1장; 생물학의 각 특성분야가 생명체 (세포)의 이해에 어떠한 기여를 하고 있는 지 ?

(1) Cell Biology; size, shape, location

1. 1600년경 crude microscope

2. modern compound microscope; bacteria (1 μm) 측정가능, 0.2 μm사이의 물체구분

    이용도--> 예) chromosome관찰; with a dye binding to DNA

                        target protein 관찰; with antibody linked to fluorescent dye (GFP)

                예) microtubules in the movement of chromosome; Ab against tubulin

3. Electron microscope

   --> very thin sections, precluding examination of living cells, high resolution (0.1 nm)

(2) Biochemistry; structure and chemistry

1. fractionation

  --> 물리, 화학적 특성에 따른 분리  예) 분자량, electric charge

2. antibody의 이용

   --> for isolating larger amounts of a protein of interest

3. tag의 이용

   --> to pull out the protein from whole cell extracts      ex) 유전자에 붙임

4. 목적;  a) how it catalyzes a chemical reaction and carry out other functions

             b) how its activity is regulated

             c) 기능과 연관된 strucure studies

(3) Genetics; damaged genes에 대한 분석--> 단백질에 대한 정확한 기능이해

1. mutation

   ** how can we isolate and maintain mutant organisms or cells ?  --> temperature-sensitive mutants

                                                                                               --> 예) 세포분열 연관 유전자 발견

       문제점; about which proteins they encode, how these proteins participate in the process

(4) Genomics; structure and expression of entire genomes

1. 이용도

     --> evolutionary processes, in following the inheritance of diseases in human families

2. DNA microarray; gene expression변이 관찰  예) disease process, development, 어떤 신호자극시

   green (no serum), red (serum)

(5) Developmental biology

 1. differnces among differentiated cells

     --> specific sets of proteins의 발현차이 때문, 서로 다른 단백질을 blue, yellow, geen dye로 tagging

 (6) 적절한 실험모델 설정

--> 조건; genomes have been sequenced or nearly so

 (7) Plasmid Vectors

1. Bacterial plasmid

   - double stranded circular DNA, 1 ~ 200 Kb

   - can replicate and inherit independently of bacterial chromosome

            depend on proteins and enzymes encoded by host

   - own genes for enzymes which are advantageous to the host      

2. Features

 (1) replication

     most cloning tasks

         - in the relaxed vectors --> a greater yield of DNA/volume

         - but, stringently controlled plasmids; use in cloning of lethal proteins which are deleterious to host                         

  (2) mobilization

      ① in usual, bacterial conjugation transmit the plasmid

      ② vectors in common use;

           ⇒ lack of 'mob' gene -> no transmission by conjugation

                                                but, a third plasmid(ColK) containing mob gene is present in cells -> possible to transmit

  (3) selective markers

      - after transformation, to identify the plasmids

      - common use; (ex) Amp, Tet, Chloramphenicol, kanamycin

        ① Ampicillin

           --> binds to many enzymes in the bacterial membrane; block the cell wall synthesis                                     

           --> Amp^R gene encodes enzyme

                                                 transport to periplasmic space -> hydrolysis of beta-lactam ring -> detoxification of drug

        ② tetracycline

           --> binds to 30S ribosomal subunit; inhibit the protein synthesis

           --> Tet^R gene encodes a membrane-associated protein

                                                prevent the antibiotic from entering into the cells

        ③  chloramphenicol

           --> binds to 50S ribosomal subunit; inhibit the protein synthesis

           --> cat (Cm^R) gene encodes a tetrameric cytosolic protein

                                              catalyze the chloramphenicol; non-binding to 50S ribosomal subunit

 (8) Gel electrophoresis

(종류) ① agarose                            ② polyacrylamide

           -- 1-10ng DNA                      -- separation of small fragments (5-500 bp)

           -- 0.2 - 50 kb                         -- 1 bp difference

(A) agarose gel electrophoresis

   ⇒ seaweed, a linear polymer

   ⇒ contaminated with polysaccharides, salts, proteins

   ⇒ low-melting gel (10-500 bp); usually high conc (4-10%), so purified DNA probably contaminated

 ** factors on movement

   1) DNA size ; inversely proportional to log₁₀bp (Fig 6-1)

    2) agarose conc.

   3) DNA conformation ; supercoiled, nicked, linear

   4) voltage

   5) base composition and temp. ; no-effect (4^oC-30^oC)

                                                  (but low-melting gel run at 4^oC)

   6) EtBr ; ~ 15% reduced

   7) buffer ; if H₂O --> slow moving

                   if 10X buffer --> heat and gel melting

                 TAE :  ⇒ buffering capacity is low and need change                                     **

                            ⇒ migration is fast (~10%)

                             ⇒ supercoiled form resolution is good

                 TBE & TPE : more expensive

                 alkaline buffer (50mM NaOH/1mM EDTA): for ssDNA, first melt in water

(B) DNA recovery from agarose gels

    ---> problem

          1) presence of inhibitors

          2) inefficient recovery ; ∥~50% 이상, in less than 5 kb

                                             ∥ no satisfactory more than 5 kb (size)

                                             ∥ no satisfactory, less than 500ng DNA (amount)

   ---> methods

          1) electrophoresis onto DEAE-cellulose membrane

          2) electroelution into dialysis bag

          3) low-melting gel

   (1) DEAE-cellulose membrane

      ⇒ simultaneously many samples, constant high yield (0.5-5 kb)

      ⇒ high purity (microinjection)

      ⇒ using high ionic strength buffer, eluted

      ⇒ 15 kb > fragment is not available

   (2) dialysis bag

       ⇒ for the large fragment (5 kb > )

   (3) low-melting gel

       ⇒ less producible, direct ligation after digestion

(C) polyacrylamide gel

   ---> a monomer

                 ↓free radicals (TEMED, ammonium persulfate)

         polymerization

                 ↓methylenebisacrylamide (1/29)

         cross-linking, gel

    ---> advantages

          1) resolution is more good

          2) accommodate larger quantities of DNA

          3) purified DNA is so pure

    ---> kinds

           1) non-denaturing gel ; can't discern the size of dsDNAs (10% difference)

           2) denaturing gel ; in presence of urea, formamide

                                      for isolation of ssDNA labeled probe

                                      for analysis of the products of DNA sequencing reactions

3장; 단백질 구조, 기능과 연관된 실험적 분석

(1) How the activites of cellular proteins are regulated ?

1. 이유; 단백질별 수명이 다양함  예) cyclin (분), lens of the eye (나이)

2. Ubiquitination & Lysosomal degradation

a) Lysosomal degradation --> for extracellular proteins and aged or defective organelles

b) ubiquitination  --> for proteins whose life spans are tightly controlled

                               for proteins that becomes misfolded

                            주요인자:   recognition sequence; Arg-X-X-Leu-Gly-X-Ile-Gly-Asp/Asn

                                            phosphorylation (cyclin), exposure of hydrophobic seq (misfolded protein in ER)  

                             예) immune system -- viral proteins of virus-infected cells

 (2) Disease implicated in misfolded proteins

   --> proteolytic degradation --> presence of insoluble proteins in various organs

          예) Alzheimer's disease, Parkinson's disease, mad cow disease

               -- β-amyloid protein 축적

(3) Purification methods of proteins

1. centrifugation

   원리; size and density

   종류;  ① differential centrifugation; separation of soluble proteins from insoluble materials

            ② rate-zonal centrifugation; size(mass) 이용 --> sucrose solution, for separating of many different types of

                                                     polymers and particles

       ③ equilibrium density-gradient centrifugation; for separating of DNA or organelles

 2. electrophoresis

    원리; charge:mass ratio

 ① SDS-PAGE

     --> rate is determined by pore sizes and the strength of the electric field

     --> SDS효과; chain length, not shape, is a sole determinant

② two-dimensional gel electrophoresis

   목적: 아주 유사한 분자량을 가진 단백질의 분리  예) 41KDa & 42KDa

   원리: charge and mass

           charge에 따른 분리 --> pH gradient, mass에 의한 분리 --> 전기영동

   용도: 세포분화/미분화, 암/정상세포간의 단백질 차이분석, 1000 proteins을 동시에 분석

3. chromatography

   원리; mass, charge, binding affinity에 의한 분리

① gel filtration

   beads --> polyacrylamide, dextran, agarose

   무거운 것일수록 빨리 빠져나옴

② ion-exchange chromatography

   beads --> positive charge or negative charge

③ affinity chromatography

 beads --> covalently attached with ligand

 elution --> by adding of excess of ligand or changing of salt concentration or pH

(4) identification of the interested proteins

Assays; simple, fast, minimal error, no degradation of proteins of interest, small amounts of materials (sensitivity)

① chromogenic and light-emitting enzyme reactions

     chromogenic --> 반응중 기질의 색깔변화 추적

     light-emitting  --> luciferase linked to antibody

② western blotting

    원리; 전기영동의 분리능력 + 항체 특이성 + enzyme assay의 sensitivity

(5) 방사선 동위원소의 실험적 이용

 specific activity = the amount of radioactivity per unit of material

 shorter the half-life is higher specific activity --> 이유; shorter time of incorporation, smaller cell sample

 in general, biological activity between labeled and unlabeled molecules is identical except for ¹²⁵I-labeled molecules

 detection; autoradiography, counter (Geiger counter, scintillation counter), phosphoimager

 Pulse-Chase experiment; for tracing the location of intracellular proteins

                                     for tracing the transformation of metabolite into others over time

(6) Mass spectrometry; for measuring the mass of proteins

1 X 10^-15 mole, 200,000 MW, 0.1% error

4장; Basic molecular genetic mechanisms

(1) How we can differentiate ds-DNA from ss-DNA ?

     T_m ?

    denaturation; breakage of H-bonding and others,  when; replication and transcription

(2) How we can measure the G:C content of DNA ?

(3) How do you know that DNA is circular and supercoiled ?

 topoisomerase I --> induce and release supercoiling

 topoisomerase II -->   ?

(4) How do we know that activator bound to enhancer interacts with RNA polymerase to initiate the transcription ?

  예) σ⁵⁴-RNA polymerase and its activator NtrC (nitrogen regulatory protein C)

         NtrB  --> NtrC phosphorylation --> binding to enhancer of GlnA gene --> interacts with σ⁵⁴-RNA polymerase

(5) How an eukaryote increases the efficiency of translation ?

    --> by polysomes and rapid ribosome recycling (= circular structure of mRNA)

                                     ex) PABP1(polyA-binding protein) + eIF4G

 -- Model for a circular structure & polysome

(6) How do we know that DNA replication has a semiconservative replication mode ?

   --> growing in the presence of ¹⁵N and ¹⁴N

(7) How we can determine that DNA is bidirectionally replicated ?

5장; Biomembranes & Cell architecture

(1) membrane상의 lipid와 단백질은 lateral diffusion을 행한다.

   --> 10⁷ times/sec, several μm/sec

   --> fluoresence recovery after photobleaching (FRAP) technique

 (2) lipid rafts (=microdomain)

      --> membrane lipid (cholesterol, sphingolipid)와 protein은 특정부위에 움직이지 않고 한정되어 있을 수 있음

      --> How the rafts are destroyed?   ① methyl-β-cyclodextrin; depletion of cholesterol

                                                        ② filipin; sequestering of cholesterol

(3) How different cell types are purified ?

--> fluorescence activated cell sorter ( FACS); by measuring the emitted fluorescent light and scattered light

                negative charges propotional to the amount of fluorescence

      그밖의 용도; ① DNA/RNA 양측정 ② shape/size 측정

--> 실제 실험결과 (green;anti-CD3, red;Thy1.2.)

 (4) How the specific organelle is purified ?

    (예) membraneous organelles; coated vesicles, GLUT4-containing vesicles

     --> organelle-specific membrane protein에 대한 항체이용

     --> by low-speed centrifugation or metallic beads coated with antibody

(5) Visualization of cell architecture (by microscope)

  --> how the live cells can be measured ?   without staining      

      원리; refractive index, thickness

      ① phase contrast microscopy ② differential interference contrast microscopy

 ③ fluorescence microscopy (in fixed cells)

   --> for localization of proteins within a cell

   --> flurochrome; rhodamine and Texas red (Red), Cy3 (orange), fluorescein (green)

   --> 예) GLUT2

④ fluorescence microscopy (in live cells)

   --> introduction of genes such as GFP (green), CFP (blue), YFP (yellow)

   --> 예) mouse embryo

  ** Yellow --> green

  --> 예) 세포내 Ca²⁺와 H⁺양의 측정; ion-sensitive fluoresent dye 이용

            ⓐ fura-2 ; Ca²⁺ sensitive dye

                          fura-2 + ethanol -> fura-2 ester -> lipophilic -> in cytosol, hydrolysis of ester -> no transfer

            ⓑ SNARF-1 ; H⁺ sensitive dye

 ⑤ confocal scanning

 limitations of conventional fluorescence microscopy  -->  1) destroy materials during processing of cutting a section

                                                                                    2) fluorescence light emitted from molecules above and below

                                                                                        the plane of focus

⑥ deconvolution microscopy

  --> same image-sharpening effect, but through a different process

⑦ transmission electron microscopy (TEM)

  --> very thin and fixed section (50nm), only a small part of a cell

  --> detection of specific proteins in the thin sections ;  use of electron-dense gold particles

metal shadowing; for detection the shape and its component of a cell

steps; 2) making a metal film, 3) stabilization of replica

⑧ scanning electron microscopy (SEM)

  --> only 10nm thickness, surface of unsectioned metal-coated specimens

6장; Cells into Tissues

 (1) Cadherins

    --> 6 subfamilies, 100 이상 proteins, classical cadherins (E-, P-, N-)

    --> cell-cell adhesion, signaling, tissue differentiation

    --> Ca²⁺ dependent adhesion

   --> E-cadherin의 역할을 규명하기 위한 실험방법; media change, antibody 이용

 ① Structure of cadherins

    cadherin domain; for Ca2+ binding (-> rigidify the cadherin oligomers) and cell-cell adhesion (3 cadherin domains 관여)

How cadherins mediate the cell-cell interactions ?

  --> by cis or trans interactions

Where the C-terminal of cadherins is linked with ?

  --> actin cytoskeleton and cytosolic adaptor proteins

  --> in tumors; defect in this interaction

② tight junction

   --> impermeability of water-soluble substances

  structure; Fig 6-9, JAM (junction adhesion molecule)

Experiment for demonstration of impermeability of water-soluble substances in tight junction

  --> use of lanthanum hydroxide (electron-dense colloid)

    --> importance of Ca²⁺ in the formation and integrity of tight junctions

          if a low conc. of Ca²⁺ in a chamber -- freely movement of fluids and salts

          if a high conc. of Ca²⁺ in a chamber -- no freely movement of fluids and salts

③ fibronectin

  --> a matrix protein, cell migration and differentiation, wound healing, a dimer protein (C-terminal linked)

         6 functional regions, 3 types repeats

   --> intergrin binding sequence of a fibronectin repeat type III; Arg-Gly-Asp (RGD) sequence

   --> Model for fibronectin binding to integrin

          synergy region and other RGD-containing proteins enhance the binding

   --> colocolization of integrins and actin filaments; red (anti-actin), green (anti-integrin)

 ④ Hybridoma technique; for prepartion of monoclonal antibodies

    How do we make a hybridoma ?

     --> fusion; viral glycoproteins, PEG

     --> selection media; HAT medium

    Use of monoclonal antibodies; affinity chromatography, immunofluorescence microscopy, therapeutic tools

  --> HAT medium containing hypoxanthine, aminopterin, thymidine

       myeloma cells (HGPRT^-) --> die

 7장; Transport across Membranes

 (1) How we study the function of transport proteins ?

      ① liposome 이용

      ② transfection into cells that normally do not express transport proteins

(2) How the transmembrane electric potential can be arised ?

     --> use of potentiometer

 (3) How is membrane electric potential in animal cells ?

      --> many K⁺ channels (outward), a few Na⁺, Cl^-, Ca²⁺ channels

      --> negative charges on the inside, positive charges on the outside

     K⁺ channels: nongated (not affected by membrane potentials or small signaling molecules

     --> 실제 membrane potential = -70 mV (by presence of Na⁺channels (inward))

     --> Na⁺/K⁺ ATPase

** in plant and fungal cells

    --> by by transport of H⁺ outward

    How the membrane potentials are measured ?

(4) Patch clamping technique

   --> to measure ion movements through single channels

   --> in whole cells or isolated membrane patches

** patch-clamping tracing; measure the time for opening or closing of channels

** How novel ion channel proteins are characterized ?

     --> in vitro transcription --> oocyte expression --> patch-clamping

(5) How the cytosolic pH is regulated ?l

   --> in metabolism; produce excess H⁺ ions from H₂CO₃

   --> How the excess H⁺ ions are removed from cytosol ?

         ① Na⁺HCO₃^-/Cl^- antiporter; HCO₃^- (inward) -> CO₂ + OH^-  (by carbonic anhydrase)

                                                  OH^- + H⁺ = H₂O

        ② Na⁺/H⁺ antiporter

** How cells cope the excess of OH^- under a certain circumstance ?

    --> HCO₃^-/Cl^- antiporter 사용하여 HCO₃^-를 세포밖으로 방출

(5) water movement

 aquaporins; water channel proteins, a tetramer

                   each subunit = 6 membrane-spanning α-helix

How the waters can be passed through the channels ? --> constriction by conserved hydrophilic Aas of side chain

                                                                                    and carbonyl groups

                                                                             --> by H-bonding between water and amino groups of side chain

** evidence of aquaporins as water channels

(6) Action potentials in nerve cells

  morphology of neurons; ① cell body ② dendrite ③ axon ④ axon terminal

 ① cell body

    --> nucleus, synthesis of all neuronal proteins and membranes

 ② axon

   --> conduction of action potentials, -60 mV (in resting), +50 mV (in stimulus)

 ③ axon hillock

   --> origin of action potentials

(7) Channel inactivation in voltage-gated K⁺ channels

 --> soon after opening, spontaneously closing the channels

K⁺ channel; 4 α subunits + 4 β subunits

** Ball and chain model for inactivation

    --> balls; positive charged, N-terminals

    --> 아래 그림; blocking한 상태

 ** experiment for demonstrating the ball domains as an inactivating segment

      --> mutant; lacking the ball domain

      --> in connecting chain experiment; shorter makes more rapid in the activation

 (8) How nerve cells make an action potential ?

** postsynaptic neurons receive signals from many presynaptic neurons

    green (postsynaptic), orange-red (presynaptic)

 ** generation of action potentials

     --> combine of excitatory receptors with inhibitory receptors --> into axon hillock (summed together)

     --> generate action potentials through threshold potential (all or nothing fashion)

9장; Molecular genetic techniques and Genomics

 (1) How do we study the essential genes in yeast ?

    --> conditional mutation; in haploid cells

                                         Tem. sensitive mutants 이용 (23^oC 자라고, 36^oC 안자람)

                                         To isolate the genes involved in cell cycle

 (2) complementation test

     --> to see whether different recessive mutations are in the same gene or not

 (3) double mutants 이용 (I)

    --> to deduce the order in which proteins function

           ① biosynthetic pathway

           ② signaling pathway

 (4) double mutants 이용 (II)

    --> to see how proteins interact with one another

           ① suppressor mutation; 다른 한쪽에서의 변이가 처음의 변이를 억제하는 효과

           ② synthetic lethal mutation; 다른 한쪽에서의 변이가 more severe효과를 주는 경우

                                                   어느한쪽에서의 변이가 효과가 없는 경우 (redundancy)

 (5) Plasmid vector를 이용한 DNA cloning 원리

(6) λ phage vector를 이용한 cDNA library 제조

(7) λ phage vector를 이용한 cDNA library 로부터 interest clone을 조사하는 방법

(8) shuttle vector를 이용한 yeast genomic library 제조

(9) yeast genomic library를 이용한 functional complementation

     --> mutation을 야기한 유전자의 분석에 많이 이용

 클로닝한 DNA fragment의 분석;

 (10) electrophoresis를 통한 DNA size별 구분

  (11) DNA sequencing; Sanger method

       --> termination에 사용되는 dideoxyribonucleoside triphosphate의 구조

     --> sequencing의 원리 및 결과

   (12) PCR (polymerase chain reaction)

    --> a million-fold after 20 cycles

  (13) target gene cloning by PCR

       --> primer 제조시 enzyme site를 삽입, 10kb 이상의 fragment를 주입할 수 있음

  (14) southern blot

     --> detection of single specific DNA from DNA mixture

  (15) northern blot

    --> detection of single specific RNA from RNA mixture

    --> ex) 적혈종양세포 분화과정에 있어서 β-globin mRNA 발현분석

 (16) E. coli expression system

      --> recombinant protein의 제조,  for production of a large amount of protein

           ex) G-CSF, factor VIII, insulin, growth hormone

      --> lac promoter 및 IPTG 이용

 (17) Mammalian cell expression system

      --> to overcome the post-translational modifications (glycosylation, hydroxylation)

      --> by lipid or electroporation

      -->  neo^r ; neomycin phosphotransferase

(18) epitope tagging

  --> add a short Aas recognized by monoclonal antibody against the short fragment

  --> 용도; to study the intracellular locolization of proteins

                ex) AP1 adaptor protein (involved in clathrin-coated vesicle formation)

(19) microarray DNA

   --> to see the gene expression pattern during specific physiological responses or developmental processes

   --> ① DNA microarray; ~1kb coding fragment/spot

          ② DNA chip; ~20bp oligonucleotide/spot

          ex) glucose or ethanol하에서 발현되는 효모유전자 pattern 분석

 (20) cluster analysis

    --> genes showing a similar gene expression in a single DNA microarray can be different in the biological function    

    --> to examine the closely related, co-regulated gene expression

           (ex) check the expression pattern after serum addition

                   --> red; incease, green; decrease, black; no change

                   --> cholesterol synthesis, cell cycle, immediate-early genes, signaling and angiogenesis, wound healing

 (21) homologous recombination in yeast

      --> primers; use of flanking sequences of target sequence

      --> selection; kanMX (resistance for G418)

(22) gene-knockout in mice

   ① knockout mutation in ES cells

       --> two selectable markers; neo (resistance for G418), tk (sensitive for ganciclovir)

   ② production of knock-out mice

      --> injection of ES cells (brown) into 4.5 days blastocyst (black)

(23) gene-knockout in specific tissues

     --> to examine the effect of knock-out mutation in the specific tissues or specific stage in development

     --> loxP-Cre recombination technique; loxP (a site-specific recombination site), Cre (enzyme for recombination in loxP)

     --> 용도; NMDA (N-methyl-D-aspartate) glutamate receptor in the hippocampus -- important in learning and memory

(24) dominant-negative mutation

     --> 정의;  cause a loss-of-function in heterozygotes

     --> 장점; avoid the difficulty of obtaining the homozygous knockout mice

                   use of cultured animal cells

                   problem of related or similar functional genes

  --> transgenic mice 제조; randomly inserted, nonhomologous recombination

                                        regulated promoter

 (25) RNA interference (RNAi)

       --> for inactivating of a specific gene

       --> use of double-strand RNA, detection through in situ hybridization

             (ex) C. elegans, Drosophila, plants, zebrafish, spiders, Xenopus, mice

       --> how; by specialized RNA-processing enzymes

             function; defense against viruses, for regulation of certain endogenous genes

(26) DNA polymorphism

    ① Restriction fragment length polymorphism (RFLP)

       --> 그림a; mutations cause to change a restriction enzyme site

       --> pedigree 조사; linkage of a certain allele to the inherited trait or disease

     ② single nucleotide polymorphism (SNP)

     ③ simple sequence repeats (SSR, microsatellites); 1, 2, or 3 base sequence repeat number

         --> detected by PCR analysis and DNA sequencing

10장; Molecular structure of genes and chromosomes

 (1) Simple-sequence DNA (= satellite DNA) is located at the specific sites of chromosomes

      --> fluorescence in situ hybridization (FISH)

      --> satellite DNA; 14-500 bp, tandom repeats of 20-100 kb

      --> centromere, telomere, chromosome arm

 (2) DNA fingerprinting

     --> repeat number of simple-sequence DNA is different among individuals

            why; by unequal cossing-over (Fig 10-6)

    --> minisatellite DNA; 15-100 bp, tandom repeats of 1-5 kb

                                    ① southern blotting; different minisatellite probes (a, b, c)

                                    ② PCR; primers for flanking sequences of minisatellites

 (3) Role of RNA intermediate in the transposition

     --> in the LTR retrotransposon; reverse transcriptase, integrase

     --> 오른쪽 panel; transposed된 Ty element에서 삽입된 intron을 볼 수 없음

(4) chromatin condensation

    --> transcribed gene is higher than the untranscribed one in histone acetylation

    --> DNase sensitive

         ex) erythrocyte is active in the globin gene expression

 (5) chromosome scaffold

   --> non-histone proteins also act as a chromosome scaffold in histone-depleted chromosomes

   --> DNA loop structure in the chromosome

         증거 (Fig10-25); A, B and C--> scaffold associated region (SAR)

  --> interphase chromosome is located within a specific, restricted regions of nucleus

          ex) human chromosome 7

 (6) chromosome band patterning

     --> G bands; staining with Giemsa reagent after mild heat or proteolysis, detection of low G + C content

            p; short arm, q; long arm

    chromosome painting (multicolor FISH)

        --> for detection of translocation that banding pattern analysis does not reveal the difference

              ex) chronic myelogenous leukemia; philadelphia chromosome의 존재

  (7) important functional elements for chromosome replication and inheritance

      ① ARS (autonomously replicating sequence); replication origin

      ② CEN (centromere); for mitotic segregation

      ③ TEL (telomere); for mitotic segregation

 (8) mitochondria DNA detection

     --> ethidium bromide (red), DiOC6 (green, for mitochondria)

     --> yellow; indicates the mtDNA

11장; transcriptional control of gene expression

(1) characterization of transcription-control sequence of genes

     --> 5' deletion experiment

     --> reporter system; ① lacZ (β-galactosidase) ② luciferase  ③ GFP

 (2) separation of three kinds of eukaryotic RNA polymerases

      --> diffrence in salt conc. in elutes, α-amanitin (cyclic peptide, 8Aas, mushroom) sensitivity; poly II is strong sensitive

 (3) CTD (carboxyl-terminal domain) of eukaryotic RNA polymerase II

      --> in the highly transcribed genes; phosphorylated (red)

      --> CTD; heptapeptide (Tyr-Ser-Pro-Thr-Ser-Pro-Ser), important to viability

 (4) Detection of an initiation site of RNA transcript

      --> nuclear run-off assay in the presence of ³²P-labeled ribonucleoside triphosphates

 (5) Linker scanning mutation

     --> to pinpoint the exact regulatory sequence (promoter-proximal elements) for gene expression

 (6) Detection of enhancer (S1 nuclease protection assay)

      --> SV40 DNA contains an enhancer for gene transcription

      --> C (control); erythrocytes, 1 and 2; fibroblasts

 (7) protein-DNA interaction

① DNase I footprinting

   --> to see the protein binding sequence or for purification of DNA-binding proteins

   --> NE ( absence of proteins), O (presence of proteins), FT (flow-through fraction)

 ② electrophoretic mobility shift assay (EMSA)

     --> for more quantitative analysis of DNA-binding proteins

  (8) in vitro transcription

     --> to confirm whether the purified transcription factors have a transcriptional activity

     --> adenovirus DNA does not have a SP1 binding site

 (9) in vivo transfection assay

     --> to check whether the cloned gene of purified transcription factors is really able to transcribe

     --> X is a cloned gene

  (10) Detection of functional domains in transcription factors (ex; GAL4) such as activator

        --> from this experiment, two functional domains of GAL4 are discovered

 (11) mechanism of gene activation through exchage of chromosomal conformation

      --> in yeast mating type; HMLα or HMRa with heterochromatin structure move into MAT with euchromatin

      --> α and a type is determined

   ** repressor proteins involved in silencing; RAP1 and SIR proteins

        --> colocalization of telomeres with SIR3

 (12) Histone deacetylation

       --> heterochromatin, untranscribed genes

       ** chromatin immunoprecipitation; to see the pattern of chromosomes

                                                        acetylation sites; N-terminal lysine residues of histones

    ** mechanism of histone deacetylation and acetylation

          ① histone deacetylation; inactivation, Ume6, Sin3, RPD3 (deacetylase)

          ② histone acetylation; activation, Gcn4, Gcn5 (acetylase)

 (13) translocation of homodimeric glucocorticoid receptor (GR)

      --> from cytosol to nucleus in the presence of dexamethasone

** hormone-dependent gene activation

12장; Post-transcriptional control and nuclear transport

 (1) RNA splicing

    --> DNA:mRNA hybridization reveals the presence of introns

     --> splicing sites; poly (T) (red), DAPI (blue), SR protein (green)

     --> splicing is occurred at discreted areas of nucleus

 (2) Role of nuclear localization signal sequence (NLS)

    --> NLS origin; come from T antigen of SV40 (wt type is present at a nucleus, mt type is at cytoplasm)

    --> basic residues are rich (Pro-Lys-Lys-Lys-Arg-Lys-Val)

      ** cytosolic pyruvate kinase + NLS of SV40 T antigen --> movement into nucleus

 (3) Requirement of cytoplasmic proteins for nuclear transport

    --> cytoplamic proteins; Ran, NTF2 (nuclear transport factor 2), importinα and β

    --> experiment; digitonin treatment causes a permeabilization of plasma membrane, bu intact in nuclear envelopes

                            ** fluorescent protein + NLS

    **  nuclear import model

 (3) nuclear export

    --> cell fusion experiment; HeLa cells + Xenopus cells

           green (for human hnRNP C), red (for human hnRNP A1)    

   --> NES (nuclear export signal); ①  leucine-rich sequence    ② a 38-residue sequence    ③ a sequence in hnRNP K

   **  nuclear export model; exportin1 + Ran.GTP --> + NES of cargo proteins

  (4) Role of 3'UTR of mRNA in the protein targeting

       --> β actin is localized in the leading edges, α actin is localized into the perinuclear regions of myotubes

       --> β-galactosidase (5') + (3') actin regions in pannels

 16장; Moving proteins into membranes and organelles

 (1) protein sorting pathways

     --> secretory pathway, nonsecretory pathway

  (2) secretory protein localization in the lumen of ER

      --> pulse-labeling experiment; generation of rough microsomes

 (3) hydrophobic N-terminal sequence (ER signal sequence) is associated with microsomes

     --> cell-free experiments

     --> ER signal sequence; 1-2 positive charged Aas + 6-12 hydrophobic core sequence 

  (4) ER translocation

    ** Model for ER translocation

        --> SRP (signal recognition particle)

        --> translocon; 3 different proteins (Sec61α, Sec61β, Sec61γ)

   --> translocated proteins are contacted with Sec61α among Sec61 complex

          ; use of modified Lys-tRNA binding to the light-activated cross-linking reagent

  (5) Resolution of the topology of membrane proteins from their sequences

     --> positive; hydrophobic portion, negative; polar portion

 (6) mitochondrial matrix-targeting sequences

     --> matrix-targeting sequence; at the N-terminal, 20-25 Aas,

                                                   hydrophobic Aas + basic Aas + hydroxylated Aas (Ser, Thr)

   ** Model for mitochondrial protein targeting

         --> cytosolic HSP70; for unfolding, import receptor, import pore (Tom40, Tim23/17, Tim44)

               matrix HSP70; ATP hydrolysis cause to pull out the translocated protein

(7) Role of mitochondrial matrix-targeting sequences in the protein targeting

       ** cell-free translocation assay;

           matrix-targeting sequences of alcohol dehydrogenase + a spacer sequence + dihydrofolate reductase (DHFR)

     --> in the presence of chaperones; unfolding causes the translocation

           in the presence of methotrexate; folding causes the non-translocation

     --> (c) ; presence of (b) using antibody against DHFR

 (8) peroxisomal proteins

   --> Zellweger syndrome; defect in transport of peroxisomal matrix proteins

   --> for peroxisomal matrix proteins; Pex10, Pex12, Pex2

   --> for peroxisomal membrane proteins; Pex3, Pex16

   ** peroxisomal biogenesis

      --> Pex19 act as a receptor

      --> PTS1 (peroxisome targeting sequence) sequence; C-terminal, PTS2 sequence; N-terminal

17장; Vesicular traffic, secretion, endocytosis

 (1) secretory and endocytic pathways of proteins

      --> cisternal progression; a nonvesicular process

      --> retrograde transport; ER-or Golgi-resident proteins

 (2) Use of GFP fusion proteins that is sensitive to Temp.

      --> for observation of secretory proteins

      --> ex) glycoprotein of vesicular stomatitis virus (VSVG)

 (3) Compartment-specific oligosaccharide modification

    --> (Man)₈(GlcNac)₂

    --> Assay for a glycoprotein transport from ER to cis-Golgi

    --> pulse-labeling experiment; label in the nonpermissive Tem. (40^oC) and treated with endoglycosidase D (cis-Golgi

                                                 specific)

 (4) Sec mutants reveal the stages of secretory pathway

     --> 5 kinds of sec yeast mutants

     --> characterization of accumulated proteins when the mutant is shifted from permissive to nonpermissive Temp.

 (5) Protein transport through Golgi compartments

      --> cell-free transport assays; fibroblast lacking N-acetylglucosamine transferase I + VSV G protein

 (6) Basic mechanism underlying vesicle budding and fusion

      --> (a) budding,  (b) fusion

      --> in vitro budding reaction; polymerization of the coat proteins (dark regions)

 (7) Role of GTP-binding proteins in the assembly of vesicle coats

    --> 3 kinds of vesicles; ① COPII; ER->Golgi  ② COPI; between Golgi, cis-Golgi->ER  

                                       ③ clathrin; membrane, trans-Golgi->endosome

   ** Model for vesicle assembly and disassembly

         (1) Sar1 (small GTP-binding protein; a regulatory function. Ras like) + Sec12 (exchange factor)

         (2) Sec23/24 binding to GTP-Sar1

         (3) Cargo protein binding to Sec23/24

         (4) Sec13/31 binding --> completion of a coat complex

         (5) GDP-Sar1 formation by Sec23

         (6) Coat disassembly

   --> mutant version of Sar1 in the GTP hydolysis

          ; no disassembly of coat and unable to fuse with target membranes

            ex) addition of a nonhydrolyzable GTP analog

 (8) Role of dynamin for pinching off of clathrin vesicles

      --> GTP hydolysis; energy release and for contraction of the vesicle neck

      --> in case of COPI and COPII vesicles, no-requirement of dynamin

     evidence; use of a nonhydrolyzable GTP analog, GTP-γ-S and gold-tagged anti-dynamin antibody

(9) Proteolytic cleavage of some membrane and secretory proteins after leaving the Trans-Golgi

    kinds; some membrane and soluble secretory proteins

             (ex) lysosomal enzymes, influenza hemagglutinin, albumin, insulin, glucagon, yeast α-mating factor

  ** in case of insulun

           --> immature secretory vesicles (closed arrowheads) and vesicles budding from trans-Golgi (arrow)

           --> contain only proproteins, not mature proteins

 ** proteolytic cleavage of albumin (a) and insulin (b)

(10) Receptor-mediated endocytosis

      --> LDL particle + ferritin

    ** Model for receptor-mediated endocytosis

 ** binding model between LDL particle and LDL receptor

  ** dissociation of endocytosed receptor-ligand complexes

      --> in late endosomes

      --> asialoglycoprotein-specific antibody conjugated with gold particles

            ex) LDL receptor; 1 turn/10-20 min

 (11) Synaptic vesicle fusion and recycling

synaptic vesicles; located in active zone, contains a Ca²⁺-binding protein

** Model for synaptic vesicle recycling

 ** synapsin-containg fibrous proteins help the localization of synaptic vesicles in active zones