dr. nabil mtiraoui, m.sc, ph.d chemical basis of inheritance unit vi mlt, fams, tu
TRANSCRIPT
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.