Download Cellular Control

Cellular Control
Module 3 A2 Biology
Context
The way that DNA codes for proteins is
central to our understanding of how cells
and organisms function
 The way in which cells control chemical
reactions determines the ways in which
organisms grow, develop and function

Objectives/Learning Outcomes
State that genes code for polypeptides
 Be able to explain the meaning of the term
Genetic Code
 Describe with the aid of diagrams, the way
in which a nucleotide sequence codes for
the amino acid sequence in a polypeptide

Model of DNA



Watson and Crick worked out the structure of DNA in 1953
The secondary structure of DNA helix similar to the
secondary structure of proteins. The structure of the protein
alpha-helix was only held together by hydrogen bonds
detected by X-ray diffraction studies.
Research by E. Chargaff showed:

a) The concentration ofAdenine equals that of thymine
 b) The concentration of guanine equals that of cytosine.



X-ray diffraction data showed a helical pattern repeating
every 34 nm with 10 subunits per turn occurs. Each subunit
takes the same amount of space occupied by a unit of a
single nucleotide.
Watson and Crick used this information to develop the
double helix as a model for DNA helix.
The double helix of DNA helix of two right-handed
polynucleotide chains that are wrapped around the same
axis.
DNA






2 strands of nucleotides
Deoxyribose-phosphate backbone
Nitrogenous bases as rungs
A-T,C-G (complementary base pairs)
Hydrogen bonds between bases
DNA can be copied over and over
because of the complementary base
pairing

The sequence of bases along a length of
DNA provides the code for a sequence
of amino acids. These amino acids are
the start of a polypeptide or protein.
 About
20 amino acids
 A length of DNA that codes for one
polypetide is called a gene
Genetic
Fingerprinting
Genetic Fingerprinting
There are approximatley 4000 genes
 Only 2% of the total DNA consists of
genes
 Rest is non-coding sequences called
introns
 Each intron is 60-100,000 bases long
 A single gene can have up to 50 introns
between its exons (coding regions)
 No known function of introns

Minisatellites

Within the Non-coding region are short
sequences of bases
 Core


sequences
Repeated over and over again
Repeating regions
 Minisatellites
or
Variable number tandem repeats


Different individuals have different numbers of
repeated core sequences
Greater the number of repeats=longer
minisatellite
Genetic Fingerprinting
Number of repeats is different
 Each individual has 50-100 different
types of minisatellites
 Chances of 2 individuals having
matching minisatellites is miniscule

 Exception???

Chose minisatellites that show the
most variation and you have DNA
profiling
Making a DNA Fingerprint

Extraction
 Sample
of cells
 Extract DNA with mixture of water
saturated phenol and chloroform (proteins
percipitate out)
 Only 0.5cm3 of blood, 0.005cm3semen or
one hair root
Making a DNA Fingerprint

Digestion
 Add

restriction enzymes to DNA
WHY??
 Recognise
sequences close to but not
within minisatellite region
 Left with DNA of different lengths
Making a DNA Fingerprint

Separation
 Fragments
separated according to size
by electrophoresis
 Place DNA fragments in agarose gel
 Pass an electric current through gel
 DNA carries negative charge

Move to + electrode
 Which
fragments will move quicker?
Making a DNA Fingerprint
Smaller fragments move quicker
 Fragments are separated into bands

-
+
BUT NOT YET VISIBLE AT THIS STAGE
Making a DNA Fingerprint

To show the band a probe is added
 Single
strand of complementary DNA
 Which cannot attach to the existing double
strand of DNA


Separate the strands into single strands by
immersing gel into alkaline solution
Use technique called Southern blotting
to transfer single strands onto a nylon
membrane
Making a DNA Fingerprint
Put thin nylon sheet over gel
 Cover with absorbent towels

 This
draws DNA onto nylon by capillary
action

Fix onto membrane with UV light
Making a DNA Fingerprint

Hybridisation
A
radioactive probe is used to bind onto and
reveal the location of a certain type of
minisatellite
 Probe consists of single strand of DNA
complementary to the core sequence
 Excess probes washed away
 Process can be repeated with different probes
 Place X-ray film over nylon membrane
 At last a DNA Profile
Making a DNA Fingerprint




Analysing
Visual inspection carried out to check for a
match
If suspected match and automated
scanning system is used to calculate the
length of the DNA fragments denoted by
the bands.
Odds are then calculated as to the likely
hood of the person committing the crime
Genetic Fingerprinting
Genetic Fingerprinting

Problems with contamination
 Until
1989 DNA was thought to be indisputable
 Forensic samples are rarely pure blood

Contamination with DNA from bacteria and fungi
 Delay
in collection of the sample means the DNA
could have decomposed resulting in longer or
shorter fragments being produced
 Ions in contaminants could affect the charge on
DNA fragments
Discoverer of genetic fingerprinting calls for universal DNA testing
By Andrea Babbington
Sunday, 18 February 2001
The discoverer genetic fingerprinting says the entire population should be DNA
tested in order to combat serious crime.
.
Hep C may benefit from genetic fingerprinting, research says
September 28, 2010
Genetic fingerprinting may predict who will benefit from early hepatitis
C treatment and who will clear the virus spontaneously, new research
shows.
VITAL DNA samples were left in a police fridge next to a halfeaten takeaway meal, it was revealed today.
Cambridgeshire cops were slammed for failing to properly store
crucial forensic evidence.
And they were warned their shoddy practices could have led to
miscarriages of justice.
Polymerase Chain Reaction
Polymerase Chain Reaction
•A minute quantity of DNA can increased a billion fold or more
• PCR involves the repeated replication of DNA in a test-tube
• The amount of DNA is replicated with each cycle
• If after 2 cycles there are four strands of DNA how many
strands would there be after
• 10 cycles
• 20 cycles
Mutations
Objectives/Learning Outcomes
State that mutations cause changes to the
sequences of nucleotides in DNA
molecules
 Explain how the mutations effect proteins
and ultimately organisms

Mutations
CAN THE BIG RED HEN LAY ONE EGG
CAN THE BIG RED MEN LAY ONE EGG
CAN ATH EBI GRE DHE NLA YON EEG G
CAN HEB IGR EDHE NLA YON EEG G
Mutations
CAN THE BIG RED HEN LAY ONE EGG
Point mutation
CAN THE BIG RED MEN LAY ONE EGG
Insertion
CAN ATH EBI GRE DHE NLA YON EEG G
Deletion
CAN HEB IGR EDHE NLA YON EEG G
Here is a sequence of DNA bases on a
piece of DNA coding strand
ATGTTTCCTGTTTACCATCGC
Mutation 1
ATGTTTCCTGTTAAATAACATCGC
Mutation 2
ATGTTTCCTATTAAATACCATCGC
Mutation 3
ATTTTTCCTGTTAAATACCATCGC
Mutation 4
ATGTTCCTGTTAAATAACATCGC
ATGTTTCCTGTTTACCATCGC
For each strand, work out:
Mutation 1
1. The sequence of amino acids
ATGTTTCCTGTTAAATAACATCGC
2. The type of mutation (point
mutation, insertion/deletion)
3. The sequence of bases on the
template DNA strand
Mutation 2
ATGTTTCCTATTAAATACCATCGC
4. The sequence of bases on the
mRNA strand
5. The tRNA anticodons for each
mRNA,
Mutation 3
ATTTTTCCTGTTAAATACCATCGC
Mutation 4
ATGTTCCTGTTAAATAACATCGC
Mutations
Sickle cell anaemia
 Melanin in skin
 Cystic fibrosis
 Huntington disease

Not all mutations are harmful, some have no effect (neutral)
whilst others can be harmful or beneficial
Lac Operon
Week 13
The E. coli lac operon and its regulator gene.
© Pearson Education Ltd 2009
This document may have been altered from the original
Week 13
How the lac operon works by stopping RNA polymerase binding to the promoter
region when lactose is absent from the growth medium
© Pearson Education Ltd 2009
This document may have been altered from the original
Week 13
How the lac operon works when lactose is present
© Pearson Education Ltd 2009
This document may have been altered from the original
Halfterm essays
Working out the genome
 Is genetic engineering right?
 Genetic fingerprinting applications
 Mutations and evolution

Gene Technology
Recombinant DNA







Isolate the gene, e.g gene for insulin
Cut open a vector that will help in the transfer
of the gene to fast growing cells
‘Stick’ the gene into cut vector
Put the vector back into fast growing cells
Find the cells that have successfully taken up
the human gene
Grow transformed cells using a fermenter
Isolate and purify the human protein made by
these cells
Isolate the gene
Find the amino acid sequence for the
target protein
 Work out the base sequence that would
code for the protein
 Make gene from artifical DNA (cDNA)

or

Use mRNA molecules carrying the code to
make artificial genes
mRNA + DNA nucleotides
 ---- cDNA + mRNA

or
Use DNA probes to find the gene
 Then use restriction endonucleases to
cut out the gene

 Recognition
site
 Blunt ends or sticky ends
Splicing- inserting the gene



The gene must be inserted into a
cell we can use to make the protein
Vectors (carriers) carry a gene into
a cell (bacterium or yeast) that will
make the protein.
Plasmids and bacteriophages are
used to do this
Plasmids
• Cut genes and cut
plasmids are mixed
together
• The sticky ends join
together- Ligation
•controlled by enzyme
Ligase
Bacteriophage
Viruses that infect
bacterial cells
 Can be used to infect the
bacteria with the gene
required

Culturing host cells
Not all cells take up the genes
 Need to able to select bacteria that have
taken up the gene.
 Use plasmids that carry genes that are
resistant to antibiotics
 When grown with media containing
antibiotics only those with the gene will
then grow

Use of recombinant technology

Make a list of uses of recombinant DNA
from your text books.
Genetic Control
Objectives

Explain the genetic control of protein
production in Prokaryotes
Control of protein production
Most genes are only expressed in certain
cell types and under certain circumstances
 In prokaryotes gene expression is
controlled by other regions of DNA that lie
close to the code for the amino acid
sequence of the protein
 The whole structure is called an operon

Escherichia Coli (E.Coli)


2 French scientist discovered that E.Coli could respire
glucose and lactose (found in milk)
Has genes that code for synthesis of 2 enzymes that help
the digestion and absorption of Lactose
Lactose
 Lactose

β
Permease
Allows cell to take up lactose
galactosidase
(Lactase)
 Catalyses the hydrolyses of lactose to glucose and galactose
Use the video clips and your
copy of the lac operon to
make your own notes.
Keywords
Operon
A length of DNA that contains the base
sequence that codes for the proteins, known as
Structural genes, and also other base sequences that
determine whether or not the gene will be switched on. A
Transcription factor is a protein that binds to DNA and
switches genes on or off.
lac operon in E.Coli
Escherichia Coli





Respires glucose but can survive in a medium of lactose
Lactose is a disaccharide made of galactose and glucose.
If a bacterium is grown on a medium containing only
glucose it doesn’t produce β-galactosidase or lactose
permease.
The genes that code for them are not expressed- they are
switched off!
If the bacterium is transferred to a medium containing
only lactose then the genes are switched on and βgalactosidase and lactose permease are produced again.
lac operon in E.Coli
lac operon in E.Coli
Promoter, where
RNA polymerase
must bind to begin
transcription of mRNA
Operator,
if
nothing is bound
to the operator
then the promoter
is available for
RNA polymerase
to bind
Longest length makes up
the structural genes which
code for –
β-Galactosidase (Z) which
catalyses the hydrolysis of
lactose to glucose and
galactose
-lactose permease (Y)transports lactose into the
cell
Regulator gene, the regulator DNA codes for a protein called a Repressor
Protein. The repressor protein has 2 binding sites one to fit the Operator,
this prevents RNA polymerase binding whilst the other site binds with
lactose, when bound it changes the shape of the repressor protein so that it
no longer fits onto the Operator DNA so it would free RNA polymerase to
transcribe
cAMP






Protein activation can be controlled by molecules e.g.
hormones and sugars
Some of these molecules work by binding to cell
membranes and triggering the production of cAMP
cAMP activates proteins inside the cell altering its
3D structure
This can change the active site of an enzyme making it
more or less active
Some proteins are needed for transcription to occur are
activated by cAMP which alters their 3D structure
Glucose reduces the amount of cAMP in the bacterial
cell
Not mentioned in Spec but…..





When glucose is available E.Coli will use this sugar in
preference to other sugars
When it finds glucose and lactose in equal amounts it
represses the use of lactose by suppressing the lac
operon
Glucose reduces the amount of cAMP in the bacterial
cell
Remember cAMP can activate proteins
The lack of cAMP has an inhibitory effect on an
additional protein that increases the production of βgalactosidase
 Glucose
reduces the amount of cAMP
in the bacterial cell
Remember cAMP can activate proteins
 A transcription control protein

Past paper question
1. The bacterium Escherichia coli uses glucose as
a respiratory substrate. In the absence of
glucose, E. coli can use lactose.
Explain how lactose induces the enzyme
system involved in its uptake and metabolism.
Homeobox genes
All animals have similar genes that control
the development of their general body
plan.
 Plants and fungi also have them
 Homeobox genes function by switching on
or off whole sets of other genes that bring
about processes resulting in the formation
of a particular part of the body

Apoptosis

Enzymes break down the cell cytoskeleton. The cytoplasm becomes
dense with organelles tightly packed.

The cell surface membrane changes and small bits called blebs
form

Chromatin condenses and the nuclear envelope breaks. DNA
breaks into fragments. The cell breaks into vesicles.

The vesicles are taken up by phaocytosis. The cellular debris is
disposed of and does not damage any other cells or tissues.The
whole process occurs very quickly.