LIF 101 Topic (1)

Laws of inheritance Part I

02 Feb 2022
 A short Background: Historical ideas
about heredity/inheritance

Preformation Blending inheritance

N. Hartsoecker in 1695 19th Century
First scientific theory explaining
heredity – Mendel’s laws
What happened when you toss a
coin with head as P & tail as p
 Gregor Johann Mendel (1822–1884)
Priest in Augustinian Monastery in Brno,
Czechoslovakia
• His work with peas laid the foundation
for genetics.   He was a University
educated priest He studied math and
science.  He returned to the monastery
and taught high school. He took care of
the monastery’s garden.

• He was curious why some plants were

like their parents & others were not. He
cross pollinated the pea plants by
brushing pollen from the stamens of one
plant to the pistil of another.

• Peas normally self pollinate so he had

to take the stamens that make pollen off
About Mendel the second plant.
 Flower:
Plant organ for sexual
reproduction, that produces
gametes (sex cells) and are
necessary for fertilization to
occur
Note that there
are specialized
organs for
reproduction
 First design an experiment
What is true
breeding?

Does purple
flowered
progeny mean
Filial blending of
inheritance?

Does this
results
discard
blending of
inheritance?
What are P,
F1 and F2
 Second : Make assumptions based on the observations
§Heritable features are characters
(coloration) and their variations are
traits (purple or white).

§Traits are determined by factors. ‘P’

for purple, ‘p’ for white Let’s assume
traits are always in pairs: PP, Pp or pp

§ When two factors are present in the

same individual one is seen physically
– this is thus dominant (purple) while
the other one (white) is recessive

• Characteristics “seen” in an individual

constitutes its phenotype (appearance).
Characteristic “present” in an individual
cter, constitutes its genotype.
Note the terms: chara
nt,
trait, factors, domina
recessive, phenotype,
genotype. • Let’s now see if these assumptions
terms
Can you define these help explain the results
now?
Third : Explain the results based on the observations &
assumptions made

Explanation based on first laws of

probability:

The first Law of probability states that the

results of one chance event have no effect
on the results of subsequent (or another)
chance event. Thus, the probability of
obtaining heads the second time you flip it
remains at ½.

The second law of probability states that

the chances of two independent events
white happening together is the product of their
individual probability
Punnett square
 Third step contd:
Interpret results
Interpretations are based on the
assumptions made : factors
represent traits, there are two
factors for each trait in each plant
– one maternally derived and the
other paternally derived

Starting with PP and pp plants in the

second generation (F2) we will end up
with 3: 1 phenotypic ratio when the
factors (P, p) segregate from each
other during gamete formation

Note this term segregation:

Questions: What segregates? When
?
What is the evidence of their
segregation?
Genotypic and phenotypic ratios in F2 generations
Let’s recall Mendel’s of law of segregation

1. Allele (factor) pairs segregate

(separate) during gamete formation,
and randomly unite at fertilization .
Let’s consider inheritance of two different pairs of fact
What Which one is
does R Character : texture of the seeds dominant?
and r Traits smooth (R ) and rough ( r)
mean?

Character : color of the seeds

Traits yellow (Y) and green (y)

If a plant is true breeding smooth and yellow –

how can we represent its genotype?

RRYY
If a plant is true breeding rough and green – how can we
represent its genotype?

rryy
7/22
 What will be the consequence if a true
breeding smooth and yellow plant is
crossed with another true breeding plant
which is rough and yellow?

P1 RRYY X rryy

8/22
 First,
find the
types of
gamete
s that
are
possible

Second,
speculate
the
outcome if
factors of
original
parent
segregate
together
9/22

Notice, however, that These results demands explanation

this inheritance follows based on an another law of
the law of segregation inheritance: namely, the law of
independent assortment
 Law of Independent assortment
(This law tells how different Mendelian factors are
distributed following segregation)

The Law of Independent Assortment

states that alleles of different genes
assort independently of one another
during gamete formation.

Alleles: each of two or more alternative

forms of a gene that arise by mutation
 Your homework:
P1 Explain the
applications of
the first and
second laws of
F1 probability during
the formation of
the gametes of
F2
the F1 parent

The dihybrid ratio

 All wild type
F1

X =

Wild type
vestigial

female gamete/egg
Male gamete/sperm
Vg/Vg vg/vg
Vg/vg Vg/vg

white Vg/vg Vg/vg

curled
 F1
Wild type

Heterozygous
Vg/vg Vg/vg

Vg vg

Vg Vg/vg
Vg/Vg

vg Vg/vg vg/vg
Q1

In Drosophila, vestigial (partially formed) wings (vg) are recessive to normal Long wing (vg
+ ), and the gene for this trait is autosomal. Similarly, the gene for white (mutant) eye (w) is
recessive to red (normal) eye (w + ), and the gene for this trait is on the X chromosome.
(Marks: 11) a) Suppose a homozygous white eye, long winged female fly is crossed with a
homozygous redeyed vestigial-winged male. Answer the following:

(Part a: 1+2+2=5)

I) Gametes of the parental (P1) generations:

Male__________________ Female________________

II) Draw a Punnett square to derive the genotypes and phenotypes of F1 progeny:

III) Summarize F1 progeny phenotypes you have now derived

Male: _________________________________,
Female:________________________________

rrvv x RRVV

RrVv
Q2

In Mendel's monohybrid cross of purple-flowered and white-flowered

peas, all members of the F1 generation were of the ____ phenotype
because their genotype was ____ at the flower-color locus.

a.white-flowered ... homozygous recessive

b.white-flowered ... heterozygous
c.purple-flowered ... homozygous recessive
d.purple-flowered ... homozygous dominant
e.purple-flowered ... Heterozygous

Q3

F1 offspring of the monohybrid cross AA X aa are

• a. All AA
• b. All Aa
• c. All aa
• d. ½ AA and ½ aa
Q4

Which of the following statements is not one of Mendel’s

tenets?

a. There are discrete units of inheritance called factors

B. Organisms inherit two factors, one from each parent.

C. Different factors assort independently into gametes.

D. Characteristics of the parents blend together in their

offspring.
 Slide series 2
LIF101AA
Laws of Inheritance Part
II
04_02_22 FRIDAY

1/21
 RECAP Assumption
:
What is
assumed here? For each
trait there is
Law of segregation one pair of
alleles

Alleles (factors) representing a given trait

segregate (separate) during gamete formation,
and randomly unite at fertilization.

An interpretation based on
How this first & second law of
inference was probability:
drawn? Each gamete receive only
one of the two alleles
(factors)

How to read ? Example

http://en.wikipedia.org/wiki/Mendelian_inheritance 2/21
 RECAP

First law
of
First law of probability
probability

Second law Second law

of of
probability probability

3/21
The First Law of Probability states that the results of one chance event have no effect
on the results of subsequent chance events.
Second Law of Probability, which states that the probability of independent chance
events occurring together is the product of the probabilities of the separate events.
This is also called a
monohybrid cross.
Why??

RECAP

4/21
 RECAP

Test cross

Purple X White
Pp pp

Explain the
design/purpos
e of this cross
??
What does this
cross reveal?

5/21
 Your home
work
§ Explain why did Mendel took two
contrasting factors to demonstrate this rule of
inheritance, namely, the law of segregation?

§ What will be the consequence if these

factors do not segregate over succeeding
generations ?

§ What is the modern term for Mendelian

factors?

§ Why should these factors be in pairs at all?

6/21
Let’s consider inheritance of two different pairs of fact
What Which one is
does R Character : texture of the seeds dominant?
and r Traits smooth (R ) and rough ( r)
mean?

Character : color of the seeds

Traits yellow (Y) and green (y)

If a plant is true breeding smooth and yellow –

how can we represent its genotype?

RRYY
If a plant is true breeding rough and green – how can we
represent its genotype?

rryy
7/21
 What will be the
consequence if a true
breeding smooth and yellow
plant is crossed with another
true breeding plant which is
rough and yellow?
P1 RRYY X rryy

8/21
 First, 9/21
find the
types of
gamete
s that
are
possible

Second,
speculate
the
outcome if
factors of
original
parent
segregate
together

Notice, however, that

this inheritance follows These results demands explanation based on
the law of segregation an another law of inheritance: namely, the
law of independent assortment
 Law of Independent assortment
(This law tells how different Mendelian factors are
distributed following segregation)

The Law of Independent Assortment

states that alleles of different genes
assort independently of one another
during gamete formation.

10/21
 Your homework:
P1 Explain the
applications of
the first and
second laws of
F1 probability during
the formation of
the gametes of
F2
the F1 parent

The dihybrid ratio

11/21
What is the consequence of the operation
of law of independent assortment in our
own inheritance ?

12/21
 New Questions

§ How many factors are there in a pea plant?

§ Are these factors real?

§ Where are they located ?

13/21
 New Topic
Chromosomal basis of Mendelian inheritance
Mendel explained inheritance in pea in 1865 – it was not called a law in
those days – it was hardly noticed. The physical basis of Mendelian
inheritance became evident when chromosomes were discovered and
their transmission in somatic cells (by Mitosis) and Gametes (Meiosis) was
understood in the early 1900s
Summary of our current
understanding

14/21
1. How many sets of chromosome do we carry? 23

2. How many versions (alleles) of a gene can we carry if every

chromosome is in pair?

3. What happens during the gamete formation (meiosis) ? Do

the gene pairs stay together?

4. What happens then to the paternal and maternal sets of

genes that we inherited ?

15/21
§ Use the following words to complete the sentence below
allele(s), diploid, gamete(s), gene, haploid

A ______organism
haploid contains two _____
gene of each ______
allele in all
cells except its _________which
gamete are _________
dipoid and contain
only one.

16/21
§ Use the following words to complete the sentence below.
dominant, homozygous, non-identical, heterozygous, identical

All non-reproductive cells contain two alleles of each gene. When

those alleles are ____________, the cell is said to be
_____________. When those alleles are _____________,
the cell is said to be _______________, in which case the cell’s (or
organism’s) phenotype is dictated by the _____________ allele.

17/21
§ The tyrosinase gene, TYR, is necessary for production of
the skin pigment melanin in humans. A recessive allele,
tyr, of this gene causes the most frequent form of
albinism. Homozygotes (tyr / tyr) do not produce
melanin, while heterozygotes (TYR / tyr) are unaffected.
What is the probability that a child born to a couple,
both heterozygous for (TYR/tyr) will be albino?

§ Your answer should be based on use of the correct

symbols

§ Define the terms used in the above question

18/21
1. One gene has alleles A and a. another has allele B and b.
For each genotype, what type(s) of gametes will form?
Assume that independent assortment occurs.
2. a. AABB
b. AaBB
Which one of the
c. Aabb
Which one of parental genotypes
d. AaBb is a product of a
the parents is
not a hybrid? monohybrid cross?

Parental Gamete types

genotype formed
Which set of
AABB AB gametes display
AaBB AB (1/2) & aB(1/2) evidence of
independent
Aabb Ab (1/2), ab(1/2) assortment?
AaBb AB (1/4), Ab (1/4) Explain why
aB (1/4), ab (1/4) there are four
types of
gametes
19/21
 Home work

2. What will be the genotypes of offspring from the following

matings? Indicate the frequencies of each genotype among

a. AABB x aaBB
b. AaBB X AABb
c. AaBb X aabb
d. AaBb X AaBb

Step 1: In each case, first identify the types gametes formed by each parent

Step 2: Place these gametes at the designated position in a Punnett square

Step 3. Display the genotypes of the products of fusion of these various gametes
in the Punnett square

Step 4. Describe the phenotypes of the genotype of the progeny and their ratios
20/21
 Next class

Chromosomal basis of Mendelian inheritance:

Meiosis – production of gametes

Somatic cell division – Mitosis

Applications of Mendelian inheritance

Also come prepared with the reading materials I

would provide in advance

21/21
 Does all these Mendelian understanding make
sense to our life?

Why marriage
amongst near relative
is not a good idea?

What is inbreeding?
What is
consanguineous
marriage

What is inbreeding?

Extra slides
 Can you inherit genes that may give rise to a disease later
on?
If you have inherited a mutated copy of either gene from a parent, you have a
higher risk of breast cancer. On average, a woman with a BRCA1 or BRCA2 gene
mutation has up to a 7 in 10 chance of getting breast cancer by age 80. This risk is
also affected by how many other family members have had breast cancer

Breast Cancer Gene Testing Kits

The first FDA-approved breast cancer gene The test kit is supplied by 23andMe, a
company that is popular for producing reports that identify a person’s ancestry and
countries of origin

Extra slides
 Extra slides

Cancer prevention treatment

On February 16, 2013, the 37-year-old Jolie underwent a preventive double
mastectomy after learning she had an 87 percent risk of developing breast cancer due to a
defective BRCA1 gene. 

Her maternal family history warranted genetic testing for BRCA mutations: her mother,
actress Marcheline Bertrand, had breast cancer and died of ovarian cancer, while her
grandmother died of ovarian cancer. Her aunt, who had the same BRCA1 defect, died of
breast cancer three months after Jolie's operation. The mastectomy, which lowered her
chances of developing breast cancer to under five percent.

Two years later, in March 2015, after annual test results indicated possible signs of early
ovarian cancer, she underwent a preventive removal of an ovary and its fallopian tube, as
she had a fifty percent risk of developing ovarian cancer due to the same genetic anomaly.
 LIF101AA

Lecture 3
Topic chromosomal basis of inheritance: mitosis, meiosis
09_02_2022

a. Examine the physical basis of transmission of Mendelian factors (or

genes). Namely the chromosomes, the carriers of DNA (the genetic
material). We will briefly cover the physical features of the
chromosomes.

b. We would also examine how duplication of chromosomes and their

distribution takes place in somatic (body) (MITOSIS) and in
reproductive/ gametes/sperms/ova cells (MEIOSIS).

c. We will then examine how transmission of chromosomes to the

daughter (mitosis) and germ (meiosis) cells follows the Mendelian
principles.
 New Topic
Chromosomal basis of Mendelian inheritance
Mendel explained inheritance in pea in 1865 – it was not called a law in
those days – it was hardly noticed. The physical basis of Mendelian
inheritance was clear when chromosomes were discovered and their
transmission in somatic cells (by Mitosis) and Gametes (Meiosis) was
understood in the early 1900s

Summary of our current understanding

 RECAP: The life cycle of a cell
Cell cycle consists of 2 major phases

– Interphase, where
chromosomes
duplicate
and cell parts
are made
– The mitotic
phase, when
nuclear division
occurs

Figure 8.5
 INTERPHASE PROPHASE
Centrosomes Early mitotic Centrosome Fragments Kinetochore
(with centriole pairs) spindle of nuclear
Chromatin envelope

Centrosome Spindle
Nucleolus Nuclear Plasma Chromosome,
envelope membrane consisting of two microtubules
sister chromatids
Figure 8.6
 METAPHASE ANAPHASE TELOPHASE AND CYTOKINESIS

Metaphase Cleavage Nucleolus

plate furrow forming

Spindle Daughter Nuclear

chromosomes envelope
forming
Figure 8.6 (continued)
X chromosome
DNA, Chromatin and Chromosome

This is where we see

chromosome as
discrete entities
under microscope
 Mother
• When the cell cell

divides, the sister

chromatids
separate Chromosome
duplication

– Two daughter
cells are Centromere Sister
chromatids
produced
Daughter
– Each has a cells
complete and
identical set of Chromosome
distribution
chromosomes to
daughter
cells

Figure 8.4C
 Meiosis –
A process to generate haploid sets
of chromosomes

Gamete carry haploid set of

chromosomes
 MITOSIS MEIOSIS
MEIOSIS I
PARENT CELL Site of
(before chromosome replication) crossing over

PROPHASE PROPHASE I
Tetrad formed
Duplicated Chromosome Chromosome by synapsis of
chromosome replication replication homologous
(two sister chromatids) 2n = 4 chromosomes

Chromosomes Tetrads
METAPHASE align at the align at the METAPHASE I
metaphase plate Metaphase plate

ANAPHASE I
ANAPHASE Sister chromatids Homologous
TELOPHASE I
TELOPHASE separate during chromosomes
anaphase separate
during
anaphase I; Haploid
sister n=2
chromatids
remain together
2n 2n No further Daughter
cells of MEIOSIS II
chromosomal meiosis I
Daughter cells replication; sister
of mitosis chromatids
separate during
anaphase II n n n n

Figure 8.15
Daughter cells of meiosis II
 MEIOSIS I: Homologous chromosomes separate

INTERPHASE PROPHASE I METAPHASE I ANAPHASE I

Centrosomes Sister chromatids

(with centriole pairs) Microtubules Metaphase remain attached
Sites of crossing over attached to plate
kinetochore
Spindle

Nuclear Chromatin Sister Tetrad Centromere Homologous

envelope chromatids (with kinetochore) chromosomes separate

Note the separation of paired

chromosomes & NOT that of
chromatids
 MEIOSIS II: Sister chromatids separate

TELOPHASE I TELOPHASE II
PROPHASE II METAPHASE II ANAPHASE II
AND CYTOKINESIS AND CYTOKINESIS

Cleavage furrow

Haploid daughter
Sister chromatids cells forming
separate

This time
chromatids
separate
 Gametes have a single set of chromosomes

• Haploid gametes are produced by a special sort

of cell division called meiosis
• Which occurs only in reproductive organs,
ovaries and testes
• Purpose of meiosis is to produce sperm and egg
and reduce the diploid set of chromosomes to a
haploid set
 MEIOSIS Hallmarks

• Meiosis involves 2 cell divisions

• Meiosis produces 4 cells from 1 parental cell
• Each of the 4 daughter cells has 23 individual
chromosomes rather than 23 pairs of chromosomes
• Meiosis reduces the chromosome number from
diploid to haploid
• Meiosis, like mitosis, is preceded by chromosome
duplication
– However, in meiosis the cell divides twice to form
four daughter cells
 § Which one is a
chromosome and where
are the chromatid?

§ How do we count
chromosome number?

§ How many ‘sets’ of

chromosomes are seen
here?

§ Idea of diploid ‘2n’

and haploid ‘n’ sets

A karyotype (ORDERED ARRANGEMENT) of metaphase chromosomes (

in homologous pairs)
 Goals of Pedigree Analysis

§ Determine the mode of inheritance:

dominant, recessive, partial
dominance, sex-linked, autosomal
etc.

§ Determine the probability of an

affected offspring for a given cross.
Symbols used in pedigree analysis:
Male Sex
unspecified
Female
Affected
Mating individuals

Heterozygotes
Parents & for autosomal
Children: recessive
1 boy; 1 Carrier of sex
girl linked recessive
(in order
of birth) Deat
Dizygotic h
(non Abortion or
identical still birth
Twins)
Propositus
Monozygotic
(identical
Twins) Consanguineous
marriage
 Pedigree Analysis
Marriage
Normal Normal male
female
I One these
parent was
therefore a
carrier
1st born
II
Affected
Siblings Is the mutant
allele dominant
or recessive?
Pedigree
Analysis
 A Pedigree

Ww ww
I
1 2

II
1
ww 2
ww 3
ww 4 5
Ww 6
Ww
Ww

III
1 2 3 4 5 6 7 8 9 10
ww ww ww Ww ww ww Ww Ww ww Ww
 Sex-linked inheritance
The inheritance of genes located on
the sex chromosome
Autosomal vs. sex-linked traits

§ Autosomal traits are caused by genes on

autosomes (chromosome 1-22)
– autosomal recessive or dominant traits
/diseases

§ Sex-linked traits are caused by genes on the

sex chromosomes (X or Y)
– X-linked recessive or dominant traits
/diseases
 Albinism :
Autosomal recessive inheritance

Why this
inheritance is
called
autosomal?
 Achondroplasia:
Autosomal dominant inheritence

Why this called

autosomal dominant
dominant ?
Sex Determination in humans

Parents
 In such an
inheritance
pattern what is
Father the ready sign
of sex-
XA Y linkage?

XA Y

Daughter Son
X XXA XY
Mother affected normal
XX Daughter Son
X XXA XY
affected normal

X-linked dominant
 X-linked recessive disease
Female carrier* mates with normal male
Sperm
FxM XN Y
XNXA XNY

XN F M
XNXN XNY
normal normal
Eggs

XA F M
XAXN XAY
carrier affected

• Half* her daughters will be carriers

• Half* her sons will be affected

*on average
X-linked recessive
disease
Affected male mates with normal female
Sperm
FxM
XNXN XAY XA Y

XN
F M
XNXA XNY
carrier normal
Eggs

XN
F M
XNXA XNY
carrier normal

• All his daughters will be carriers

• None of his sons will be affected
Pedigree Illustrating Inheritance
Pattern of an X-Linked dominant
mutation
 Pedigree Illustrating X-Linked
Recessive Inheritance Pattern

Great grand mother

Grand mother
Nani
Grand Uncle

Grand
daughter
Uncle (mama)

Nephew
Hemophilia : X-linked recessive

Empress of India 1876

Queen Victoria (1819-1901)

passed haemophilia A
on to many of her descendants
 Hemophilia: The Royal Disease

The inheritance of the X-linked recessive condition hemophilia

in the royal families of Europe
 Edward Victoria
(Duke of Kent) (Princess of Saxe-Coburg)

Victoria
Albert
(Queen of England)

Arthur
Victoria Frederick Alice Louis Alfred Helena Louise Leopold Helen Beatrice Henry

Wihelm II Sophie George V Alix Nikolas II of Russia Alfonso XII Spain Eugenic

George VI Waldemar Henry Alexic Alfonso Gonzal

o

Queen Elizabeth Prince Philip

Normal Male

Normal Female

Hemophilic Male

Carrier Female

? Male died in infancy

Possible hemophilic
Some X-linked recessive human
diseases

• Hemophilia A
• Hemophilia B
• Red-green color blindness
• Duchenne muscular dystrophy
• Retinitis pigmentosum (one of many
loci)
• Lesch-Nyhan Syndrome
• Many others
Some X-linked dominant human
diseases

• Incontinentia Pigmenti
• Hypophosphatemic Rickets
• Charcot-Marie-Tooth
disease
• Chondrodysplasia Punctata
 Hypertrichosis pinnae auris : Y-linked

XY XX

XX XY XX XX XY XY XX

XX XX XY XY

As only males have a Y chromosome, the genes

are simply passed from father to son. Every son
of the father will be affected. Very few Y-linked
disorders are know except infertility
Inheritance of eye coloration in fruit fly, Drosophila:
An example of sex-linked inheritance

Normal Red Mutant white

 First : Observe
No eye coloration : white eyed

Normal eye coloration : red

eyed
How do we name the gene and what are its
alleles??
Gene name white

Normal Allele : white (+) red pigmentation

Mutant allele : white no pigmentation

Which one is recessive and which one is dominant?

 Then test and infer
w+w+ ww W+
W

86
w+w w+w w+w w
 Problem
A yellow body-colored female fruit fly was
mated to a male with normal body color
(brown).

All the female progeny were brown (normal)

while all the male progeny were yellow.
What does this inheritance pattern signify?

Solve this
problem
Significance of chromosome numbers in
in heritance

§ What if individual chromosomes do not

separate during meiosis? Error in
chromosome number
 The Culprit: Non-disjunction

Older women are more likely than younger women to

evidence chromosome damage and meiotic
irregularities
Pre-natal Diagnosis: Amniocentesis
Will it be possible to
detect a defective
gene by
amniocentesis?

Fetal cells present in

amniotic fluid are then
cultured and analyzed for
chromosomes (karyotyping)
and other biochemical tests
 This baby has
one X
chromosome. Yet
it is a female
baby. Why?

Monosomy: Turner’s Syndrome: 44+X

Individual chromosome segregation going wrong during
meiosis
Why down’s
syndrome does
How can not suggest
errors in abnormal sex
chromosomal linked
segregation inheritance?
result in
Down’s
syndrome?

Trisomy of chromosome . 21: Down’s

Syndrome: 45+XY/45+XX
Other disorders: Trisomy-13 (47:+13), Trisomy-18 (47:+18),
Klinefelter Syndrome (44+XXY), Triple X syndrome (44+XXX), XYY
trisomy (44+XYY)
People suffering from
Down syndrome can
lead a reasonably
normal life
 LIF101AA
Lecture 5
Wednesday 16 and Friday 18 Feb, 2022

Flow of genetic information & genetic code

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

 Let’s first appreciate the problem of
transfer of information from DNA
(genes) to protein

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

 Consider the case of Sickle Cell Anemia?
 A serious condition in which red blood
cells can become sickle-shaped
 Normal red blood cells are smooth and
round. They move easily through blood
vessels to carry oxygen to all parts of the
body.
 Sickle-shaped cells don’t move easily
through blood. They’re stiff and sticky
and tend to form clumps and get stuck in
blood vessels.
 The clumps of sickle cell block blood flow
in the blood vessels that lead to the
limbs and organs. Blocked blood vessel
can cause pain, serious infection, and
organ damage.
 While the defect is seen in the protein,
sickle cell anemia is produced by changes
in gene sequence

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

 Normal and Sickled Red Blood Cells in Blood Vessels

Figure B shows abnormal, sickled red blood cells clumping and

blocking the blood flow in a blood vessel. The inset image shows a cross-
section of a sickled red blood cell with abnormal strands of
hemoglobin.

Figure A shows normal red blood cells flowing freely

in a blood vessel. The inset image shows a cross-
section of a normal red blood cell with normal
hemoglobin.
Source from http://www.nhlbi.nih.gov/health/dci/Diseases/Sca/SCA_WhatIs.html
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
 Questions?

• What could be the reason(s) for the wrong

message from DNA that resulted in a
wrong protein (hemoglobin)?
• How ‘information’ encoded in the DNA of
the nucleus is transferred to the cytoplasm
• How does this information ‘translate’ into
protein??

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

 Central dogma of Molecular biology
RECAP सिद्धांत
Nature of information flow
Flow of information
Flow of information: nucleic Nucleic acid to
acid to nucleic acid protein

Replication

Transcription Translation

DNA RNA protein

Reverse transcription

Why there is no loss We will now explore this flow of

of information information: that is, process of
during replication? decoding the genetic message

How much of the

information in DNA
goes to RNA??
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 Flow of genetic information
A gene is transcribed into RNA
which is then translated into the polypeptide

DNA

Transcription

RNA

Translation

Protein

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 FLOW OF GENETIC INFORMATION FROM DNA TO
RNA TO PROTEIN
§ Gene (DNA) is a linear sequence of many
nucleotides

§ DNA is transcribed into a linear sequence

of RNA
§ RNA is translated into a linear sequence of
amino acids – polypeptides (protein)
DNA RNA Protein
Entire business of flow of (polypeptide chain
genetic information thus
depends on constructing
(transferring information) one
kind of polymer to another

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 Transcription – Step I

T G C A T A G C G C A T

Template DNA Strands unzip locally

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 Transcription – Step II

A C G T A T C G C G T A DNA

U G C A U A G C G C A U mRNA
5’ 3’

One of the DNA strands serve as a template

and copied as mRNA (messenger RNA)

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 A C G T A T C G C G T A
DNA
T G C A T A G C G C A T

RNA U G C A U A G C G C A U

mRNA is then releases

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

                                                                         

Notice the difference between ribose and deoxyribose in

the figure above. In ribose, carbon atom #2 carries one
hydroxyl group (colored red). In deoxyribose, carbon
atom #2 carries a hyrogen atom instead of a hydroxyl
group.

Chemical structure of RNA

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 In place of
thymine in
DNA

Cytosine Thymine Uraci

l
DNA/RNA DNA RNA

Pyrimidine bases
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 Transcription

RNA polymerase RNA nucleotides

T C C A A T
A U
C T
T

G
U
A

G
C
C A U C C A
C
G A
T A G G T T A

Direction of transcription
Template Strand
of DNA
Newly made RNA

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 § In the nucleus, the DNA helix unzips
§ And RNA nucleotides line up along one
strand of the DNA, following the base
pairing rules
§ As the single-stranded messenger RNA
(mRNA) peels away from the gene
§ The DNA strands rejoin

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 Steps in transcription of a gene
RNA polymerase

DNA of gene

Promoter
DNA Terminator
DNA

1 Initiation

Area shown

2 Elongation In Figure 10.9A

Growing
RNA
3 Termination

Completed RNA
RNA
polymerase

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 How does DNA/RNA code for protein?
Strand to be transcribed

T A C T T C A A A A T C

DNA
A T G A A G T T T T A G

Codon -
Transcription
a sequence of
nucleotides that
codes for an A U G A A G U U U U A G

amino acid RNA

Start Stop
Translation
condon condon

Met Lys Phe

Polypeptide

STEP III TRANSLATION

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 Dictionary of genetic code

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

 How can four nucleotides code for
20 amino acids?

§If one nucleotide codes for one amino acid then a

maximum of four amino acid could be coded

§ If two nucleotide code for one amino acid then a

maximum of 16 amino acid could be coded

§ If three nucleotides code for one amino acid then

a maximum of 64 codons can be generated, while
the number of amino acids are only 20

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 The dictionary of genetic code (64 potential codons)

5’-3’
A dictionary
offers
translation
from one
language to
the other.

Which two
languages
are being
translated
in a genetic
dictionary?

Codons are
shown in RNA
to make it
easy to follow
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 Hallmarks of genetic
code
§ Triplet : three consecutive nucleotides code of
one amino acid

§ Redundant : meaning not unique (there

are more than one codon for most amino
acids)
§ Universal: All organisms bacteria to
human use the same genetic
codes/dictionary
§ Comma-less: meaning consecutive
codons are readout into an amino acid
sequence without gaps

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 Many amino acids
are specified by
more than one
codon, meaning
thereby that
codons redundant

Codons specifying
the same amino
acid are called
synonyms

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 How does an universal genetic code impact
Biotechnology??
What would have
been the case if
bacterial genetic code
were to be different
from human?

What do we
understand when we
mean when we say
that genetic code is
universal?

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

 Three codons direct chain termination

Ø Three codons, UAA, UAG, and UGA signify

chain termination.
Ø They are not read by tRNAs but by proteins
called release factors (RF1 and RF2 in
bacteria and eRF1 in eukaryotes).

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 Three Rules

Ø Codons are read in a 5’ to 3’ direction.

Ø Codons are non-overlapping and the
message contains no gaps.
Ø The message is translated in a fixed
reading frame which is set by the initiation
codon.

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 Mutation versus genetic code

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 What Are Mutations?

• Changes in the nucleotide sequence of DNA

• May occur in somatic cells (aren’t passed to
offspring), but could cause diseases, such
as cancer
• May occur in gametes (eggs & sperm) and
be passed to offspring

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 Nature of mutations

1. Missense mutation: An alternation that

changes a codon specific for one amino acid to
a codon specific for another amino acid.

2. Nonsense or stop mutation: An alternation

causing a change to a chain-termination
codon.

3. Frameshift mutation: Insertions or deletions

of one or a small number of base pairs that
alter the reading frame.

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 Point Mutation
§ Change of a single nucleotide
§ Includes the deletion, insertion,
or substitution of ONE
nucleotide in a gene

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 Types of Gene/Point Mutations
§ Substitutions
§ Insertions
§ Deletions

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 Frameshift Mutation
§ Inserting or deleting one or
more nucleotides
§ Changes the “reading frame”
like changing a sentence
§ Proteins are then built
incorrectly

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 Gene Mutation
Animation

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Impact of insertion or deletion of a nucleotide

THE FAT CAT ATE THE BIG RAT

Delete C THE FAT ATA TET HEB IGR AT

Insert A THE FAT ATA ATE THE BIG RAT

Frame shift mutation

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 Example Point Mutation

• Sickle Cell anemia is

the result of one
nucleotide
substitution
• Occurs in the
hemoglobin gene

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 Nature of base
pair substitution
leading to
missense
mutation

Can a base
substitution
also result in a
nonsense
mutation??
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Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
 Some practical
consideration from
this demographic data

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

 Question

If an mRNA of human beta-globin

gene is added to bacterial cell
free extract then human beta
globin polypeptide is formed
(translated)
What does this observation signify
with respect to genetic code?

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

 The amino acid sequence shown in the following table was obtained from the central
region of a particular polypeptide chain in the wild type and several mutant bacterial
strains
Codon

1 2 3 4 5 6 7 8 9

a. Wild type phe leu pro thr val thr thr arg trp
b. Mutant 1 phe leu his his gly asp asp thr val
c. Mutant 2 phe leu pro thr met thr thr arg trp
d. Mutant 3 phe leu pro thr val thr thr arg
e. Mutant 4 phe pro pro arg
f. Wild type phe leu pro ser val thr thr arg trp

Hint: For each mutant, first examine the nature of the change at the
protein level and then extrapolate to DNA level to decide if the
change represents base pair substitution (leading to missense, non-
sense mutations) or deletion/insertion leading to frameshift
mutation. You then provide the final solution by assigning the
codons (with the help of genetic code dictionary) to the wild type
amino acid sequence and then working out the codons for the
mutant proteins by considering the nature of the mutations that
might have
Copyright © 2005 Pearsonoccurred
Education, Inc. Publishing as Benjamin Cummings
 Key players in decoding the genetic message

DNA

Messenger-RNA

Transfer RNA

Amino
acid
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
 Transfer RNA

Leucine

Anti-codon

Codon

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 How genetic code is readout in the cell

DNA A C G T A T C G C G T A
T G C A T A G C G C A T

3’ U G C A U A G C G C A U 5’
mRNA
3’
Amino
acid
5’ Anti-Codon
5’ 3’

tRNA C A G
G U C
3’ 5’

Codon
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Codon-anticodon pairing of two tRNA Leu molecules

5’- 3’ 5’- 3’

CUG Codon CUC

GAC Anti- codon GAG
3’- 5’ 3’- 5’
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 Problem
In mutant strain X of E. coli, a leucine tRNA that recognizes
the codon 5’-CUG-3’ in normal cell has been so altered that
is now recognizes the codon 5’-GUG-3’.

A missense mutation (lets call this mutation Y) that affects

amino acid 10 of a particular protein is suppressed in
mutant X cells (that is when the cells are mutant for both X
and Y, which now appears wild type)

a. What mutational even has occurred in mutant X cells?

b. What amino acid would normally be present at position
10 of the protein (without the missense mutation)?
c. What amino acid is put in at position 10 if the missense
mutations is not suppressed ?
d. What amino acid is put in at position 10 if the missense
mutations is suppressed ?

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 How the genetic code was cracked?

The use of artificial mRNAs and the availability of

cell-free systems for carrying out protein synthesis
began to make it possible to crack the code

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 Codon Assignments from Repeating Copolymers

Organic chemical and enzymatic

techniques were used to prepare
synthetic poly-ribonucleotides
with known repeating sequences.

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 Experimental Results:

Ø UUU codes for phenylalanine.

Ø CCC codes for proline.
Ø AAA codes for lysine.
Ø The guanine residues in poly-G firmly hydrogen
bond to each other and form multistranded
triple helices that do not bind to ribosomes.

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

copolymer Codons Amino Acids Codon
Recognized Incorporated Assignment
(CU)” CUC|UCU|CUC … Leucine 5’-CUC-3’
Serine UCU
(UG)” UGU|GUG|UGU… Cystine UGU
Valine GUG
(AC)” ACA|CAC|ACA… Threonine ACA
Histidine CAC
(AG)” AGA|GAG|AGA… Arginine AGA
Glutamine GAG
(AUC)” AUC|AUC|AUC… Polyisoleucine 5’-AUC-3’
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Recombinant DNA technology
Genetic Engineering

LF101AA
23-02-22
 Recap: Griffith’s experiment
Bacterial transformation

What does transformation signify? What opportunities does this

An incoming bacterial factor (DNA ) observation offer?
could replace an endogenous factor Could an incoming DNA ( foreign
gene) be made to stay in a cell?
 Some background information
§ Prokaryotes are haploid: for example,
bacteria E. coli – it has only one chromosome

§ Bacterial chromosome is circular

§ Bacterial chromosome represent a single

unit of replication Bacterial
chromosome Plasmids
§ Bacterial cell can carry extra chromosomal
replicating bodies, called plasmids
 Transformation by a plasmid (vector)
DNA

Host bacterium

Host bacterium
Transformed by a
plasmid

The transformed host

bacterium and it progeny
would express the gene
(marker) carried by the
plasmid: for instance,
antibiotic resistance
What if the plasmid (vector) DNA is
made into a recombinant DNA first
before transformation?
 Definitions of recombinant DNA
(rDNA) and genetic engineering

§ Recombinant DNA: A new DNA molecule created

by joining together two or more DNA fragments
not normally associated with each other: for
example a DNA from human and bacteria

§ Genetic engineering: The end product of genetic

engineering is an organism that has been
genetically modified using recombinant DNA
technology without taking recourse to its natural
course of mutation/ evolutionary changes etc
Recombinant DNA: definition

A unique piece of DNA artificially

created by joining them together
Recombinant DNA:
Vocabulary
§ Vector
§ Recombinant DNA
§ Restriction
enzyme
§ Ligase
§ Transformation
§ Selection
§ Gene clone
 Cloning Vectors

§ A cloning vector is a DNA molecule that has

an origin of replication and is capable of
replicating in a bacterial cell.

§ Most vectors are genetically engineered

plasmids or phages.
 What is a
Cloning vector
Plasmids replicon?

• Plasmids: Extra-chromosomal genetic elements that

are capable of autonomous replication (replicon)
What does these green
area on the plasmids
signify? What are the
A marker (gene) that can advantages of
be readily detected – having a cloning
antibiotic resistance, for vector
example

Transformation

Step 1 Step2
 Characteristics of plasmid cloning
vector :

§ Small size (<15Kb) for optimal efficiency of

transformation in bacteria
• Origin of replication-allows plasmid to
replicate in the bacteria
• Unique restriction enzyme sites for cloning
• Carries selectable markers
Plasmid: it’s a circular DNA molecule
containing:
§ a multiple cloning site or
MCS
§ an antibiotic resistance gene
§ an origin of replication
MCS

ampR

Ori
 Plasmids as a cloning vector
This is the
DNA
(green)
Origin of
that has
replicatio been
n cloned in
Ampicilli
the
n plasmid
resistanc vector
e
§A DNA vector
§Self replicating
§ Carries
selection markers
Two most critical enzymes for
recombinant DNA technology :
§ Cutting enzyme:
Restriction endonuclease
§ Stitching enzymes : Ligase

Where do we find
the cutting enzyme
Lytic life cycle of a virulent phage, such as T2

The story of how restriction enzymes were discovere

 The story of how restriction enzymes were discovered
Restriction endonucleases are part of a bacterium’s defense
against invaders
Restriction-modification systems
allow the bacterium to distinguish
self from non-self DNA
Restriction: bacterial endonucleases cleave
both strands of foreign DNA at specific
recognition sites

Modification: bacteria protect their own

DNA by adding a methyl group to the
recognition sites in their own DNA Electron micrograph
bacteriophage infecting a
bacterium

Type II restriction enzymes are widely used in molecular biology:

enzymes cleave, but do not modify, their specific recognition sites
The story of how
restriction enzymes
were discovered
Discovery of
Restriction
endonucleases

Host 1 Host 1

Modification Restriction

Host 1
Modification
Methylase

Host 1
 REs with 6-nucleotide recognition sites (6-cutters)
are widely used in molecular biology

Sites would randomly be expected every 1/4096 nucleotides

Recognition sites are often

palindromes

Restriction Enzyme Strain of origin Recognition site

EcoRI E. coli (strain RY13) GAATTC
Hind III H. influenza AAGCTT
BamHI B. amyloliquefaciens GGATCC
 Utilities of Restriction enzyme: Precise, defined, and reproducible
cuts that can be ligated again to create recombinant DNA

Results of gel electrophoresis: production of linear DNA

fragments of different sizes by different restriction
endonucleases

Plasmid DNA 10
9
8
7
6
5
At how many points did 4
EcoRI restriction enzyme cut 3
the circular plasmid DNA , if
2
one finds two linear pieces
of DNA : one 7 kilo bases 1
and the other 3 kilo bases
long, an what is what is the
length of the original
circular plasmid DNA ?
Restriction Maps

• A restriction map is a
linear (or circular) map of
the order and distances
of restriction
endonuclease cut sites in
a segment of DNA
• Each DNA fragment, no
matter what size, has its
own unique restriction
map
• Restriction maps are
useful in comparing DNA
fragments to look for
regions of identity
 Restriction Mapping

• Restriction endonucleases helps build

maps of linear and circular molecules
HaeII
BamHI
EcoRI
HaeIII
Restriction map
of a plasmid
AA plasmid
plasmic

HindIII
HaeII
What is the benefit of
having a restriction map
for a given piece of DNA?
 What are the utilities of the RE cuts?
Step 1 Example of EcoRI cut

Is this a cut or G A A T T C Will these two strand

a nick C T T A A G of DNA stay
together or fall apart
Step 2
These
G A A T T C snap to
C T T A A + G create
sticky end

Step 3
Ligated

Ligated
G A A T T C
C T T A A G
What can we now do with the RE cut DNAs?
 Answer the following

§ What are RE cuts?

§ Why are the RE cuts unique??
§ What are the utilities of the RE
cuts?
§ What can we now do with the
RE cut DNAs?
The first cloning experiment done by Boyer and Cohen
 You can call this as a
passenger/foreign DNA
that is being carried to
the bacterial host cells
by the plasmid vector
(carrier)
 Basic Cloning Process
1. Plasmid is cut open with a restriction enzyme that
leaves an overhang: a sticky end

2. Foreign DNA is cut with the same enzyme.

3. The two DNAs are mixed. The sticky ends anneal

together, and DNA ligase joins them into one
recombinant molecule.

4. The recombinant plasmids are transformed into E. coli

5. Cells carrying the plasmid are selected by adding an

antibiotic: the plasmid carries a gene for antibiotic
resistance.
 RECAP
Plasmid and DNA cloning

Origin of This is the

replicatio DNA (green)
that has
n
Ampicilli been cloned
in the
n plasmid
resistanc vector
e
§A DNA vector
§Self replicating
§ Carries selection
markers
Transformed bacterial clone § It can be made into a
would then represent a gene recombinant DNA and
clone as well. thus used to clone genes

Can you now tell the

meaning of a gene clone?
Restriction enzymes are extensively used in molecular biology. Below are
the recognition sites of two of these enzymes, BamHI and BclI.

BamHI, cleaves after the first G:

5’ GGATCC 3’
3’ CCTAGG 5’
Q:1
Does cleavage by BamHI results in a 5’ or 3’ overhang? What is the
sequence of this overhang?
Ans: 5’ G 3’
3’ CCTAG 5’
Cleavage by BamHI leaves a 5’ overhang

b. BclI cleaves after the first T:

Does cleavage by BclI result in a 5’ or 3’ overhang? What is the

sequence of this overhang?
Ans: 5’ T 3’
3’ ACTAG 5’
Cleavage by BclI leaves a 5’ overhang
Q2. Below is a map for the plasmid pGEN101 (total length=20kb). Using this
map as a guide give the number of restriction fragments along with their
associated lengths that would result from digesting pGEN101 with the
restriction enzymes EcoRI, BamHI, and a combination of EcoRI+BamHI.

Ans: Digest performed:

EcoRI 20 kb
BamHI 2 kb, 6 kb, 12 kb
EcoRI+BamHI 2 kb, 4 kb, 6 kb, 8 kb
Q3. Two freshmen college students performed the following set of
restriction digests on a newly isolated plasmid pBLA230. The reaction that
they carried out along with the fragment obtained in single and double
digestion were:
HpaI 26 kb
HindIII 13 kb, 6 kb, 4 kb, 3 kb
HpaI+HindIII 7 kb, 6 kb, 6 kb, 4 kb, 3 kb

Using this information construct the restriction map of pBLA230.

ANSWER
 All the videos
YouTube video for gene cloning cartoons displayed here are
useful for
https://www.youtube.com/watch?v=sjwNtQY appreciating genetic
LKeUlink for gene cloning
Khan academy YouTube engineering/gene
cloning/recombinan
https://www.youtube.com/watch?v=5ffl- t DNA technology
0OYVQU

MIT open courseware : Speaker - Eric Langer

Course: fundamental of biology
Topic : Basic mechanisms of cloning I and II

https://www.youtube.com/watch?v=CdAgz
k5tQhs
https://www.youtube.com/watch?v=reYwbn
uhFU0

Dr. Eric Lander, Director of the Broad Institute of MIT and Harvard,
speaks at the Mouse Genome Sequencing Press Conference on
December 4, 2002
 If you have understood
the topic covered then
you would be able to
Question: answer this question

If you are provided with plasmid DNA and also

human DNA, will you be able to create a recombinant
DNA?

Can this DNA be cloned?

Where has molecular cloning (genetic engineering) led us to

How to interpret the human genome |

Alisha Holloway |
TEDxClaremontColleges
https://www.youtube.com/watch?v=Mph3GfDKM
-o

Watching such videos will

help you connect with the
impact of the genetic
engineering on understanding
human health and diseases
 LIF 101AA

Topic Application of Recombinant DNA Technology:

Genetic detection and DNA finger printing

Wednesday 02 March 2022

 Idiom मुहावर When we search for
Looking for a needle in a haystack! say a 10,000 bp long
DNA/gene in the 3.0
×109 bp long human
genome, we are
essentially searching a
needle from a
haystack

Southern blot:
How to identify a desired piece of DNA from the total
genome
 Background information # 1

REs with 6-nucleotide recognition sites (6-cutters)

are widely used in molecular biology

Sites would randomly be expected every 1/4096 nucleotides

(1/46)
Actual sizes vary widely with average of ~4000 bp

Recognition sites are often

palindromes

Restriction Enzyme Recognition site

EcoRI GAATTC
Hind III AAGCTT
BamHI GGATCC
 Background information # 2
Gel electrophoresis: a technique to separate DNA by size
§ DNA is negatively charged due to its sugar-phosphate backbone
§ Thus, when placed in an electrical field, DNA migrate towards the
positive pole
§ Agarose gel is used to slow the movement of the DNA, ,which separate
by size
0 hr
§ Smaller the DNA fragment, longer does it migrated in an agarose gel

1 hr later
 Background information # 2 contd
Molecular weight Restriction digested
marker DNA fragment genomic DNA. It
appears as a smear
since the fragment of
all sizes are present
here

Restriction digested
fragments of genomic How do we then
Restriction digested DNA from an eukaryote search out a DNA
fragments of DNA vector fragment of our
interest from this
restriction digested
fragment?
 Background information # 3
Single stranded DNAs can hybridize with
complementary sequences

Non-pairing interactions Non-pairing interactions

Background information # 3
 Steps in Southern blot

This DNA probe (for example, cloned

fragment of globin gene) is labeled
(radioactively)

https://www.youtube.com/watch?v=GPVf_AWMY
Z4
Recap
Example Point Mutation
• Sickle Cell disease is the result of
one nucleotide substitution

• Occurs in the hemoglobin gene

The sixth position in the

normal beta chain has
glutamic acid, while sickle
beta chain has valine. This is
the sole difference between
the HbS an allele of Hbb
-globin gene, mRNA, and polypeptide showing the normal Hb-
A sequences and the mutant Hb-S sequences
DdeI is a restriction
enzyme that recognizes a
sequence 5’ CTNAG 3’
(where N stands for any
nucleotide)

This mutant sequence

cannot be recognized by
DdeI
Detection of sickle-cell gene by the DdeI restriction
fragment length polymorphism in southern blot

This is detection of the

restricted fragments of the Hb
gene by radioactive probe by
a technique called southern
blot.

What does this band represent?

Why it is absent in normal
individuals and why it is
present as a single band sickle
cell individuals
 Examples of DNA Molecular Testing

Sickle-cell anemia is an example:

a. A single base-pair change in the b-globin gene results in
abnormal hemoglobin, Hb-S, rather than the normal Hb-
a. Hb-S molecules cause sickling of red blood cells.
b. The Hb-S mutation is an AT-to-TA base pair change in the
6th codon of b-globin, resulting in a valine rather than a
glutamic acid, and also eliminating a DdeI restriction
enzyme site .
A practical use of Southern blot: genetic
testing
Prenatal diagnosis

Newborn screening.

Carrier (heterozygotes) detection

Amniocentesis:
A method to
collect fetal cells
 Recap

Background Information
Beta-thalassemia is a form recessive genetic disorder that reduces the
production of hemoglobin. In acute cases it is lethal.
www.ygyh.org/thal/inherited (your genes your health)
Observation
Baby with Beta-thalassemia is born to a couple who were otherwise
normal
Question
How will you explain this observation?

Answer: Progeny of (T/t X T/t) would thus be: (T/T, T/t and t/t)

§ Can the birth of the unfortunate

thalassemic baby be avoided?

§ Can the carrier individuals be detected?

 Concept of DNA Molecular Testing
1. Designing DNA molecular tests requires knowledge of gene mutations
that cause a disease, derived from sequencing the gene involved.
2. Often a disease is caused by a variety of mutations, complicating its
study. The breast cancer genes BRCA1 and BRCA2 are examples:
a. Normal BRCA1 and BRCA2 genes control cell growth in breast and ovarian
tissue.
b. Mutations in the BRCA1 and BRCA2 genes can lead to cancer. Hundreds of
mutations in these genes have been identified.
c. Each BRCA1 or BRCA2 mutation confers a different risk of developing
cancer, ruling out a single DNA molecular test to assess an individual’s
breast cancer risk associated with these genes.
3. Genetic testing reveals the presence of a mutation associated with a
genetic disease.

Why this image

makes sense here??
 DNA finger printing
Short tandem repeats (STR)  A short tandem repeat is a microsatellite,
consisting of a unit of two to thirteen nucleotides repeated hundreds of times in a
row on the DNA strand.

Since each one of

Satellite DNA consists of us inherit different
very large arrays of combination of
tandemly repeating, maternal and
non-coding DNA.  paternal satellite
repeats, we are all
unique. These
Satellite repeats
are thus out DNA
finger prints
 Fig. 8.10 Procedure for DNA typing as used for a
paternity case

Is he the
real
 Other Applications of DNA Typing
1. Examples of DNA typing used to analyze present-
day samples include:
a. Forensic analysis in criminal cases. DNA samples from
victims and suspects are compared
b. Population studies to determine variability in groups of
people.
c. Proving horse pedigrees for registration purposes.
d. Forensic analysis in wildlife crimes, allowing body parts of
poached animals to be matched and used as evidence.
e. Detection of pathogenic E. coli strains in foods
f. Detection of genetically modified organisms (GMOs) in
bulk or processed foods, based on the presence of inserted
sequences.
 LIF101AA
FRIDAY 04_03_2022

üGene regulation in prokaryotes

 Flow of genetic information
A gene is transcribed into RNA
which is then translated into the polypeptide

DNA
RECAP
Transcription

RNA

Translation

Protein
 Transcription

RECAP

RNA polymerase RNA nucleotides

T C C A A T
A U
C T
T

G
U
A

G
C
C A U C C A
C
G A
T A G G T T A

Direction of transcription
Template Strand
of DNA
Newly made RNA
 How does DNA/RNA code for protein?

Strand to be transcribed
RECAP
T A C T T C A A A A T C
DNA
A T G A A G T T T T A G

Codon -
a sequence Transcription
of nucleotides
that codes for A U G A A G U U U U A G
an amino acid RNA
Start Stop
Translation
condon condon

Met Lys Phe

Polypeptide
 But how will RNA polymerase
decide which one to transcribe
from hundreds and thousands of
genes in our genome??

Gene regulation
Regulation of the synthesis of a
gene’s transcript and its protein
product is termed as gene
regulation.
 1.5% of human genome codes for protein
Between 20,000-25,000 estimated protein coding
genes are present in human genome

Why control gene expression?

• Each of our cells carry entire genetic instructions for our

growth, development and metabolic functions.

• Some of these genes are needed to be expressed all the

time: those regulating our cellular respiration, for
instance. These are also called house keeping genes

• Other genes are not expressed all the time.

• These are switched “on” and “off” on demand

 Promoter:
RNA Pol binding site in
DNA sequence upstream A basic scheme
of transcription start
point

RNA
polymerase Gene

Direction of transcription
Promoter

Transcriptional Regulation is mediated by

controlling the access of the RNA polymerase to the
promoter
The promoters of genes transcribed by RNA polymerase II consist of a
core promoter and a regulatory promoter that contain consensus
sequences.

A point to remember:
Like transcription initiation
We will not go in to these there are also
details either transcription termination
mechanisms
 Negative Regulation Positive Regulation

RNA Polymerase RNA Polymerase

Binding of X
Promoter repressor An activator
blocks the help RNA Pol
binding of the bind to the
Repressor RNA Pol to the Activator promoter
promoter

X
Recall what is a
prokaryote.

Two broad strategies for regulation of prokaryotic transcription

 A simple design of gene regulation: ‘supply on demand’

In prokaryotes gene regulation takes place in

response to the environment
Glucose
§ E. coli can use either glucose,
which is a monosaccharide, or
lactose, which is a disaccharide

§ However, lactose needs to be

hydrolysed (digested) first

§ So the bacterium prefers to use

glucose when it is available. Lactose

§ On the other hand, when only

lactose is available it turns “ON”
the genes and, therefore, the
enzymes required for lactose
breakdown
Glucose Galactose
 Regulation of the lac Z gene – an example of negative
regulation
( lacZ gene codes for b-galactosidase enzyme that breaks
down lactose )
REPRESS
What can be OR
X
the inducer INDUCE
of the lac Z R
gene? INACTIVE
REPRESS X
OR

Answer: OPERATO
lactose R:
Binding
• When lactose site
is for
absent in the medium, the lacZ gene is switched-
repressor
off: that is, no mRNA is transcribed and no proteins are made.
• In the presence of lactose in the medium, lacZ gene is turned on:
that is, mRNA is transcribed and the polypeptide b-galactosidase is
synthesized
 Illustration of a NEGATIVE INDUCIBLE
Question:1 REGULATION gene expression TRANSCRIPTION
Repressor is a
protein: should Regulation of lac Z gene
This is also called
there be a an inducible model
separate gene for of gene regulation.
repressor? RNA POl WHY?
Question:2
How will lac Z gene REPRESSOR
be regulated if a ACTIVE
gene coding for X
repressor is INDUCER
mutated? REPRESSOR
Question:3 INACTIVE
What will be the
consequence if X
Repressor binding site is
operator sequence is called
altered? OPERATOR
Question:4
What will happen if
promoter sequence itself is
altered?
 Further, lacZ gene is co-regulated along with
two other genes called lac A and lac Y

§ A set of gene co-regulated under one promoter are called

operon. Thus, an operon represents a group of genes that
are transcribed at the same time.

§ They usually control an important biochemical process.

§ They are only found in prokaryotes.

 The lac Operon

P O lacZ lacY lacA

mRNA 5’ 3’

How many
messenger RIBSOSOME BINDING SITE
RNA are made
by the lac
operon?
 The lac Operon

P O lacZ lacY lacA

mRNA 5’ 3’

Proteins
b-galactosidase Permease Transacetylase

How many
Why would you consider the lac operon a polypeptide
smart system? (proteins) are made
by the lac operon?
 Regulation of the lac Operon by the
repressor
What is this ?

P O lacZ lacY lacA

X NO mRNA Note that there are

two binding sites in
the repressor one for
lactose the operator (DNA)
and the other for
the inducer (lactose)

P O lacZ lacY lacA

X NO mRNA

In the presence of lactose in the medium, lactose binds to the repressor.

The lactose-repressor complex then leaves the operator.
 Regulation of the lac Operon by the
repressor

P O lacZ lacY lacA

mRNA

Once “Inducer-Repressor” complex moves away from

the Operator (O). RNA polymerase can then bind to
the promoter (P) and initiate transcription of the lac
Operon
 About the lac I gene that codes for the repressor

P O lacZ lacY lacA

X NO mRNA
lac repressor
lac
repressor The lac repressor binds to the operator and
inhibits transcription of the lac operon.

Question:
Should the lac I gene be
always active (constitutive)
or sometimes active
(inducible)?
Question
Lac repressor has two binding sites: one for
binding with ‘Operator’ and the other for
the ‘Inducer’. What will be the consequence
if a mutation alters its ‘inducer binding’ site
The story of conjugation in bacteria
 The Lac operon, an example of a transcriptionally regulated system.
 
The most direct way to control the expression of a gene is to regulate its rate of
transcription; that is, the rate at which RNA polymerase transcribes the gene into
molecules of messenger RNA (mRNA).       
 
E. coli break lactose down using two (there are 3, but our problem set only addressed two)
enzymes: b-galactosidase, which is encoded by the LacZ gene, and permease, which is
encoded by the LacY gene. These genes and the regions that regulate them are called the
Lac operon.

Other important players in the lac operon are:

Operator (LacO) : the binding site for the repressor

Promoter (LacP) the binding site for RNA polymerase
Repressor (LacI) the gene encoding for the lac repressor protein- in the absence of lactose
the repressor protein binds to the operator and blocks binding of RNA polymerase at
promoter

 The genotypes are written in order of: repressor (i)

promoter (p)
operator (o)
 LacZ (z) 
LacY (y)

 i+p-o+z+y+
 Concept of partial diploidy in bacteria

Haploid

Haploid E. coli E. coli with a plasmid

Partially
Haploid

E. coli with a plasmid, which

carried part of the E. coli
DNA/GENES
If the organism in question is a partial diploid, the two chromosomes are
written side by side, separated by a slash. Good copies of genes are indicated
by a + sign.

Example: i+p-o+z+y+/i-p+o+z-y+
 How to solve a lac operon question.
 
Look at the promoter first - do you have a good copy of the promoter (p+)? If not, RNA
polymerase cannot get transcription started and that chromosome is a bust.

If you do have p+, next look at how the repressor and the operator interact.

Here are a few scenarios:

i+p+o+ :  the repressor binds to the operator in the absence of lactose and inhibits
transcription of the z and y genes. BUT, when lactose is around, it kicks the repressor off
of the operator, and the z and y genes are expressed (look to see whether you have good
copies of z and y, i.e. z+ and y+).

isp+o+   : this is a super repressor- lactose cannot kick it off, so transcription of z and y

FAILS whether lactose is around or not.

is p+oc  :  the super repressor meets the constitutive operator. The constitutive operator
deflects any repressor (even a super repressor) and transcription OCCURS whether lactose
is around to not.
One last thing to remember is that while p and o are regulatory regions and only affect
the genes that they are sitting right next to (they act in cis), i codes for a diffusible
regulatory protein, so it can affect both chromosomes (it can act in trans) of  partial
diploid. We didn’t have any examples of this in our problem set problem, so here is one:
What would you expect in a partially diploid case as below?

 is p- oc z- y+/i - p+o+ z+ y-  :

In this case, the first chromosome is a bust because of a bad
promoter. The second chromosome has a bad repressor, but the
protein produced by the super repressor from the first chromosome
will bind to the operator of the second chromosome. In this case,
beta-galactosidase is produced in the presence of lactose, but not
in the absence of lactose. There is no functional LacY,
so permease is never produced.

 is p- oc z- y+ (ON THE PLASMID)

i - p+ o+ z+ y- (ON THE MAIN CHRMOSOME)

What will be the net consequence if

lactose is added in the medium