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Sub topics :

Search for genetic material---nucleic acid or protein/DNA or RNA?Griffiths Transformation ExperimentAverys Transformation ExperimentHershey-Chase Bacteriophage ExperimentTobacco Mosaic Virus (TMV) ExperimentNucleotides - composition and structureDouble-helix model of DNA - Watson & CrickOrganization of DNA/RNA in chromosomes

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Search for the genetic material:

Stable source of informationAbility to replicate accuratelyCapable of change

Timeline of events:

1890Weismann - substance in the cell nuclei controls development.1900Chromosomes shown to contain hereditary information, later shown to be composed of protein & nucleic acids.1928Griffiths Transformation Experiment1944Averys Transformation Experiment1953Hershey-Chase Bacteriophage Experiment1953Watson & Crick propose double-helix model of DNA1956Gierer & Schramm/Fraenkel-Conrat & Singer

Demonstrate RNA is viral genetic material.

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Frederick Griffiths Transformation Experiment - 1928

transforming principle demonstrated with Streptococcus pneumoniae

Griffith hypothesized that the transforming agent was a IIIS protein.

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Oswald T. Averys Transformation Experiment - 1944

Determined that IIIS DNA was the genetic material responsible for Griffiths results (not RNA).

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Bacteriophage = Virus that attacks bacteria and replicates by invading a living cell and using the cells molecular machinery.

Structure of T2 phage

DNA & protein

Hershey-Chase Bacteriophage Experiment - 1953

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Life cycle of virulent T2 phage:

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T2 bacteriophage is composed of DNA and proteins:Set-up two replicates:Label DNA with 32PLabel Protein with 35S

3.Infected E. coli bacteria with two types of labeled T2

4.32P is discovered within the bacteria and progeny phages, whereas 35S is not found within the bacteria but released with phage ghosts.

Hershey-Chase Bacteriophage Experiment - 1953

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Using P32

Is DNA the genetic material?

When centrifuged, phage protein coats remain in the supernatant while bacteria form a pellet

The pellet is radioactive, but the supernatant is not.

Did DNA enter the bacteria?

Bacteria grown in normal non-radioactive media

T2 grown in P32 containing media incorporate P32 into their DNA

Blending causes phage protein coat to fall off

T2 attach to bacteria and inject genetic material

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Using S35

Is protein the genetic material?

When centrifuged, phage protein coats remain in the supernatant while bacteria form a pellet

The supernatant is radioactive, but the pellet is not.

Did protein enter the bacteria?

Bacteria grown in normal non-radioactive media

T2 grown in S35 containing media incorporate S35 into their proteins

Blending causes phage protein coat to fall off

T2 attach to bacteria and inject genetic material

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Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment - 1956

Fraenkel-Conrat & Singer - 1957

Used 2 viral strains to demonstrate RNA is the genetic material of TMV

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Conclusions about these early experiments:

Griffith 1928 & Avery 1944:

DNA (not RNA) is transforming agent.

Hershey-Chase 1953:

DNA (not protein) is the genetic material.

Gierer & Schramm 1956/Fraenkel-Conrat & Singer 1957:

RNA (not protein) is genetic material of some viruses.

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STORES AND PASSES ON GENETIC INFORMATION FROM ONE GENERATION TO ANOTHER .

DNA :

DEOXYRIBONUCLEIC ACID

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Nucleotide = monomers that make up DNA and RNA (Fig.8)

Three components

1. Pentose (5-carbon) sugar

DNA = deoxyribose

RNA = ribose

(compare 2 carbons)

2. Nitrogenous base

Purines

Adenine

Guanine

Pyrimidines

Cytosine

Thymine (DNA)

Uracil (RNA)

3. Phosphate group attached to 5 carbon

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Nucleotides are linked by phosphodiester bonds to form polynucleotides.

Covalent bond between the phosphate group (attached to 5 carbon) of one nucleotide and the 3 carbon of the sugar of another nucleotide.

This bond is very strong, and for this reason DNA is remarkably stable. DNA can be boiled and even autoclaved without degrading!

The ends of the DNA or RNA chain are not the same. One end of the chain has a 5 carbon and the other end has a 3 carbon.

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A Nucleotide
Adenosine Mono Phosphate (AMP)

Nucleotide

Nucleoside

OH

O

CH2

Sugar

H

H

H

OH

N

N

N

N

NH2

Base

P

O

OH

HO

O

Phosphate

2

3

4

5

1

H+

-

Pyrimidines

Purines

N

N

NH

N

NH2

O

Guanine

N

N

Adenine

N

N

NH2

O

O

N

N

NH2

N

NH2

Cytosine

O

O

NH

O

O

NH

Uracil

(RNA)

CH3

N

N

N

N

Thymine

(DNA)

-

+

+

+

-

-

Base Pairing
Guanine And Cytosine

O

O

N

N

H

N

N

H

Cytosine

N

N

N

N

H

O

N

H

H

Guanine

Base Pairing
Adenine And Thymine

O

O

N

CH3

N

N

H

+

-

Thymine

N

N

N

N

H

N

H

-

+

Adenine

Base Pairing
Adenine And Cytosine

O

O

N

N

H

N

N

H

Cytosine

-

+

-

N

N

N

N

H

N

H

-

+

Adenine

Base Pairing
Guanine And Thymine

O

O

N

CH3

N

N

H

+

-

Thymine

N

N

N

N

H

O

N

H

H

Guanine

+

+

-

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5 end

3 end

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James D. Watson & Francis H. Crick - 1953

Double Helix Model of DNA

Two sources of information:

Base composition studies of Erwin Chargaffindicated double-stranded DNA consists of ~50% purines (A,G) and ~50% pyrimidines (T, C)amount of A = amount of T and amount of G = amount of C (Chargraffs rules)%GC content varies from organism to organism

Examples:%A%T%G%C%GC

Homo sapiens31.031.519.118.437.5

Zea mays25.625.324.524.649.1

Drosophila27.327.622.522.545.0

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James D. Watson & Francis H. Crick - 1953

Double Helix Model of DNA

X-ray diffraction studies - Rosalind Franklin & Maurice Wilkins

Conclusion :

DNA is a helical structure with

distinctive regularities, 0.34 nm & 3.4 nm.

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Double Helix Model of DNA: Six main features

Two polynucleotide chains wound in a right-handed (clockwise) double-helix.Nucleotide chains are anti-parallel: 5 3

3 5

Sugar-phosphate backbones are on the outside of the double helix, and the bases are oriented towards the central axis.Complementary base pairs from opposite strands are bound together by weak hydrogen bonds.

A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds).

e.g.,5-TATTCCGA-3

3-ATAAGGCT-5

Base pairs are 0.34 nm apart. One complete turn of the helix requires 3.4 nm (10 bases/turn).Sugar-phosphate backbones are not equally-spaced, resulting in major and minor grooves.

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Denaturation and Renaturation

Heating double stranded DNA can overcome the hydrogen bonds holding it together and cause the strands to separate resulting in denaturation of the DNAWhen cooled relatively weak hydrogen bonds between bases can reform and the DNA renatures

TACTCGACATGCTAGCAC

ATGAGCTGTACGATCGTG

Double stranded DNA

TACTCGACATGCTAGCAC

ATGAGCTGTACGATCGTG

Double stranded DNA

Renaturation

TACTCGACATGCTAGCAC

ATGAGCTGTACGATCGTG

Denatured DNA

Denaturation

Single stranded DNA

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Denaturation and Renaturation

DNA with a high guanine and cytosine content has relatively more hydrogen bonds between strandsThis is because for every GC base pair 3 hydrogen bonds are made while for AT base pairs only 2 bonds are made Thus higher GC content is reflected in higher melting or denaturation temperature

Intermediate melting temperature

Low melting temperature

High melting temperature

TGCTCGACGTGCTCG

ACGAGCTGCACGAGC

67 % GC content -

TACTAGACATTCTAG

ATGATCTGTAAGATC

33 % GC content -

TACTCGACAGGCTAG

ATGAGCTGTCCGATC

50 % GC content -

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Yeast Alanine tRNA

RNA (A pairs with U and C pairs with G)

Examples:

mRNAmessenger RNA

tRNAtransfer RNA

rRNAribosomal RNA

snRNAsmall nuclear RNA

RNA secondary structure:

single-stranded

Function in transcription

(RNA processing) and translation

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Organization of DNA/RNA in chromosomes

Genome = chromosome or set of chromosomes that contains all the

DNA an organism (or organelle) possesses

Viral chromosomes1. single or double-stranded DNA or RNA

2. circular or linear

3. surrounded by proteins

TMV T2 bacteriophage bacteriophage

Prokaryotic chromosomes

1. most contain one double-stranded circular

DNA chromosome

2. others consist of one or more chromosomes

and are either circular or linear

3. typically arranged in arranged in a dense

clump in a region called the nucleoid

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Problem:

Measured linearly, the Escherichia coli genome (4.6 Mb) would be 1,000

times longer than the E. coli cell.

The human genome (3.4 Gb) would be 2.3 m long if stretched linearly.

Solutions:

Supercoiling DNA double helix is twisted in space about its

own axis, a process is controlled by

topoisomerases (enzymes).

(occurs in circular and linear DNA molecules)

2.Looped domains

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More about genome size:

C value=total amount of DNA in the haploid (1N) genome

Varies widely from species to species and shows no relationship to structural or organizational complexity.

ExamplesC value (bp)

48,502

T4 168,900

HIV-1 9,750

E. Coli 4,639,221

Lilium formosanum 36,000,000,000

Zea mays 5,000,000,000

Amoeba proteus 290,000,000,000

Drosophila melanogaster 180,000,000

Mus musculus 3,454,200,000

Canis familiaris 3,355,500,000

Equus caballus 3,311,000,000

Homo sapiens 3,400,000,000

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Eukaryotic chromosome structure

Chromatincomplex of DNA and chomosomal proteins

~ twice as much protein as DNA

Two major types of proteins:

Histonesabundant, basic proteins with a positive charge

that bind to DNA

5 main types: H1, H2A, H2B, H3, H4

~equal in mass to DNA

evolutionarily conserved

Non-histonesall the other proteins associated with DNA

differ markedly in type and structure

amounts vary widely

>> 100% DNA mass

Packing of DNA into chromosomes:

Level 1Winding of DNA around histones to create a nucleosome structure.Level 2Nucleosomes connected by

strands of linker DNA like

beads on a string.

Level 3Packaging of nucleosomes into

30-nm chromatin fiber.

Level 4Formation of looped domains.

Figs. 2.25-29

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The Central Dogma
of Molecular Biology

Cell

1998 Timothy G. Standish

DNA

mRNA

Transcription

Polypeptide

(protein)

Translation

Ribosome

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More about different types of DNA you should know about:

Centromeric DNA (CEN)Center of chromosome, specialized sequences function with the microtubles and spindle apparatus during mitosis/meiosis.Telomeric DNAAt extreme ends of the chromosome, maintain stability, and consist of tandem repeats. Play a role in DNA replication and stability of DNA.Unique-sequence DNAOften referred to as single-copy and usually code for genes.Repetitive-sequence DNAMay be interspersed or clustered and vary in size.

SINEsshort interspersed repeated sequences (100-500 bp)

LINEslong interspersed repeated sequences (>5,000 bp)

Microsatellites short tandem repeats (e.g., TTA|TTA|TTA)

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Chromosomes vs Genes

A chromosome constitutes an entire DNA molecule + proteinProtein = histonesSupercoiled DNA in nucleosomesHumans contain 46 such molecules (23 pairs)44 somatic chromosomes2 sex chromosomes (X +Y)

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Chromosomes vs Genes

Genes constitute distinct regions on the chromosomeEach gene codes for a protein productDNA -> RNA-> proteinDifferences in proteins brings about differences between individuals and species

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How do chromosomes become double stranded?

During the life of the cell, each chromosome of DNA makes a copy of itselfThis must occur prior to cell division to insure each daughter cell gets a complete set

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Extrachromosomal DNA

is DNA located or maintained in a cell apart from the chromosomes.

Outside the nucleus, DNA is found in cytoplasmic organelles, such as mitochondria (both plants and animals) and in chloroplast (found only in green plants and photosynthetic protists).

The genes in mitochondria (mt) and chloroplast (cp) are also known as Extrachromosomal genes, non Mendelian genes, cytoplasmic genes, organellar genes or extranuclear genes.

DNA can also be found in the plasmids of bacteria.

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TUGAS : PRESENTASI

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