LIF 101 16th October 2016

MaleFemale

Mating

Parents &Children:1 boy; 1 girl(in order of birth)Dizygotic(non identical Twins)

Monozygotic(identicalTwins)

SexunspecifiedAffectedindividuals

Heterozygotesfor autosomal recessiveCarrier of sex linked recessive

Death

Abortion or still birth

Propositus

Consanguineousmarriage

Symbols used in pedigree analysis:RECAP

Pedigree Analysis

Normal female Normal maleMarriage

1st born

Siblings

Affected

I

II

One these parent was therefore a

carrier

Is the mutant allele dominant or recessive?

RECAP

Albinism : Autosomal recessive inheritance

Why this inheritance is called autosomal?

RECAP

Achondroplasia: Autosomal dominant inheritence

Why this called autosomal dominant

dominant ?

RECAP

Sex Determination in humans

Parents

RECAP

Female carrier* mates with normal male

FXNXN

Y

XN

XA

• Half* her daughters will be carriers• Half* her sons will be affected

F x MXNXA XNY

Eggs

Sperm

XN

MXNY

normal normal

MXAY

affected

FXAXNcarrier

*on average

X-linked recessive diseaseRECAP

Pedigree Illustrating X-Linked Recessive Inheritance Pattern

Grand daughter

Uncle (mama)

Grand Uncle

Nephew

Great grand mother

Grand motherNani

Hemophilia : X-linked recessive

Queen Victoria (1819-1901) passed haemophilia A

on to many of her descendants

Empress of India 1876

Haemophilia leads to a severely increased risk of prolonged bleeding from common injuries, or in severe cases bleeds may be spontaneous and without obvious cause.

Haemophilia A is a genetic deficiency in clotting factor VIII which causes increased bleeding and usually affects males. About 70% of the time it is inherited as an X-linked recessive trait, but around 30% of cases arise from spontaneous mutations.

Victoria (Princess of Saxe-Coburg)

Edward(Duke of Kent)

Victoria (Queen of England) Albert

Victoria Frederick

Wihelm II Sophie George V

George VI

Queen Elizabeth

Prince Philip

Alice Louis Alfred Helena LouiseArthur

Leopold Helen Beatrice Henry

Nikolas II of RussiaAlix

Waldemar Henry

Alfonso XII Spain Eugenic

Alfonso Gonzalo

Alexic

Normal Male

Normal Female

Hemophilic Male

Carrier Female

Male died in infancyPossible hemophilic?

Inheritance of eye coloration in fruit fly, Drosophila:An example of sex-linked inheritance

Normal Red Mutant white

12

Then test and infer w w

w+w

W+ w+w+

w+w w+w

W

w

Problem TEST: A yellow body-colored female fruit fly was

mated to a male with normal body color (brown).

RESULT: All the female progeny were brown (normal) while all the male progeny were yellow.

EXPLAIN: What does this inheritance pattern signify?

Display this cross

What if individual chromosomes do not separate during meiosis? Error in chromosome number

Significance of chromosome numbers in in heritance

The Culprit: Non-disjunction

Older women are more likely than younger women to evidence chromosome damage and meiotic

irregularities

Pre-natal Diagnosis: Amniocentesis

Fetal cells present in amniotic fluid are then cultured and analyzed for chromosomes (karyotyping) and other biochemical tests

Will it be possible to detect a defective

gene by amniocentesis?

Monosomy: Turner’s Syndrome: 44+X

This baby has one X

chromosome. Yet it is a

female baby. Why?

Individual chromosome segregation going wrong during meiosis

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)

Why down’s syndrome does

not suggest abnormal sex

linked inheritance?

How can errors in chromosomal

segregation result in Down’s

syndrome?

People suffering from Down syndrome can lead a reasonably normal life

Genes and, chromosomes and some interesting facts about our inheritance

These cats are known as ‘Calico cats’ marked by their patchy coat (fur) coloration . The

coloration gene (or fur pigmentation gene) is present on the X chromosome (X-linked)

Coat coloration in XB/Xb cats

B= orange alleleb= Black allele

Possible Female genotypes

Possible male genotypes

Female cell where X carrying orange

allele is inactivated

Female cell where X chromosome carrying black allele allele is inactivated

However, one of the X chromosomes is randomly inactivated in female cat body

cells

Explain why male cats

have uniform coat

color

Explain why these female cats have uniform coat color

Explain why these female cats have patchy coat color

Note the fur/coat coloration in male and female cats

How will you name the gene that regulate eye

coloration?

How will you decide which one is recessive

and which one is dominant ?

What will be the consequence if the while gene is X-linked and Drosophila female and male have XX and XY sex chromosome constitution?

What types and proportions of F2 progeny are expected the white

gene were located on an autosome?

P1

Predict the phenotypic ratios in the progeny if a Drosophila female, heterozygous for white mutation is mated with a white eye-colored male ?

Do you think inheritance of white gene in Drosophila follows Mendelian laws?

Do you think inheritance of white gene display a chromosomal basis of inheritance? Which chromosome?

What if individual chromosomes do not separate during meiosis? Error in chromosome number

What if entire sets of chromosome do not separate? Error in chromosome sets

Significance of chromosome numbers and sets

Polyploidy in plants Colchicine inhibits chromosome segregation

In nature 3n, 4n, 6n and 8n plants are very common

Many varieties of wheat, cotton, banana, tobacco , strawberries etc are polyploids

Polyploidy results in bigger grain or fruit sizes and high yield which is beneficial

Thus, polyploidy is also artificially induced in plants by breeders using colchicine.

Polyploidy in plants

The cultivated strawberry is an octaploid hybrid derived from hybridization of two

natural octaploids.

Potato flowers. Potato is an autotetraploid.

This supermarket banana is most likely the common variety Cavendish, an autotriploid of Musa acuminata

Seeds of bread wheat. Wheat is a allohexaploid. Its genome consists of three ancestral parental genomes

DNA as a genetic material

Griffith’s experiment 1927:discovery of transforming factor (TF))

Non-virulent strain does

not kill

Heating kills virulent strain

Heat-killed virulent strain can transfer its

virulence “factor” to a non-virulent

strain

First historical demonstration of an chemical entity for genetic inheritance

Dies

Dies

Lives

Lives

Virulent

Non-virulent

Virulent, heat killed

Non-virulent + heat killed

Virulent strain kills the host

Pneumococcal infection

What does this heat killing signify with respective genetic inheritance

What is genetic transformation?

Which one was genetically transformed? Mouse or the bacteria?

Avery, MacLeod, McCarty

• Purified the transforming factor (TF) to investigate its properties.

• Salient points:– Treatment with protease did not destroy

TF– Treatment with RNase did not destroy TF– Purified TF did not have sugars

• Thus TF should be DNA Do these terms transforming factors, Mendelian factors and

DNA carry similar meaning ?

What about Mendelian alleles? Are these too transforming factors, Mendelian factors?

What is there in transforming or Mendelian factor? And now in DNA?

DNA carries digitized information Four digits are used to write genetic

codes

Watson and Crick proposed structure of DNA 1953

Thus, to understand the nature of genetic information we need to understand the basic DNA structure and how information flows:

Within an individual.

From one generation to the other

Within a cell

Journal Nature in its 171st volume on pages 737–738 ( 25 April 1953)

DNA is a very large molecule made up of a long chain of sub-units

The sub-units are called nucleotides

Each nucleotide is made up of

a sugar called deoxyribose

a phosphate group -PO4 and

an organic base

DNA molecule RECAP

A few general features you need to remember about DNA structure

Chargaff’s RuleChargaff’s Rule

• AdenineAdenine must pair with ThymineThymine

• GuanineGuanine must pair with CytosineCytosine

• Their amounts in a given DNA molecule will be about the sameabout the same.

G CT A

RECAP

DNA Double HelixDNA Double Helix

P

P

P

O

O

O

1

23

4

5

5

3

3

5

P

P

PO

O

O

1

2 3

4

5

5

3

5

3

G C

T A

H-bonds

H-bonds

RECAP

´

DNA strands run anti-parellel

´

Mark the features of DNA that makes it ideal as a genetic

materials

Sequence in one strand serves as the

template for the sequence in the

other

Sequence of nucleotides in DNA strands could store

genetic information

RECAP

DNA RNA protein Transcription Translation

Replication

Reverse transcription

Central dogma of Molecular biology सि�द्धांत

In subsequent lectures we will explore this flow of

information the decoding process

Flow of information

Nucleic acid to protein

Flow of information: nucleic acid to nucleic

acid

Nature of information flow

Why there is no loss of

information during

replication? How much of the information in DNA goes to

RNA??

Replication: synthesis of daughter DNA from parental DNA Transcription: synthesis of RNA using DNA as the template Translation: protein synthesis using mRNA molecules as the

template Reverse transcription: synthesis of DNA using RNA as the template

Flow of genetic information

Test your understanding

How many DNA molecules are present in a metaphase chromosome?

How many DNA molecules are present in a chromatid?

Are the DNA sequences of two sister chromatids are identical?

How many DNA molecules are there in a human sperm?

Question

Which aspect of information flow a sperm represent ?

Which aspect of information flow does these sister chromatids

symbolize ?

Approximate gene numbersChromosome 1 : 4220Chromosome 2 : 1,491

Chromosome 3 : 1,550

RECAP

Why did the author say that human genome is written in 23 chapters?

Why not 46?

Does a cell read these chapters one

by one - in a sequence?

How many chapters are there in our book of life?? And how many words/sentences are there in each chapter??

What is implied when one says that genetic information is being

readout?

Here ‘book of life’ is a metaphor

लाक्षणि�क रूप �ेउपमा

AUTOBIOGRAPHY OF A SPECIES IN 23 CHAPTERS

What is a gene/ Mendelian factor:

a letter, a word or

a sentence?

Next lecture:We would examine the nature of genetic information and how it flows within a cell

Slide series 6

Flow of genetic information: Three aspects -

Transcription, Translation&

Genetic code

Let’s first appreciate the challenge of transfer of information from DNA

(genes) to protein

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

Normal and Sickled Red Blood Cells in Blood Vessels

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.

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.

Source from http://www.nhlbi.nih.gov/health/dci/Diseases/Sca/SCA_WhatIs.html

• What could be the reason(s) for the wrong message from the DNA that produced 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??

Questions?

DNA RNA protein Transcription Translation

Replication

Reverse transcription

Central dogma of Molecular biology सि�द्धांत

We will now explore this flow of information: that is,

process of decoding the genetic message

Flow of information

Nucleic acid to protein

Flow of information: nucleic acid to nucleic

acid

Nature of information flow

Why there is no loss of

information during

replication? How much of the information in DNA goes to

RNA??

RECAP

Flow of genetic information A gene is transcribed into RNA

which is then translated into the polypeptide

DNA

Transcription

RNA

Protein

Translation

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)

Entire business of flow of genetic information thus depends on constructing (transferring information) one kind of polymer from

another

DNA RNA Protein(polypeptide chain

Transcription – Step I

Template DNA Strands unzip locally

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

Transcription – Step II

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

One of the DNA strands serve as a template and copied as mRNA

(messenger RNA)

mRNA

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

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

DNA

RNA

mRNA is then releases

                                                                       

Chemical structure of RNA

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.

Cytosine

Thymine

Uracil

Pyrimidine bases

DNA/RNA DNA RNA

In place of thymine in DNA

Transcription

RNA polymerase RNA nucleotides

Direction of transcription Template

Strand of DNANewly made RNA

TC

A T C C A A TT

GG

CC

AATTGGAT

G

U

C A U C C A AU

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

Steps in transcription of a geneRNA polymerase

DNA of gene

PromoterDNA Terminator

DNA

Area shownIn Figure 10.9A

GrowingRNA

Completed RNARNApolymerase

1 Initiation

2 Elongation

3 Termination

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

A T G A A G T T T T A G

A U G A A G U U U U A G

Transcription

Translation

RNA

DNA

Met Lys PhePolypeptide

Startcondon

Stopcondon

Strand to be transcribed

How does DNA/RNA code for protein?

Codon - a sequence of nucleotides that

codes for an amino acid

STEP III TRANSLATION

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

The dictionary of genetic code (64 potential codons)

आनुवान्शि�की कोड की शब्दकोश 5’-3’

A dictionary

offers translation from one language

to the other. Which two

languages are being translated

in a genetic

dictionary?

Why do we need

this translation

?

Where does it take

place?

What is the name

of this process?

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

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?

DNA

Messenger-RNA

Transfer RNA

Amino acid

Key players in decoding the genetic message

Transfer RNA

Leucine

Codon

Anti-codon

How genetic code is readout in the cell

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

DNA

3’ U G C A U A G C G C A U 5’ mRNA

5’

3’

5’G U C

3’

C A G

3’5’

tRNA

Amino acid

Anti-Codon

Codon

Many amino acids are specified by more than one codon-degeneracy

Codons specifying the same amino acid are called synonyms

Connect the three points about genetic codes: redundancy, degeneracy and synonymous

CUG CUC

Codon-anticodon pairing of two tRNA Leu molecules

5’- 3’ 5’- 3’Codon

GAC3’- 5’

Anti- codon GAG3’- 5’

Three codons direct chain termination

Three codons, UAA, UAG, and UGA signify chain termination.

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

Three Rules

Codons are read in a 5’ to 3’ direction. Codons are non-overlapping and the

message contains no gaps. The message is translated in a fixed

reading frame which is set by the initiation codon.

FATCATATETHERAT

Meaning and consequences of non-overlapping & comma-less reading of the triplet codons

FATATATETHERAT

Let’s consider a metaphor of a normal

DNA sequence

What is the

problem here?

What is the

consequence?

Mutation: a change in genetic code

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. Frame-shift mutation: Insertions or deletions of one or a small number of base pairs that alter the reading frame.

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

Codon Assignments from Repeating Copolymers

Organic chemical and enzymatic techniques were

used to prepare synthetic poly-ribonucleotides with known

repeating sequences.

Experimental Results:

UUU codes for phenylalanine. CCC codes for proline. AAA codes for lysine. The guanine residues in poly-G firmly

hydrogen bond to each other and form multistranded triple helices that do not bind to ribosomes.

copolymer Codons Recognized

Amino Acids Incorporated

Codon 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’

What Are Mutations?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

Nature of point 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.

Point Mutation Change of a single nucleotide Includes the deletion,

insertion, or substitution of ONE nucleotide in a gene

Types of Gene/Point Mutations

Substitutions InsertionsDeletionsFrameshift

Frameshift Mutation

Inserting or deleting one or more nucleotides

Changes the “reading frame” like changing a sentence

Proteins built incorrectly

Gene Mutation Animation

Problems THE FAT CAT ATE THE BIG RAT

THE FAT ATA TET HEB IGR AT Delete C

THE FAT ATA ATE THE BIG RAT Insert A

Frame shift mutation

RBC Structure

of Hb

Primary sequence of Hb –first 26 amino acid

RECALL

Example Point Mutation

• Sickle Cell disease is the result of one nucleotide substitution

• Occurs in the hemoglobin gene

Problem

Before the true nature of the genetic coding process was fully understood, it was proposed that the message might be read in overlapping triplets. For example, the sequence of GCAUC might be read as GCA CAU AUC

G C A U C

Think of test that would negate this possibility

Consider a gene that specified the structure of hemoglobin. Arrange the following events in the most likely sequence in which they would take place

a. Anemia is observed

b. The shape of the oxygen binding site is altered

c. An incorrect codon is transcribed into hemoglobin mRNA

d. The ovum (female gamete) receive a high radiation dose

e. An incorrect codon is generated in the DNA of the hemoglobin gene

f. A mother (an X-ray technician) accidentally steps in front of an operating X-ray generator

g. A child dies h. The oxygen-transport capacity of the body is severely impairedi. A wrong tRNA is attached j. Nucleotide pair substitution occurs in the DNA of the gene for hemoglobin

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?

Question

Home assignment 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

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 argf. Wild type phe leu pro ser val thr thr arg trp

1 2 3 4 5 6 7 8 9

Codon

For each mutant, say what change has occurred at the DNA level, whether the change is a base pair substitution or frame shift mutation, and in which codon the mutation occurred

Home assignment 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 ?

Slide series 7

Gene regulation - Prokaryote- Eukaryotes

Gene regulation

Regulation of the synthesis of a gene’s transcript and its protein

product is termed as gene regulation.

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: 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

Transcriptional Regulation is mediated by controlling the access of the RNA polymerase to

the promoter.

RNA polymerase Gene

Promoter Direction of transcription

Basic schemePromoter:

RNA Pol binding site in DNA sequence

upstream of transcription start point

The promoters of genes transcribed by RNA polymerase II consist of a core promoter and a regulatory promoter that contain

consensus sequences.

Not all the consensus sequences shown are found in all promoters.

A point to remember:Like transcription

initiation there are also transcription termination

mechanisms

We will not go in to these details either

Negative Regulation

Activator

RNA Polymerase

X

Repressor

Binding of repressor blocks the

binding of the RNA Pol to the

promoter

Positive Regulation

X

RNA Polymerase

An activator help RNA Pol bind to the promoter

Two broad strategies for regulation of prokaryotic transcription

In prokaryotes gene regulation takes place in response to the environment

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.

On the other hand, when only lactose is available it turns “ON” the genes and therefore the enzymes required for lactose breakdown

Glucose

Glucose Galactose

Lactose

When lactose is absent in the medium, the lac Z gene is switched-off . That is, no mRNA is transcribed and no proteins are made.-In the presence of lactose in the medium, lac Z gene is turned on. That is, mRNA is transcribed and the proteins are made.

X

X

REPRESSOR

INDUCER

INACTIVE REPRESSOR

OPERATOR:Binding site for repressor

Regulation of the lac Z gene – an example of negative regulation ( lacZ gene codes for -galactosidase enzyme that breaks down

lactose ) What can

be the inducer of the lac Z

gene?

Answer:lactose

X

Illustration of a NEGATIVE REGULATION gene expression

INDUCIBLE TRANSCRIPTION

X

REPRESSOR ACTIVE

INDUCERREPRESSOR

INACTIVE

Regulation of lac Z gene This is also called

an inducible model of gene

regulation. WHY?

Repressor binding site

OPERATOR

Question:1Repressor is a protein: should

there be a separate gene for repressor?

Question:2How will lac Z gene be regulated if a gene coding for

repressor is mutatedQuestion:3

What will be the consequence if

operator sequence is altered?Question:4

What will happen if promoter sequence is

altered

ConceptMutations need always directly alter the target gene. There are

alternative ways of altering gene expression, other than mutations within the gene

RNA POl

Further, lac Z 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’

RIBSOSOME BINDING SITE

How many messenger

RNA are made by the lac operon?

P O lacZ lacY lacA

Proteinsb-galactosidase Permease Transacetylase

The lac Operon

mRNA 5’ 3’

How many polypeptide

(proteins) are made by the lac operon?

Why would you consider the lac operon a smart system?

In the presence of lactose in the medium, lactose binds to the repressor. The lactose-repressor complex is unable to bind to the

operator.

P O lacZ lacY lacA

X NO mRNA

lactose

P O lacZ lacY lacA

X NO mRNA

Regulation of the lac Operon by the repressor

Note that there are two binding sites in the repressor one for the operator and the other for

the inducer

Once “Inducer-Repressor” complex moves away from the Operator (O) RNA polymerase can bind to the

promoter (P) and initiate transcription of the lac Operon

P O lacZ lacY lacA

mRNA

Regulation of the lac Operon by the repressor

P O lacZ lacY lacA

lac repressor

X NO mRNA

lac repressorThe lac repressor binds to the operator and

inhibits transcription of the lac operon.

About the lac I gene that codes for the repressor

Question: Should the lac I gene be

always active (constitutive) or sometimes active

(inducible)?

QuestionLac 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

Eukaryotic Gene Regulation:

Most eukaryotic genes are controlled individually and have regulatory sequences that are much more complex than those of the lac operon.

All of the cells in a multi-cellular organism carry the complete genetic code in their nucleus, but only a few of the available genes can be expressed in the appropriate cells of different tissues.

Complex regulation allows for this specificity.

Why is gene regulation in eukaryotes more complex than in prokaryotes?

Gene regulation determined the complexities of the eukaryotic

organisms

Eukaryotic DNA bind with a variety of protein.

DNA and a group of proteins called ‘histones’ form the primary chromatin structure.

Chromatin in turn complexes with a large array of ‘non-histone’ proteins

chro

mat

inch

rom

osom

e

Incr

easin

g co

mpl

exity

of h

ighe

r ord

er st

ruct

ure

GGGCGG

CCAAT

TATA

mRNA

-200 bp -100 bp -30 bp

Promoter proximal elements Promoter

Region upstream of the transcription start site in higher eukaryotes

Promoter proximal elements

Regulatory protein

Promoter Transcribed region of a gene

RNA polymerase

Regulatory elements

Promoter

RNA transcript

DNA

Regulatory elements and promoter of an eukaryotic gene

The effect of specific point mutations in the proximal regulatory elements and promoter on β-globin gene transcription. Each line represents the transcription level of in a mutant relative to that of a wild-type β-globin gene. Red arrows shows some the mutations that affect gene transcription. The black dots represent nucleotides for which no mutation had been generated.

Rela

tive

tran

scrip

tion

leve

l Functional role of the regulatory elements and

promoter Question:Can changes

in the sequence of regulatory sequences too cause

gene mutation?

Applications of the understanding of the gene regulation

What if lacZ gene is fused with the promoter of another gene?

lacZ

A different promoter

Background information Genes expression is regulated during development – the colored stripes represent the areas where a certain gene (named even-skipped) is expressed . Note a total of total

seven stripes

Normal 7 stripes of even-skipped gene in Drosophila embryo

Stripe 2 module

Regulatory elements of a gene determine its expression in specific locations

Now a bacterial lacZ gene fused to only stripe 2 module of

even skipped regulatory element and injected back into

the fly embryo

Even skipped regulatory region

What does this finding

reveal?

Recombinant plasmid containing rat growth hormone fused to mouse metallothionein regulatory region in a bacterial plasmid vector

This recombinant plasmid,was injected into the mouse oocytes.

Promoter function seen in transgenic mice.

Mt-1 (metallothionein) gene is induced by heavy metal

A mouse derived from the eggs injected with growth hormone gene (left) and a normal littermate (right). The expression of a ‘transgene' mostly depends on the regulatory sequences which have been used in the design of the gene construct. A classical example is the transgenic giant mice in which the expression of a growth hormone (GH) gene is driven by the metallothionein promoter

Key conclusions: Genes regulate functions- GH Growth Promoters drive gene expression Promoter of each gene each gene is uniquely regulated

Which one is the mother?