Dr. Nabil MTIRAOUI, M.Sc, Ph.D

CHEMICAL BASIS OF INHERITANCE

Unit VI

MLT, FAMS, TU

Molecular Genetics

The branch of genetics that deals with hereditary transmission and variation on the molecular level.

Deals with the expression of genes by studying the DNA sequences of chromosomes

The study of the molecular structure of genes, involving DNA and RNA.

DNA Structure & PropertiesLecture 11

I. DNA’s Discovery & Structure

What is a Genetic “Factor”?

From Mendel: we now accepted that there was genetic

transmission of traits. Traits are transmitted by “factors”

Organisms carry 2 copies of each “factor” The question now was: what is the factor that

carries the genetic information?

Requirements of Genetic Material

Must be able to replicate, so it is reproduced in each cell of a growing organism.

Must be able to control expression of traits Traits are determined by the proteins that act within us Proteins are determined by their sequences

Therefore, the genetic material must be able to encode the sequence of proteins

It must be able to change in a controlled way, to allow variation, adaptation, thus survival in a changing environment.

Chromosomes – The First Clue

First ability to visualize chromosomes in the nucleus came at the turn of the century construction of increasingly powerful microscopes the discovery of dyes that selectively colored

various components of the cell Scientists examined cellular nuclei and

observed nuclear structures, which they called chromosomes

Observation of these structures suggested their role in genetic transmission

Implications

Chromosomes behaved like Mendel’s “factors” Mendel's hereditary factors were either located on

the chrs or were the chromosomes themselves. Proof chromosomes were hereditary factors –

1905: The first physical trait was linked to the presence

of specific chromosomal material conversely, the absence of that chromosome meant

the absence of the particular physical trait. Discovery of the sex chromosomes

"X" and "Y." distinguished from other chromosomes and from

each other

What Carries the Genetic Information?

Chromosomes are about 40% DNA & 60% protein. Protein is the larger component

Protein molecules are composed of 20 different subunits

DNA molecules are composed of only four Therefore protein molecules could encode more

information, and a greater variety of information protein had the possibility for much more diversity than

in DNA Therefore, scientists believed that the protein in

chromosomes must carry the genetic information

1. A History of DNA

F. Griffiths (1928)

Tried to determine what genetic material was made of.

The Transforming Principle

Fredrick Griffith - 1928 Discovered that different strains of the bacterium

Strepotococcus pneumonae had different effects on mice One strain could kill an injected mouse (virulent) Another strain had no effect (avirulent) When the virulent strain was heat-killed and injected into

mice, there was no effect. But when a heat-killed virulent strain was co-injected with

the avirulent strain, the mice died. Concluded that some factor in the heat killed bacteria

was transforming the living avirulent to virulent? What was the transforming principle and was this the

genetic material?

Griffiths’ Experiment

Griffiths’ ExperimentPneumococcus bacteria on mice

2 STRAINS

S-typeSmooth colonies

Virulent

R-typeRough colonies

Avirulent

Innoculate into mice Innoculate into mice

Dead from pneumonia

Not killed

Avery, MacCleod & McCarthy (1944)

Tried purifying the transforming principle to change R-type Pneumococcus to S-type

The Transforming Principle is DNA

Avery, Macleod, & McCarty – 1943 Attempted to identify Griffith’s “transforming

principle” Separated the dead virulent cells into fractions

The protein fraction DNA fraction

Co-injected them with the avirulent strain. When co-injected with protein fraction, the mice lived with the DNA fraction, the mice died

Result was IGNORED Most scientists believed protein was the genetic material. They discounted this result and said that there must have

been some protein in the fraction that conferred virulence.

The Hershey-Chase Experiment

Hershey & Chase – 1952 Performed the definitive

experiment that showed that DNA was the genetic material.

Bacteriphages = viruses that infect bacteria

Bacteriphage is composed only of protein & DNA

Inject their genetic material into the host

The Hershey-Chase Experiment

The Experiment

Prepared 2 cultures of bacteriophages Radiolabeled sulphur in one culture

there is sulphur in proteins, in the amino acids methionine and cysteine

there is no sulphur in DNA Radiolabeled phosphorous in the second culture

there is phosphorous in the phosphate backbone of DNA none in any of the amino acids.

So this one culture in which only the phage protein was labeled, and one culture in which only the phage DNA was labeled.

Experiment Summary Performed side by side experiments with

separate phage cultures in which either the protein capsule was labeled with radioactive sulfur or the DNA core was labeled with radioactive phosphorus.

The radioactively labeled phages were allowed to infect bacteria.

Agitation in a blender dislodged phage particles from bacterial cells.

Centrifugation pelleted cells, separating them from the phage particles left in the supernatant.

Results Summary

Radioactive sulfur was found predominantly in the supernatant

Radioactive phosphorus was found predominantly in the cell fraction, from which a new generation of infective phage was generated.

Thus, it was shown that the genetic material that encoded the growth of a new generation of phage was in the phosphorous-containing DNA.

Chargaff’s Rule

Chargaff’s rule is a rule about DNA,

Chargaff’s Rule

Once DNA was recognized as the genetic material, scientists began investigating its mechanism and structure.

Erwin Chargaff – 1950 discovered the % content of the 4 nucleotides was the

same in all tissues of the same species percentages could vary from species to species.

He also found that in all animals (Chargaff’s rule): %G = %C

%A = %T This suggested that the structure of the DNA was

specific and conserved in each organism. The significance of these results was initially

overlooked

Watson and Crick shared the 1962 Nobel Prize for Physiology and Medicine with Maurice Wilkins. Rosalind Franklin died before this date.

The Double Helix: Watson & Crick

The Double Helix: Watson & Crick

James Watson and Francis Crick – 1953 Presented a model of the structure of DNA. It was already known from chemical studies that

DNA was a polymer of nucleotide (sugar, base and phosphate) units.

X-ray crystallographic data obtained by Rosalind Franklin, combined with the previous results from Chargaff and others, were fitted together by Watson and Crick into the double helix model.

Two types of nucleic acid can be recognized:

deoxyribonucleic acid (DNA) and ribonucleic acid

(RNA).

DNA is mostly found in the nucleus where it forms the

principal substance of the chromosomal material, the

chromatin. In addition to DNA, chromatin contains

proteins, mainly histones, and little RNA.

2. Chemical Bases in DNA

In prokaryotes, DNA is present in a single

chromosome in the nucleoid.

Little DNA is also found in mitochondria and in

chloroplasts.

Many viruses are made up of DNA, mostly double

stranded, but some are single stranded.

2. Chemical Bases in DNA

3. Primary Structure: Nucleotide & Nucleoside

The addition of a pentose sugar to a base produces a nucleoside .

If the sugar is ribose, a ribonucleoside is produced; if the sugar is 2-deoxyribose, a deoxyribonucleoside is produced

Addition of phosphate group to nucleoside produces nucleoside mono-phosphate (NMP) like AMP or CMP or a nucleotide

3. Primary Structure: Mononucleotide

PURINESPURINES1. Adenine (A)Adenine (A)

2. Guanine (G)Guanine (G)

PYRIMIDINESPYRIMIDINES3. Thymine (T)Thymine (T)

4. Cytosine (C)Cytosine (C) T or C

3. Primary Structure: Nitrogenous Bases

A or G

3. Primary Structure: Dinucleotide

3. Primary Structure: Polynucleotide

3’-End

1’

OI

O=P-O-CH2IO-

O

2’3’4’

5’

1’

OI

O=P-O-CH2IO-

O

2’3’4’

5’ H

N

N

NN

N

HH

HH

N

N

NN

N

HH

H

1’

OI

O=P-O-CH2IO-

O

OH

2’3’4’

5’

1’

OI

O=P-O-CH2IO-

O

OH

2’3’4’

5’

NH2

N

N

OH

H

NH2

N

N

OH

H

NH2

N

N

OH

H

NH2

N

N

OH

H

1’

OI

O=P-O-CH2IO-

O

2’3’4’

5’

1’

OI

O=P-O-CH2IO-

O

2’3’4’

5’

NH2

HN

N

NO

N NH2

HN

N

NO

N

H

NH2

HN

N

NO

N NH2

HN

N

NO

N

HH

N OH

HO

N

N

OH

H3C

N OH

HO

N

N

OH

H3C

1’

OI

O=P-O-CH2IO-

O

2’3’4’

5’

1’

OI

O=P-O-CH2IO-

O

2’3’4’

5’ N OH

HO

N

N

OH

H3C

N OH

HO

N

N

OH

H3C

1’

OI

O=P-O-CH2IO-

O

2’3’4’

5’

1’

OI

O=P-O-CH2IO-

O

2’3’4’

5’ N OH

HO

N

N

OH

H3C

N OH

HO

N

N

OH

H3C

1’

OI

O=P-O-CH2IO-

O

2’3’4’

5’

1’

OI

O=P-O-CH2IO-

O

2’3’4’

5’

5’-EndThymine

Adenine

Cytosine

Guanine

3’ 5’ Phosphodiester bond

A

3. Primary Structure: Polynucleotide

4. Secondary structure: double helical structure

The 2 strands are twisted about each other, coiled around a common axis, forming a right- handed double helix.

The hydrophilic sugar- phosphate backbone of each chain lies on the outside of the molecule. The hydrophobic nitrogenous bases project inwards from the outer sugar-phosphate framework, perpendicular to the long axis of the helix and are stacked one above the other. The stacking of bases is held by hydrophobic bonds .This helps in holding the helical structure.

The nitrogenous bases of the 2 strands meet each other near the central axis of the helix where they become connected by hydrogen bonds between the amino, or imino, hydrogen and the ketonic oxygen atoms. The hydrogen bonding between the bases helps to hold the 2 strands of the DNA together.

GuanineThymine Adenine Cytosine

4. Secondary structure: double helical structure

A nitrogen-containing ring structure called a base. The base is attached to the 1' carbon atom of the pentose. In DNA, four different bases are found:

two purines, called adenine (A) and guanine (G)

two pyrimidines, called thymine (T) and cytosine (C)

*A always pairs with T : two hydrogen bonds

*C always pairs with G : three hydrogen bonds

4. Secondary Structure: Chargaff’s Rule

The 2 strands of the double helical molecule are antiparallel, i.e., they run in opposite direction; one runs in the 5’ to 3’ direction, while the other runs in the 3’ to 5’ direction.

4. Secondary Structure: Direction of Strands

B conformation (B-DNA): The most common form of DNA. The minor groove and major groove,

are of different widths on the outside of DNA.

A-DNA: Forms under conditions of low salt and

low humidity. There can be transient shifts from B to A

form.

Z-DNA: Consists of alternating purines and

pyrimidines Found infrequently. Z-DNA is:

long and thin Left-handed, Phosphate backbone has a zig-zag

appearance.

5. DNA Conformations

6. Key Features of a DNA molecule

7. Key Features of a RNA molecule

DNA is the carrier

of genetic

information, which

is stored in the

form of a nucleotide

sequence. DNA has

2 important

functions:

“replication” and

“transcription”.

Replication

DNA

Transcription

RNA

Translation

Protein

8. Biochemistry of DNA

9. What is Gene ? The gene, the basic

units of inheritance; it is a segment within a very long strand of DNA with specific instruction for the production of one specific protein. Genes located on chromosome on it's place or locus.

9. What is Gene ?

A gene in relation to the double helix structure of DNA and to a chromosome (right). Introns are regions often found in eukaryote genes that are removed in the splicing process (after the DNA is transcribed into RNA): only the exons encode the protein. This diagram labels a region of only 40 or so bases as a gene. In reality most genes are hundreds of times larger.