ 1968 (H5N9) showed a strong hemagglutination inhibiting (HI) antibody response 6 weeks after immunization in the presence of Freund’s adjuvant. 

      Expression of the HA ectodomain (the segment of the protein spanning outside of the viral envelope) fused to a KDEL peptide, to enhance accumulation through retention in the endoplasmic reticulum, and a poly-histidine purification tag has also been utilized as a mean for facilitating the production of recombinant influenza antigens in plants. Examples include the HA ectodomains from a human seasonal influenza strain (A ⁄ Wyoming ⁄ 03 ⁄ 03 (H3N2) (6) and highly pathogenic avian strains A⁄ Indonesia ⁄ 5 ⁄ 05 (7), A⁄ Bar-headed Goose ⁄ Qinghai ⁄ 1A ⁄ 05 and A⁄ Anhui ⁄ 1 ⁄ 05 (8). In immunogenicity studies in mice, HA ectodomains have been shown to induce significant HI responses when administered in conjunction with Quil A. However, although such composition induced a strong immune response with doses as low as 1 lg (A ⁄ Anhui ⁄ 1 ⁄ 05 (H5N1)) (8), two doses were required to obtain an HI antibody response. A ferret study further showed that three doses of 45 lg adjuvanted with Quil A were required to confer protection against a lethal challenge with the homologous strain of H5 ectodomain (7). Together, the above mentioned immunogenicity studies, performed with HA fragments in fusion with a carrier protein or with other peptides, provided strong indications that plant-made influenza antigens can be produced by agroinfiltration and that these HA fragments induce hemagglutination inhibition antibody response in model animals. However, the high dosage and multiple injections required to induce a protective immune response in ferrets from immunization with the ectodomain of H5 (A ⁄ Indonesia ⁄ 5 ⁄ 05 (H5N1)) suggests that a higher order of antigen organization is required for optimal stimulation of a protective immune response. More recently, attempts at producing the entire H5 protein (from strain A⁄ Vietnam ⁄ 1203 ⁄ 04 (H5N1)) or its HA1 domain by transient or stable transformation of N. benthamiana were reported unsuccessful as they led to only detectable accumulation of the mature H5 or HA1 domain (10). Fragments of 34 or 27 kDa from the HA1 domain, or fusions of the HA1 domain with 26 kDa fragment from a human or a mouse heavy chain constant region were reported to accumulate at higher levels. In an immunogenicity study in mice, two doses of 10 lg of the 34 kDa fragment from the antigenic region of H5 from influenza A⁄ Vietnam ⁄ 1203 ⁄ 04 (H5N1), administered in the presence of alum-CpG as adjuvant, induced high H5 specific antibody titers but failed to induce significant HI antibody titers (10), again highlighting the need for a higher degree of organization of multivalent antigens to stimulate a protective immune response.

       A similar strategy was recently proposed in the international patent application WO2007⁄ 011904 for the presentation of influenza M2e universal epitope onto CPMV particles. Cowpea plants rubbed with RNA1 and chimeric RNA2 encoding a CP-M2e fusion produced chimeric CPMV particles comprising CP-M2e (5). Similarly, expression of a chimeric cucumber mosaic virus (CMV) capsid protein fused to the M2e epitope in N. benthamiana using a potato virus X (PVX) expression vector also led to the production of chimeric CP-M2e. However, assembly of the capsid proteins into chimeric viral particles was not demonstrated in this study (4). Capsid proteins of the PVX expression vector have also been used for the display of H-2Db-restricted epitope from the influenza NP from strain A⁄ PR ⁄ 8 ⁄ 34 (2). In this study, the epitope coding sequence was fused to the CP gene of the PVX vector, creating chimeric PVX particles displaying the NP epitope. An immunogenicity study in mice, designed to evaluate the cellbased immune response, showed that when administered in the presence of incomplete Freund’s adjuvant, 50 lg of chimeric viral particles displaying the NP epitope activated ASNENMETM-specific CD8+ IFN-c secreting cells. Although chimeric viral particles antigen presentation platforms benefit from the display of selected antigenic epitopes in a multivalent fashion, this strategy also bear some intrinsic drawbacks. Only a limited number of antigenic epitopes of small size (less than 25 amino acids) can be displayed using this system. Therefore, it is easier for rapidly evolving viruses like influenza to evade the immune response induced by such vaccines by replacing a few amino acids in the selected immunogenic region. Conformational epitopes may also not fold properly, thereby inducing the production of antibodies that will not recognize the cognate native epitope. Finally, the use of chimeric plant viral particles as presentation devices will require regulatory acceptance in themselves in addition to the regulatory hurdles faced by new manufacturing platform.

 Conclusions and future prospects

 With the world's population at over 6.4 billion (http://esa.un.org) , majority of the poor need affordable technological solutions to health. Protection from viral infections is currently the most difficult area to address through drug development. Progress in plant genetic engineering has opened novel opportunities to use plants as bioreactors for safe and cost effective production of   Influenza vaccine antigens.

      As is clear from several examples cited in this review, the production of recombinant proteins in plant systems has a great potential. Recent developments in this area have significantly increased its utility and enabled various groups to explore the possibility of producing vaccine antigens from a variety of plants, which can be directly or indirectly used to develop commercial processes. Transgenic plants that can produce biologically active proteins or subunit oral vaccines and antibodies have been developed, though the applications of these technologies are at least a decade away.

      The need to establish safety, efficacy and functional equivalence of the vaccine antigens should guide future development and research in plant based preventive and therapeutic technologies.

 References

 1.                                  Cardineau, G., Mason, H.S., Vaneck, J.M., Kirk, D.D. and Wamsley, A.M. (2004)                           Vectors and cells for preparing immunoprotective compositions derived from transgnic plants. International Patent application WO2004 0098533.

 

2.                                  Lico, C., Mancini, C., Italiani, P., Betti, C., Boraschi, D., Benvenuto, E. and Baschieri, S. (2009) Plant-produced potato virus X chimeric particles displaying an influenza virus-derived peptide activate specific CD8+ T cells in mice. Vaccine, 27, 5069–5076.

 

3.                                  Nathalie , L., Brian J, W., Sonia ,T., Emanuele, M., Miche`, D., Giulia, L., Louis-P, V. (2010) Preclinical and Clinical Development of Plant-Made Virus-Like Particle Vaccine against Avian H5N1 Influenza. PLoS ONE, 1-12.  

 

4.                                  Nemchinov, L.G. and Natilla, A. (2007) Transient expression of the ectodomain of matrix 2 (M2e) of avian influenza A virus in plants. Prot. Expression and Purif., 56, 153–159.

 

5.                                 Rasochova, L., Radam, J.M., Phelps, J.P. and Dang, N. (2007) Recombinant flu vaccines. International Patent application WO2007 0011904.

                                

6.                                  Shoji, Y., Chichester, J.A., Bi, H., Musiychuk, K., de la Rosa, P., Goldschmidt, L., Horsey, A., Ugulava, N., Palmer, G.A., Mett, V. and Yusibov, V. (2008) Plant-expressed HA as seasonalinfluenza vaccine candidate. Vaccine, 26, 2930–2934.

 

7.                                 Shoji, Y., Bi, H., Musiychuk, K., Rhee, A., Horsey, A., Roy, G., Green, B., Shamloul, M., Farrance, C.E., Taggart, B., Mytle, N., Ugulava, N., Rabindran, S., Mett, V., Chichester, J.A. andYusibov, V. (2009a) Plant-derived hemagglutinin protects ferrets against challenge infection with the A Indonesia 05 05 strain of influenza. Vaccine, 27, 1087–1092.

 

 

8.                                 Shoji, Y., Farrance, C.E., Bi, H., Shamloul, M., Green, B., Manceva, S., Rhee, A., Ugulava, N., Roy, G., Musiychuk, K., Chichester, J.A., Mett, V. and Yusibov, V. (2009b) Immunogenicity of hemagglutinin from A Bar-headed Goose Qinghai 1A 05 and A Anhui 1 05 strains of H5N1 influenza viruses produced in Nicotiana benthamiana plants. Vaccine, 27, 3467–3470.

 

9.                                 Siddharth T., Praveen C., Verma , P.,  Rakesh T. (2009) Plants as bioreactors for the production of vaccine antigens. Biotechnology Advances 27 ,  449–467

 

10.                             Spitsin, S., Andrianov, V., Pogrebnyak, N., Smirnov, Y., Borisjuk, N., Portocarrero, C., Veguilla, V., Koprowski, H. and Golovkin,M. (2009) Immunological assessment of plant-derived avian fluH5 HA1 variants. Vaccine 27, 1289–1292.

+ نوشته شده توسط گرشاسب ریگی چشمه علی در دوشنبه 1390/07/04 و ساعت 10:44 |

How Cells Read the Genome: From DNA to Protein

Only when the structure of DNA was discovered in the early 1950s did it become clear how the hereditary information in cells is encoded in DNA's sequence of nucleotides. The progress since then has been astounding. Fifty years later, we have complete genome sequences for many organisms, including humans, and we therefore know the maximum amount of information that is required to produce a complex organism like ourselves. The limits on the hereditary information needed for life constrain the biochemical and structural features of cells and make it clear that biology is not infinitely complex.

In this chapter, we explain how cells decode and use the information in their genomes. We shall see that much has been learned about how the genetic instructions written in an alphabet of just four “letters”—the four different nucleotides in DNA—direct the formation of a bacterium, a fruit fly, or a human. Nevertheless, we still have a great deal to discover about how the information stored in an organism's genome produces even the simplest unicellular bacterium with 500 genes, let alone how it directs the development of a human with approximately 30,000 genes. An enormous amount of ignorance remains; many fascinating challenges therefore await the next generation of cell biologists.

The problems cells face in decoding genomes can be appreciated by considering a small portion of the genome of the fruit fly Drosophila melanogaster (Figure 6-1). Much of the DNA-encoded information present in this and other genomes is used to specify the linear order—the sequence—of amino acids for every protein the organism makes. As described in Chapter 3, the amino acid sequence in turn dictates how each protein folds to give a molecule with a distinctive shape and chemistry. When a particular protein is made by the cell, the corresponding region of the genome must therefore be accurately decoded. Additional information encoded in the DNA of the genome specifies exactly when in the life of an organism and in which cell types each gene is to be expressed into protein. Since proteins are the main constituents of cells, the decoding of the genome determines not only the size, shape, biochemical properties, and behavior of cells, but also the distinctive features of each species on Earth.

One might have predicted that the information present in genomes would be arranged in an orderly fashion, resembling a dictionary or a telephone directory. Although the genomes of some bacteria seem fairly well organized, the genomes of most multicellular organisms, such as our Drosophila example, are surprisingly disorderly. Small bits of coding DNA (that is, DNA that codes for protein) are interspersed with large blocks of seemingly meaningless DNA. Some sections of the genome contain many genes and others lack genes altogether. Proteins that work closely with one another in the cell often have their genes located on different chromosomes, and adjacent genes typically encode proteins that have little to do with each other in the cell. Decoding genomes is therefore no simple matter. Even with the aid of powerful computers, it is still difficult for researchers to locate definitively the beginning and end of genes in the DNA sequences of complex genomes, much less to predict when each gene is expressed in the life of the organism. Although the DNA sequence of the human genome is known, it will probably take at least a decade for humans to identify every gene and determine the precise amino acid sequence of the protein it produces. Yet the cells in our body do this, thousands of times a second.

The DNA in genomes does not direct protein synthesis itself, but instead uses RNA as an intermediary molecule. When the cell needs a particular protein, the nucleotide sequence of the appropriate portion of the immensely long DNA molecule in a chromosome is first copied into RNA (a process called transcription). It is these RNA copies of segments of the DNA that are used directly as templates to direct the synthesis of the protein (a process called translation). The flow of genetic information in cells is therefore from DNA to RNA to protein (Figure 6-2). All cells, from bacteria to humans, express their genetic information in this way—a principle so fundamental that it is termed the central dogma of molecular biology.

Despite the universality of the central dogma, there are important variations in the way information flows from DNA to protein. Principal among these is that RNA transcripts in eucaryotic cells are subject to a series of processing steps in the nucleus, including RNA splicing, before they are permitted to exit from the nucleus and be translated into protein. These processing steps can critically change the “meaning” of an RNA molecule and are therefore crucial for understanding how eukaryotic cells read the genome. Finally, although we focus on the production of the proteins encoded by the genome in this chapter, we see that for some genes RNA is the final product. Like proteins, many of these RNAs fold into precise three-dimensional structures that have structural and catalytic roles in the cell.

We begin this chapter with the first step in decoding a genome: the process of transcription by which an RNA molecule is produced from the DNA of a gene. We then follow the fate of this RNA molecule through the cell, finishing when a correctly folded protein molecule has been formed. At the end of the chapter, we consider how the present, quite complex, scheme of information storage, transcription, and translation might have arisen from simpler systems in the earliest stages of cellular evolution

 Figure 6-1. Schematic depiction of a portion of chromosome 2 from the genome of the fruit fly Drosophila melanogaster . This figure represents approximately 3% of the total Drosophila genome, arranged as six contiguous segments. As summarized in the key, the symbolic representations are: rainbow-colored bar: G-C base-pair content; black vertical lines of various thicknesses: locations of transposable elements, with thicker bars indicating clusters of elements; colored boxes: genes (both known and predicted) coded on one strand of DNA (boxes above the midline) and genes coded on the other strand (boxes below the midline). The length of each predicted gene includes both its exons (protein-coding DNA) and its introns (non-coding DNA) (see Figure 4-25). As indicated in the key, the height of each gene box is proportional to the number of cDNAs in various databases that match the gene. As described in Chapter 8, cDNAs are DNA copies of mRNA molecules, and large collections of the nucleotide sequences of cDNAs have been deposited in a variety of databases. The higher the number of matches between the nucleotide sequences of cDNAs and that of a particular predicted gene, the higher the confidence that the predicted gene is transcribed into RNA and is thus a genuine gene. The color of each gene box (see color code in the key) indicates whether a closely related gene is known to occur in other organisms. For example, MWY means the gene has close relatives in mammals, in the nematode worm Caenorhabditis elegans, and in the yeast Saccharomyces cerevisiae. MW indicates the gene has close relatives in mammals and the worm but not in yeast. (From Mark D. Adams et al., Science 287:2185–2195, 2000. © AAAS.)

 Figure 6-2. The pathway from DNA to protein. The flow of genetic information from DNA to RNA (transcription) and from RNA to protein (translation) occurs in all living cells.

 From DNA to RNA

Transcription and translation are the means by which cells read out, or express, the genetic instructions in their genes. Because many identical RNA copies can be made from the same gene, and each RNA molecule can direct the synthesis of many identical protein molecules, cells can synthesize a large amount of protein rapidly when necessary. But each gene can also be transcribed and translated with a different efficiency, allowing the cell to make vast quantities of some proteins and tiny quantities of others (Figure 6-3). Moreover, as we see in the next chapter, a cell can change (or regulate) the expression of each of its genes according to the needs of the moment—most obviously by controlling the production of its RNA.

 Figure 6-3. Genes can be expressed with different efficiencies. Gene A is transcribed and translated much more efficiently than gene B. This allows the amount of protein A in the cell to be much greater than that of protein B.

 Portions of DNA Sequence Are Transcribed into RNA

The first step a cell takes in reading out a needed part of its genetic instructions is to copy a particular portion of its DNA nucleotide sequence—a gene—into an RNA nucleotide sequence. The information in RNA, although copied into another chemical form, is still written in essentially the same language as it is in DNA—the language of a nucleotide sequence. Hence the name transcription.

Like DNA, RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds (Figure 6-4). It differs from DNA chemically in two respects: (1) the nucleotides in RNA are ribonucleotides—that is, they contain the sugar ribose (hence the name ribonucleic acid) rather than deoxyribose; (2) although, like DNA, RNA contains the bases adenine (A), guanine (G), and cytosine (C), it contains the base uracil (U) instead of the thymine (T) in DNA. Since U, like T, can base-pair by hydrogen-bonding with A (Figure 6-5), the complementary base-pairing properties described for DNA in Chapters 4 and 5 apply also to RNA (in RNA, G pairs with C, and A pairs with U). It is not uncommon, however, to find other types of base pairs in RNA: for example, G pairing with U occasionally.

 Figure 6-4. The chemical structure of RNA. (A) RNA contains the sugar ribose, which differs from deoxyribose, the sugar used in DNA, by the presence of an additional -OH group. (B) RNA contains the base uracil, which differs from thymine, the equivalent base in DNA, by the absence of a -CH3 group. (C) A short length of RNA. The phosphodiester chemical linkage between nucleotides in RNA is the same as that in DNA.

 Despite these small chemical differences, DNA and RNA differ quite dramatically in overall structure. Whereas DNA always occurs in cells as a double-stranded helix, RNA is single-stranded. RNA chains therefore fold up into a variety of shapes, just as a polypeptide chain folds up to form the final shape of a protein (Figure 6-6). As we see later in this chapter, the ability to fold into complex three-dimensional shapes allows some RNA molecules to have structural and catalytic functions.

Figure 6-5. Uracil forms base pairs with adenine. The absence of a methyl group in U has no effect on base-pairing; thus, U-A base pairs closely resemble T-A base pairs (see Figure 4-4).

 Figure 6-6. RNA can fold into specific structures. RNA is largely single-stranded, but it often contains short stretches of nucleotides that can form conventional base-pairs with complementary sequences found elsewhere on the same molecule. These interactions, along with additional “nonconventional” base-pair interactions, allow an RNA molecule to fold into a three-dimensional structure that is determined by its sequence of nucleotides. (A) Diagram of a folded RNA structure showing only conventional base-pair interactions; (B) structure with both conventional (red) and nonconventional (green) base-pair interactions; (C) structure of an actual RNA, a portion of a group 1 intron (see Figure 6-36). Each conventional base-pair interaction is indicated by a “rung” in the double helix. Bases in other configurations are indicated by broken rungs.

 Transcription Produces RNA Complementary to One Strand of DNA

All of the RNA in a cell is made by DNA transcription, a process that has certain similarities to the process of DNA replication discussed in Chapter 5. Transcription begins with the opening and unwinding of a small portion of the DNA double helix to expose the bases on each DNA strand. One of the two strands of the DNA double helix then acts as a template for the synthesis of an RNA molecule. As in DNA replication, the nucleotide sequence of the RNA chain is determined by the complementary base-pairing between incoming nucleotides and the DNA template. When a good match is made, the incoming ribonucleotide is covalently linked to the growing RNA chain in an enzymatically catalyzed reaction. The RNA chain produced by transcription—the transcript—is therefore elongated one nucleotide at a time, and it has a nucleotide sequence that is exactly complementary to the strand of DNA used as the template (Figure 6-7).

Figure 6-7. DNA transcription produces a single-stranded RNA molecule that is complementary to one strand of DNA.

Transcription, however, differs from DNA replication in several crucial ways. Unlike a newly formed DNA strand, the RNA strand does not remain hydrogen-bonded to the DNA template strand. Instead, just behind the region where the ribonucleotides are being added, the RNA chain is displaced and the DNA helix re-forms. Thus, the RNA molecules produced by transcription are released from the DNA template as single strands. In addition, because they are copied from only a limited region of the DNA, RNA molecules are much shorter than DNA molecules. A DNA molecule in a human chromosome can be up to 250 million nucleotide-pairs long; in contrast, most RNAs are no more than a few thousand nucleotides long, and many are considerably shorter.

The enzymes that perform transcription are called RNA polymerases. Like the DNA polymerase that catalyzes DNA replication (discussed in Chapter 5), RNA polymerases catalyze the formation of the phosphodiester bonds that link the nucleotides together to form a linear chain. The RNA polymerase moves stepwise along the DNA, unwinding the DNA helix just ahead of the active site for polymerization to expose a new region of the template strand for complementary base-pairing. In this way, the growing RNA chain is extended by one nucleotide at a time in the 5′-to-3′ direction (Figure 6-8). The substrates are nucleoside triphosphates (ATP, CTP, UTP, and GTP); as for DNA replication, a hydrolysis of high-energy bonds provides the energy needed to drive the reaction forward (see Figure 5-4).

 Figure 6-8. DNA is transcribed by the enzyme RNA polymerase. The RNA polymerase (pale blue) moves stepwise along the DNA, unwinding the DNA helix at its active site. As it progresses, the polymerase adds nucleotides (here, small “T” shapes) one by one to the RNA chain at the polymerization site using an exposed DNA strand as a template. The RNA transcript is thus a single-stranded complementary copy of one of the two DNA strands. The polymerase has a rudder (see Figure 6-11) that displaces the newly formed RNA, allowing the two strands of DNA behind the polymerase to rewind. A short region of DNA/RNA helix (approximately nine nucleotides in length) is therefore formed only transiently, and a “window” of DNA/RNA helix therefore moves along the DNA with the polymerase. The incoming nucleotides are in the form of ribonucleoside triphosphates (ATP, UTP, CTP, and GTP), and the energy stored in their phosphate-phosphate bonds provides the driving force for the polymerization reaction (see Figure 5-4). (Adapted from a figure kindly supplied by Robert Landick.)

The almost immediate release of the RNA strand from the DNA as it is synthesized means that many RNA copies can be made from the same gene in a relatively short time, the synthesis of additional RNA molecules being started before the first RNA is completed (Figure 6-9). When RNA polymerase molecules follow hard on each other's heels in this way, each moving at about 20 nucleotides per second (the speed in eukaryotes), over a thousand transcripts can be synthesized in an hour from a single gene.

 Figure 6-9. Transcription of two genes as observed under the electron microscope. The micrograph shows many molecules of RNA polymerase simultaneously transcribing each of two adjacent genes. Molecules of RNA polymerase are visible as a series of dots along the DNA with the newly synthesized transcripts (fine threads) attached to them. The RNA molecules (ribosomal RNAs) shown in this example are not translated into protein but are instead used directly as components of ribosomes, the machines on which translation takes place. The particles at the 5′ end (the free end) of each rRNA transcript are believed to reflect the beginnings of ribosome assembly. From the lengths of the newly synthesized transcripts, it can be deduced that the RNA polymerase molecules are transcribing from left to right. (Courtesy of Ulrich Scheer.)

 Although RNA polymerase catalyzes essentially the same chemical reaction as DNA polymerase, there are some important differences between the two enzymes. First, and most obvious, RNA polymerase catalyzes the linkage of ribonucleotides, not deoxyribonucleotides. Second, unlike the DNA polymerases involved in DNA replication, RNA polymerases can start an RNA chain without a primer. This difference may exist because transcription need not be as accurate as DNA replication (see Table 5-1, p. 243). Unlike DNA, RNA does not permanently store genetic information in cells. RNA polymerases make about one mistake for every 104 nucleotides copied into RNA (compared with an error rate for direct copying by DNA polymerase of about one in 107 nucleotides), and the consequences of an error in RNA transcription are much less significant than that in DNA replication.

Although RNA polymerases are not nearly as accurate as the DNA polymerases that replicate DNA, they nonetheless have a modest proofreading mechanism. If the incorrect ribonucleotide is added to the growing RNA chain, the polymerase can back up, and the active site of the enzyme can perform an excision reaction that mimics the reverse of the polymerization reaction, except that water instead of pyrophosphate is used (see Figure 5-4). RNA polymerase hovers around a misincorporated ribonucleotide longer than it does for a correct addition, causing excision to be favored for incorrect nucleotides. However, RNA polymerase also excises many correct bases as part of the cost for improved accuracy.

 Cells Produce Several Types of RNA

The majority of genes carried in a cell's DNA specify the amino acid sequence of proteins; the RNA molecules that are copied from these genes (which ultimately direct the synthesis of proteins) are called messenger RNA (mRNA) molecules. The final product of a minority of genes, however, is the RNA itself. Careful analysis of the complete DNA sequence of the genome of the yeast S. cerevisiae has uncovered well over 750 genes (somewhat more than 10% of the total number of yeast genes) that produce RNA as their final product, although this number includes multiple copies of some highly repeated genes. These RNAs, like proteins, serve as enzymatic and structural components for a wide variety of processes in the cell. In Chapter 5 we encountered one of those RNAs, the template carried by the enzyme telomerase. Although not all of their functions are known, we see in this chapter that some small nuclear RNA (snRNA) molecules direct the splicing of pre-mRNA to form mRNA, that ribosomal RNA (rRNA) molecules form the core of ribosomes, and that transfer RNA (tRNA) molecules form the adaptors that select amino acids and hold them in place on a ribosome for incorporation into protein (Table 6-1).

Table 6-1. Principal Types of RNAs Produced in Cells


 

TYPE OF RNA

FUNCTION


 

 

mRNAs

messenger RNAs, code for proteins

rRNAs

ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis

tRNAs

transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids

snRNAs

small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNA

snoRNAs

small nucleolar RNAs, used to process and chemically modify rRNAs

Other noncoding RNAs

function in diverse cellular processes, including telomere synthesis, X-chromosome inactivation, and the transport of proteins into the ER


 

Each transcribed segment of DNA is called a transcription unit. In eukaryotes, a transcription unit typically carries the information of just one gene, and therefore codes for either a single RNA molecule or a single protein (or group of related proteins if the initial RNA transcript is spliced in more than one way to produce different mRNAs). In bacteria, a set of adjacent genes is often trans-cribed as a unit; the resulting mRNA molecule therefore carries the information for several distinct proteins.

Overall, RNA makes up a few percent of a cell's dry weight. Most of the RNA in cells is rRNA; mRNA comprises only 3–5% of the total RNA in a typical mammalian cell. The mRNA population is made up of tens of thousands of different species, and there are on average only 10–15 molecules of each species of mRNA present in each cell.

 Signals Encoded in DNA Tell RNA  Polymerase Where to Start and Stop

To transcribe a gene accurately, RNA polymerase must recognize where on the genome to start and where to finish. The way in which RNA polymerases perform these tasks differs somewhat between bacteria and eukaryotes. Because the process in bacteria is simpler, we look there first.

The initiation of transcription is an especially important step in gene expression because it is the main point at which the cell regulates which proteins are to be produced and at what rate. Bacterial RNA polymerase is a multisubunit complex. A detachable subunit, called sigma (σ) factor, is largely responsible for its ability to read the signals in the DNA that tell it where to begin transcribing (Figure 6-10). RNA polymerase molecules adhere only weakly to the bacterial DNA when they collide with it, and a polymerase molecule typically slides rapidly along the long DNA molecule until it dissociates again. However, when the polymerase slides into a region on the DNA double helix called a promoter, a special sequence of nucleotides indicating the starting point for RNA synthesis, it binds tightly to it. The polymerase, using its σ factor, recognizes this DNA sequence by making specific contacts with the portions of the bases that are exposed on the outside of the helix (Step 1 in Figure 6-10).

After the RNA polymerase binds tightly to the promoter DNA in this way, it opens up the double helix to expose a short stretch of nucleotides on each strand (Step 2 in Figure 6-10). Unlike a DNA helicase reaction (see Figure 5-15), this limited opening of the helix does not require the energy of ATP hydrolysis. Instead, the polymerase and DNA both undergo reversible structural changes that result in a more energetically favorable state. With the DNA unwound, one of the two exposed DNA strands acts as a template for complementary base-pairing with incoming ribonucleotides (see Figure 6-7), two of which are joined together by the polymerase to begin an RNA chain. After the first ten or so nucleotides of RNA have been synthesized (a relatively inefficient process during which polymerase synthesizes and discards short nucleotide oligomers), the σ factor relaxes its tight hold on the polymerase and eventually dissociates from it. During this process, the polymerase undergoes additional structural changes that enable it to move forward rapidly, transcribing without the σ factor (Step 4 in Figure 6-10). Chain elongation continues (at a speed of approximately 50 nucleotides/sec for bacterial RNA polymerases) until the enzyme encounters a second signal in the DNA, the terminator (described below), where the polymerase halts and releases both the DNA template and the newly made RNA chain (Step 7 in Figure 6-10). After the polymerase has been released at a terminator, it reassociates with a free σ factor and searches for a new promoter, where it can begin the process of transcription again.

  Figure 6-10. The transcription cycle of bacterial RNA polymerase. In step 1, the RNA polymerase holoenzyme (core polymerase plus σ factor) forms and then locates a promoter (see Figure 6-12). The polymerase unwinds the DNA at the position at which transcription is to begin (step 2) and begins transcribing (step 3). This initial RNA synthesis (sometimes called “abortive initiation”) is relatively inefficient. However, once RNA polymerase has managed to synthesize about 10 nucleotides of RNA, σ relaxes its grip, and the polymerase undergoes a series of conformational changes (which probably includes a tightening of its jaws and the placement of RNA in the exit channel [see Figure 6-11]). The polymerase now shifts to the elongation mode of RNA synthesis (step 4), moving rightwards along the DNA in this diagram. During the elongation mode (step 5) transcription is highly processive, with the polymerase leaving the DNA template and releasing the newly transcribed RNA only when it encounters a termination signal (step 6). Termination signals are encoded in DNA and many function by forming an RNA structure that destabilizes the polymerase's hold on the RNA, as shown here. In bacteria, all RNA molecules are synthesized by a single type of RNA polymerase and the cycle depicted in the figure therefore applies to the production of mRNAs as well as structural and catalytic RNAs. (Adapted from a figure kindly supplied by Robert Landick.)

 Several structural features of bacterial RNA polymerase make it particularly adept at performing the transcription cycle just described. Once the σ factor positions the polymerase on the promoter and the template DNA has been unwound and pushed to the active site, a pair of moveable jaws is thought to clamp onto the DNA (Figure 6-11). When the first 10 nucleotides have been transcribed, the dissociation of σ allows a flap at the back of the polymerase to close to form an exit tunnel through which the newly made RNA leaves the enzyme. With the polymerase now functioning in its elongation mode, a rudder-like structure in the enzyme continuously pries apart the DNA-RNA hybrid formed. We can view the series of conformational changes that takes place during transcription initiation as a successive tightening of the enzyme around the DNA and RNA to ensure that it does not dissociate before it has finished transcribing a gene. If an RNA polymerase does dissociate prematurely, it cannot resume synthesis but must start over again at the promoter.

How do the signals in the DNA (termination signals) stop the elongating polymerase? For most bacterial genes a termination signal consists of a string of A-T nucleotide pairs preceded by a two-fold symmetric DNA sequence, which, when transcribed into RNA, folds into a “hairpin” structure through Watson-Crick base-pairing (see Figure 6-10). As the polymerase transcribes across a terminator, the hairpin may help to wedge open the movable flap on the RNA polymerase and release the RNA transcript from the exit tunnel. At the same time, the DNA-RNA hybrid in the active site, which is held together predominantly by U-A base pairs (which are less stable than G-C base pairs because they form two rather than three hydrogen bonds per base pair), is not sufficiently strong enough to hold the RNA in place, and it dissociates causing the release of the polymerase from the DNA, perhaps by forcing open its jaws. Thus, in some respects, transcription termination seems to involve a reversal of the structural transitions that happen during initiation. The process of termination also is an example of a common theme in this chapter: the ability of RNA to fold into specific structures figures prominently in many aspects of decoding the genome.

Figure 6-11. The structure of a bacterial RNA polymerase. Two depictions of the three-dimensional structure of a bacterial RNA polymerase, with the DNA and RNA modeled in. This RNA polymerase is formed from four different subunits, indicated by different colors (right). The DNA strand used as a template is red, and the non-template strand is yellow. The rudder wedges apart the DNA-RNA hybrid as the polymerase moves. For simplicity only the polypeptide backbone of the rudder is shown in the right-hand figure, and the DNA exiting from the polymerase has been omitted. Because the RNA polymerase is depicted in the elongation mode, the σ factor is absent. (Courtesy of Seth Darst.)

Transcription Start and Stop Signals Are Heterogeneous in Nucleotide Sequence

As we have just seen, the processes of transcription initiation and termination involve a complicated series of structural transitions in protein, DNA, and RNA molecules. It is perhaps not surprising that the signals encoded in DNA that specify these transitions are difficult for researchers to recognize. Indeed, a comparison of many different bacterial promoters reveals that they are heterogeneous in DNA sequence. Nevertheless, they all contain related sequences, reflecting in part aspects of the DNA that are recognized directly by the σ factor. These common features are often summarized in the form of a consensus sequence (Figure 6-12). In general, a consensus nucleotide sequence is derived by comparing many sequences with the same basic function and tallying up the most common nucleotide found at each position. It therefore serves as a summary or “average” of a large number of individual nucleotide sequences.

One reason that individual bacterial promoters differ in DNA sequence is that the precise sequence determines the strength (or number of initiation events per unit time) of the promoter. Evolutionary processes have thus fine-tuned each promoter to initiate as often as necessary and have created a wide spectrum of promoters. Promoters for genes that code for abundant proteins are much stronger than those associated with genes that encode rare proteins, and their nucleotide sequences are responsible for these differences.

Like bacterial promoters, transcription terminators also include a wide range of sequences, with the potential to form a simple RNA structure being the most important common feature. Since an almost unlimited number of nucleotide sequences have this potential, terminator sequences are much more heterogeneous than those of promoters.

We have discussed bacterial promoters and terminators in some detail to illustrate an important point regarding the analysis of genome sequences. Although we know a great deal about bacterial promoters and terminators and can develop consensus sequences that summarize their most salient features, their variation in nucleotide sequence makes it difficult for researchers (even when aided by powerful computers) to definitively locate them simply by inspection of the nucleotide sequence of a genome. When we encounter analogous types of sequences in eukaryotes, the problem of locating them is even more difficult. Often, additional information, some of it from direct experimentation, is needed to accurately locate the short DNA signals contained in genomes.

Promoter sequences are asymmetric (see Figure 6-12), and this feature has important consequences for their arrangement in genomes. Since DNA is double-stranded, two different RNA molecules could in principle be transcribed from any gene, using each of the two DNA strands as a template. However a gene typically has only a single promoter, and because the nucleotide sequences of bacterial (as well as eukaryotic) promoters are asymmetric the polymerase can bind in only one orientation. The polymerase thus has no option but to transcribe the one DNA strand, since it can synthesize RNA only in the 5′ to 3′ direction (Figure 6-13). The choice of template strand for each gene is therefore determined by the location and orientation of the promoter. Genome sequences reveal that the DNA strand used as the template for RNA synthesis varies from gene to gene (Figure 6-14; see also Figure 1-31).

Having considered transcription in bacteria, we now turn to the situation in eucaryotes, where the synthesis of RNA molecules is a much more elaborate affair.

 Figure 6-12. Consensus sequence for the major class of E. coli promoters. (A) The promoters are characterized by two hexameric DNA sequences, the -35 sequence and the -10 sequence named for their approximate location relative to the start point of transcription (designated +1). For convenience, the nucleotide sequence of a single strand of DNA is shown; in reality the RNA polymerase recognizes the promoter as double-stranded DNA. On the basis of a comparison of 300 promoters, the frequencies of the four nucleotides at each position in the -35 and -10 hexamers are given. The consensus sequence, shown below the graph, reflects the most common nucleotide found at each position in the collection of promoters. The sequence of nucleotides between the -35 and -10 hexamers shows no significant similarities among promoters. (B) The distribution of spacing between the -35 and -10 hexamers found in E. coli promoters. The information displayed in these two graphs applies to E. coli promoters that are recognized by RNA polymerase and the major σ factor (designated σ70). As we shall see in the next chapter, bacteria also contain minor σ factors, each of which recognizes a different promoter sequence. Some particularly strong promoters recognized by RNA polymerase and σ70 have an additional sequence, located upstream (to the left, in the figure) of the -35 hexamer, which is recognized by another subunit of RNA polymerase.

 Figure 6-13. The importance of RNA polymerase orientation. The DNA strand serving as template must be traversed in a 3′ to 5′ direction, as illustrated in Figure 6-9. Thus, the direction of RNA polymerase movement determines which of the two DNA strands is to serve as a template for the synthesis of RNA, as shown in (A) and (B). Polymerase direction is, in turn, determined by the orientation of the promoter sequence, the site at which the RNA polymerase begins transcription.

 Figure 6-14.Directions of transcription along a short portion of a bacterial chromosome. Some genes are transcribed using one DNA strand as a template, while others are transcribed using the other DNA strand. The direction of transcription is determined by the promoter at the beginning of each gene (green arrowheads). Approximately 0.2% (9000 base pairs) of the E. coli chromosome is depicted here. The genes transcribed from left to right use the bottom DNA strand as the template; those transcribed from right to left use the top strand as the template.

 Transcription Initiation in Eukaryotes Requires Many Proteins

In contrast to bacteria, which contain a single type of RNA polymerase, eukaryotic nuclei have three, called RNA polymerase I, RNA polymerase II, and RNA polymerase III. The three polymerases are structurally similar to one another (and to the bacterial enzyme). They share some common subunits and many structural features, but they transcribe different types of genes (Table 6-2). RNA polymerases I and III transcribe the genes encoding transfer RNA, ribosomal RNA, and various small RNAs. RNA polymerase II transcribes the vast majority of genes, including all those that encode proteins, and our subsequent discussion therefore focuses on this enzyme.

Table 6-2. The Three RNA Polymerases in Eucaryotic Cells


 

TYPE OF POLYMERASE

GENES TRANSCRIBED


 

RNA polymerase I

5.8S, 18S, and 28S rRNA genes

RNA polymerase II

all protein-coding genes, plus snoRNA genes and some snRNA genes

RNA polymerase III

tRNA genes, 5S rRNA genes, some snRNA genes and genes for other small RNAs


 

Although eucaryotic RNA polymerase II has many structural similarities to bacterial RNA polymerase (Figure 6-15), there are several important differences in the way in which the bacterial and eukaryotic enzymes function, two of which concern us immediately.

  1. While bacterial RNA polymerase (with σ factor as one of its subunits) is able to initiate transcription on a DNA template in vitro without the help of additional proteins, eukaryotic RNA polymerases cannot. They require the help of a large set of proteins called general transcription factors, which must assemble at the promoter with the polymerase before the polymerase can begin transcription.
  2.  Eukaryotic transcription initiation must deal with the packing of DNA into nucleosomes and higher order forms of chromatin structure, features absent from bacterial chromosomes.

 Figure 6-15. Structural similarity between a bacterial RNA polymerase and a eucaryotic RNA polymerase II. Regions of the two RNA polymerases that have similar structures are indicated in green. The eukaryotic polymerase is larger than the bacterial enzyme (12 subunits instead of 5), and some of the additional regions are shown in gray. The blue spheres represent Zn atoms that serve as structural components of the polymerases, and the red sphere represents the Mg atom present at the active site, where polymerization takes place. The RNA polymerases in all modern-day cells (bacteria, archaea, and eucaryotes) are closely related, indicating that the basic features of the enzyme were in place before the divergence of the three major branches of life. (Courtesy of P. Cramer and R. Kornberg.)

 RNA Polymerase II Requires General Transcription Factors

The discovery that, unlike bacterial RNA polymerase, purified eukaryotic RNA polymerase II could not initiate transcription in vitro led to the discovery and purification of the additional factors required for this process. These general transcription factors :

  1. help to position the RNA polymerase correctly at the promoter,
  2. aid in pulling apart the two strands of DNA to allow transcription to begin,
  3. and release RNA polymerase from the promoter into the elongation mode once transcription has begun.

The proteins are “general” because they assemble on all promoters used by RNA polymerase II; consisting of a set of interacting proteins, they are designated as TFII (for transcription factor for polymerase II), and listed as TFIIA, TFIIB, and so on. In a broad sense, the eukaryotic general transcription factors carry out functions equivalent to those of the σ factor in bacteria. Figure 6-16 shows how the general transcription factors assemble in vitro at promoters used by RNA polymerase II.

The assembly process starts with the binding of the general transcription factor TFIID to a short double-helical DNA sequence primarily composed of T and A nucleotides. For this reason, this sequence is known as the TATA sequence, or TATA box, and the subunit of TFIID that recognizes it is called TBP (for TATA-binding protein). The TATA box is typically located 25 nucleotides upstream from the transcription start site. It is not the only DNA sequence that signals the start of transcription (Figure 6-17), but for most polymerase II promoters, it is the most important. The binding of TFIID causes a large distortion in the DNA of the TATA box (Figure 6-18). This distortion is thought to serve as a physical landmark for the location of an active promoter in the midst of a very large genome, and it brings DNA sequences on both sides of the distortion together to allow for subsequent protein assembly steps. Other factors are then assembled, along with RNA polymerase II, to form a complete transcription initiation complex (see Figure 6-16).

After RNA polymerase II has been guided onto the promoter DNA to form a transcription initiation complex, it must gain access to the template strand at the transcription start point. This step is aided by one of the general transcription factors, TFIIH, which contains a DNA helicase. Next, like the bacterial polymerase, polymerase II remains at the promoter, synthesizing short lengths of RNA until it undergoes a conformational change and is released to begin transcribing a gene. A key step in this release is the addition of phosphate groups to the “tail” of the RNA polymerase (known as the CTD or C-terminal domain). This phosphorylation is also catalyzed by TFIIH, which, in addition to a helicase, contains a protein kinase as one of its subunits (see Figure 6-16, D and E). The polymerase can then disengage from the cluster of general transcription factors, undergoing a series of conformational changes that tighten its interaction with DNA and acquiring new proteins that allow it to transcribe for long distances without dissociating.

Once the polymerase II has begun elongating the RNA transcript, most of the general transcription factors are released from the DNA so that they are available to initiate another round of transcription with a new RNA polymerase molecule. As we see shortly, the phosphorylation of the tail of RNA polymerase II also causes components of the RNA processing machinery to load onto the polymerase and thus be in position to modify the newly transcribed RNA as it emerges from the polymerase.

Figure 6-16. Initiation of transcription of a eukaryotic gene by RNA polymerase II. To begin transcription, RNA polymerase requires a number of general transcription factors (called TFIIA, TFIIB, and so on). (A) The promoter contains a DNA sequence called the TATA box, which is located 25 nucleotides away from the site at which transcription is initiated. (B) The TATA box is recognized and bound by transcription factor TFIID, which then enables the adjacent binding of TFIIB (C). For simplicity the DNA distortion produced by the binding of TFIID (see Figure 6-18) is not shown. (D) The rest of the general transcription factors, as well as the RNA polymerase itself, assemble at the promoter. (E) TFIIH then uses ATP to pry apart the DNA double helix at the transcription start point, allowing transcription to begin. TFIIH also phosphorylates RNA polymerase II, changing its conformation so that the polymerase is released from the general factors and can begin the elongation phase of transcription. As shown, the site of phosphorylation is a long C-terminal polypeptide tail that extends from the polymerase molecule. The assembly scheme shown in the figure was deduced from experiments performed in vitro, and the exact order in which the general transcription factors assemble on promoters in cells is not known with certainty. In some cases, the general factors are thought to first assemble with the polymerase, with the whole assembly subsequently binding to the DNA in a single step. The general transcription factors have been highly conserved in evolution; some of those from human cells can be replaced in biochemical experiments by the corresponding factors from simple yeasts.

Figure 6-17. Consensus sequences found in the vicinity of eucaryotic RNA polymerase II start points. The name given to each consensus sequence (first column) and the general transcription factor that recognizes it (last column) are indicated. N indicates any nucleotide, and two nucleotides separated by a slash indicate an equal probability of either nucleotide at the indicated position. In reality, each consensus sequence is a shorthand representation of a histogram similar to that of Figure 6-12. For most RNA polymerase II transcription start points, only two or three of the four sequences are present. For example, most polymerase II promoters have a TATA box sequence, and those that do not typically have a “strong” INR sequence. Although most of the DNA sequences that influence transcription initiation are located “upstream” of the transcription start point, a few, such as the DPE shown in the figure, are located in the transcribed region.

 Figure 6-18. Three-dimensional structure of TBP (TATA-binding protein) bound to DNA. The TBP is the subunit of the general transcription factor TFIID that is responsible for recognizing and binding to the TATA box sequence in the DNA (red). The unique DNA bending caused by TBP—two kinks in the double helix separated by partly unwound DNA—may serve as a landmark that helps to attract the other general transcription factors. TBP is a single polypeptide chain that is folded into two very similar domains (blue and green). (Adapted from J.L. Kim et al., Nature 365:520–527, 1993.)

 

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