Chapter 15 재조합 DNA 기술 (Recombinant DNA Technology) 1 Cloning a Gene 1. DNA 2. Restriction enzyme Cohesive (sticky) ends 3. Plasmid vector DNA ligase 4. Transformation 2 Restriction Endonuclease (제한효소) EcoRI Specific nucleotide sequence Sticky ends DNA ligase (phosphodiester bond) 3 Synthesis of Complementary DNA Eukaryotic mRNA Exon and intron Splicing Messenger RNA (mRNA) Reverse transcriptase (RT) Complementary DNA (cDNA) 4 Gel Electrophoresis of DNA 5 Southern Blotting for DNA Identification 1. Isolation 2. Digestion by restriction endonuclease 3. Separation by gel electrophoresis 6 Southern Blotting for DNA Identification 4. Transfer 5. Hybridization with a radioactive probe 6. Exposure to X‐ray film 7 Polymerase Chain Reaction Deoxyribonucleotides Taq DNA polymeras DNA primers 1. Heating for melting (95°C) 2. Cooling for annealing (60°C) 3. DNA synthesis (75‐80°C) 8 Plasmids • Replicate autonomously and easy to purify • Requirements for vectors – An origin of replication – A selectable marker – A multicloning site (MCR) • Site that allows gene to be cloned to placed into the plasmid vector – Insertional inactivation • Lac Z gene (‐galatosidease) • Turn X‐gal into a blue color 9 Specific Interaction of Polyhistidines with Nickel Metal Ions Polyhistidines Nickel 10 Construction and Purification of a Polyhistidine‐tagged Protein 11 Construction and Purification of a Polyhistidine‐tagged Protein Bind to nickel column Wash Release with imidazole 12 Chapter 16 미생물 유전체학 (Microbial Genomics) 13 DNA Sequencing • Sanger DNA sequencing – Most commonly used method – Referred to as the chain‐termination DNA sequencing method – Uses dideoxynucleoside triphosphates (ddNTP) 14 Sanger Method of DNA Sequencing 15 Automated Sanger Method of DNA Sequencing 다른 색으로 형광 염색이 되어 있는 ddNTP를 사용 16 16 Pyrosequencing • (DNA)n + dNTP ‐> (DNA)n+1 + PPi by DNA polymerase • PPi + adenosine phosphosulfate (APS) ‐> ATP + SO42‐ by ATP sulfurylase • ATP + luciferin + O2 ‐> AMP + PPi + oxyluciferin + CO2 + light by luciferase 17 DNA Microarray 18 Proteomics (단백질체학) • The study of the proteome • The entire collection of proteins that an organism produces • Provides information about genome function not available from mRNA studies 19 Two Dimensional Gel Electrophoresis First dimension • Isoelectric focusing • pH gradient determines isoelectric point Second dimension • Electrophoresis (SDS‐ PAGE) • Separation by molecular weight 20 Tandem Mass Spectrometry (순차적 질량분석법) 21 Chromatin immunoprecipitation (크로마틴 면역침전법) 22 Chapter 17 미생물의 분류와 진화의 다양성 (Microbial Taxonomy and the Evolution of Diversity) 23 Taxonomy (분류학) • Science of biological classification • Classification (분류) – Arrangement of organisms into groups • Nomenclature (명명) – Assignment of names • Identification (동정) – Determination to which a new isolate belongs 24 Polyphasic Taxonomy (다면분류) • Determine the genus(속) and species(종) of a newly discovered prokaryote • Phenotypic analysis (표현론적 분류) • Phylogenetic analysis (계통발생학적 분류) – Evolutional distance (진화적 거리) • Genetic analysis (유전적 분류) 25 Three Domains(영역) • Bacteria (진정세균) • Archaea (고세균) • Eukarya (진핵생물) • Small subunit (SSU) rRNA nucleotide sequences • Within domain(영역) – Phylum(문), class(강), order,(목) family(과), genus (속), and species (종) 26 Binomial System of Nomenclature (이명법) • Each organism has two names – Genus name (속명) – italicized and capitalized (e.g., Escherichia) – Species epithet (종명) – italicized but not capitalized (e.g., coli) • Can be abbreviated after first use (e.g., E. coli) 27 Classical Characteristics (고전적인 특징) • • • • • Morphological (형태적) Physiological (생리적) Biochemical (생화학적) Ecological (생태적) Genetic (유전적) 28 Molecular Characteristics (분자생물학적 특징) • Nucleic acid base composition (핵산 염기조성) – G+C content – Melting temp • Nucleic acid hybridization (핵산혼성화) – Sequence homology • Nucleic acid sequencing (핵산서열분석) – Small subunit ribosomal RNAs 29 Molecular Characteristics (분자생물학적 특징) • Genomic fingerprinting (유전체 지문법) – Restriction fragment length polymorphism (RFLP, 제한효소 단편 길이 다형성) – 16S ribosomal RNA – 특정유전자를 PCR로 증폭 – 특정 제한효소로 절단 – 젤 전기영동으로 분석 • Amino acid sequencing 30 Restriction Fragment Length Polymorphism (RFLP) 31 Bergey’s Manual of Systematic Bacteriology (버지의 세균계통학편람) • 1권 – 고세균, 진화상으로 멀리 떨어진 진정세균과 광영양 진정세균 (19장) • 2권 – 프로테오박테리아 (20장) • 3권 – G+C 함량이 낮은 그람양성 세균 (21장) • 4권 - G+C 함량이 높은 그람양성 세균 (22장) • 5권 – 플랑토미세테스, 클라미디아, 스피로헤타, 박테로이드, 베루코마이크로비아 (19장) 32 Chapter 18 고세균 (Archaea) 33 고세균의 일반적인 특징 • 진핵생물과의 공통점 – 유전자의 복제, 전사, 번역을 담당하는 단백질을 암호화하는 유전자들 • 진정세균과의 공통점 – 대사과정에 관여하는 단백질을 암호화하는 유전자 • 고세균의 예 – 메탄생성, 황산염환원, 극호염성, 세포벽이 없는 고세균, 극호열성 황 대사균 • 병원성 고세균은 없음 34