II HISTORY A Frederick Griffith 1928 Transformation Beadle

II. 분자생물학 실험적 HISTORY A. 생명체의 유전현상 : 유전물질, 이중나선, 유전암호, 유전자 발현 조절 Frederick Griffith (1928) : Transformation Beadle & Tatum (1941) : One gene-one enzyme hypothesis Avery, Macleod, & Mc. Carthy (1944) : T/P는 genetic material (DNA) Lederberg & Tatum (1946) : Conjugation Erwin Chargaff (1950) : Purine = Pyrimidine Wilkins & Franklin (1950) : X-ray diffraction study James Watson & Francis Crick (1953) : DNA는 double helix Meselson & Stahl (1958) : Semi-conservative DNA replication Nirenberg & Khorana (1961) : Genetic code Jacob & Monod (1961) : Protein expression regulation (Operon) Genetic engineering era (1970) : Plasmid, restriction enzyme, transformation methods

1928, 그리피스의 실험 (Frederick Griffith's experiment) “Transforming principle”

Beadle & Tatum (1941) : One gene – One enzyme

Oswald Avery, Colin Macleod, Maclyn Mc. Carthy (1944) : 유전물질 입 증 Transforming principle (T/P)은 DNA라고 결론

Lederberg & Tatum (1946) : Conjugation (접합) The F+ plasmid undergoes rolling circle replication Hfr conjugation

Erwin Chargaff (1950) : Purine기 합 (A, G) = Pyrimidine기 합 (C, T)

$Wilkins & Franklin (1950) : X-ray diffraction study$

Wilkins & Franklin (1950) : X-ray diffraction study

Zinder & Lederberg (1952) : Transduction (형질도입 )

허시-체이스 실험 (Hershey-Chase experiment, 1952) Transduction is viral-mediated horizontal transfer of genetic material. Bacteriophage를 통한 DNA 는 유전물질임을 입증 : 자손 에게 ½ 전달 (P 32)

James Watson & Francis Crick (1953) : Double helix

Meselson & Stahl (1958) : Semi-conservative DNA replication

Nirenberg & Khorana (1958) : Genetic code

Jacob & Monod (1961) : Protein expression regulation (Lac operon)

13. Genetic engineering era (1970) : 재조합 DNA 기술, 세포융합, 핵치환기술 (1) Recombinant DNA Technology : Stanley Cohen & Herbert Boyer (1973)

Herbert Boyer Robert Swanson

1) Plasmid & Vector : Joshua Nirenberg (1952)/Jacob & Wollman (1955) A. Cloning Vector

Multi. Cloning Site

B. Expression Vector - Promoter : RNA polymerase가 결합하는 부위 -35 region (TTGACA) & -10 region (TATAAT) - Shine-Dalgarno sequence : ribosome 30 S subunit의 16 S r. RNA 결합부위 m. RNA : 5’UA AGGAGG UGAUC 3’ 16 S r. RNA : 3’AU UCCUCC ACUAG 5’ - 개시코돈 : ATG (open reading frame), 용도에 따라 signal sequence (분비벡터) - Multicloning site (MCS) : 여러 종류의 제한효소가 각각 한 부위 - Terminator : 전사종결부위 (Rho factor dependent & independent terminator)

A. Restriction enzymes B. 자르는 방식 - Endonuclease Phosphodiester 결합 절단 4~6염기 인식 : 1/256, 1/4096 Palindromic sequence - 5’-protruding end - 3’-protruding end - Blunt end

D. T 4 DNA ligase : 1966, B. Weiss & C. C. Richardson 작용 기작 - DNA의 5’-phosphate 기와 3’-OH 간의 phosphodiester bond를 연결 해 주는 역할 * cohesive 및 blunt end의 연결 * 에너지원 : ATP 및 NAD

T 4 DNA Ligase - MW 68 k. Da - DNA 또는 RNA의 5’-P 와 3’-OH - Energy원 : ATP E. coli DNA Ligase - c. DNA 합성 시 사용 - Only DNA의 5’-P 와 3’-OH - Energy원 : NAD (Nicotinamide Adenine Dinucleotide)

Ligation reaction - Cohesive end ligation * vector : insert DNA molar ratio = 1 : 3 * 12℃ - 15℃ effective (37℃ exonuclease activity) - Blunt end ligation * vector : insert DNA molar ratio = 1 : 1 * 23℃ more effective Ligase unit - 1 weiss unit = 0. 2 unit * Pyrophosphate 1 nmole의 P 32 가 37℃, 20 분 동안 γ, β-32 P ATP로 교환을 촉매하는 효소의 양 - 0. 015 weiss unit of T 4 DNA ligase * Phage λ DNA (5 μg)의 Hind. III fragment를 16℃, 30 분 동안 50% 를 결합하는 활성