高中職科學教師尖端科技研究經驗培育計畫－高中科學教師生物科技研究經驗培育計畫

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師，尖端科技研究經驗培育營暑期課程內容及上課時間如下：週次月日星期時間地點上課內容及方式 1 7 10 四 1:30-5:30 敬業樓 202 電腦教室

Introduction of databases and information retrieval: NCBI,

EBI, PDB

講述、討論、上機實作

1 7 11 五 1:30-5:30

Search for homologs and sequence analysis: BLAST,

alignment, and building a phylogenetic tree 講述、討論、上機實作

2 7 17 四 1:30-3:30

科學館3 樓會議室

Protein structure analysis and modeling: Deep View and

Swiss model 講述、討論、上機實作

2 7 17 四 3:30-5:30

The Structural Basis of Protein Function

講述、討論

2 7 18 五 1:30-5:30

Protein Purification and Analysis

講述、討論 3 7 24 四 1:30-5:30 Molecular cloning

講述、討論

3 7 25 五 1:30-5:30

Techniques of molecular and cellular biology 科學館

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體並轉殖到大腸桿菌菌株。結合生物反應器以液態方式大量培養後，並利用高壓均質機打破細胞後，利用離心機蒐集細胞水溶液的部分後，再接續蛋白質純化與結晶的工作。

蛋白質濃度測量

(7)

析時間，故較慢離開層析管柱。

蛋白質結晶

蛋白質晶體培養成長

目前所知最佳的晶體培養條件為： 0.15M NaCl + 0.1M Bicine + 19% PEG550

Solution總體積為1000µL ，Protein：Solution＝1：1

同步輻射蛋白質結晶學

將蛋白質結晶帶至同步輻射中心BL13B1 做 X-ray 繞射，得到繞射點數據。並利用同步輻射中心的BLU-ICE 監控內部的機器和 HKL2000 進行繞射光譜的蒐集與評估，稱為 data process。將晶體以 loop 自 drop 中撈起後，就可以用低溫鉗架上儀器。繞射前需做晶體的調整，調整時需注意有無在粗、微調監控螢幕的中心，還可以觀察是否有晶體裂開的現象，將晶體移至無裂開或是晶型較完整的地方。

生物資訊學

BLAST ( Basic Local Alignment Search Tool )

(8)

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(10)

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實際操作部分 實驗架構流程圖如下

純化 E. coli 中的 PGRMC1

(1) 將 E. coli 和破菌緩衝液(20mM Tris，pH7.0 )震盪溶解，並加入 PMSF(蛋白脢抑制劑)和 DNase1，利用 French press 將 E.coli 細胞壁打破，以超高速離心機離心，分離出 Soluble Part 和Membrane Part。

(12)

His-tag 在轉譯出的 PGRMC1_human protein 上，使得 PGRMC1_human 帶有 His-tag，可和此層析管柱的過度金屬-鎳產生親合性吸引，藉此將目標蛋白質留在管柱中，再用 imidazole 將目標蛋白沖提出來，完成第一步純化；利用 SDS-PAGE 檢視 PGRMC1_human 有無成功被化出來，以進入下一階段層析純化。 (3) 將前步驟純化出的蛋白質溶液以 Superdex 200 層吸管柱進一步純化 Superdex 200 管柱屬於膠體過濾法，利用分子量的不同，大分子無法進入膠球內空隙而首先流析出來，而小分子物質則進入膠球內空隙，而產生較長的流動距離及流析時間，故較慢離開層析管柱，利用此種特性，可將目標蛋白更進一步精製純化。 (4) 利用 SDS-PAGE 檢視 PGRMC1_human 有無成功被純化出來。 (根據文獻 PGRMC1 分子量約為 25~28kDa) PGRMC1 膜蛋白表現在真核系統-酵母菌中

z PCR (Polymerase Chain Reaction)製備 PGRMC1

與中研院化學所陳長謙院士實驗室林素卿博士共同合作，由林博士提共含有 PGRMC1 的轉殖基因載體。設計兩組引子(primer)，分別為 primer1

(5'TAATTAATgCggCCgCATggCTgCCgAggATgTgg 3')與 primer2 (5'

gCCgACTAgTCCATCATTTTTCCgggCACTCTCATC 3')，與抽出含 PGRMC1 的質體(plasmid) 進行PCR 反應，得到不含 His-tag 的 PGRMC1-FLAG fusion protein 產物。

z Double Digested by restriction enzyme

(13)

圖二 pESC-LEU vector Map

z Ligation

利用 dsDNA 於 OD260 nm有特殊吸收，測得vector：insert =34 fmol/µL：253 fmol/µL，以

vector ：insert ＝1µL：1µL 為比例，再加入 Ligase(Violet)於 16℃下 ligation overnight。其中含有2 個樣品：樣品 1 為 vector + insert + ligase；樣品 2 為 vector + ligase，藉此檢查 vector 有無以限制酶作用完全。

利用 dsDNA 於 OD260 nm有特殊吸收，測得vector：insert =34 fmol/µL：253 fmol/µL，以

vector ：insert ＝1µL：1µL 為比例，再加入 Ligase(Violet)於 16℃下 ligation overnight。其中含有2 個樣品：樣品 1 為 vector + insert + ligase；樣品 2 為 vector + ligase，藉此檢查 vector 有無以限制酶作用完全。

z Transformation 至 E.coli (DH5α)

將構築好的 plasmid 以熱休克方式(Heat shock)，轉型進入 E. coli

(14)

z 限制酶切解確認轉型成功

將抽出的 plasmid 以限制酶 NotΙ 與 SpeΙ 分別在 37℃作用 2 小時，跑洋菜膠 DNA 電泳，根據NCBI 網站的 PGRMC1_human 胺基酸序列為 588bp，

發現在約600bp 處有 insert (PGRMC1) band 出現。

z PGRMC1 DNA 定序

(15)

梁。如何讓高中教師掌握尖端科技之脈動，傳達科學的本質，提升學生探索科學的能力，就是計劃的目標。這次屏東教育大學是以生物科技為主軸，提供高中教師課餘時間參與大學實驗室進行專題研究；我是參與陳皇州教授實驗室酵蛋白質純化專題探討，計畫內容可區分兩大部分：第一部分蛋白質純化課程，由屏教大生物化學系教授親自授課，讓老師們更了解目前大學所做關於蛋白質研究的基礎理論；第二部份屬於純化實作部分，所學的內容這些都可以提供教師回到學校後教導高中生，啟發有志從事生物化學的學生方向。 第一部分：蛋白質純化課程 蛋白質結構講解酵素純化三步驟粗蛋白、部分純化、均質酵素蛋白質純化與分析的原理

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B 液：分離膠體衝溶液(resolution solution) 配製1.5 M Tris-HCl buffer（pH = 8.8）以 0.45 µm 濾膜過濾。 C 液：焦集膠體緩衝溶液(staking solution) 配製0.5 M Tris-HCl buffer（pH = 6.8）以 0.45 µm 濾膜過濾。 D 液：10X 電泳槽緩衝溶液 (Tris-glycine buffer) 將30.3 g Tris 加入 144.4 g 甘胺酸(glycine)與 10 g SDS 溶於 1L 水中。 E 液：樣品緩衝溶液(sample buffer) 5 mL 甘油(glycerol)加上 1.5 g SDS，6.5mL 分離膠體衝溶液以及 0.02 g Bromophenol blue，使用時加上 2.5 mL 硫醇基乙醇 (β-mercaptoethanol)，加二次水至總體積為 50 mL。 F 液：起始劑(Initiator solution；簡稱 APS)

取30 mg 過硫酸銨(ammonium persulfate)加入 300 µL 二次水配成 10(w/v)%的溶液。 G 液：十二磺酸溶液(sodium dodecyl sulfate：簡稱 SDS)

將SDS 配成 10(w/v)%的溶液。 H 液：染色液(Vesterberg staining solution)

將0.25 g 的 Coomassie brilliant blue R-250 加入 112.5 mL 甲醇以及 25 mL 醋酸，加水稀釋至250 mL。 I 液：退染液(Destaining solution) 400 mL 甲醇再加上 100 mL 醋酸，加水稀釋至 1L。 J 液：蛋白質標準品液(Marker) 由Amersham 購得低分子量標準品(LMW)，每瓶含有 575 µg 蛋白質，加入 200 µL 樣品緩衝溶液(E 液)，加以分裝保存於－20oC 冰箱。標準品其規格如下：

Protein Mr Source Amount (µg)

Phosphorylase 　 97,000 rabbit muscle 67 Albumin 66,000 bovine serum 83 Ovalbumin 45,000 chicken egg

(18)

Carbonic anhydrase 30,000 bovine

erythrocyte 83 Trypsin inhibitor 20,100 soybean 80

　-Lactalbumin 14,400 bovine milk 116

電泳動力源(Electrophoresis Power Source)

水平膠體電泳：GE-100 可選擇電壓 50 或 100V。

垂直膠體電泳：Bio-Rad 數位式調控電壓並設有計時器。

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行政院國家科學委員會專題研究計畫成果報告附件

承先啟後 -- 高中科學教師生物科技研究經驗培育計畫 (教師培育)

(24)

在分子及細胞生物技術上，本計劃之課程內容著重於分子選殖之

原理、技術與衍生應用，結合概念闡訴及操作細節討論，讓參與之高

中教師能對此領域具深度了解。內容同時設計實作課程，利用實際操

作課程講解之技術，以模組形式進行整套相關之實驗，讓參與之高中

教師不只是紙上談兵，而是真實體驗並進行實驗流程，同時藉由實驗

問題深入探討相關之可能應用，加強延伸思考之基礎。課程內容大綱

如下（詳細課程資料如後附之講義內容，資料亦可於針對本計劃建構

之網站觀看，網址

http://cclearn.npue.edu.tw/tuition/lynnfarh-web/npue_web/）

：

講解課程：

Restriction enzyme and ligase

Vectors

Protein production and purification

Gene Identification

Post-genome analysis

Engineering plants

(25)

Molecular cloning

(26)

• gene cloning, molecular

cloning

• cloned DNA, insert DNA,

target DNA, foreign DNA

• recombinant DNA, DNA

How ?

(27)

Molecular cloning

Restriction

enzyme and

(28)

Breaking

Breaking and

joining DNA using

joining

restriction enzymes

(29)

Restriction

Restriction enzyme

•

• type II restriction endonucleases

type II

• specific sequence (

recognition site)

recognition site

•

(30)

DNA cleavage by

type II restriction

enzymes

(31)

(32)

EcoRI

• bacteria:

Escherichia (genus)

E

coli (species)

co

• strain:

R type (RY13)

R

•

(33)

(34)

The structure of the restriction enzyme

BamHI bound to DNA

highly conserved catalytic

core – 5 stranded β-sheet

flanked by 2 α-helices

scanning

(35)

(36)

phosphoamide

bond

(37)

the optimum temperature

is 37

(38)

(39)

The basics of cloning into a plasmid vector containing

a

(40)

The ligation of vector DNA that has

been treated with

phosphatase to

phosphatase

(41)

Molecular cloning

Vectors

(42)

Why Vectors?

(43)

Key concepts 1

• Vectors are

autonomously replicating DNA molecules that

autonomously replicating

(44)

Key concepts 2

• DNA fragments cloned into vectors can be isolated in

(45)

Key concepts 3

• Vectors are based on naturally occurring DNA sequences

that have been

modified and combined to serve particular

modified

(46)

Key concepts 4

• The choice of vector depends chiefly upon the size of the

(47)

Key concepts 5

•

• Vectors have been

Vectors have been

developed for a variety of specific

purposes

purposes, including the production of single-stranded DNA,

(48)

Escherichia coli

(

₍

E. coli

_{E. coli}

)

₎

•

• gram

gram

-

negative, motile, rod-shape

negative

(49)

Prokaryote: Escherichia coli

• could be cultured on the simple medium (

easy to grow)

easy to grow

• the cell generation time at 37℃ is about 22 min (in enriched

liquid culture) –

rapid doubling time

(20

–

30 min)

• Its

genetics are well understood

genetics

• Lab strains are generally

safe and contain mutations that do

safe

not allow them to escape the lab environment

• one of the most studied organism (

fully mapped and

sequenced genome

sequenced genome)

• extra-chromosomal copies of DNA can be exploited to

carry

foreign DNA fragments

(50)

Bacterial

conjugation – transfer of genetic

conjugation

information

• F (fertility) genes

• pilus

• fertility factor

–

F

episome (99159 bp) – extra-chromosomal plasmid

episome

–

Hfr – integrated into genome

Hfr

–

(51)

~ carry foreign

DNA fragment

(52)

Vectors

•

• autonomously replicating DNA sequences that

autonomously replicating

can be used to

carry foreign DNA fragments

• plasmids or bacteriophage lambda (λ)

•

• cloning vector

cloning vector

•

(53)

(54)

Plasmids

(55)

Plasmid DNA

• A plasmid is a DNA molecule separate from the chromosomal

DNA and capable of

autonomous replication. It is typically

autonomous replication

circular

and double

-

stranded. It usually occurs in bacteria, sometimes in

stranded

eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces

cerevisiae). Size of plasmids

varies from 1 to over 400 kilobase pairs

varies

(56)

Plasmids

• often carry a gene that encodes

resistance to either

resistance

antibiotics or heavy metals

antibiotics or heavy metals, or that produces DNA

restriction and modification

restriction and modification enzymes

•

• relaxed plasmids

relaxed

–

multiple copies (10-200) / cell

• replicate using host derived proteins

•

(57)

What

’

s your name ?

•

• pBR322

pBR322

–

p – plasmid

–

BR – who developed the plasmid

–

322 – the number of their stock collection

• pUC

(58)

(59)

Where

Where can we put our insert into

(60)

(61)

(62)

insertional

(63)

dis

disadvantages

double screening procedure

↓

(64)

pUC plasmids

high copy number

high copy number –

G → A mutant ~

upstream of the

initiation site of RNAI

blue

-

white

screening

(65)

2686 bp

• LacZ’ (α peptide) + LacZα (ω peptide

encoded by chromosomal DNA) → active

hybrid

β

-

galactosidase

•

(66)

The activation of the

lac operon in

lac operon

E. coli

• β-galactosidase →

glucose and galactose

• permease

• operator

• promoter

(67)

α

-

complementation

and

(68)

(69)

…a number of examples to show

the diversity of plasmid use

(70)

if

No suitable restriction enzyme

_No

(71)

PCR

P

(72)

(73)

The

products formed

products

during the

first 3 cycles

(74)

(75)

without

without having to use restriction

(76)

CreR

(77)

Gene shuffling between plasmids using

recombination

13bp 8bp 13bp

(78)

“

one strand at a time

” mechanism of exchange

”

•

• Cre

Cre

-

lox (2 component

lox

only)

–

Cre

is encoded by

phage P1

–

lox

site

–

widely used as genetic

engineering tool

•

• Cre

Cre

exists in 2 distinct

green

(79)

(80)

Bacterial transformation

(81)

3 methods for the transformation of cells

• chemical transformation

• electroporation

(82)

(83)

(84)

Molecular cloning

Protein production and

(85)

(86)

Regulatable

promoters

•

(87)

The activation of the

lac operon in

lac operon

E. coli

•

• β

β

-

galactosidase →

galactosidase

glucose and galactose

• permease

• operator

• promoter

• repressor

• CAP binding site and

CAP (

catabolite

c

a

(88)

The effect of the concentrations of

glucose,

glucose

lactose, and

lactose

cAMP

cAMP in the growth medium on the level of transcription

from the E. coli

lac

promoter

• E. coli

lac

promoter

–

turn off

(

lac

repressor protein)

–

(89)

The effect of the concentrations of

glucose,

glucose

lactose, and

lactose

cAMP

cAMP in the growth medium on the level of transcription

from the E. coli

lac

promoter

• CAP (catabolite activator protein) ＋ promoter ~

increases the affinity of

increases

the promoter for RNA polymerase

(90)

The effect of the concentrations of

glucose,

glucose

lactose, and

lactose

cAMP

cAMP in the growth medium on the level of transcription

(91)

tac

(92)

(93)

Protein purification

(94)

tac

(95)

(96)

(97)

Affinity chromatography

(98)

simplify the purification

simplify the purification of recombinant

(99)

(100)

(101)

Cleavage of fusion proteins

• unsuitable for clinical use

• may affect biological function

• oligonucleotide linker recognized by a specific

non

bacterial

protease

(102)

histidine-tagged protein (His-tag)

• 6 or 8 His (either N- or C-terminus)

• nickel

nitrilo

n

triacetic

t

acid column

a

(103)

The binding of proteins

tagged with multiple

histidine

histidine residues to Ni

2+

_{-NTA resin}

• immobilized metal ion affinity chromatography (IMAC)

(104)

(105)

The purification of proteins tagged with

GST

• glutathione S-transferases

(GSTs) - ~26 kDa dimeric

protein

• glutathione (GSH) –

(106)

The purification of

proteins tagged with

MBP

(107)

IMPACT

IMPACT system

•

• Intein m

I

mediated

p

purification with

an

affinity c

a

chitin

binding

tag

t

•

• protein self

protein self

-

splicing

• intein and extein

N

C

4

(108)

(109)

(110)

(111)

immunoaffinity

chromatographic purification

(112)

(113)

(114)

Gene Identification

(115)

I am

here!!

(116)

How ?

• DNA sequence

• Protein sequence

(117)

Screening by

nucleic acid

_{nucleic acid}

hybridization

• need to

know something about DNA sequence

know

•

(118)

Screening for DNA sequences by

nucleic acid hybridization

(119)

(120)

(121)

probe

• γ-

32 _{P-ATP – polynucleotide}

kinase (for end-labelled)

• α-

32 _{P-dCTP – incorporated}

(122)

(123)

(124)

(125)

(126)

The

degeneracy of the genetic code

degeneracy

Met

-

Ile

-

Trp

-

Pro

-

Phe

5

5 ’

’

-

ATG

-

ATA

-

TGG

-

CCA

-

TTC

-

3

3 ’

’

C C T

T T

G

5

(127)

(128)

Southern blotting

alkali

denatured

BLOCK (salmon sperm DNA)

UV cross

_{or heat}

-

linking

probe

(129)

(130)

…

gene isolated from one organism has been used as

gene

a hybridization probe to detect a

homologous gene

homologous

(131)

Bacteria or Phage

• colony hybridization screening

• λ plaques

–

clear background

–

can be left several times

(132)

(133)

Immunoscreening

(134)

(135)

(136)

polyclonal

polyclonal and

monoclonal antibody

_{monoclonal antibody}

(137)

epitope

•

• continuous

continuous

epitope

–

primary amino acid sequence

•

• discontinuous

discontinuous

epitope

–

(138)

Immunoscreening

Immunoscreening of a λ

phage library

lacZ

(139)

(140)

Immuno

(141)

Immuno

(142)

(143)

functional

complementation

(144)

Two

(145)

fusion

protein

fusion

protein

-

protein

interaction

The two

(146)

Plasmids to generate the DNA binding and activation

domain fusions required for the 2-hybrid screen

Bait

(147)

Reporter gene

(148)

The results of a typical 2

-

hybrid screen

host

–

GAL4 and

GAL80 genes

(149)

One-hybrid screening

identification of proteins

that regulate the promoter

of known genes

(150)

Three-hybrid screening

do

not

interact

directly

(151)

Three-hybrid screening

do

not

interact

directly

(152)

Reverse 2

(153)

The construction of plasmids producing

different prey proteins for use in a

genome-

genome

-

wide two

wide two-

-hybrid

hybrid screen

with different common tail

–

for

2nd round of PCR

common sequence

for

homologous recombination

(154)

A

genome

-

wide two

-

hybrid screen to identify

hybrid

(155)

Post

(156)

--

ome

_ome

• transcript

ome

–

the mRNA content of a cell

–

different genes, different stage, different factors…

• prote

ome

–

the protein content of a cell

• metabol

ome

–

(157)

What happen?

• normal cells → cancerous cells

–

what genes are turned

on or

on

off ?

off

–

what proteins are made ?

(158)

Global changes in gene expression

Microarrays

(159)

The construction of a DNA microarray

• 25,000 ~ 30,000 genes in humans

–

individually analyzing the expression (mRNA levels) of all genes…

• massive numbers of genes

–

yeast sporulation ~ 1000 genes (1/6)

• a small subset of genes

(160)

The construction of a DNA

microarray

• rapidly analyzing the expression of all

genes within a genome

–

providing a fixed single strand

fixed single strand of DNA to

which labelled cDNA fragment can bind

–

DNA fragments are physically attached to

an inert support (chip)

(161)

Difference between 2 system

•

• Affymetrix chips

Affymetrix

chips

–

chemically synthesized single-stranded DNA oligonucleotides ( ~ 25

bases )

–

attached to a quartz surface using photolabile agents and

photolithographic techniques

–

500,000 / 1.28 cm

2

_chip

• ••

Stanford chips (microarray)

–

constructed by robotic spotting of DNA fragments

–

from PCR amplification (every individual gene)

–

dried by heating to 100

℃

for 2 s, fixed by UV cross

--

-

linking, and then

denatured (95

(162)

(163)

(164)

Difference between 2 system

• ••

Affymetrix

chips

–

chemically synthesized single

--

-

stranded DNA

oligonucleotides

( ~ 25

bases )

–

attached to a quartz surface using

photolabile

agents and

photolithographic techniques

–

500,000 / 1.28 cm

22 2

_chip

•

• Stanford chips (microarray)

Stanford chips (microarray)

(165)

(166)

(167)

Identifying genes whose

expression differs

expression

(168)

Flowers

Leaves

AAAAA

_AAAAA

AAAAA

_AAAAA

mRNA isolation

DNA microarray probe preparation

(169)

Flower

Leaf

DNA microarray hybridization

(170)

(171)

Laser excitation

Cy5: ~650 nm

Cy3: ~550 nm

Image overlay

No changes

(172)

(173)

(174)

Cy3

labeled cDNA from etiolated seedlings

Cy5

labeled cDNA from green leaves

(175)

The characterization of malignant melanomas

based on their gene expression profile

• expression

differences between

differences

normal and cancerous cells

•

• normal human breast epithelial

normal

cells and breast

cancer cells

cancer

•

• may be involved in the process

may

of cellular proliferation

cellular proliferation during

cancer growth

•

(176)

The mechanism of

antisense inhibition

antisense

and

RNA interference

• antisense RNA

–

ex. tomato polygalacturonase

–

tetracycline

inducible promoter

inducible

• RNA interference (RNAi)

–

nematode C. elegans

–

co-injection of

double

-

stranded

RNA

•

(177)

Structural genomics

Structural genomics to solve

protein structures based

only on

only

the knowledge of

gene sequence

• protein structure

–

X-ray crystallography

–

nuclear

n

magnetic

m

resonance (NMR)

r

• E. coli expression vector

–

with tag (ex. his-tag)

–

purification

•

• in vitro transcription/translation system

in vitro

–

T7 or SP6 polymerase promoter

(178)

E

(179)

Transgenic plants

are simply plants that contain a gene that

has been artificially inserted into that plants, instead of bein

g

inserted through natural way.

The gene that is inserted into the crop is called a

(180)

轉殖技術

•

• 載體媒介轉殖法

載體媒介轉殖法

(vector

-

mediated gene transfer)

• 農桿菌載體

• 病毒載體

• 跳躍子載體

•

• 非載體媒介轉殖法

非載體媒介轉殖法

(vector

-

free gene transfer)

(181)

農桿腫瘤菌

Agrobacterium

tumefaciens

• Agrobacterium tumefaciens is a

Gram-

negative soil phytopathogen

.

• Agrobacterium has the

broad host range

.

• Agrobacterium affect most dicotyledonous

dicotyledonous

plants

in nature, resulting in crown gall

crown gall

tumors

tumors at the soil-air junction upon tissue

(182)

The mechanism of crown gall formation

different Ti

direct the

synthesis of

different

opines

(183)

The

Ti

(

t

umor

i

nducing)

plasmid

carried by

Agrobacterium

•

• 2 Cloning strategies

2 Cloning strategies

•

• co

co

-

integration

vectors

–

homologous recombination

with a small vector plasmid

•

• binary vectors

binary vectors

–

not need to be physically

associated with

vir genes

vir

23

23 kbp

kbp

left and right border – 25 bp

imperfect direct repeats

(184)

(185)

Fin.

(186)

Protein expression and western

blotting

(187)

實驗時程

• 1st week Competent cells preparation and transformation

↓

• 2nd week Protein induction

↓

• 3rd week SDS PAGE analysis

↓

• 4th week Western transfer

↓

(188)

(189)

SDS

(190)

Western blotting

(191)

(192)

3 major methods for the transformation

of cells

• chemical transformation

• electroporation

(193)

(194)

勝任細胞的製作

(195)

(196)

Let

(197)

(198)

Expression in E. coli

• many advantages…

• easily broken for the harvesting of the proteins

•

• prokaryotic organism

pro

–

NO

splicing

–

(199)

(200)