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Introduction to Molecular Biology. Siripan Limisrichaikul. Basic Molecular Biology. ความเป็นมา ที่มาของความรู้ เข้าใจในการทดลองเพื่อพิสูจน์สิ่งต่างๆ Definition : genetic materials, genome, gene, Central Dogma : กระบวนการขั้นตอนทั้งหมดของพันธุกรรม Replication Transcription / genetic code

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Introduction to Molecular Biology

Siripan Limisrichaikul


Basic Molecular Biology

  • ความเป็นมา ที่มาของความรู้ เข้าใจในการทดลองเพื่อพิสูจน์สิ่งต่างๆ

  • Definition: genetic materials, genome, gene,

  • Central Dogma : กระบวนการขั้นตอนทั้งหมดของพันธุกรรม

    • Replication

    • Transcription / genetic code

    • Translation


Terminology

Molecular biology:

study of biology at a molecular level.

Genetics:

study of the effect of genetic differences on organisms.

Biochemistry:

study of the chemical substances and vital processes in living organism.


Molecular

Biology

Genetics

Biochemistry

Relationships

Genes

Functions

Proteins


Timeline of Molecular Biology

http://snhs-plin.barry.edu/Genetics_Lab/genetics_labS05.htm

http://www.genome.gov/Images/press_photos/highres/38-300.jpg


R

R’

R’

R

Parent

F1; filial

1865 : Gregor Mendel

  • The principle of segregation ; allele

  • Law of independent assortment

  • Law of dominance


1928 : Frederick Griffith

  • Pneumococcus : cause pneumonia (die)

    • Rough surface type ( R ) ; non-virulence

    • Smooth surface type ( S ) ; Virulence Die

    • death Smooth (dS) ; non-virulence

    • Mix ( R ) + (ΔS) ; Virulence Die

Substancefrom cell can transform to another cell.


#4

#1

#2

#3

(R+ ΔS)

(S)

(R)

(ΔS)

S ; Virulence Die

ΔS ; heat to kill S

R ; non-virulence

ΔR ; heat to kill R

http://www.nature.com/scitable/topicpage/Isolating-Hereditary-Material-Frederick-Griffith-Oswald-Avery-336HOMEWORK


1944 : Oswald Avery, Colin Maloed, Maclyn Mc carthy


(ΔS)

RNAse

Protease

RNAse

Protease

DNAse

DNAse

Add (R)


1944 : Oswald Avery, Colin Maloed, Maclyn Mc carthy

  • (R+ ΔS) + Treated with

    1. RNase (destroy RNA),

    2. Protease (destroy protein)

    3. DNase (destroy DNA)

  • S type reborn again in 1&2

  • NoS type in 3

+ DNA, Protein

+ DNA, RNA

+ RNA, Protein

the transforming substance is DNA


1952 : Hershey & Chase

  • T2 phage is a virus that infects bacteria

  • Radioactive label

    • 32P-DNA

    • 35S-protein

  • In progeny,

    32P- >>35S-labeled phage

アニメ

Genetic material is DNA

NOT Protein

http://www.nature.com/scitable/topicpage/Isolating-Hereditary-Material-Frederick-Griffith-Oswald-Avery-336


DNA : genetic materials

  • Cellular genetic material

    • (RNA for RNA virus)

  • Genetic material = information in organism and be transferred to next generation

  • Vital and Essential for cell

“DNA carries the heritable information”


1953 : James Watson, Francis Crick, et al

ROSALIND FRANKLIN, UK…ONE DAY LATE

  • Building block of DNA;

    • Nitrogenous base

    • Sugar

    • Phosphate

  • X-ray diffraction : double helix

  • Purine must pair with Pyrimidine

    • Proportion of G = C

    • Proportion of A = T


  • 1958 Mathew Melselson & Franklin Stahl

    • Semiconservative replication of DNA

  • 1958 Paul C Zamecnik

    • Transfer RNA (tRNA)

  • 1961 Sidney Brenner, Francois Jacob, Mathew Melselson

    • Messenger RNA (mRNA)

  • 1966 Marshall Nirenberg

    • Finished unraveling the genetic code

  • 1977 Walter Gilbert & Frederick Sanger

    • DNA sequencing

  • GENOMICS ERA

    • Human genome projects (HGP)

    • Post-HGP

  • further study in “Application Mol Biol”


Central Dogma

Classical Central Dogma (Crick, Nature 1958) :

The “Central Dogma” deals with detailed residue by residue transfer of sequential information… such information cannot be transferred from protein to either proteins or nucleic acid

3 major molecules are DNA, RNA, and Protein

www.scfbio-iitd.res.in/tutorial/orf.html


Central Dogma : Eukaryote ≠ Prokaryote

GENOTYPE

PHENOTYPE


DNA Replication

DNA  DNA

Transcription

DNA  RNA

Translation

RNA  Protein

RNA Replication

RNA  RNA

Reverse Transcription

RNA  DNA

Self replication protein

Protein  Protein

Central Dogma

General

Special


Special Central Dogma

  • RNA Replication :

    primitive viruses (HepB, Dengue, flu, rabies)

    • RNA  RNA

  • Reverse Transcription :retroviruses (HIV)

    • RNA  DNA

  • Self Protein Replication :prions

    • Protein  Protein


General

Special

Special Central Dogma


Genetic code : code for functional molecule (protein)

  • Information or language of organisms.

  • A mapping between tri-nucleotide (triplets) sequences, calledcodons, and amino acids

  • Code is not universal.

  • Not all genetic information is stored using the genetic code. Some are regulatory sequences, intergenic segments, and chromosomal structural, epigenetics

Further study in gene expression


Terminology

  • Genome = total hereditary information in living organism

    • Genomes = gene (coding) + noncoding

    • Genomes = Nuclear DNA + mitochondrial DNA

    • Genomics ≠ Genetics (study of genes or gr. of genes

  • Gene = DNA, coding region

  • Chromosome = packing structure of genome in cell

    = genome + protein


genome

chromosome

gene

DNA

protein


References

- หัทยา กาวีวงศ์ อณูพันธุศาสตร์(2005) เชียงใหม่: ห้างหุ้นส่วนจำกัด บุญชัยการพิมพ์

- นเรศร สุขเจริญ, และคณะ อณูชีววิทยาทางการแพทย์ (2541)กรุงเทพฯ : บริษัท เท็กซ์ แอน เจอร์นัล พับลิเคชั่น จำกัด

- Watson, JD., etal.(1953) Molecular structure of Nucleic Acids, Nature, 171:737-8.

- Alberts, B., et al. The cell. (2002) New York and London: Garland Science

- Lewin,B. (2008) Gene IX, Jones and Barlett, London

- O’Connor,C. (2008) Isolating hereditary material: Frederick Griffith, Oswald Avery, Afred Hershey and Martha Chase. Nature Education1(1).

Animation

- http://www.nature.com/scitable/topicpage/Isolating-Hereditary-Material-Frederick-Griffith-Oswald-Avery-336

-http://highered.mcgraw-hill.com/sites/0072835125/student_view0/animations.html#

- http://www.genome.gov/Images/press_photos/highres/38-300.jpg


Chemical structure of nucleotide

  • Chemical structure of nucleotide/genome

  • Biomedical important

    • Drugs

    • Diseases

    • Treatments

    • Genetic engineering applications

FINAL examination


Topics

1. Structure and numbering: sugar, base, phosphate

2. Distinguish between purine, pyrimidine base, conformation

3. Nomenclature ofnucleoside, nucleotide, polynucleotide and nucleic acid

4. DNA andRNA: base paring, calculate

5. Biomedical important :

nucleotide analog drug : Chemotherapy, Antiviral agent

Diseases

Treatment

Genetic engineering applications


CH2

5’

S

3’

OH

X

5-membered ring sugar = pentose

O

1’

4’

numbering

2’

RIBOSE ; X = OH

DEOXYRIBOSE ; X = H


N

B

CH2

5’

S

3’

OH

X

Nucleoside = sugar + base

Structure & Nomenclature

B = nitrogenous base

[pyrimidine / purine]

OH

OH

O

beta N glycosidic linkage

1’

4’

2’

X = H; deoxyribose :: DNA

X = OH; ribose :: RNA


Nitrogenous base [pyrimidine / purine]

aromatic ring structure

absorb UV. light atmax = 260 nm

conc. of DNA or RNA a # nucleotide

http://66.102.7.104/search?q=cache:kz9t9IvdHnYJ:wine1.sb.fsu.edu/BCH4053/Lecture18/Lecture18.htm+lactam+ring+structure+DNA&hl=th


CH3

Pyrimidine

nucleoside

thymidine

Purine

nucleoside


Base conformation

(a) syn-adenosine (b) anti-adenosine


OH

Phosphoester bond

B

CH2

5’

S

O

P

O

O

3’

x

OH

Nucleotide = nucleoside + phosphate

OH

N

o

1’

4

2’

X = H; deoxyribose :: DNA

X = OH; ribose :: RNA

Phosphate bond; High energy bond


OH = nucleotide, RNA

H = deoxynucleotide, DNA

Nucleotide

  • anti-

  • purine/pyrimidine


Base formula

Base

(x=H)

Nucleoside (x=sugar)

Nucleotide

(x=sugar phosphate)

Cytosine

(C)

Cytidine

(C)

Cytidine monophosphate

(CMP)

Thymine

(T)

Thymidine

(T)

Thymidine monophosphate

(TMP)

x

Uracil

(U)

Uridine

(U)

Uridine monophosphate

(UMP)

x

x

Guanine

(G)

Guanosine

(G)

Guanosine monophosphate

(GMP)

x

Adenine

(A)

Adenosine

(A)

Adenosine monophosphate

(AMP)

x

Nomenclature

Pyrimidine

Purine


Nomenclature

NMP; Nucleosidemonophosphate

building blockfor RNA synthesis

dNMP; deoxy nucleosidemonophosphate

building block for DNA synthesis

N = A or G or T or U or C


5’

B

B

B

S

S

S

H

OH

O

O

H

H

P

P

P

O

O

O

O

O

O

O

O

O

CH2

CH2

CH2

5’

5’

5’

3’

3’

3’

3’

Polynucleotide

Nucleic acid

Phosphodiester bond

or

Phosphate linkage

RiboNucleic Acid ; RNA DeoxyriboNucleic Acid ; DNA


RNA

Monomer of Ribonucleotide : ATP,GTP,UTP,CTP

Mostly Single Strand RNA ; ds in some virus

Type of RNA ;

mRNA,tRNA,rRNA

snRNA (small nuclear RNA)

hnRNA= pre-mRNA

Heterogeneous nuclear RNA


Ribonucleotide monomer

NTP has a unique role as a high-energy molecule

ATP: "energy currency" for the cell

GTP: major energy source for protein synthesis

CTP: essential metabolite in phospholipid synthesis

UTP: used in formation of activated intermediates in carbohydrate biosynthesis


5’

3’

3’

5’

Complementary base A=T, C=G

DNA

Anti-parallel


Base pairing

Purine

Pyrimidine

Thymine /

Uracil

Adenine

Cytosine

Guanine

N

O

H

C

Hydrogen bond


Double strand DNA

5’

3’

A

T

T

C

C

G

A

G

C

G

T

A

C

G

T

A

A

C

T

G

G

C

T

A

A

G

G

C

T

C

G

C

A

T

G

C

T

T

G

A

C

C

G

3’

5’

Length =

21 base pairs ; 42 bases

Composition

A = T

G = C

A+G = T+C


Erwin Chargaff’s

Composition of DNA double strand

@ T = A and C = G ; A/T = C/G = 1

@ purine ( A + G ) = pyrimidine ( C + T )

@ A + T not equal to C + G


= 9

= 9

= 12

= 12

A

T

C

G

Problem

DNA = 21 base pairs = 42 bases

If………… G = 12 base, A,C,T=?

G = C = 12 bases ,

A+T = (42-24) = 18 bases

A = T = 9 bases


Clinical importance; anti-metabolite drugs

1. Chemotherapy / anti-cancer agents

2. Antiviral agents

PRINCIPLE: NUCLEOTIDE analog

- competitive with the real one

- interfere DNA/RNA synthesis


Nucleoside Analogues


2.2 Non-nucleoside inhibitors:

Foscarnet = pyrophosphate analogue

selectively inh viral DNA pol.& RT at the PP-binding site prevent removal of PO4-PO4HSV CMV – resist to acyclovir, HIV – inhibit RT

3. Block reverse transcriptase (RT)- for HIV, HBV

3.1 nucleoside analogues:

Azidothymidine (Zidovudine, retrovir, AZT)

dideoxythiacytidine =3TC (lamivudine)

Adefovir (nucleotide analoque)


Anti-metabolites

Folate antagonists

Purine nucleotide antagonists

Pyrimidine nucleotide antagonists


Mechanism ofPurine & Pyrimidine antagonists

Principle; similar structure to nucleotides

1. Inhibit production of nucleoside

2. Inhibit base incorporation in DNA synthesis

No DNA synthesis

No Cell proliferation

Antitumor / Antiviral


x

Structure of purine base & its antagonists

SH

x

x

Adenine

6-MP

fludarabine

SH

x

6-TG

Guanine


6-MP

6-TG

fludarabine

Anti-metabolites

Purine antagonists

Purine ; A, G

Purine nucleoside

Purine nucleotide

DNA synthesis

6-MP = 6-Mercaptopurine

6-TG = 6-Thioguanine


F

x

x

x

OH

Cytosine

Anti-metabolites

Pyrimidine antagonists

5-FU

T

U

Cladribine

Ara-C


2. Inhibit dTMP production

Thymidilate synthase

dTMP

DNA

dUMP

5-FU,

5-FdUMP

Mechanism of 5-FU

  • Inhibit RNA synthesis

  • - mRNA

  • - tRNA

  • - rRNA

No transcription

No translation


Chemotherapeutic agents

  • Agents that interfere with Nucleic acid synthesis

  • Antimetabolite [trimethoprim, sulphonamide]

  • Nucleotide analogue (inh viral replication)

    • AZT (azidothymidine=chemical name=zidovudine =generic name)

    • Ganciclovir

    • Acyclovir

    • Vidarabine

  • Purine antagonist [6-MP, 6-TG, Fudarabine phosphate, cladribine, pentostatin]

  • Pyrimidine antagonist [5-FU, ARA-C, azacitidine]


เอกสารอ้างอิง

  • นเรศร สุขเจริญ, อภิวัฒน์ มุทิรางกูร, ยง ภู่วรวรรณ อณูชีววิทยาทางการแพทย์ บ.เท็กซ์ แอนด์ เจอร์นัล พับลิเคชั่น จำกัด กรุงเทพฯ ๒๕๔๑(ISBN: 947-639-283-2)

  • มนตรี จุฬาวัฒนทล และคณะ. ชีวเคมี คณะวิทยาศาสตร์ มหาวิทยาลัยมหิดล ๒๕๔๒(ISBN: 974-86639-8-1)

  • AlbertB., Bray D, Lewis J., Raff M, Roberts K, Watson JD. Molecular biology of the cell, 3rd ed.Garland publishing Inc. NY (ISBN: 0-8153-1619-4)

  • Hames BD., Hooper NM., Houghton JD. Instant notes in biochemistry. BIOS Scientific publishers limited, 1997. University of Leeds, UK (ISBN: 981-3083-37-9)

  • http://66.102.7.104/search?q=cache:kz9t9IvdHnYJ:wine1.sb.fsu.edu/ BCH4053/Lecture18/Lecture18.htm+lactam+ring+structure+DNA&hl=th

  • (available at14Sept07)


OH

Cytosine

Arabynosylcytosine, Cytarabine, Ara-C

Inhibit DNA synthesis

Ara-C


cAMP, and cGMP are important regulators of cell metabolism and are found in virtually all cells


In the Lactam form, the ring nitrogens can serve as H-bond donors, and the keto oxygens serve as H-bond acceptors, when interacting with other molecules

Purines and pyrimidines can undergo a keto-enol tautomeric shift. The keto tautomer is called a lactamring, wheres the enol tautomer is a lactimring. The lactam (keto) tautomer is the predominant form at neutral pH.


Questions

1.The process by which RNA is made from DNA:1) synthesis2)translation3) transcription4) replication

3. Adenosine always pairs with………………..

5. The DNA molecule is held together by:……..hydrogen bonds

9. The sugar found in DNA is:…………..

13. RNA differs from DNA in that:1) it has a different kind of sugar2) it is single stranded3) it has uracil 4) all of these


  • Nucleic acids function in:

  • Immunity

  • Structural support

  • Chemical transmission

  • Hereditary information

  • The components of nucleic acids are:

  • A 6 carbon sugar, phosphate group, nitrogenous base

  • A 6 carbon sugar and various R groups

  • A 5 carbon sugar and a nitrogenous base

  • A 5 carbon sugar, phosphate group, nitrogenous base


WHAT’S FUNCTIONAL MOLECULE IN OUR BODY?

PROTEINs

CARBOHYDRATEs

LIPIDs


Central Dogma of Molecular biology

What’s a building block for DNA/RNA?


Energy storage ;

ATP, GTP, UTP, CTP

NAD+, NADP+, FAD and coenzyme A

Coenzymes ;

Second messengers;

cAMP,cGMP

Intermediate in biosynthesis;

glycogen,glycoprotein

Building block for

DNA, RNA

Functions of nucleotides ;


R

S

ΔS

ΔS+R


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